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Fixed Mirror
Transducer
MEMS Mirror
Plastic Film
Photoacoustic
To m o g r a p h y
HUABEI JIANG
Photoacoustic
To m o g r a p h y
HUABEI JIANG
University of Florida, Gainesville, FL, USA
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To Yonghong, Max, and Michael
Contents
Preface.......................................................................................................................xi
About the Author.................................................................................................... xiii
v
vi Contents
Bibliography.......................................................................................................... 273
Preface
Photoacoustic tomography (PAT) is an emerging biomedical imaging modality that
combines the high-contrast and spectroscopic-based specificity of optical imaging
with the high spatial resolution of ultrasound imaging in a single modality. By lis-
tening to light, PAT detects tissue-absorbed photons ultrasonically through the pho-
toacoustic effect. Since ultrasonic scattering is two to three orders of magnitude
weaker than optical scattering in tissue, PAT breaks the 2–4 mm spatial resolution
limit associated with pure optical tomography such as diffuse optical tomography
(DOT) for deep tissue imaging, or the ~1 mm depth limit associated with confo-
cal and multiphoton microscopy and optical coherent tomography (OCT). In PAT,
tissue is excited with a short (typically a few nanoseconds) laser pulse (focused or
unfocused); the subsequent laser-induced transient photoacoustic waves in the range
of 1–100 MHz, due to the transient thermoelastic expansion of light-absorbing
components in tissue, are detected by wideband unfocused or focused ultrasound
transducer(s). Unique advantages of PAT are that functional or biochemical parame-
ters such as deoxy-hemoglobin (HbR), oxy-hemoglobin (HbO2), water (H2O), lipids,
and so forth along with vasculature and blood flow can be imaged in high resolution.
In addition, highly specific molecular PAT can be realized through the use of molec-
ular contrast agents. Finally, PAT can be made portable for bedside applications, is
economical, and uses non-ionization radiation.
PAT has found its potential clinical applications in several areas. In breast imag-
ing, PAT offers the submillimeter-resolution ability to quantitatively image the high
optical contrast generated through the presence of blood, water, and lipid, which are
the predominant transformations associated with malignancy. Clinical studies con-
ducted at multiple institutions and countries have repeatedly shown that 2-to-11 and
higher absorption contrasts exist in breast cancers that can be imaged by PAT. These
studies suggest that PAT has the potential to detect breast tumors at early stages.
Application of PAT to joint imaging has been recently explored, offering an oppor-
tunity for early detection and monitoring of progressive diseases including osteoar-
thritis and rheumatoid arthritis. In this case, the optical contrast is produced through
the degraded articular cartilage and the increased water content and turbidity in
the synovial cavity. Other clinical applications of PAT also are starting to appear,
including intravascular imaging and intraoperative imaging during cancer treatment.
While brain imaging and electrophysiology play a central role in neuroscience
research and in the evaluation of neurological disorders, a single noninvasive modal-
ity that offers both high spatial and temporal resolution is currently not available.
PAT may become such a neuroimaging modality at least in animals. In Chapter 8,
I show an acute epilepsy rat model in which PAT can noninvasively track seizure
brain dynamics with both high spatial and temporal resolution and at a depth that is
clinically relevant. The noninvasive yet whole surface and depth capabilities of PAT
actually seeing what is happening during ictogenesis in terms of seizure onset and
spread.
xi
xii Preface
The concept of PAT emerged in the mid-1990s, and the field of PAT is now rap-
idly moving forward. Several reviews and edited books on PAT have been published.
However, I have decided to write a book that presents a detailed and comprehensive
treatment of PAT. It appears timely to produce such a book for the first time in this
field. The book is essentially a collection of the research work that the author and
his colleagues have been pursuing over the past decade. This actually presents a
unique feature of the book as it allows the principles of PAT and its applications
to be treated in a systematic way. In addition, the collection covers almost every
aspect on PAT from mathematics, image reconstruction methods, instrumentation,
and phantom/animal experiments to clinical applications. I believe this book will be
particularly useful for graduate students and researchers who wish to enter the field
of PAT.
In Chapter 1, the fundamentals of PAT are presented including the theory on photo-
acoustic effect, various image reconstruction methods, and instrumentation. Chapter
2 describes various advanced methods for quantitative PAT, which allow the recov-
ery of tissue optical absorption coefficients and/or acoustic properties. Experimental
validations of the reconstruction methods are also presented here. The develop-
ment of several image-enhancing schemes including both software and hardware
approaches are discussed in Chapter 3. Chapter 4 describes array-based PAT systems
that are the foundation for the realization of 2D, 3D, and 4-D PAT. Photoacoustic
microscopy (PAM), a variation of PAT, is detailed in Chapter 5. The recent devel-
opment of multimodality methods is presented in Chapter 6. These works describe
the combinations of PAT/PAM with other imaging methods including DOT, fluores-
cence molecular tomography (FMT), ultrasound imaging and OCT, which represent
new directions in the field of PAT. Chapter 7 focuses on the discussion of contrast
agents–based molecular PAT where both nontargeted and cell receptor–targeted
methods are included. Chapter 8, the final chapter, describes the clinical applications
and animal studies in the areas of breast cancer detection, osteoarthritis diagnosis,
seizure localization, intravascular imaging, and image-guided cancer therapy.
I wish to thank my previous and current colleagues whose contributions to the
works presented have made this book possible. I would also like to thank the staff
at CRC Press, especially associate editor Ashley Gasque and project coordinator Jill
Jurgensen, who have worked diligently at bringing this book to fruition. Finally, my
wife, Yonghong, and my family made it all worthwhile.
Huabei Jiang
Gainesville, Florida
About the Author
Huabei Jiang, PhD, is J. Crayton Pruitt Family professor in the Department of
Biomedical Engineering at the University of Florida. He has published more
than 300 peer-reviewed scientific articles and patents. Dr. Jiang is a fellow of the
Optical Society of America (OSA), a fellow of the International Society of Optical
Engineering (SPIE), and of the American Institute of Medical and Biological
Engineering (AIMBE).
xiii
1 Fundamentals of
Photoacoustic
Tomography
1.1 PHOTOACOUSTIC EFFECT
1.1.1 Empirical Description
The basic physical principles behind PA effect can be demonstrated using some
approximate solutions. For generality, a 3D model using lateral dimensions x and y and
depth z needs to be considered. Biological tissue is homogenously irradiated with a
short laser pulse of energy E (J) on an area A (m2), and the photon density or radiant
exposure (J/m2) on the surface can be approximately stated as,
I ( x , y, z ) ≈ Φ 0 e − eff z (1.2)
1
2 Photoacoustic Tomography
where μeff is the effective optical attenuation coefficient, which is a function of opti-
cal absorption coefficient, a and reduced scattering coefficient, s ,
eff = 3 a ( a + s ) (1.3)
It should be noted that the photon density here can be accurately calculated
using the photon diffusion equation with any kind of source distribution. In PA
effect, the stress generation depends on the locally absorbed light energy density,
Ψ that is the product of optical absorption coefficient, a and photon density, Φ
(Norton and Vo-Dinh 2003),
Ψ= Φ (1.4)
a
With mass density, ρ (kg/m3) and specific heat, Cp (J/(kg°C)), the absorbed light
energy leads to a temperature increase (°C),
T ( x , y, z ) = Ψ( x , y, z )/(ρ.C p ) (1.5)
Additionally due to laser excitation, the absorbed light energy also gives rise to a
fractional volume expansion dV/V,
κ = C p /(ρvs2Cv ) (1.7)
where Cp and Cv are, respectively, the specific heat capacities at constant pressure
and volume, and vs is the acoustic velocity. If the laser excitation is in both the ther-
mal and stress confinements, the fractional volume expansion is negligible and the
local pressure rise can be obtained from Equation (1.6),
p0 = β T/κ (1.8)
p0 = β a Φ/κρCv = Γ a Φ (1.10)
Fundamentals of Photoacoustic Tomography 3
For water and diluted aqueous solution, Γ can be estimated by the following
empirical equation,
If the laser pulse width is much shorter than the thermal relaxation time, τth , the
excitation is said to be in thermal confinement, and heat conduction is negligible dur-
ing the laser excitation. The thermal confinement condition is written,
Example
A laser pulse with energy E = 30 mJ, pulse length = 10 ns, beam radius r = 1 cm
and wavelength = 550 nm irradiates human blood vessel. The radiant exposure is,
Then we can obtain the temperature increase and initial pressure (assuming ρ =
1 g/cm3 and Cp= 4 Jg–1K–1),
To obtain this pressure, stress confinement with a region d = 1/μa = 400 μm and
sound velocity vs = 1.5 μm/ns needs to be satisfied: τp = 10 ns < d/vs = 266 ns.
4 Photoacoustic Tomography
1.1.2 Rigorous Theory
The empirical description presented in Section 1.1.1 is intended to illustrate the PA
effect and certainly cannot be used for the purpose of imaging. The PA wave genera-
tion and propagation in biological tissue is rigorously described by the Helmholtz-
like wave equation. To derive the PA wave equation, three general elastic mechanical
and fluid dynamic equations are responsible for the PA generation and propagation:
Newton’s law of motion, equation of continuity, and thermal elastic equation:
∂
ρ(r ) V (r , t ) = − p(r , t ) (1.14)
∂t
1 ∂ ∂
⋅ V (r , t ) = − 2
p(r , t ) + β T (r , t ) (1.15)
ρ(r ) vs (r ) ∂t ∂t
∂
ρ(r )C p T (r , t ) = H (r , t ) (1.16)
∂t
where V is particle velocity and H is the laser excitation source term which can be
written as H = ΨI(t), where I(t) is the temporal illumination function. Considering
Equations (1.14)–(1.16) and eliminating V, we obtain
1 1 ∂2 β ∂
ρ(r ) ⋅ p(r , t ) − 2 2
p(r , t ) = − H (r , t ) (1.17)
ρ(r ) vs (r ) ∂t C p ∂t
2 1 ∂2 β ∂
p(r , t ) − p(r , t ) = − H (r , t ) (1.18)
2
v (r ) ∂t 2
s C p ∂t
2 ∂2 β ∂
p(r , t ) − p(r , t ) = − H (r , t ) (1.19)
v ∂t 2
2
0 C p ∂t
where v0 is the constant acoustic velocity within the entire problem domain. Denoting
the following Fourier transform expression for acoustic pressure,
+∞
P(r , ω ) =
∫ p(r , t) exp(−iωt) dt (1.20)
−∞
Fundamentals of Photoacoustic Tomography 5
and taking the Fourier transform on Equation (1.18) with respect to variable t, we obtain
the PA wave equation in frequency-domain (assuming H = ΨI (t ) = Ψδ(t − t0 ) ):
2 βΨ(r )
P(r , ω ) + k 2 P(r , ω ) = ik vs (1.21)
Cp
2 βΨ(r )
P(r , ω ) + k02 P(r , ω ) = ik0 v0 (1.22)
Cp
where k0 = ω/v0 . If both the acoustic velocity and attenuation are heterogeneously
distributed, the frequency-domain PA wave equation is then described as:
2 βΨ(r )
P(r , ω ) + k02 (1 + O) P(r , ω ) = ik0 v0 (1.23)
Cp
where O is a coefficient that depends on both acoustic velocity and attenuation as follows:
∑ w S (t + δ )
i
i i i
S (r , t ) = (1.25)
∑w i
where S(r, t) is the image output at a particular spatial point r within the imaging
domain, Si(t) is the time-resolved acoustic signal from the ith receiver, δi is the
6 Photoacoustic Tomography
delay applied to this signal, and wi is an amplitude weighting factor, which is used
to enhance the beam shape, to reduce sidelobe effects, or to minimize the noise
level. Note that δi = |Ri – r|/v is just the time needed for the acoustic signal to travel
from point r to point Ri, where Ri is the spatial location of the ith receiver and v is
the assumed constant acoustic velocity. The summed signal is typically normalized
to make the output independent of the actual set of transducers. This delay-and-sum
algorithm has been tested and evaluated using considerable phantom and in vivo
experiments (Hoelen and de Mul 2000, Manohar et al. 2007).
2 βΨ(r ) (1.26)
P(r , ω ) + k02 P(r , ω ) = ik0 v0
Cp
Fundamentals of Photoacoustic Tomography 7
where the following second-order absorbing boundary conditions have been applied
(Jiang et al. 2006):
∂2 P
P ⋅ nˆ = ηP + γ (1.28)
∂φ2
where η = (−ik0 − 3/2ρ + i3/8 k0 ρ2 )/(1 − i/k0 ρ) and γ = (−i/2 k0 ρ2 )/(1 − i/k0 ρ) , N is the
total number of nodes of the finite element mesh, <(⋅)> indicates the integration over
the problem domain, and ≡ expresses the integration over the boundary. In both the
forward and inverse calculations, the unknown Ψ needs to be separated into real (ΨR)
and imaginary (ΨI) parts, both of which are expanded in a similar fashion to P as a
sum of unknown parameters multiplied by a known spatially varying basis function.
The matrix form of Equation (1.27) is expressed as follows:
where
∂2 ψ j
Aij = ψ j ⋅ ψ i − k02 ψ j ψ i −
∫ ηψ j + γ
∂φ2
ψ i ds (1.30)
Bi = − Ik0 v0β ∑Ψ
k
R,k ψ k ψi C p + k 0 v0 β ∑Ψ
l
I ,l ψlψi C p (1.31)
To form an image from a presumably uniform initial guess of the optical prop-
erty distribution, we use iterative Newton’s method to update ΨR and ΨI from their
starting values. In this method, we Taylor expand P about an assumed (ΨR, ΨI)
R ,Ψ
distribution, which is a perturbation away from some other distribution, (Ψ I ),
such that a discrete set of P values can be expressed as
P (Ψ I ) = P (Ψ R , Ψ I ) + ∂ P Ψ R + ∂ P
R ,Ψ Ψ I + (1.33)
∂Ψ R ∂Ψ I
considered as true data (observed or measured), and the relationship can be truncated
to yield
J χ = P o − P c (1.34)
where
and Pi o and Pic are measured and calculated acoustic field data based on the assumed
(ΨR, ΨI ) distribution data for i = 1,2,…M boundary locations. ΨR,k (k = 1,2, …, K)
and ΨI ,l (l = 1,2,…, L) are the reconstruction parameters for the optical property
profile. In order to realize an invertible system of equations for χ, Equation (1.34)
is left multiplied by the transpose of J to produce
( J T J + λI) χ = J T (P o − P c ) (1.39)
where regularization schemes are invoked to stabilize the decomposition of J T J;
I is the identity matrix, and λ is the regularization parameter determined by com-
bined Marquardt and Tikhonov regularization schemes. We have found that when
T
λ = (P o − P c ) × trace [ J J] , the reconstruction algorithm generates best results for
PAT image reconstruction. The process now involves determining the calculated
acoustic field data and Jacobian matrix. The reconstruction algorithm here uses the
hybrid regularization-based Newton method to update an initial (guess) optical prop-
erty distribution iteratively via the solution of Equations (1.29) and (1.39) so that an
object function composed of a weighted sum of the squared difference between com-
puted and measured acoustic pressures for all acoustic frequencies and optical wave-
lengths can be minimized.
2 1 ∂2 p(r, t ) βΨ (r) ∂ I (t )
p(r, t ) − 2 2
=− (1.40)
v0 ∂t Cp ∂t
N N
∂2 p j 1
∑ ∫
j =1
pj
S
ψ i ⋅ ψ j dS + ∑ j =1
∂2 t ∫ S v02
ψ i ψ j dS −
∫ ψ
l
i p ⋅ nˆ dl
βΨ ∂ I
=
∫ S C P ∂t
ψ i dS (1.41)
Bm p = 0 (1.42)
m
1 ∂ ∂ 4l − 3
Bm = ∏ +
v0 ∂t ∂r
+
2r
(1.43)
l =1
1
Bm = O 1
(1.44)
2 m +1+
r 2
∂p 1 ∂p p
B1 p = + + = 0 (1.45)
∂r v0 ∂t 2r
1 ∂p p
p ⋅ nˆ = − − . (1.46)
v0 ∂t 2r
In both the forward and the inverse calculations, the unknown coefficients Ψ need
to be expanded in a similar fashion to p as a sum of unknown parameters multiplied
10 Photoacoustic Tomography
by a known spatially varying basis function. Thus, Equation (1.41) can be expressed
as the following matrix form in consideration of Equation (1.46),
1
K ij =
∫ ψ i ⋅ ψ j dS +
2r ∫ ψ ψ dl (1.48)
l
i j
1
Cij =
v0 ∫ ψ ψ dl (1.49)
l
i j
1
M ij =
v02 ∫ ψ ψ dS (1.50)
S
i j
β ∂Ι
Bi =
Cp ∫ ψ ∑ψ Ψ
S
i
k
k k dS ⋅
∂t
(1.51)
T
∂2 p1 ∂2 p2 ∂2 p
{
p} = 2
, 2 ,…, 2 N (1.54)
∂ t ∂ t ∂ t
Note that here Newmark’s time-stepping scheme has been used for the discreti-
zation of time dimension (Smith and Griffiths 2004). The Newmark algorithm is a
commonly used implicit method for the second order propagation equations such as
Equation (1.47). In our case, we have
1
{P}t + t = {P}t + {P}t t + − α {P}t + α{P}t + t t 2 (1.56)
2
where Δt is the time interval; α and δ are the time-stepping parameters, which deter-
mine the accuracy and stability of the algorithm; {P}t ,{P}t , and {P}t are the values
Fundamentals of Photoacoustic Tomography 11
of the pressure and its derivatives at time t; and {P}t + t ,{P}t + t , and {P}t + t are the
values of the pressure and its derivatives at the subsequent instant t + Δt.
Assuming that [ K ],[C ],[ M ], α, δ and Δt are constant, and from Equations (1.47),
(1.55), and (1.56), we obtain the following formula for calculating {P}t + t from
{P}t ,{P}t , and {P}t :
1 δ 1 1 1
[K ] + [M ] + [C ] {P}t + t = [ B]t + t + [ M ] {P}t + {P}t + − 1 {P}t
α t2 α t α t2 α t 2α
δ δ δ
+ [C ] {P}t + − 1 {P}t + −1 t{P}t
α t α 2α
(1.57)
The derivatives of the pressure at the subsequent instant are computed as follows:
1 1 1
{P}t + t = 2
({P}t + t − {P}t ) − {P}t − − 1 {P}t , (1.58)
α t α t 2α
δ = 0.5 and α = 0.25 are typically used in the image reconstruction. When
δ ≥ 0.5, and α ≥ 0.25(0.5 + δ)2, the Newmark algorithm preserves unconditional
stability, which means that the amount of Δt does not affect the stability of the solu-
tion to the equation (Bath 1976).
Following the procedures described in Section 1.2.2.1, the matrix equation for
inversion can be written as
(J T
t Jt + λI ) χ = JtT ( Pt o − Ptc ) . (1.60)
∂P
where Jt is the time-dependent Jacobian matrix formed by ∂Ψ at each time step,
o ,c
Pt is the time-dependent measured and calculated acoustic field data for i =
1,2,… M boundary locations, χ = ( Ψ1 , Ψ 2 ,… Ψ N )T is the updating vector,
I is the identity matrix, and λ is the regularization parameter determined by com-
bined Marquardt and Tikhonov regularization schemes. Here the submatrix of the
Jacobian for each time step is assembled to form the full Jacobian matrix for all time
steps. Thus we just need to solve Equation (1.47) one time per iteration for the whole
time range of interest.
Chapter 5 for more discussion on PAM). Since PAM uses focused transducer, its
image can be simply formed by the A-line or B-mode image formation method
used in conventional ultrasound imaging. In this method, one-dimensional depth-
resolved images (i.e., A-line) at each transducer location are acquired and additional
raster scanning of the transducer along a transverse direction (x or y) produces the
2D images referred to as B-scan. Further raster scanning along the other transverse
direction enables the reconstruction of a 3D image. The photoacoustic images can
be displayed through cross-sectional (B-scan) images, or maximum amplitude pro-
jection (MAP) images, where a MAP image is formed by projecting the maximum
photoacoustic amplitudes along a direction (typically z) to its orthogonal plane (x–y).
Let us use an example to illustrate how one A-line image is formed. A target (hair)
is embedded in a background phantom (Figure 1.1a). Figure 1.1b presents an A-line
acoustic signal collected along z-axis crossing the center of the hair target. The depth
of an image pixel (e.g., L for the center of the hair target shown in Figure 1.1b) is
obtained by multiplying the delay time or time difference between the launching
of the laser pulse and the receiving of the acoustic signal by the assumed constant
acoustic speed in phantom/tissue (typically 1.5 mm/μs). In order to achieve the best
spatial resolution, the target should be located at the focal plane of the transducer.
Figure 1.1c gives the recovered 2D photoacoustic image of the hair target along x-z
plane.
x
Transducer
z
Water 0
0.5
Surface 1
1.5
2
L
2.5
3
Phantom Target
3.5
4
4.5
5
5.5
0.05 0 0.05 0.1
Amplitude (a.u.)
(a) (b) (c)
FIGURE 1.1 (a) Schematic showing a hair target embedded in a phantom. (b) A-line acous-
tic signal collected along z-axis crossing the center of target (dashed line along z-axis in (a)).
(c) Recovered 2D image of the hair target along x-z plane.
Fundamentals of Photoacoustic Tomography 13
Trigger
Reflecting
mirror Pulsed laser
PC
Concave lens
MCA
Rotator
RH LH
ROB LOB
Membrane
Pulser & 2 mm
receiver
Transducer 0 0.3 0.6 1
FIGURE 1.2 Schematic of a typical PAT system. The sample here is a rat. The membrane is
used to isolate the water and rat while ensuring the target (i.e., rat head) is within the imaging
domain. The insert shows one slice of tomographic photoacoustic image of the rate brain. PC:
personal computer.
1.3 INSTRUMENTATION
A typical PAT system consists of a short-pulsed laser (e.g., several nanoseconds), a
singe transducer scanning subsystem, and acoustic signal detection subsystem (see
Figure 1.2). In this system, pulsed light from a Nd:YAG, Ti:Sapphire, or diode laser
is coupled into the sample via an optical subsystem and generates acoustic pressure
wave. A transducer (1 MHz or higher central frequency) is used to receive the acous-
tic signals. The transducer and the sample are immersed in a water tank. A rotary
stage rotates the receiver relative to the center of the tank. One set of data is taken at
multiple receiver positions (e.g., 120 positions) when the receiver is scanned circu-
larly over 360°. The complex wavefield signal is first amplified by a preamplifier and
then amplified further by a pulser/receiver. A data acquisition board converts it into
digital one, which is fed to a computer. The entire data acquisition can be realized
through C programming, for example. In this system, data collection for a total of
120 measurements may require up to 2 minutes.
In the typical PAT setup shown, the mechanical scanning significantly reduces
the speed of data acquisition. The use of a transducer array has clear acquisition time
advantages over a mechanically scanned system, making real-time data acquisition
possible. In addition, a transducer array-based system eliminates the use of a water
tank and allows the direct contact of the array with the skin (of course ultrasound
gel needs to be applied as coupling medium). The major drawback of an array-based
system, however, is its significantly added cost. We will detail an array-based PAT
system in Chapter 4.
14 Photoacoustic Tomography
While the image reconstruction methods presented in Chapter 1 and the linear algo-
rithms such as backprojection and filtered backprojection seen in the literature can be
used to provide high-quality photoacoustic (PA) images, the PA images provided are
qualitative in nature as they represent only the distribution of absorbed light energy
density that is the product of the intrinsic optical absorption coefficient and extrinsic
optical fluence distribution, indicating that the absorbed energy density as an imag-
ing parameter of conventional photoacoustic tomography (PAT) is clearly not an
intrinsic property of tissue. It is well known, however, that it is the tissue absorption
coefficient that directly correlates with tissue physiological/functional information.
These physiological parameters including hemoglobin concentration, blood oxygen-
ation, and water content are critical for accurate diagnostic decision making.
While the term quantitative PAT means primarily the ability to recover the opti-
cal absorption coefficient distribution, we show in this chapter that quantitative PAT
can also extract tissue acoustic property distribution such as acoustic velocity and/or
attenuation. It is clear that neither delay-and-sum method nor linear algorithms such
as backprojection/filtered backprojection can do so. Fortunately, the iterative finite
element (FE)-based nonlinear algorithms described in Chapter 1 can be extended
to achieve these quantification abilities. Since the first report of quantitative PAT
in 2006 (Yuan and Jiang 2006), several quantitative PAT methods have been devel-
oped and experimentally validated (Yuan et al. 2006, 2007; Yin et al. 2007; Yao
et al. 2009; Yuan and Jiang 2012). Here we describe four methods for recovering
absorption coefficient and one method for simultaneous reconstruction of optical
and acoustic properties, all FE-based in frequency-domain. We use phantom experi-
ments to demonstrate the validation of each quantitative method.
15
16 Photoacoustic Tomography
in which Γ is the Grüneisen coefficient (Γ = c0β/Cp), and Φ0 is the incident laser flu-
ence at the surface of the phantom/tissue. Once ξ in Equation (2.1) is obtained, the
absolute absorbed energy density for PAT imaging can be obtained from the absolute
acoustic pressure that is the product of ξ and measured acoustic signal. In addition,
the nonlinear relationship between the optical fluence within the tissue and optical
absorption coefficient can be approximated by the photon diffusion equation and
type III Boundary Conditions (BCs) BCs as follows (Jiang 2010),
Examples
30
0.05
20 0.045
0.04
10 0.035
0.03
0
0.025
0.02
10
0.015
20 0.01
0.005
30
30 20 10 0 10 20 30
(a)
30
0.018
20 0.016
0.014
10
0.012
0 0.01
0.008
10
0.006
0.004
20
0.002
30
30 20 10 0 10 20 30
(b)
FIGURE 2.1 Reconstructed optical absorption coefficient images (units: mm–1). (a), (b), and (c):
Two targets containing 0.5, 0.25, and 0.125 nM nanoparticles in each target, respectively. (continued)
18 Photoacoustic Tomography
× 103
30 8
7
20
6
10
5
0 4
3
10
2
20
1
30
30 20 10 0 10 20 30
(c)
FIGURE 2.1 (continued) Reconstructed optical absorption coefficient images (units: mm–1).
(a), (b), and (c): Two targets containing 0.5, 0.25, and 0.125 nM nanoparticles in each target,
respectively.
Absorption Coefficient (mm–1)
0.050 0.020
0.045 0.018
0.040 0.016
0.035 0.014
0.030 0.012
0.025 0.010
0.008
0.020 0.006
0.015 0.004
0.010 0.002
0.005 0.000
–4 –2 0 2 4 –6 –4 –2 0 2
x (mm) y (mm)
(a) (b)
Absorption Coefficient (mm–1)
0.008
0.007
0.006
0.005
0.004
0.003
0.002
–4 –2 0 2 4
x (mm)
(b)
FIGURE 2.2 Recovered optical absorption profiles plotted along y = 0 mm (a), x = 0.5 mm
(b), and y = 0 mm (c) from the images shown in Figures 2.1(a)–(c), respectively.
Quantitative Photoacoustic Tomography 19
2.8 to 3.3 mm, which is in good agreement with the actual object size of 3 mm.
Moreover, the nanoparticles concentration used in our experiments (0.125, 0.25,
and 0.5 nM) gives an absorption coefficient of the object of 0.01, 0.025, and
0.05 mm–1, respectively. As can be seen from Figures 2.1 and 2.2, we are able to
obtain quantitatively resolved images in terms of the size as well as absorption
coefficient of the objects.
The reconstructed absorption coefficient image for the fourth experiment is
shown in Figure 2.3a. The recovered object size in this case was calculated to
be 5.0 mm, and the peak value of recovered absorption property is 0.072/mm,
as plotted in Figure 2.3b. Again, both values are in good agreement with the
exact values.
We also note that the absorption coefficient value of the objects is more accu-
rately recovered for the higher contrast cases (see Figures 2.1a and 2.3a) than that
for the lower contrast cases (Figures 2.1b and c). The error between the recon-
structed and exact absorption coefficient is up to 31% for the low-contrast cases.
The degradation is most likely due to the limitations associated with the simple
20
0.07
15
0.06
10
0.05
5
0.04
0
5 0.03
10 0.02
15 0.01
20
20 10 0 10 20
(a)
0.08
0.07
Absorption Coefficient (mm–1)
0.06
0.05
0.04
0.03
0.02
0.01
0.00
–0.01
–4 –2 0 2 4 6 8
x (mm)
(b)
FIGURE 2.3 (a) Recovered optical absorption coefficient image (a) (units: mm–1), (b)
Optical absorption profile along y = –5.5 mm from the image shown (a).
20 Photoacoustic Tomography
iteration procedure for calculating μa, because we have to know the strength
of incident light in advance, perform careful calibration with the nonscattering
medium, and assume the reduced scattering coefficient of the phantom known
as a constant (1.0 mm–1 for the first three cases and 3.0 mm–1 for the fourth case).
We will see that many of these limitations can be overcome using the methods
described in the following three sections.
∑(Φ
2
χ2 = (m)
i − Φ(i c ) ) (2.4)
i =1
where Φ(i m ) and Φ(i c ) are the measured and calculated optical fluence for i = 1,2,…
M boundary locations. The goal of the second step is to obtain the distribution of
optical fluence within the entire imaging domain through an optimization procedure
based on Equations (2.3) and (2.4). This simple least squares minimization scheme
can be explained as follows: Given a range of values of s , a, and S (always avail-
able empirically), we compute the χ2 error as function of s, a , and S where Φ(i m )
is from the measured optical data and Φ(i c ) is calculated from Equation (2.3). The
rationale of this scheme is based on the argument that the minimum of χ2 corre-
sponds to the effective values of s, a, and S associated with the medium of inter-
est. The desired distribution of optical fluence, Φ, is calculated from Equation (2.3)
with the optimized set of s, a, and S in place. Thus, the final distribution of a is
calculated using a = Ψ/Φ where Ψ is obtained from the conventional PAT (the first
step). We found that this division is computationally stable, because there is no pos-
sibility of having a zero or close to zero value for Φ.
Quantitative Photoacoustic Tomography 21
Examples
0.03
20
10 0.02
0
0.01
10
0
20
20 0 20
(a)
FIGURE 2.4 Reconstructed optical absorption coefficient images (units: mm–1): (a) a 1 mm-
diameter target, (b) two targets (2 and 3 mm in diameter, respectively), and (c) a 0.5 mm-
diameter target. (continued)
22 Photoacoustic Tomography
0.03
20
10 0.02
0
0.01
10
0
20
20 0 20
(b)
0.03
20
0.02
10
0 0.01
10 0
20
0.01
20 0 20
(c)
FIGURE 2.4 (continued) Reconstructed optical absorption coefficient images (units: mm–1):
(a) a 1 mm-diameter target, (b) two targets (2 and 3 mm in diameter, respectively), and (c) a
0.5 mm-diameter target.
0.04
0.5 mm
Absorption Coefficient (/mm)
0.03 1 mm
2 mm
3 mm
0.02
0.01
0.00
0.01
0.02
0 10 20 30 40 50
x (mm)
× 103
20
20
15
10
10
0
5
10
0
20
20 10 0 10 20
(a)
× 103
20
10
10
0 5
10
0
20
20 10 0 10 20
(b)
FIGURE 2.6 Reconstructed optical absorption coefficient images (units: mm–1). A 2 mm-
diameter target having an optical contrast of 2:1 (a) and 1.5:1 (b), relative to the background.
The reconstructed absorption coefficient images of the final two cases are
shown in Figures 2.6a and 2.6b, and the associated absorption coefficient profiles
are depicted in Figure 2.7. We immediately see that the different optical contrast
levels of the objects relative to the background are clearly resolvable.
We also observe that the imaging quality for the smallest target (Figure 2.4c) and
lowest contrast (Figure 2.6b) cases is degraded with stronger artifacts and over- or
underestimated target size, compared with that for the larger target and higher
contrast cases. The degradation is most likely due to the lower signal-to-noise ratio
(SNR) for the smallest target and lowest contrast cases. We also believe that the
nonuniformity, significant variation, and negative values seen in Figures 2.4 and
2.6 are primarily caused by the limited bandwidth of the transducer used, which
is directly related to the target size. This is evident from Figure 2.5 where we see
a clear trend—the smaller the target size, the larger the amplitude of the negative
values. For example, the peak negative value for the 0.5, 1.0, 2.0, and 3.0 mm target is
–0.02, –0.015, –0.007, and 0.003 mm –1, respectively. In addition, we found that
the negative value issue does not appear to be related to the contrast level. For
24 Photoacoustic Tomography
Absorption Coefficient(/mm)
0.020 1.5x
2.0x
0.016
0.012
0.008
0.004
0.000
0.004
0 10 20 30 40 50
x (mm)
FIGURE 2.7 Recovered optical absorption profiles plotted along y = 6 mm (a), and y =
6.5 mm (b) from the images shown in Figures 2.6(a) and (b), respectively.
example, the image of the 2 mm target size with 3:1 contrast (in Figure 2.5) gives
almost the same level of amplitude of negative value as the images of the 2 mm
target size with 2:1 or 1.5:1 contrast (Figure 2.7).
The assumption of homogeneous or constant absorption coefficient during the
procedure for estimating the distribution of optical fluence should not contribute
to the degradation significantly, because the heterogeneity size is small in the
cases studied here. We expect, however, that this assumption may have significant
impact on the estimation of the distribution of optical fluence if the heterogeneity
size becomes large (e.g., larger than 1 cm in diameter). In this case it would be
necessary to develop new PAT methods without such an assumption for recon-
structing the distribution of absorption coefficient (see Methods 3 and 4 below).
where E (r ) = 1/ a (r ), and S(r) is the incident point or distributed source term. For the
inverse computation, the so-called Tikhonov regularization sets up a weighted term
as well as a penalty term to minimize the squared differences between computed and
measured absorbed energy density values,
2 2
min Ψ c − Ψ o + β L[ E − E0 ] (2.7)
{
χ
where J is the Jacobian matrix formed by ∂Ψ/∂E inside the whole reconstruction
domain including the boundary zone. The practical update equation resulting from
Equation (2.8) is utilized with β = 1,
In addition to the usual Tikhonov regularization, the PAT image (absorbed energy
density map) is used both as input data and as a priori structural information to
regularize the solution so that the ill-posedness associated with such inversion can
be reduced. In our reconstruction scheme, the PAT image is first segmented into dif-
ferent regions according to different heterogeneities or tissue types. We then employ
both the distribution of absorbed energy density in the entire imaging domain and
segmented a priori structural information for optical inversion. The segmented
a priori spatial information can be incorporated into the iterative process using the
regularization filter matrix, L shown in Equation (2.9). Laplacian-type filter matrix is
employed and constructed according to the region or tissue type it is associated based
on derived priors. This filter matrix is able to relax the smoothness constraints at the
interface between different regions or tissues, in directions normal to their common
boundary so that the covariance of nodes within a region is basically realized. As
such, the elements of matrix L, Lij, are specified as follows (Yuan et al. 2007):
1 if i= j
Lij = −1/ NN if i, j one region (2.10)
0 if i, j diffrent region
26 Photoacoustic Tomography
where NN is the total node number within one region or tissue. It is noted from
Equation (2.9) that a hybrid regularization scheme that combines both Levenberg–
Marquardt and Tikhonov regularizations are utilized. The optical absorption coef-
ficient distribution is reconstructed through the iterative procedures described by
Equations (2.5), (2.6), and (2.9).
Examples
Experimental data used to validate Method 3 were collected using the PAT system
presented in Figure 1.1. For the first two experiments, we embedded two objects
with a size ranging from 2.0 to 5.5 mm in diameter in a 50.8 or 40.0 mm-diameter
solid cylindrical phantom. We then immersed the object-bearing solid phantom
into a 110.6 mm-diameter water background. The phantom materials used con-
sisted of Intralipid as scatterer and India ink as absorber with Agar powder (1–2%)
for solidifying the Intralipid and India ink solution. The background phantom had
μa = 0.01 mm–1 and μs′= 1.0 mm–1 while the two targets had μa= 0.03 mm–1 and μs′ =
2.0 mm–1 for test 1, and μa= 0.07 mm–1 and μs′ = 3.0 mm–1 for test 2. In the next two
experiments, we placed a single-target-containing phantom into the water, aiming
to test the capability of resolving target having different optical contrasts relative
to the background phantom. The target size was 1.0 and 2.0 mm in diameter for
tests 3 and 4, respectively. The target had μa= 0.03 mm–1 and μs′= 2.0 mm–1 for test
3, and μa= 0.015 mm–1 and μs′ = 2.0 mm–1 for test 4. In the image reconstructions
for the four experiments, we assumed scattering coefficient known as constant
(1.0mm–1). The initial guesses of optical absorption coefficient for the target(s) and
background medium were 0.02 mm–1 and 0.01 mm –1, respectively.
The results from the first two sets of experiments are shown in Figure 2.8, where
(a) and (b) present the reconstructed absorption coefficient images of two objects
having a size of 2.0 and 3.0 mm (test 1), and 5.5 mm (test 2) in diameter, respec-
tively, while the recovered absorbed energy density maps for experiments 1 and 2
are also plotted in (c) and (d) for comparison. We see that the objects in each case
are clearly detected. As shown in Table 2.1, the recovered absorption coefficients
20
15 0.025
10 0.02
5
0 0.015
5
10 0.01
15 0.005
20
25
20 10 0 10 20
(a)
FIGURE 2.8 Reconstructed absorption coefficient images (a, b), absorbed light energy den-
sity images (c, d). (a), (c) are for experiment 1, and (b), (d) for experiment 2. The axes (left
and bottom) illustrate the spatial scale, in mm, whereas the scale (right) records the absorbed
optical energy density in relative units, or absorption coefficient in mm–1. (continued)
Quantitative Photoacoustic Tomography 27
20 0.07
0.06
10
0.05
0 0.04
0.03
10
0.02
20
20 10 0 10 20
(b)
20 6000
5000
10 4000
0 3000
2000
10
1000
20 0
20 10 0 10 20
(c)
20
0.025
10
0.02
0.015
0
0.01
10
0.005
20
20 10 0 10 20
(d)
FIGURE 2.8 (continued) Reconstructed absorption coefficient images (a, b), absorbed light
energy density images (c, d). (a), (c) are for experiment 1, and (b), (d) for experiment 2. The
axes (left and bottom) illustrate the spatial scale, in mm, whereas the scale (right) records the
absorbed optical energy density in relative units, or absorption coefficient in mm–1.
of the target and background are quantitative compared to the exact values for both
experiments. By estimating the FWHM of the absorption coefficient profiles, the
recovered object size was found to be 1.8, 2.7, and 5.0 mm, which is also in good
agreement with the actual object size of 2.0, 3.0, and 5.5 mm for experiments 1
and 2. The reconstructed absorption coefficient images for experiments 3 and 4
are shown in Figures 2.9a and 2.9b. We immediately see that the different optical
contrast levels of the objects relative to the background are quantitatively resolved.
28 Photoacoustic Tomography
TABLE 2.1
Average Value of the Recovered Absorption Coefficient (mm –1) of Target and
Background and Target Size (mm) for Experiments 1 and 2
Targets (μa) Background(μa) Target Sizes
Test Reconstructed Exact Reconstructed Exact Reconstructed Exact
(1) Target 1 0.027 0.03 0.012 0.01 1.8 2.0
Target 2 0.028 0.03 0.012 0.01 2.7 3.0
(2) Target 1 0.067 0.07 0.011 0.01 5.0 5.5
It is important to note that our reconstruction method does not need any
calibration procedure due to the use of relative incident laser source strength
and normalized absorbed energy density distribution where the normalization is
performed by simply dividing the absorbed energy density at each nodal location
with the maximum absorbed energy density (i.e., Ψ(r ) = Ψ(r )/Ψmax). An optimiza-
tion scheme was then applied to search for the boundary conditions coefficient,
20
0.025
10
0.02
0
0.015
10
0.01
20
20 10 0 10 20
(a)
× 103
20 14
10 12
0 10
10 8
20 6
20 10 0 10 20
(b)
FIGURE 2.9 Reconstructed absorption coefficient images for experiment 3 (a) and experi-
ment 4 (b). The axes (left and bottom) illustrate the spatial scale in mm, whereas the scale
(right) records the absorption coefficient in mm–1.
Quantitative Photoacoustic Tomography 29
α and the relative source strength as described previously (Iftimia and Jiang 2000).
As such, the reconstruction of optical properties with our algorithms does not
depend on the absolute values of absorbed energy density and optical fluence as
well as the boundary parameter. For example, even though the values/scales of
the absorbed energy density for experiments 1 and 2 are very different as shown
in Figures 2.8c and 2.8d, the algorithm is still able to recover the absorption coef-
ficient images quantitatively in terms of the location, size, and absorption coef-
ficient value of the objects. In addition, our method is able to resolve the issue of
negative absorbed energy density values often seen in conventional PAT.
2 v0βΨ
p(r , ω ) + k02 p(r , ω ) = ik0 (2.11)
Cp
Ap = B (2.12)
Based on the finite element solution to Equation (2.12), we can use Marquardt–
Tikhonov regularization-based Newton method to directly recover/update absorption
coefficient from an initial guess of μa,0 by minimizing an object function composed
of a weighted sum of the squared difference between computed and measured acous-
tic pressure around the boundary area given by
Min: F = ∑( p o
i − pic )2 (2.13)
i =1
( ℑT ℑ + λI) χ = ℑT ( po − pc ), a = a ,0 + ζ χ (2.14)
N
∂ pi ∂ pi ∂Ψ k
∂ a, j
= ∑ ∂Ψ k ∂ a , j
(i = 1,2... M ; j, k = 1,2... N ) (2.15)
k =1
in which N is the nodal number of the finite element mesh in the entire problem domain
and the sensitivities of ∂p/∂Ψ in each iteration can be calculated from Equation (2.12)
based on adjoint method (see Section 3.2 for detail about this method),
∂p ∂B ∂A
[ A] = − {P} (2.16)
∂Ψ ∂Ψ ∂Ψ
∂Ψ k Φk + a,k (∂Φ k /∂ a, j ) if (k = j)
= (2.17)
∂ a, j a,k (∂Φ k /∂ a, j ) if ( k ≠ j)
And photon fluence Φ in Equation (2.17) can be computed by the finite element
solution to the following photon diffusion equation:
in which S(r) is the normalized light source term and D(r) is the diffusion coefficient.
The finite element discretized form of Equation (2.18) is written,
Examples
20 20
10 10
0 0
–10 –10
–20 –20
0.035
New scheme
0.030 Previous scheme
Absorption Coefficient (mm–1)
0.025
0.020
0.015
0.010
0.005
0.000
–0.005
–20 –10 0 10 20
x (mm)
(c)
FIGURE 2.10 Reconstructed μa image using Method 1 (a), Method 4 (b), and recovered
optical absorption profile (c) plotted along y = –7.0 mm from the images shown in Figures
2.10(a) and (b). The axes (left and bottom) illustrate the spatial scale, in mm, whereas the
color scale (right) records μa in mm–1.
Quantitative Photoacoustic Tomography 33
20
20
10
10
0 0
–10 –10
–20 –20
0.035
0.030 New scheme
Previous scheme
Absorption Coefficient (mm–1)
0.025
0.020
0.015
0.010
0.005
0.000
–0.005
–0.010
–0.015
–0.020
–0.025
–20 –10 0 10 20
x (mm)
(c)
FIGURE 2.11 Reconstructed μa images using Method 1 (a), Method 4 (b), and recovered opti-
cal absorption profile (c) plotted along y = 1.0 mm from the images in Figures 2.11(a) and (b).
10
8
0.25
6
4
0.2
2
0 0.15
2
0.1
4
6 0.05
8
10
10 5 0 5 10
FIGURE 2.12 The recovered μa image for the human hairs embedded in phantom.
34 Photoacoustic Tomography
2 v0βΨ(r )
p(r , ω ) + k02 (1 + O) p(r , ω ) = ik0 (2.21)
Cp
where O is a coefficient that depends on both acoustic speed and attenuation as fol-
lows (Jiang et al. 2006):
co2 i αv0
O= 2
−1+ (2.22)
c k0 v 2
in which v is the speed of acoustic wave in the medium/tissue and α is the acoustic
attenuation coefficient. The finite element discretization of Equation (2.21) is stated as,
Ap = B (2.23)
∂2 ψ j
− ik02 ∑ l
OI ,l ψ l ψ j ψ i −
∫ ηψ j + γ
∂φ2
ψ i ds,
v0β v0β
Bi = −ik0
Cp ∑Ψ
k
R ,k ψ k ψ i + k0
Cp ∑Ψ
l
I ,l ψ l ψ i (2.24)
Quantitative Photoacoustic Tomography 35
in which Ψ, O, and p are expressed by their real and imaginary components, Ψi is the
basis function, < > indicates integration over the problem domain; and the second-
order absorption boundary conditions are employed here (Yuan et al. 2005),
∂2 ψ n
ψ n ⋅ nˆ = ηψ n + γ (2.25)
∂2 θ
2
− ik − 3/2 ρ+ i 3/8 kρ− i/2 kρ2
where η = 1− i/kρ , γ = 1− i/kρ and θ is the angular coordinate at a radial posi-
tion ρ. To obtain a matrix equation capable of inverse solution, a combined Marquardt
and Tikhonov regularization scheme is used for the inverse calculation,
where po = ( p1o , p2o ,..., pMo )T , pc = ( p1c , p2c ,..., pM ) , and pio and pic are observed
c T
and computed complex acoustic field data for i = 1, 2…, M boundary measurement
locations, Δχ is the update vector for the optical and acoustic properties, ℑ is the
Jacobian matrix formed at the boundary measurement sites, λ is a scalar, and I is the
identity matrix. Thus here the image formation task is to update optical and acoustic
property distributions via iterative solution of Equations (2.23) and (2.26) so that a
weighted sum of the squared difference between computed and measured acoustic
pressure can be minimized.
Examples
Phantom experiments were conducted using the PAT system shown in Figure 1.1
to demonstrate this reconstruction approach. In the first two experiments, we
embedded one glycerol powder-containing target (10 mm in diameter) in a 30 mm-
diameter solid cylindrical phantom. We then immersed the object-bearing solid
phantom in a 50.8 mm-diameter water background, as displayed in Figure 2.13a.
The acoustic speed in the region of the target containing 10% (phantom 1) and
Φ = 10 2 Φ = 10
3.5 X
Φ=5 2 7
r = 1.5
Transducer
path
(a) (b) (c)
FIGURE 2.13 Experiment geometry for (a) phantoms 1 and 2, (b) phantom 3, and (c) phan-
tom 4. The dimension units are millimeters.
36 Photoacoustic Tomography
28% (phantom 2) glycerol was 1540 and 1620 m/s, respectively, while the acoustic
speed for the background was 1480 m/s. For the third experiment, a 5 mm- and
a 10 mm-diameter target (each containing 28% glycerol) were embedded into a
30 mm-diameter solid cylindrical phantom. Again the entire solid phantom was
immersed in a 50.8 mm-diameter water background, as shown in Figure 2.13b.
In the final experiment, we embedded two nanoparticle-containing objects
(3 mm in diameter) in a 50 mm-diameter solid cylindrical phantom, as plotted
in Figure 2.13c. Each target had 0.5 nM nanoparticles. We then immersed the
object-bearing solid phantom in a 110.6 mm-diameter water background. For all
the experiments, the phantom materials used consisted of Intralipid as scatterer
and India ink as absorber with Agar powder for solidifying the Intralipid and India
ink solution. The optical absorption and scattering coefficients for the phantom
were 0.004 and 1.0 mm –1, respectively. The optical absorption contrast between
the target and phantom is about 10 for experiments 1–3, and 20 for experiment 4.
Figures 2.14a and 2.14b show the reconstructed acoustic and optical images for
experiment 1, while 2.14c and 2.14d present the recovered acoustic and optical
× 106
20 1.525
1.52
10
1.515
0 1.51
10 1.505
1.5
20 1.495
20 10 0 10 20
(a)
0.16
20
0.14
10 0.12
0 0.1
0.08
10
0.06
20
0.04
20 10 0 10 20
(b)
FIGURE 2.14 Reconstructed acoustic speed (a, c) and absorbed optical energy density
(b, d) images with different acoustic contrast levels with respect to the background, (a) and
(b) for experiment 1, and (c) and (d) for experiment 2. The axes (left and bottom) illustrate the
spatial scale in millimeters, whereas the scale (right) records the acoustic speed, in mm/s, or
absorbed optical energy density, in a relative unit. (continued)
Quantitative Photoacoustic Tomography 37
× 106
1.65
20
1.6
10
1.55
0
1.5
10
1.45
20
1.4
20 10 0 10 20
(c)
20 0.25
10 0.2
0.15
0
0.1
10
0.05
20
0
20 10 0 10 20
(d)
FIGURE 2.14 (continued) Reconstructed acoustic speed (a, c) and absorbed optical energy
density (b, d) images with different acoustic contrast levels with respect to the background,
(a) and (b) for experiment 1, and (c) and (d) for experiment 2. The axes (left and bottom) illus-
trate the spatial scale in millimeters, whereas the scale (right) records the acoustic speed, in
mm/s, or absorbed optical energy density, in a relative unit.
images for experiment 2. We see that the object in each case is clearly detected.
By estimating FWHM of the acoustic property, as displayed in Figure 2.15, we
found that the recovered size of these objects ranged from 9.1 to 9.3 mm, in good
agreement with the actual object size of 10 mm. In our experiments, the 10% and
28% glycerol-containing target used gave an acoustic speed of 1540 and 1620 m/s,
respectively. As can be seen from Figure 2.15, we are able to obtain quantitatively
resolved images in terms of the acoustic property as well as the position, shape,
and size of the objects.
Figure 2.16a and 2.16b displays the reconstructed acoustic speed and optical
images for the third experiment having two targets. By estimating the FWHM of
the acoustic profiles plotted in Figure 2.17, we found that the recovered size of the
two objects was 4.6 and 9.8 mm, respectively. Again, both size values were in
good agreement with the exact values of 5 and 10 mm. As shown in Figure 2.16,
the reconstructed acoustic properties for these cases are also accurate relative to
38 Photoacoustic Tomography
1,530,000
1,525,000
1,520,000
1,510,000
1,505,000
1,500,000
1,495,000
1,490,000
15 10 5 0 5 10 15
y (mm)
(a)
1,640,000
1,620,000
1,600,000
Velocity (mm/s)
1,580,000
1,560,000
1,540,000
1,520,000
1,500,000
1,480,000
15 10 5 0 5 10 15
x (mm)
(b)
the exact value, which is further confirmed by the quantitative analysis shown in
Figure 2.17.
Figures 2.18a and 2.18b present the reconstructed acoustic and optical prop-
erties of two objects having 0.5 nM nanoparticles within each of the objects. By
estimating the FWHM of the acoustic speed plotted in Figure 2.18c, we found that
the recovered diameter size of these objects ranges from 2.8 to 3.3 mm, in good
agreement with the actual object size of 3 mm. In addition, it is noted the imaging
resolution in Figure 2.16 is not as good as that in Figure 2.18 due to the low opti-
cal contrast between the targets and phantoms for experiments 1–3. Further, from
Figures 2.14, 2.16, and 2.18 we can also see that the image quality of both opti-
cal and acoustic images appears to be similar when different acoustic contrasts
between the target and the background were used.
Quantitative Photoacoustic Tomography 39
× 106
1.62
20
1.6
10 1.58
1.56
0
1.54
10 1.52
20 1.5
1.48
20 10 0 10 20
(a)
20 0.2
10 0.15
0 0.1
10
0.05
20
0
20 10 0 10 20
(b)
FIGURE 2.16 Reconstructed acoustic speed (a) and absorbed optical energy density
(b) images for experiment 3. The axes (left and bottom) illustrate the spatial scale in millime-
ters, whereas the scale (right) records the acoustic speed, in mm/s, or absorbed optical energy
density, in a relative units.
1,650,000
1,600,000
1,550,000
Velocity (mm/s)
1,500,000
1,450,000
1,400,000
1,350,000
1,300,000
15 10 5 0 5 10 15
x (mm)
(a)
1,650,000
1,600,000
Velocity (mm/s)
1,550,000
1,500,000
1,450,000
1,400,000
–15 –10 –5 0 5 10 15
x (mm)
(b)
FIGURE 2.17 (continued) Recovered acoustic speed profiles plotted along y = 0 mm (a), y
= –8 mm (b) from the image shown in Figure 2.16(a).
× 106
50
1.6
1.55
0 1.5
1.45
50 1.4
50 0 50
(a)
0.35
50
0.3
0.25
0.2
0
1.15
0.1
0.05
50
50 0 50
(b)
FIGURE 2.18 Reconstructed acoustic speed (a) and absorbed optical energy density
(b) images for experiment 4. (c) is the recovered acoustic speed profile plotted along x = 0 mm
from the image shown in (a).The axes (left and bottom) illustrate the spatial scale in millime-
ters, whereas the scale (right) records the acoustic speed, in mm/s, or absorbed optical energy
density, in a relative units. (continued)
Quantitative Photoacoustic Tomography 41
1,620,000
1,600,000
1,580,000
1,560,000
Velocity (mm/s)
1,540,000
1,520,000
1,500,000
1,480,000
1,460,000
1,440,000
15 10 5 0 5 10 15
y (mm)
(c)
FIGURE 2.18 (continued) Reconstructed acoustic speed (a) and absorbed optical energy
density (b) images for experiment 4. (c) is the recovered acoustic speed profile plotted along
x = 0 mm from the image shown in (a).The axes (left and bottom) illustrate the spatial scale in
millimeters, whereas the scale (right) records the acoustic speed, in mm/s, or absorbed optical
energy density, in a relative units.
2.6 MULTISPECTRAL PAT
For most of the cases where the heterogeneity/object has contrast in both the optical
property and acoustic velocity, the absorbed energy density and acoustic velocity can
be recovered at a single wavelength. This is very similar to that seen in diffuse optical
tomography (DOT) where both absorption and scattering coefficients can be recon-
structed at a single wavelength when the objects have contrast in both the absorption
and scattering (Jiang 2010). For such cases, crosstalk certainly exists, but it is not strong.
This crosstalk is also dependent on the level of contrast for the two parameters. For the
experimental cases examined in Sections 2.1–2.5, the level of contrast used was rela-
tively low, which explains how the insignificant crosstalk occurred in our reconstruction.
However, for the cases where acoustic heterogeneity is quite different from the
distribution of absorbed light energy density, our simulation tests indicate that the
crosstalk cannot be minimized using one wavelength measurement. For our simula-
tion tests, a circular background region (15 mm in radius) contains three circular
targets (2 mm in radius each) positioned at 2 (top left), 9 (top right), and 6 o’clock
(bottom). Top left inclusion has contrast for velocity, while bottom and top right
inclusions have contrast for absorbed energy density. The velocity for the target
and background is 1.54 × 106 mm/s and 1.44 × 106 mm/s, respectively, while the
absorbed energy density for the background and targets is 1 mJ/mm3 and 10 mJ/mm3,
respectively. We can see from the recovered images shown in Figure 2.19 that the
absorbed energy density distribution has some crosstalk effect on acoustic veloc-
ity, while acoustic velocity distribution has no significant effect on the recovered
absorbed energy density due to its low contrast.
42 Photoacoustic Tomography
15
10
y 0
–5
–10
–15
–10 0 10
x
(a)
× 106
15
1.5
10 1.48
5 1.46
1.44
0
1.42
5
1.4
10 1.38
15 1.36
15 10 5 0 5 10 15
(b)
15
10
y 0
–5
–10
–15
–10 0 10
x
(c)
FIGURE 2.19 Reconstructed images using one wavelength measurement. (a) and (b) are
the exact and recovered acoustic velocity (mm.s–1). (c) and (d) are the exact and recovered
absorbed energy density(mJ.mm–3). (continued)
Quantitative Photoacoustic Tomography 43
15
10
10
5 8
0 6
5 4
10
2
15
10 0 10
(d)
2 v 0β a (r , λ)Φ(r , λ)
p(r , ω , λ) + k02 (1 + O) p(r , ω , λ) = ik0 (2.27)
Cp
where λ is the wavelength of incident light. In addition, according to the Beer’s law,
the wavelength-dependent tissue absorption can be expressed as
a (λ ) = ∑ ε (λ)c (2.28)
i i
i =1
where ci is the concentration and εi (λ) is the extinction coefficient of the ith chro-
mophore (HbO2, Hb or H2O) at wavelength λ. In consideration of Equation (2.28),
Equation (2.27) can be rewritten,
2 2
v0 β ∑ ε (λ)c Φ(r , λ)
i =1
i i
Thus, similar to Equations (2.14) and (2.26), the inverse solution can be obtained
by solving the following equation:
( J T J + λ I) χ = J T ( po − pc ) (2.30)
in which χ = ( O1 , O2 ,…, On , c1,1 , c1,2 ,…, c1,n , c2,1 , c2,2 ,…, c2,n , c3,1 ,
c3,2 ,…, c3,n )T is the update vector for chromophores and acoustic velocity; λ′ is
the regularization parameter determined by combined Marquardt and Tikhonov
regularization schemes; pio and pci are measured and calculated data for i = 1,2,…M
boundary locations and are written as for each acoustic frequency ω and each optical
wavelength λ,
Examples
TABLE 2.2
Chromophore Concentrations and Acoustic Velocity for Test Objects
Object Position HBO2 (μM) HB (μM) H2O(%) Acoustic Velocity (mm/s)
Background 6 2 20 1.44×106
Top right 20 12 50 1.44×106
Top left 6 12 20 1.44×106
Bottom middle 20 12 50 1.74×106
Quantitative Photoacoustic Tomography 45
15 15
12
10 10
10
5 5 8
y 6
0 0
–5 –5 4
–10 2
–10
0
–15 –15
–10 0 10 –10 0 10
x
15 15
10 10
20
5 5
y 15
0 0
–5 –5 10
–10 –10
5
–15 –15
–10 0 10 –10 0 10
x
15 15
10 50
10
5 45
5
40
y
0 0 35
–5 –5 30
–10 –10 25
20
–15 –15
–10 0 10 –10 0 10
x
15 × 106
15
1.7
10 10
5 5 1.65
y 0 0 1.6
–5 –5
1.55
–10 –10
1.5
–15 –15
–10 0 10 –15 –10 –5 0 5 10 15
x
FIGURE 2.20 Exact (left column) and reconstructed (right column) images using six opti-
cal wavelengths. The first to the fourth rows show the Hb, HBO2, H2O, and acoustic velocity
images, respectively.
46 Photoacoustic Tomography
the true locations of the targets including HbO2, HbR, H2O and acoustic velocity.
The reconstructed images shown in the right column of Figure 2.20 correspond to
HbO2, HbR, H2O and acoustic velocity, respectively. We see from Figure 2.20 that
the crosstalk errors between the chromophore concentrations and acoustic veloc-
ity have been effectively reduced using our quantitative PAT approach.
3 Image Enhancement
Software and Hardware
Approaches
47
48 Photoacoustic Tomography
for an arbitrary node i of the fine mesh which is embedded in a coarse mesh element
with nodes L1, L2, and L3, the values of OR and OI at node i become
OR ( xi , yi ) = ∑O
n =1
R , Ln φ Ln ( x , y)
OI ( xi , yi ) = ∑O I , Ln φ Ln ( x , y) (3.1)
n =1
where k and ℓ are the nodes on the coarse mesh, ϕk and φ are the basis functions
centered on nodes k and ℓ in this mesh, and the inner products are still performed
over the elements in the fine mesh. Since ϕk and ψ i are defined on the coarse mesh
and fine mesh, respectively, evaluating these inner products can be quite involved. A
way to simplify these integrations is to generate the fine mesh from the coarse mesh
by splitting the coarse elements into fine elements.
Examples
Two phantom experiments were conducted to test the dual meshing scheme. We
employed single-target-containing phantom tests, aiming to validate the accu-
racy improvement in detecting target when the dual mesh is used. The phantom
materials used consisted of Intralipid as scatter and India ink as absorber with
Agar powder (1–2%) for solidifying the Intralipid and India ink solution. The
absorption coefficient of the background phantom was 0.01 mm –1, while the
absorption coefficient of the target was 0.015 mm –1 and 0.02 mm –1 for test 1 and
test 2, respectively. Figure 3.1 shows the reconstructed absorbed energy density
images for the two cases with uniform mesh and dual mesh. We see that the tar-
get is clearly better recovered with the dual mesh (b and d) in terms of its shape
and size over that with the uniform mesh (a and c) for both cases. We also note
that the background region for both cases is more smoothly recovered using the
dual mesh scheme compared to that using the uniform mesh.
Image Enhancement Software and Hardware Approaches 49
14000
20 10000 10
12000
8000
10 5 10000
6000
8000
0 4000 0
6000
–10 2000 –5 4000
0
2000
–20 –10
–2000 0
–20 –10 0 10 20 –10 –5 0 5 10
(a) (b)
× 104
2
20 10
15000
10 5 1.5
10000
0 0 1
5000
–10 –5
0.5
0
–20 –10
0
–20 –10 0 10 20 –10 –5 0 5 10
(c) (d)
FIGURE 3.1 Reconstructed photoacoustic images. (a) and (b): The image for case 1 with
uniform and dual mesh. (c) and (d): The image for case 2 with uniform and dual mesh.
[Ξ ]T [ A]{∂ P/∂OR} = − [Ξ ]T [∂ A/∂OR ]{P} (3.5a)
[Ξ ]T [ A]{∂ P/∂Ψ R} = [Ξ ]T {∂ B/∂Ψ R} − [Ξ ]T [∂ A/∂Ψ R ]{P} (3.5b)
50 Photoacoustic Tomography
Now we can immediately tell that the left-hand side of the above equations actu-
ally gives the corresponding elements in the relative Jacobian matrix based on the
adjoint sensitivity method,
3.3 TOTAL-VARIATION-MINIMIZATION SCHEME
Measurement noises are always the major factor affecting the quantitative accuracy
of the reconstructed images. For example, errors for quantitatively recovering opti-
cal absorption coefficient can be as large as 10% for simulated data with 5% noise
added and 20% for experimental data (Jiang et al. 2006; Yao and Jiang 2009). In
an effort to reduce the noise effect and to further enhance the quantitative accuracy
of photoacoustic image reconstruction, here we consider a total-variation-minimi-
zation scheme within our FE-based reconstruction framework. Our existing recon-
struction algorithms are based on the least squares criteria (i.e., the regularized
Newton method) (see Chapters 1 and 2) that stand on the statistical argument that
the least squares estimation is the best estimator over an entire ensemble of all
possible pictures. Total variation, on the other hand, measures the oscillations of
a given function and does not unduly punish discontinuities (Dobson and Santosa
1994; van den Berg and Kleinmann 1995). Hence, one can hypothesize that a
hybrid of these two minimization schemes should be able to provide higher quality
image reconstructions. In fact, the strategy of finding minimal total variation has
proved to be successful in applications including electrical-impedance tomography
(van den Berg and Kleinmann 1995), microwave imaging (Vogel and Oman 1996),
image processing (Dobson and Santosa 1994), optimal design (Acar and Vogel
1994), and diffuse optical tomography (Paulsen and Jiang 1996).
A practical need exists for reconstruction of photoacoustic images from few mea-
surements, as this can greatly reduce the required scanning time and the number of
Image Enhancement Software and Hardware Approaches 51
ultrasound sensors placed near or on the boundary of an object to receive the laser-
induced acoustic signals. In addition, in many practical implementations of PAT the
photoacoustic signals are recorded over an aperture that does not enclose the object,
which results in a limited-angle tomographic reconstruction problem. In such cases,
the existing reconstruction algorithms, which are based on the least squares criteria
(i.e., the regularized Newton method) (see Chapters 1 and 2), often generate distorted
images with severe artifacts. Here we demonstrate that the total-variation-minimi-
zation (TVM)-based algorithm offers excellent photoacoustic image reconstruction
from few-detector and limited-angle data.
3.3.1 Mathematical Derivations
We describe the implementation of TVM within the FE-based reconstruction frame-
work in time-domain. To form an image from a presumably uniform initial guess of
the absorbed energy density distribution, we need a method of updating Ψ from its
starting value. This update is accomplished through the least squares minimization
of the following functional:
F ( p, Ψ) = ∑( p
j =1
o
j − pcj )2 , (3.9)
where poj and pcj are observed and computed acoustic field data for i = 1,2,…M
boundary locations. Using the regularized Newton method, we obtained the follow-
ing equation for updating Ψ:
where po = ( p1o , p2o ,… pMo )T , pc = ( p1c , p2c ,… pM ) , Δχ is the update vector for the
c T
absorbed optical energy density, ℑ is the Jacobian matrix formed by ∂ p/∂Ψ at the
boundary measurement sites, λ is the regularization parameter determined by com-
bined Marquardt and Tikhonov regularization schemes, and I is the identity matrix.
Two typical approaches exist for minimizing total variation: a constrained mini-
mization through the solution of the nonlinear photoacoustic (PA) equation (Rudin
et al. 1992; Paulsen and Jiang 1996) and an unconstrained minimization by addition
of the total variation as a penalty term to the least squares functional (Dobson and
Santosa 1994, 1996). From a computational standpoint, unconstrained minimiza-
tions are much easier to implement and require much fewer modifications to the
existing algorithm (Dobson and Santosa 1996). Thus here the unconstrained TVM
is used.
We now incorporate the total variation of Ψ as a penalty term by defining a new
functional (Yao and Jiang 2011):
F ( p, Ψ ) = F ( p, Ψ ) + L (Ψ ). (3.11)
52 Photoacoustic Tomography
Here
L (Ψ) =
∫ ω 2Ψ | Ψ |2 + δ 2 dx dy, (3.12)
is the penalty term, ω Ψ and δ are typically positive parameters that need to be deter-
mined numerically. The minimization of Equation (3.11) proceeds in standard fash-
ion by the differentiation of F with respect to each nodal parameter that constitutes
the Ψ distribution; simultaneously all these relations are set to zero. These steps lead
to the following nonlinear system of equations:
M
∂ F ∂ pcj
∂Ψ i
=− ∑( p
j =1
o
j − pcj )
∂Ψ i
+ Vi , (i = 1,2 N ), (3.13)
where
N N
∂ψ k ∂ψ i ∂ψ k ∂ψ i
∂L
ω 2Ψ ∑
k =1
Ψk
∂x ∂x
+ ∑Ψ
k =1
k
∂y ∂y
Vi =
∂Ψ i
=
∫ N
2
N
2
dx dy
∂ψ k ∂ψ k
ω 2Ψ ∑Ψk =1
k
∂x
+ ∑Ψ
k =1
k
∂y
+ δ2
Similarly to Equation (3.10), the following matrix equation for TVM constrained
inversion can be obtained
(ℑT ℑ + R + λI ) χ = ℑT ( po − pc ) − V , (3.14)
where
3.3.2.1 Simulations
In these simulations, a dual meshing method, as detailed in Section 3.1, is used for
fast yet accurate inverse computation. The fine mesh used for the forward calcula-
tion consisted of 3627 nodes and 7072 elements, while the coarse mesh used for the
inverse calculation had 930 nodes and 1768 elements. All the images obtained from
the method without the TVM are the results of 3 iterations, while those obtained
from the TVM-enhanced method are the results of 15 or more iterations. As men-
tioned above, the parameters ωψ and δ were determined through numerical experi-
mentation. A constant value of δ = 0.001 was sufficient for the current simulation
and experimental studies, while the value of ωψ is related to the SNR of the mea-
surements (Yao and Jiang 2011). For the simulations presented, ωψ = 0.5 for the first,
second, and third cases, and ωψ = 1.0 for the fourth case were used.
For the first simulation, the test geometry is a 30 mm-diameter circular back-
ground containing an off-center 4 mm-diameter target region. The target had
Ψ = 2.0 mJ/mm 3, while the background had Ψ = 1.0mJ/mm 3 . In this case the image
reconstruction was performed with 0, 10, and 25% noise added to the “measured”
data. Figure 3.2 gives two sets of absorbed energy density images recovered using
the reconstruction method without (Figures 3.2a, 3.2c, and 3.2e) and with the TVM
enhancement (Figures 3.2b, 3.2d, and 3.2f) under the conditions of different noise
levels. As can be seen, enhancement of the reconstruction by the incorporation of
the TVM is obvious over the method without the TVM. To provide a more quantita-
tive assessment of these images, Figure 3.3 is included, in which the reconstructed
absorbed energy density profiles are displayed along transects through the target for
the images shown in Figure 3.2. We find that the TVM-enhanced method not only
can improve the quality of the recovered images but also can decrease the sensitivity
of the method to noise effect.
The second simulation is intended to investigate how the target size affects the
TVM enhancement. In this case, no noise was added to the “measured” data, and
the diameter of the off-center target was 2 mm. Figures 3.4a and 3.4b give two sets
of absorbed energy density images recovered using the method without the TVM
(Figure 3.4a) and with the enhancement (Figure 3.4b), while Figures 3.4c and 3.4d
present the quantitative profiles of the absorbed energy density along transects
through the target for the images shown in Figures 3.2a,b and Figures 3.4a,b. Again,
considerable improvement can be observed from the reconstructed results when the
TVM is invoked compared with the method without the TVM. It is also interesting
to note that the enhancement for this case is more striking than that for the first one
where the background contained a larger target.
The third simulation aims to see how the contrast level of the absorbed energy
density between the target and the background impacts the TVM enhancement.
54 Photoacoustic Tomography
10 2 10 2
1.8
5 5
1.6
0 1.5 0
1.4
–5 –5
1.2
–10 1 –10
1
–10 0 10 –10 0 10
(a) (b)
2 10 2
10
1.8
5 5
1.6
0 1.5 0
1.4
–5 –5
1.2
–10 1 –10
1
–10 0 10 –10 0 10
(c) (d)
2.2
10 2 10 2
5 5 1.8
0 1.6
1.5 0
1.4
–5 –5
1.2
–10 1 –10
1
–10 0 10 –10 0 10
(e) (f )
FIGURE 3.2 Reconstructed absorbed energy density images from simulated data with and
without the total-variation-minimization (TVM) enhancement under different noise levels
(case 1). (a) Without the TVM, 0% noise. (b) With the TVM, 0% noise. (c) Without the TVM,
10% noise. (d) With the TVM, 10% noise. (e) Without the TVM, 25% noise. (f) With the
TVM, 25% noise. The axes (left and bottom) illustrate the spatial scale in mm, whereas the
color scale (right) records the absorbed energy density in mJ/mm3.
In this case, no noise was added to the “measured” data, and the off-center tar-
get had a diameter of 4 mm and Ψ = 1.5 mJ/mm 3 . Figures 3.5a and 3.5b present
two sets of absorbed energy density images recovered using the method with-
out the TVM (Figure 3.5a) and with the TVM enhancement (Figure 3.5b), while
Figures 3.5c and 3.5d show the quantitative profiles of the absorbed energy density
Image Enhancement Software and Hardware Approaches 55
Exact Exact
Without TVM Without TVM
2.0 2.0
With TVM With TVM
1.5 1.5
1.0 1.0
–10 –5 0 5 10 –10 –5 0 5 10
X Position/mm X Position/mm
(a) (b)
Exact
Without TVM
Absorbed Energy Density
2.0
With TVM
1.5
1.0
–15 –10 –5 0 5 10 15
X Position/mm
(c)
FIGURE 3.3 Comparison of the exact and reconstructed absorbed energy density profiles
along transect y = 0 mm for the images appearing in Figure 3.2. (a) 0% noise. (b) 10% noise.
(c) 25% noise.
along transects through the target for the images shown in Figures 3.2a,b and
Figures 3.5a,b. We can see that the images formed by incorporation of the TVM
are clearly enhanced qualitatively in visual content relative to that obtained using
the method without the TVM. We also note that the lower the contrast level, the
larger the enhancement.
In the fourth simulation, three targets having different shapes (circular, ellipti-
cal, and rectangular) were embedded in the background, and the absorbed energy
density of these targets was 2.5, 1.5, and 2.0 mJ/mm 3 , respectively. A noise level of
25% was added to the “measured” data in this case. Figure 3.6 shows the exact and
the recovered absorbed energy density images as well as the quantitative absorbed
energy density property profiles along the transect that across these targets. Again,
the improvement in image quality is apparent.
To quantitatively evaluate the reconstruction quality using the reconstruction
method with and without TVM enhancement, we use the universal quality index
56 Photoacoustic Tomography
2.2
2.2
10 10 2
2
5 1.8 5 1.8
0 1.6 0 1.6
–5 1.4 –5 1.4
1.2 1.2
–10 –10
1 1
–10 0 10 –10 0 10
(a) (b)
2.0
1.5
1.5
1.0 1.0
2.5 5.0 7.5 10.0 12.5 2.5 5.0 7.5 10.0 12.5
X Position X Position/mm
(c) (d)
FIGURE 3.4 Reconstructed absorbed energy density images from simulated data with and
without the total-variation-minimization (TVM) enhancement with different target sizes
(case 2). (a) Without the TVM, 2 mm-diameter target. (b) With the TVM, 2 mm-diameter
target. (c) The absorbed energy density profiles along the transect y = 0 mm for the images
appearing in Figures. 3.2a and 3.2b (4 mm-diameter target). (d) The absorbed energy density
property profiles along the transect y = 0 mm for the images appearing in Figures 3.4(a) and
3.4(b) (2 mm-diameter target). For (a) and (b), units of measurements on the axes are in mm
and units of measurements for numbers given at the color bar are in mJ/mm3.
(UQI) to measure the degree of similarity between the reconstructed and exact
images (Wang and Bovik 2002). We first interpreted an image f as vectors of size
N: f = ( f1 , f2 ,… f N )T , where superscript T denotes the matrix transpose, and N, the
number of image data acquired from the FE-based algorithm. We then defined image
means, variances, and covariances over the whole image domain as
N
1
fj=
N ∑ f , (3.15) k
j
k =1
N
1
j
σ =
N ∑( f
k =1
k
j
−f j 2
) , (3.16)
Image Enhancement Software and Hardware Approaches 57
1.6
1.5
10 10
1.4 1.4
5 5
1.3
0 0
1.2 1.2
–5 –5
1.1
–10 1 –10
1
–10 0 10 –10 0 10
(a) (b)
Exact Exact
Without TVM Without TVM
1.5
1.0
1.0
2.5 5.0 7.5 10.0 12.5 2.5 5.0 7.5 10.0 12.5
X Position X Position/mm
(c) (d)
FIGURE 3.5 Reconstructed absorbed energy density images from simulated data with and
without the total-variation-minimization (TVM) enhancement with different contrast levels
between the target and the background (case 3). (a) Without the TVM, 1.5:1 contrast. (b) With
the TVM, 1.5:1 contrast. (c) Absorbed energy density profiles along the transect y = 0 mm for
the images appearing in Figures. 3.2(a) and 3.2(b) (2:1 contrast). (d) Absorbed energy density
profiles along the transect y = 0mm for the images appearing in Figures 3.5(a) and 3.5(b)
(1.5:1 contrast). For (a) and (b), units of measurements on the axes are in mm and units of
measurements for numbers given at the color bar are in mJ/mm3).
2Cov{f 1 , f 0 } 2f1f 0
UQI{f 1 , f 0 } = . (3.18)
(σ1 ) 2 + (σ 0 ) 2 ( f 1 ) 2 + ( f 0 ) 2
UQI measures the image similarity between the reconstructed (f 1) and reference/
exact (f 0) images, and its value ranges between 0 and 1. The value of the UQI is
58 Photoacoustic Tomography
2.4
10 2.2 10 2.5
5 2 5
2
0 1.8
0
1.6 1.5
–5 –5
1.4
–10 1.2 –10 1
–10 0 10 –10 0 10
(a) (b)
3.0
Exact
FIGURE 3.6 Reconstructed absorbed energy density images from simulated data with and
without the total-variation-minimization (TVM) enhancement for three targets having dif-
ferent shapes (case 4). (a) Exact image. (b) Without the TVM. (c) With the TVM. (d) The
absorbed energy density profiles along the transect y = 0 mm. For (a), (b), and (c), units of
measurements on the axes are in mm and units of measurements for numbers given at the
color bar are in mJ/mm3).
closer to 1 when the reconstructed image is more similar to the exact image. We cal-
culated the UQIs for the simulation cases presented above, and the results are given
in Figure 3.7 where Figures 3.7a and 3.7b show the UQIs for the first case when a
different noise level is considered and for all four cases without added noise. In
Figure 3.7, the horizon axis indicates the case number (from cases 1 to 4, presented
above), and the vertical axis shows the value of UQI. We immediately note from
Figure 3.7 that the TVM-based method provides significantly better UQIs than that
without TVM.
3.3.2.2 Phantom Experiments
In this section phantom experimental data were used to confirm our findings from
the simulations. The experimental setup used for collecting the phantom data
was a pulsed ND: YAG laser-based single transducer (1 MHz) scanning system
described in Chapter 1. Three phantom experiments were conducted. In the first two
Image Enhancement Software and Hardware Approaches 59
1.00 1.00
0.98
0.98
0.96
0.94 0.96
UQI
UQI
0.92
0.94
0.90
0.88 Without TVM 0.92 Without TVM
With TVM With TVM
0.86
0.90
0% 10% 25% 1 2 3 4
Noise Level Case #
FIGURE 3.7 UQI calculated from the recovered images with and without TVM enhance-
ment from simulated data. (a) Case 1 when different noise levels are considered. (b) Cases 2–4.
experiments, we embedded one or two objects with a size ranging from 3.0 to 0.5mm
in a 50mm-diameter solid cylindrical phantom. The phantom materials used con-
sisted of Intralipid as scatterer and India ink as absorber with Agar powder (1–2%)
for solidifying the Intralipid and India ink solution. The absorption coefficient of the
background phantom was 0.01 mm–1, while the absorption coefficient of the target(s)
was 0.03 mm–1. In the last experiment, we used a single-target-containing phantom,
aiming to test the capability of detecting target having low optical contrasts relative
to the background phantom. In this case, the target had an absorption coefficient of
0.015 mm–1. The reduced scattering coefficients of the background phantom and
targets were 1.0 and 3.0 mm–1 for the first two experiments, and 1.0 and 2.0 mm–1 for
the last experiment.
A total of 120 receivers were equally distributed along the surface of the circular
background region. In the reconstructions, the fine mesh used for the forward cal-
culation consisted of 5977 nodes and 11,712 elements, while the coarse mesh used
for the inverse calculation had 1525 nodes and 2928 elements. The reconstructed
images were the results of 3 and 15 iterations for the method without and with the
TVM, respectively. For these experimental cases, ω Ψ = 1.0 and δ = 0.001 for the
first and third cases and ω Ψ = 2.0 and δ = 0.001 for the second case were used.
Here we note that in the experimental situation, the effect of ultrasonic transducer
response should be considered in the PAT image reconstruction because the trans-
ducer mechanical-electrical impulse response as well as the transducer aperture
effect may introduce significant errors in the forward model. To reduce these effects
in our calculations, we applied the normalized acoustic pressure distribution in the
reconstruction and used the appropriate initial parameters obtained from a search-
ing method (Jiang 2010).
Figure 3.8 shows the reconstructed absorbed energy density images for all the
three experimental cases, and Figure 3.9 presents quantitative absorption coefficient
profiles along transects through one target for the images shown in Figure 3.8. We
see that considerably enhanced images are achieved with the TVM, especially when
60 Photoacoustic Tomography
1.5
20 20
1 1
10 10
0 0.5 0
0.5
–10 –10
0
0
–20 –20
–20 –10 0 10 20 –20 –10 0 10 20
(a) (b)
1 1
20 20
0.8
10 0.5 10
0.6
0 0 0.4
0
–10 –10 0.2
0
–20 –0.5 –20
1
20 20
0.8 0.8
10 0.6 10 0.6
0 0.4 0 0.4
0.2
–10 0.2
–10
0
0
–20 –0.2 –20
FIGURE 3.8 Reconstructed absorbed energy density images from the three phantom exper-
iments. (a) Case 1 without the TVM. (b) Case 1 with the TVM. (c) Case 2 without the TVM.
(d) Case 2 with the TVM. (e) Case 3 without the TVM. (f) Case 3 with the TVM. Units of
measurements on the axes are in mm and units of measurements for numbers given at the
color bar are mJ/mm3.
Image Enhancement Software and Hardware Approaches 61
–0.4
–0.4
–20 –10 0 10 20 –20 –10 0 10 20
X Position/mm X Position/mm
(a) (b)
With TVM
Absorbed Energy Density
0.4 With TVM
0.8
0.2
0.4
0.0
0.0
–0.2
–20 –10 0 10 20 –20 –10 0 10 20
X Position/mm X Position/mm
(c) (d)
FIGURE 3.9 Recovered absorbed energy density profiles along (a) y = –7.0 mm crossing the
3 mm-diameter target for experimental case 1, (b) y = 8.0 mm crossing the 2 mm-diameter
target for experimental case 1, (c) y = 1.0 mm for experimental case 2, and (d) y = 6.5 mm for
experimental case 3.
the target is small (case 2), or when the contrast level between the target and the
background is low (case 3).
Because there is no true or exact image available for the experimental cases, we
use the contrast-to-noise ratio (CNR) to quantitatively evaluate the experimental
results. To compute CNR, we selected two regions of interest (ROIs) (each has the
same size). One region is in the target (referred as t-ROI) and the other is in the back-
ground (denoted as b-ROI). The CNR is defined as
f (t ) − f ( b )
CNR = , (3.19)
σ(b)
where f (t ) and f ( b ), respectively, denote the mean over the t-ROI and b-ROI, and σ( b )
denotes the variances over the b-ROI. We used solid- and dashed-line circles to express
62 Photoacoustic Tomography
1
20 20
1 0.8
10 1 10
0.6
0 0.5 0 3 0.4
2
–10 –10 0.2
0 0
–20 –20
–20 –10 0 10 20 –20 –10 0 10 20
(a) (b)
30
Without TVM
20 0.8 With TVM
10 0.6 20
4
CNR
0 0.4
0.2 10
–10
0
–20
0
–20 –10 0 10 20 1 2 3 4
Region #
(c) (d)
FIGURE 3.10 CNR calculated for the recovered images using the method with and without
TVM. (a)–(c) Images shown in Figures. 3.8(b), 3.8(d), and 3.8(f) with the ROIs marked (four
pairs of ROIs; solid-line-circle: t-ROI; dashed-line-circle: b-ROI). (d) CNR computed for the
4 pairs of ROIs shown in (a)–(c). For (a), (b), and (c), units of measurements on the axes are
mm and units of measurements for numbers given at the color bar are mJ/mm3.
the t-ROI and b-ROI for the three experimental cases. We calculated the CNR for each
pair of the t- and b-ROIs using Equation (3.19) and present the results in Figure 3.10.
We see that the CNR obtained using the method with TVM is clearly larger than that
using the method without TVM, indicating that the TVM-based method is less sensi-
tive to the noise effects.
20 20
1 1
10 10
0 0.5 0 0.5
–10 –10
0 0
–20 –20
1
20 20 0.8
0.8
10 10 0.6
0.6
0.4
0 0.4 0
0.2 0.2
–10 –10 0
0
–20 –0.2 –20 –0.2
20 20
1 1
10 10
0 0.5 0
0.5
–10 –10
0 0
–20 –20
–20 –10 0 10 20 –20 –10 0 10 20
(e) (f )
1
20 1 20
0.8
10 0.8 10
0.6 0.6
0 0
0.4 0.4
–10 0.2 –10 0.2
–20 0 –20 0
FIGURE 3.11 Reconstructed photoacoustic images based on few-detector data for case 1.
(a) 120 detectors, without TVM. (b) 60 detectors, without TVM. (c) 30 detectors, without
TVM. (d) 15 detectors, without TVM. (e) 120 detectors, with TVM. (f) 60 detectors, with
TVM. (g) 30 detectors, with TVM. (h) 15 detectors, with TVM.
Image Enhancement Software and Hardware Approaches 65
20 20 0.6
10 0.5 10 0.4
0 0 0.2
0 0
–10 –10
–0.2
–20 –0.5 –20
–0.4
–20 –10 0 10 20 –20 –10 0 10 20
(a) (b)
0.6 0.5
20 20
0.4
10 10
0.2
0 0 0 0
1
20 20 0.8
0.6
10 0.5 10
0.4
0 0 0.2
0 0
–10 –10
–0.2
–20 –0.5 –20 –0.4
20 20 0.6
0.6
10 10 0.4
0.4
0 0 0.2
0.2
–10 –10 0
0
–0.2
–20 –0.2 –20
–20 –10 0 10 20 –20 –10 0 10 20
(g) (h)
FIGURE 3.12 Reconstructed photoacoustic images based on few-detector data for case 2.
(a) 120 detectors, without TVM. (b) 60 detectors, without TVM. (c) 30 detectors, without
TVM. (d) 15 detectors, without TVM. (e) 120 detectors, with TVM. (f) 60 detectors, with
TVM. (g) 30 detectors, with TVM. (h) 15 detectors, with TVM.
66 Photoacoustic Tomography
1
20 20
0.8 0.8
10 0.6 10 0.6
0 0.4 0 0.4
0.2 0.2
–10 –10
0 0
–20 0.2 –20
–0.2
–20 –10 0 10 20 –20 –10 0 10 20
(a) (b)
1
20 20 0.8
0.6
10 10
0.5 0.4
0 0 0.2
0
–10 0 –10
–0.2
–20 –20 –0.4
–20 –10 0 10 20 –20 –10 0 10 20
(c) (d)
1
20 20
0.8 0.8
10 10 0.6
0.6
0 0.4 0 0.4
0.2 –10 0.2
–10
0 0
–20 –20
–0.2
–20 –10 0 10 20 –20 –10 0 10 20
(e) (f )
20 20 0.8
0.8
10 0.6 10 0.6
0 0.4 0 0.4
–20 0 –20 0
FIGURE 3.13 Reconstructed photoacoustic images based on few-detector data for case 3.
(a) 120 detectors, without TVM. (b) 60 detectors, without TVM. (c) 30 detectors, without
TVM. (d) 15 detectors, without TVM. (e) 120 detectors, with TVM. (f) 60 detectors, with
TVM. (g) 30 detectors, with TVM. (h) 15 detectors, with TVM.
Image Enhancement Software and Hardware Approaches 67
1.5
20 20
1 1
10 10
0 0.5 0 0.5
–10 –10
0 0
–20 –20
0.5
–20 –10 0 10 20 –20 –10 0 10 20
(a) (b)
20 20
1
10 0.5 10
0 0 0.5
0
–10 –10
0
–20 –0.5 –20
1.5
0.8
20 20
1 0.6
10 10
0.4
0 0.5 0
0.2
–10 0 –10 0
FIGURE 3.14 Reconstructed photoacoustic images based on limited-angle data for case 1.
(a) 120 detectors over 360°, without TVM. (b) 60 detectors over 180°, without TVM. (c) 30
detectors over 90°, without TVM. (d) 120 detectors over 360°, with TVM. (e) 60 detectors
over 180°, with TVM. (f) 30 detectors over 90°, with TVM.
1.00 1.00
0.75 0.75
UQI
UQI
0.50 0.50
1.00
0.75
UQI
0.50
0.25
W/o TVM
W/ TVM
0.00
360° 180° 90°
Scanning Degree
(c)
FIGURE 3.15 UQIs calculated from the recovered images for the three cases based on few-
detector data without TVM (a) and with TVM (b) and for case 1 based on limited angle data
with and without TVM.
in tissue, it has well-known limitations (Firbank et al. 1996; Hielscher et al. 1998;
Dehghani et al. 1999). For example, it fails to describe accurately light propagation
in medium where its absorption coefficient is not much smaller than the scattering
coefficient, or in so-called void-like regions where the absorption and scattering coef-
ficients are very low. Another situation where the diffusion approximation is not accu-
rate enough is in modeling light propagation in media of small volume, because of
the small optical distance between sources and measurements (Ren et al. 2007). In
addition, the variable anisotropy factor, g, also affects the photon migration in tissue,
which is not fully considered in the DE (Klose et al. 2002). In the area of diffuse
optical tomography (DOT), it has been shown that improved reconstruction of optical
properties can be obtained using RTE or higher-order diffusion equations (Klose et
al. 2002; Xu et al. 2003; Ren et al. 2007; Yuan et al. 2009).
Here we describe a RTE-based qPAT method that allows for quantitative recov-
ery of absorption coefficient images of heterogeneous media using tomographic
photoacoustic measurements. In this method, acoustic measurements from the con-
ventional PAT are combined with the RTE to separate the product of absorption
coefficient and optical fluence. We demonstrate our reconstruction algorithm using
Image Enhancement Software and Hardware Approaches 69
considerable simulated data under various practical scenarios and validate it using a
series of tissue-like phantom experiments.
( ⋅ + s + a )φ(r , ) = s
∫ S n −1
φ(r , )Θ( , ) d + q(r , ), (3.20)
where s = s (r ) and a = a (r ) are the scattering and absorption coefficients of
the
medium, respectively; φ (r , ) is the radiance; and q (r , ) is the source term;
∈ S n−1 denotes a unit vector in the direction of interest. Here S n−1 is the angular
direction; n = 2 or 3 denotes the physical domain, which is considered isotropic in
the sense that the probability of scattering between two directions depends only on
the relative angle between those directions. R is the spatial domain, and ∂R denotes
its boundary. The kernel Θ( , ) is the scattering phase function describing the
probability
density that a photon with an initial direction will have a direc-
tion after a scattering event. Here we assume that the scattering phase function
depends only on the angle between the incoming and outgoing directions, and thus
Θ( , ) = Θ( ⋅ ). (3.21)
1 1 − g2
Θ( ⋅ ) = 2
. (3.22)
2π (1 + g − 2 g cos γ )
where γ is the angle between the input direction and output direction . The
anisotropy factor, g (−1 < g < 1), defines the shape of the probability density. It is
known that g is between 0.7 and 0.99 for most tissues (Wnek and Bowlin 2004).
The boundary conditions (BC) for the RTE assume that no photons travel in an
inward direction at the boundary ∂R, that is,
φ(r , ) = 0, ⋅ nˆ < 0 for all r ∈∂ R (3.23)
where n̂ is the outward unit normal on ∂S. This BC, also known as free surface BC
or vacuum BC, implies that once a photon escapes the domain, it does not reenter it.
70 Photoacoustic Tomography
The BC can be modified to include a boundary source φ0 (r , ) at the source posi-
tion εi ∂R, and can be written as follows (Tervo et al. 1999):
φ 0 (r , ), r ∈∪ i εi ⋅ nˆ < 0
φ(r , ) = (3.24)
0, r ∈∂ S \∪i ε i ⋅ nˆ < 0
The solution to the RTE with the above boundary conditions is existent and unique
(Duderstadt and Martin 1979; Qatanani et al. 2007). To obtain the Galerkin weak form
of the RTE, Equation (3.20) is multiplied by a set of locally spatially varying Lagrangian
test/basis function, ψ j = ψ j (r , ) and integrated over the physical domain, R and the
angular directions, S n−1, yielding the finite-element discretization of Equation (3.20):
N
− ∑φ ∫ ∫
j =1
j
R S n −1
ψ j (r , ) ⋅ ψ i (r , ) d dr
N
+ ∑ φ ∫ ∫
j =1
j
∂R S n −1
( ⋅ nˆ )+ ψ j (r , )ψ i (r , ) d dS
N
+ ∑φ ∫ ∫
j =1
j
R S n −1
( a + s )ψ j (r , )ψ i (r , ) d dr (3.25)
N
− ∑φ ∫ ∫
j =1
j
R S n −1
s
∫ S n −1
Θ( ⋅ )φ(r , ) d ψ i (r , ) d dr
N
= ∑ φ ∫ ∫
j =1
0
j
∂R S n −1
( ⋅ nˆ )− ψ j (r , )ψ i (r , ) d dS
+
∫∫ R s n −1
ψ i (r , )q(r , ) d dr
where φ0j (r , ) is the source
term at the boundary, ( ⋅ nˆ ) ± denotes the positive
and the negative parts of ( ⋅ nˆ ) , and q(r , ) is the actual light source numerically
placed inside the domain.
In addition, φ(r , ) needs to be expanded as the sum of coefficients multiplied by
the Lagrangian basis function:
N Nn Na
φ(r , ) = ∑ j =1
ψ j (r , )φ j = ∑∑ψ
nj =1 mj =1
nj (r )ψ mj ( )φnj ,mj (3.26)
where ψ nj (r ) and ψ mj ( ) are the nodal spatial and angular basis functions; φnj ,mj is
the radiance at the spatial nodal point, nj, and direction, mj; Nn is the number of spa-
tial nodes of the mesh; and Na is the number of angular directions. It is known that the
ray effect may disturb the standard FE-techniques when solving the RTE, since it can
Image Enhancement Software and Hardware Approaches 71
produce oscillating results, or it can visually be seen as “photon rays” radiating from
the source into the direction of the discretization angles (Kanschat 1998; Tarvainen
et al. 2005). To overcome the ray effect, the streamline diffusion modification (SDM)
is used in theFE-solution of the RTE. In the SDM, the test function is of the form
(ψ j (r , ) + δ ⋅ ψ j (r , )) instead of the standard form of a test function (ψ j (r , )).
The parameter δ is the “smoothing” parameter, which is a spatially varying constant
that depends on the local absorption and scattering (Kanschat 1998).
Equation (3.25) can be rewritten in matrix form:
[ K1 + K1,sdm + K 2 + K 3 + K 3,sdm + K 4 + K 4,sdm ]{φ} = [ B1 ]{φ0 } + {B2} (3.27)
where
2π
∂ψ ni (r ) ∂ψ (r )
K1ij = −
∫ R
ψ nj (r )
∫ 0
cos θ
∂x
+ sin θ ni
∂y
ψ mj (θ)ψ mi (θ) d θ dr
2π
∂ψ ni ( r ) ∂ψ ( r ) ∂ψ nj ( r ) ∂ψ ( r )
K1,ijsdm =
∫ ∫ R
δ
0
cos θ
∂x
+ sin θ ni
∂y
cos θ
∂x
+ sin θ nj
∂y
ψ mj ( θ ) ψ mi ( θ ) dθdr
2π
K 2ij =
∫ ∂R
ψ nj (r )ψ ni (r )dS
∫ 0
(nˆ x cos θ + nˆ y sin θ)+ ψ mj (θ)ψ mi (θ) d θ
2π
K 3ij =
∫ R
( a + s )ψ nj (r )ψ ni (r ) dr
∫ 0
ψ mj (θ)ψ mi (θ) d θ
2π
∂ψ ni (r ) ∂ψ (r )
K 3,ij sdm =
∫ R
δ( a + s )ψ nj (r ) dr
∫0
cos θ
∂x
+ sin θ ni
∂y
ψ mj (θ)ψ mi (θ) d θ
2π 2π
K 4ij = −
∫ R
s ψ nj (r )ψ ni (r )dr
∫0
ψ mi (θ) d θ
∫0
ψ mj (θ )Θ(θ ⋅ θ ) d θ
2π
∂ψ ni (r ) ∂ψ (r )
K 4,ij sdm = −
∫ R
δ s ψ nj ( r )dr
∫ 0
cos θ
∂x
+ sin θ ni
∂y
ψ mi ( θ ) d θ
2π
∫0
ψ mj (θ )Θ(θ ⋅θ ) d θ
2π
B1ij =
∫ ∂R
ψ nj ( r ) ψ ni ( r )dS
∫ 0
(nˆ x cos θ + nˆ y sin θ)− ψ mj (θ)ψ mi (θ) d θ
2π
B2i =
∫∫
R 0
q(r , θ)ψ mi (θ) d θψ ni (r )dr
{φ} = {φ1 , φ2 ,φ N n N a }T ;{φ0 } = φ10 , φ20 ,φ0N n N a { }
72 Photoacoustic Tomography
where the outward unit normal is denoted by nˆ = (nˆ x , nˆ y ), S n−1 = S 1 the unit cir-
cle, = (cos θ,sin θ) a point on S 1 , i = N a (ni − 1) + mi , j = N a (nj − 1) + mj , and
(i, j = 1,… N n N a ) .
2 v0βΨ(r )
p(r , ω ) + k02 p(r , ω ) = ik0 ; (3.28)
Cp
where p is the pressure wave; k0 = ω/v0 is the wave number described by the angu-
lar frequency, ω, and the speed of acoustic wave in the medium, v0; β is the ther-
mal expansion coefficient; Cp is the specific heat; Ψ is the absorbed energy density
that is product of absorption coefficient, μa, and optical fluence, Φ (i.e., Ψ = a Φ );
po = ( p1o , p2o ,… pMo )T , pc = ( p1c , p2c ,… pM ) , and pio and pic are observed and com-
c T
puted complex acoustic field data for i = 1,2,⋯M boundary locations; Δχ is the update
vector for the absorbed optical energy density; ℑ is the Jacobian matrix formed by
∂ p/∂Ψ at the boundary measurement sites; λ is the regularization parameter deter-
mined by combined Marquardt and Tikhonov regularization schemes; and I is the
identity matrix. Thus here the image formation task is to update absorbed optical
energy density distribution via iterative solution of Equations (3.28) and (3.29) so that
an object function composed of a weighted sum of the squared difference between
computed and measured acoustic data can be minimized.
The second step is to determine the μa distribution by the following iterative solu-
tion procedure using finite element method (see Section 2.1): (a) choose an initial
value for μa, e.g., a = 0.0001 mm −1; (b) compute the optical fluence Φ using the
RTE; (c) calculate the absorbed energy density by Ψ c = a Φ ; (d) compute the error
between the measured Ψ (from the first step) and calculated Ψ c , and μa is updated
using a = Ψ/Φ; and (e) if the error is sufficiently small, then the iterative calcula-
tion stops; otherwise repeat steps (b) to (d) until a small error is reached. This pro-
cedure converges within about 10 to 20 iterations generally. In the reconstruction,
Image Enhancement Software and Hardware Approaches 73
Source
FIGURE 3.16 Test geometry used for the simulations described in Section 3.4.3.1.
μs, and g are assumed as constant, while the incident laser source strength and the
absorbed energy density Ψ are estimated in advance.
100
µa = 0.1 mm–1
0.01
µa = 1.0 mm–1
1E–4
1E–6
1E–8 RTE
DE
1E–10
20 30 40 50 60 70 80
Position (mm)
(a)
Photon Intensity (Photons/mm2)
0.1
0.01 RTE
DE
20 30 40 50 60 70 80
Position (mm)
(b)
FIGURE 3.17 Comparison of the diffusion and transport calculations: (a) influence of
μa /μ′ ;
s (b) influence of an almost scattering- and absorption-free heterogeneity.
observed inside the target, while the two models agree well outside the target
region. We also observe significant differences between the two calculations for
the third simulation although the diffusion approximation holds everywhere in the
media for this case. We note that all these results shown in Figure 3.17 agree well
with those published in the literature involving transport calculations (Klose et al.
2002, Aydin et al. 2004).
Image Enhancement Software and Hardware Approaches 75
FIGURE 3.18 Test geometry used for the simulations described in Section 3.4.3.2.1.
10 10
0.02 0.02
5 5
0 0.015 0 0.015
–5 –5
0.01 0.01
–10 –10
–10 –5 0 5 10 –10 –5 0 5 10
(a) (b)
10 10
0.02 0.02
5 5
0 0.015 0 0.015
–5 –5
0.01 0.01
–10 –10
–10 –5 0 5 10 –10 –5 0 5 10
(c) (d)
10 10
0.02 0.02
5 5
0 0.015 0 0.015
–5 –5
0.01 0.01
–10 –10
–10 –5 0 5 10 –10 –5 0 5 10
(e) (f )
FIGURE 3.19 Reconstructed absorption images from simulated data using the RTE-based
(a, c) and DE-based (b, d) methods. (a) and (b): μa /μ′s = 0.01. (c) and (d): μa /μ′s = 1.0. (e) and
(f): μa /μ′s = 0.01 with 10.0% noise level.
with 10.0% noise added to the measured data. We see that both methods can recover
the absorption images qualitatively for the two cases, while it is clear that the RTE-
based method has less noise sensitivity than the DE-based method. Quantitative
absorption coefficient profiles along a cut-line (Y = 0 mm) through the target for the
images shown in Figure 3.19 are presented in Figures 3.20a,b. We immediately note
that both methods agree well for a small a / s ratio when the absorption coefficient
is much smaller than the reduced scattering coefficient (Figure 3.20a), while the
absorption coefficient is considerably overestimated using the DE-based method for
a large a / s ratio (Figure 3.20b). We performed several additional reconstructions
Image Enhancement Software and Hardware Approaches 77
0.03 0.03
Recovered Absorption Coefficient
0.02 0.02
0.01 0.01
50
PAT + RTE
40 PAT + DE
Relative Error (%)
30
20
10
FIGURE 3.20 Recovered absorption coefficient profiles along y = 0 mm for the images
shown in Figure 3.19(a): μa /μ′s = 0.01. (b): μa /μ′=
s 1.0. (c): Relative errors of the recovered
absorption coefficient of the target over different μa /μ′s ratios.
with different μa /μ′s ratios, and present the relative errors of the recovered absorption
coefficient of the target for these cases in Figure 3.20c. It can be seen that the error
for the DE-based method increases as the μa /μ′s ratio increases and can be as large as
>40% when μa /μ′s = 1.0. In these cases, the RTE-based method always gives accurate
reconstruction with a largest error of 0.1%.
Group 2 simulation is intended to investigate how the g factor affects the recov-
ered absorption coefficient quantitatively when the μa /μ′s ratio is small (μ′s = 1.0mm–1).
Figures 3.21a and 3.21b present the recovered absorption coefficient images for g =
0.75 using the RTE- and DE-based methods, respectively, while Figure 3.21c shows
the absorption property profiles along the cut-line y = 0 mm for the images shown
in Figures 3.21a and 3.21b. We can see that the DE-based method can qualitatively
reconstruct the absorption image, but significantly underestimate the absorption coef-
ficient values. Relative errors of the recovered absorption coefficient of the target for
several cases with different g factors are given in Figure 3.21d where we see that the
DE-based method underestimates the absorption coefficient values for all the cases,
while the RTE-based method recovers these values accurately. It is interesting to
78 Photoacoustic Tomography
10 10
0.02 0.02
5 5
0 0.015 0 0.015
–5 –5
0.01 0.01
–10 –10
–10 –5 0 5 10 –10 –5 0 5 10
(a) (b)
Recovered Absorption Coefficient
Exact 0
PAT + RTE
0.02 PAT + DE
Relative Error (%)
–10
–20
–30
0.01
–40 PAT + RTE
PAT + DE
–50
–15 –10 –5 0 5 10 15 0.00 0.25 0.50 0.75
X Position (mm) g factor
(c) (d)
FIGURE 3.21 Reconstructed absorption images from simulated data with g = 0.75 using
the RTE (a) and DE (b). (c) Absorption coefficient profiles along Y = 0mm for the images
shown in (a) and (b). (d) Relative errors of the recovered absorption coefficient of the target
over different g factors.
note that the error due to the DE-based estimation linearly increases as the g factor
increases.
Group 3 simulation aims to see how the domain size impacts the reconstructions.
In these cases we significantly decrease the domain size from R = 15mm to R = 1.5 mm,
while keeping the ratio R/r as constant, g = 0 and μ′s = 1.0 mm–1. Figures 3.22a and
3.22b show the recovered absorption coefficient images for R = 1.5 mm using the RTE-
and DE-based methods, respectively, while Figure 3.22c gives the absorption property
profiles along the cut-line Y = 0 mm. Figure 3.22d provides the relative errors of the
recovered absorption coefficient of the target with changed domain size. As can be seen
from Figure 3.22, while the DE-based method can reconstruct the absorption images
qualitatively for a small domain size, it always overestimates the recovered absorption
coefficient values for small size domains, and the error can be as large as 35% when the
domain size is just couple of millimeters in diameter. We note that there is almost no
error for reconstructing the absorption coefficient using the RTE-based method.
Image Enhancement Software and Hardware Approaches 79
1 1
0.02 0.02
0.5 0.5
0 0.015 0 0.015
–0.5 –0.5
0.01 0.01
–1 –1
0.030 50
Recovered Absorption Coefficient
Exact PAT+RTE
PAT+RTE 40 PAT+DE
0.025 PAT+DE
Relative Error (%)
30
0.020
20
0.015
10
0.010 0
FIGURE 3.22 Reconstructed absorption images from simulated data with R = 1.5 mm
using the RTE (a) and DE (b). (c) Absorption coefficient profiles along Y = 0 mm for the
images shown in (a) and (b). (d) Relative errors of the recovered absorption coefficient of the
target over different domain size.
3.4.3.2.2 Experiments
The PAT setup employed is described in Chapter 1. Here a Ti-Sapphire laser (Altos,
Bozeman, Montana) with a pulse repetition rate of 10 Hz and a pulse width of <10 ns
at 880 nm was used in the experiments. The laser beam was expanded to a beam
with 3.8 cm in diameter and delivered to the top surface of a cylindrical solid phan-
tom. The solid phantoms used in our experiments were made of Intralipid, India
ink, distilled water and Agar power, among which Intralipid served as the scatter-
ing media and the India ink served as the absorption media. In the experiments,
we embedded a single cylindrical target (centered or slightly off-centered) with
various size and optical properties in a cylindrical background having different
size and optical properties. Table 3.1 lists the dimensions and optical properties of
the target and the background used for the five phantom experiments designed for
the comparative study of the RTE- and DE-based qPAT. A mesh consisting of 1301
80 Photoacoustic Tomography
TABLE 3.1
Optical Properties and Size of the Target and the Background Used in the
Phantom Experiments
Background Targets
nodes and 2480 triangular elements along with 64 angular directions was used for
the reconstructions.
Figures 3.23a to 3.23j present the reconstructed absorption coefficient images
for the five phantom experiments using the RTE-based (left column of Figure 3.23)
and DE-based (right column of Figure 3.23) algorithms, respectively. Consistent
with the simulation findings shown in the previous section, we immediately see that
the target can be clearly imaged by the two methods for all the cases. The major
difference between the two methods lies in the ability of quantitatively resolv-
ing absorption coefficient. This difference in quantifying absorption coefficient is
clearly demonstrated in Figure 3.24, which shows the recovered absorption prop-
erty profiles along a transect through the center of the target and background for
the images shown in Figure 3.23. We see that the DE-based method either under-
estimates or overestimates the absorption coefficient of the target for a decreased
small domain size or a large μa /μ′s ratio/increased g factor, while the RTE-based
method always provides accurate reconstruction of the absorption coefficient for
all the cases. Again, these observations are consistent with the findings from the
simulations.
We found that the average value of the recovered absorption coefficient of target
is 0.0511 mm–1, 0.0486 mm–1, 0.0489 mm–1, 0.0486 mm–1, and 0.0492 mm–1 using
the RTE-based method, which is in excellent agreement with the actual value of
0.05 mm–1, while the DE-based method gives a recovered target absorption coef-
ficient of 0.0452 mm–1, 0.0386 mm–1, 0.0565 mm–1, 0.0751 mm–1, and 0.0369 mm–1
for the experimental cases. We list the relative errors of the recovered absorption
coefficient of the target for the five experiments in Table 3.2. It is clear that the
DE-based method presents significant errors for the cases examined especially when
the domain size is decreased and g factor is nonzero (case 2), g factor is increased
(case 5), or the domain size is decreased and the μa /μ′s ratio is increased along with a
nonzero g factor (case 4). We also note that the RTE-based method gives an error of
below 3% for all the cases.
Image Enhancement Software and Hardware Approaches 81
5 5
0.05 0.05
0.04 0.04
0 0.03 0 0.03
0.02 0.02
0.01 0.01
–5 –5
–5 0 5 –5 0 5
(a) (b)
–1 0 1 –1 0 1
(c) (d)
5 5
0.05 0.05
0.04 0.04
0 0.03 0 0.03
0.02 0.02
0.01 0.01
–5 –5
–5 0 5 –5 0 5
(e) (f )
5 5
0.05 0.05
0.04 0.04
0 0.03 0 0.03
0.02 0.02
0.01 0.01
–5 –5
–5 0 5 –5 0 5
(i) (j)
FIGURE 3.23 Reconstructed absorption coefficient images (mm–1) from the five sets of
phantom experimental data using the RTE (a, c, e, g, i) and DE (b, d, f, h, j): (a) and (b) case 1,
(c) and (d) case 2, (e) and (f) case 3, (g) and (h) case 4, (i) and (j) case 5.
82 Photoacoustic Tomography
(a) (b)
0.07 0.10
0.07
Absorption Coefficient (mm–1)
Exact
0.06 PAT+RTE
PAT+DE
0.05
0.04
0.03
0.02
0.01
0.00
–4 –3 –2 –1 0 1 2 3 4
y Position (mm)
(e)
FIGURE 3.24 Recovered absorption coefficient profiles along (a) x = 0 mm for the images
shown in Figures 3.23(a) and 3.23(b). (b) x = –0.25 mm for the images shown in Figures 3.23(c)
and 3.23(d). (c) x = –0.5 mm for the images shown in Figures 3.23(e) and 3.23(f). (d) x = –1.0 mm
for the images shown in Figures 3.23(g) and 3.23(h). (e) x = 0.0 mm for the images shown in
Figures 3.23(i) and 3.23(j).
Image Enhancement Software and Hardware Approaches 83
TABLE 3.2
Relative Errors of the Recovered Absorption
Coefficient of the Target for the Five
Experimental Cases
Cases DE-Based Algorithm RTE-Based Algorithm
1 –9.6% 2.2%
2 –22.8% –2.8%
3 13.2% –2.2%
4 50.2% –2.8%
5 –26.2% –1.6%
3.5 PARALLEL COMPUTATION
PAT image reconstruction, especially full 3D reconstruction, requires significant
computational times. Given the large problem size, three major areas of the algo-
rithm are costly (see Chapter 1): (1) computation of the forward solution, (2) buildup
of the Jacobian matrix, which requires calculation of the derivatives of acoustic field
with respect to optical/acoustic properties at the receiver sites, and (3) computation
of a full matrix equation for the inverse solution. Because of the massively parallel
structure of these types of image reconstruction algorithms, parallel computing can
be adapted for 3D PAT reconstruction.
Parallel computing is basically the concurrent execution of many computations
using many processors. In a parallel computing scheme, the memory request and
the computation tasks for a computation assignment can be spread on multiple pro-
cessors as shown in Figure 3.25. Here we give a detailed description on the parallel
computing based 3D PAT algorithm. In the frequency-domain FE-based PAT algo-
rithm, the forward calculation of acoustic field and the determination of Jacobian
A computation assignment
… …
CPU CPU CPU CPU CPU CPU CPU CPU
FIGURE 3.25 Schematic of parallel computing with a combination of both shared memory
and distributed memory.
84 Photoacoustic Tomography
matrix J are based on the forward matrix A at each frequency ωi, ordered from ω1
to ωKwith total K frequency elements. The matrix A in the forward calculation is
usually a symmetric sparse matrix and stored in memory by banded storage or com-
pressed storage strategy, requiring less memory than the Jacobian matrix J and the
Hessian matrix JTJ, which is usually a full matrix. Herein, we spread the forward
calculation of acoustic field and the determination of Jacobian matrix on distributed
processors (total number is (Q + 1) by the frequency element ωi, where the whole
matrix A under certain frequency elements is stored in the memory of the specifi-
cally assigned processor. As shown in flowchart of the high-performance photo-
acoustic tomography (Figure 3.26), the matrix A at frequency ωi is stored in the
memory of a processor determined by Mod(i − 1, L), where L is the average number
No
Because the Hessian matrix H is symmetric, we store only the upper triangle
of the Hessian matrix spread over the processors. In the calculation of element Hij,
communication and data exchange
may
be involved among the distributed proces-
sors, except that the vector {J j } and {J j } are both stored in the memory of the same
processor. Since the Hessian matrix is stored dispersedly, the inverse reconstruction
requires a parallel solving method, where parallelized Cholesky decomposition is
used in the high-performance PAT. While the parallelized code can improve the
computation speed at least by two-order of magnitude compared to the un-paral-
lelized code for 3D image reconstruction, it is still computational demanding. For
example, a 3D image reconstructed using a finite element mesh of 41,323 tetrahedral
elements and 7519 nodes required ~120 minutes per iteration for a total of 10 itera-
tions on a computation cluster with 10 processors (each of the processors worked at
2.2 GHz with 2 GB memory).
3.6 VARIABLE-THICKNESS MULTILAYERED
POLYVINYLIDENE FLUORIDE TRANSDUCER
In PAT, the ultrasound transducer is a critical component as it receives the wide-
band acoustic waves generated in tissue by the pulsed laser. The sensitivity of the
transducer determines largely the signal-to-noise ratio (SNR), while its bandwidth
86 Photoacoustic Tomography
defines the resolution. Transducers currently used for PAT are largely based on
three types of piezoelectric materials including piezoelectric ceramics (PZT), sin-
gle crystal (PMN-PT), and polyvinylidene fluoride (PVDF). Among these materi-
als, PVDF owns some advantages. PVDF has a high mechanical damping ability
and a unique permittivity, resulting in a high bandwidth. In addition, the acoustic
impedance (Z0) of PVDF is 2.7 MRays, which is much lower than that of PZT
(>30 MRays). Hence it provides a good coupling efficiency to human tissue or com-
monly used ultrasound gels. PVDF is flexible and can be easily fabricated in convex
shapes replacing the acoustic lens commonly used in PZT or PMN-PT. We note,
however, that the sensitivity of the PVDF transducers currently used for PAT is
not comparable to the PZT and PMN-PT based transducers (Manohar et al. 2004,
Ermilov et al. 2009, Xi et al. 2012), leading to limited SNR especially when targets
are deeply located within tissues. In order to improve the sensitivity of the PVDF
transducers, several transducer designs based on the same-thickness PVDF layers
including folded transducer (FT), Barker-coded transducer (BCT), and switchable
Barker-coded transducer (SBCT) have been suggested in the field of ultrasound
imaging (Zhang and Lewin 1995, Fukuda et al. 2006, Nakazawa et al. 2007). While
the sensitivity is improved, there is no significant improvement in bandwidth from
these designs. Here we describe a design of PVDF transducer with variable-thick-
ness layers to provide improvement in both the sensitivity and bandwidth for pho-
toacoustic imaging.
The design of our multilayered transducer is shown in Figure 3.27a where the
PVDF films (metalized film sheets, measurement specialties) having three different
thicknesses (28 µm, 52 µm, and 110 µm) are sandwiched by electrodes. The fabrica-
tion procedure of this transducer is as follows: (1) shaping the lateral dimension of
Electrode –
PVDF (28 µm) Trigger Mirror
+ Nd: YAG
+ Transparent glue
PVDF (52 µm)
–
– Rotator
PVDF (110 µm) 2D stage
+
PC
Backing
(a) Pulser/receiver
Lens
Amplifier
Trigger Transducer
Transducer
Oscilloscope (c)
(b)
FIGURE 3.27 (a) Configuration of the multilayered PVDF transducer. (b) Schematic of
the circular-scanning based photoacoutic imaging system. (c) Schematic of the pulse/echo
evaluation system.
Image Enhancement Software and Hardware Approaches 87
the three PVDF films to 10 mm × 3 mm; (2) removing the unneeded silver coating
from both sides of each film to ensure electric insulation between the positive and
negative electrodes; (3) stacking/gluing the films/backing material together using
ultrasound transparent epoxy (EPO-TEK 301-2, Epoxy Technology); (4) connecting
coaxial cables to the electrodes using silver epoxy (E-solder 3022. VonRoll USA,
Inc.); and (5) sealing the whole transducer with ultrasound transparent epoxy.
The multilayered transducer fabricated is tested using a circular scanning pho-
toacoustic imaging system (Figure 3.27b). A laser beam generated from a pulsed
Nd:YAG laser (532 nm) was redirected through the center of the rotator and
expanded by a lens to illuminate an imaging area of 6.25 cm2. A 2D linear stage
(NLS4 Series, Newmark Systems) was used to optimize the position of transducer.
A water-tank with a through hole in the bottom sealed with transparent membrane
was used to provide coupling for ultrasound transmission. The signal received by
the PVDF transducer was amplified by two low-noise amplifiers and digitalized/
stored by a data acquisition card at 50MHz sampling rate. A pulse/echo test system
(Figure 3.27c) was used to evaluate the bandwidth of the multilayered transducer.
The transducer was driven by a pulser/receiver (5073PR, Panametrics-NDT), and a
piece of metal with a thickness of 5 mm was placed in front of the transducer to act
as a reflector. The reflected pulse was received by the transducer, amplified by the
internal amplifier of the pulser/receiver and recorded using an oscilloscope.
Before the performance evaluation and in vivo experiments, the transducer was
calibrated through an experiment with a hair-embedded solid phantom. The solid
phantom was a mixture of Intralipid (scatter), India ink (absorber), and 2% Agar
powder (solidifier). The absorption and reduced scattering coefficients of the phan-
tom were 0.007 mm–1 and 1.0 mm–1. A human hair was inserted in the solid phantom,
which was illuminated by the light beam. Figures 3.28a, 3.28b, and 3.28c show the
acoustic signals from different layers of the transducer where we note a time shift
1000 1000
28 um T1 = 0.6 52 um T2= 0.66
PA Signal
PA Signal
0 0
–1000 –1000
0 0.4 0.8 1.2 0 0.4 0.8 1.2
Time (us) Time (us)
(a) (b)
PA Signal
W/o Cali
0 0
–1000 –1000
0 0.4 0.8 1.2 0 0.4 0.8 1.2
Time (us) Time (us)
(c) (d)
FIGURE 3.28 Photoacoustic signals of the 28µm- (a), 52 µm- (b), and 110 µm-thick (c) layer
transducer from a hair. (d) Photoacoustic signals with and without the calibration.
88 Photoacoustic Tomography
between these signals, caused by the different distance of these three layers from the
target. Hence, calibration of this time shift is needed before further evaluating the
performance of the transducer. Assuming that the arrival times of the signals are T1,
T2, and T3 for the layers of 28, 52, and 110 µm, respectively, and setting the arrival
time for the 28 µm-thick layer as the baseline, we can then calculate the time shift of
the other two layers relative to T1 by the following relationships:
Δt1 = T2 − T1 (3.32a)
Δt2 = T3 − T1 (3.32b)
Thus, the photoacoustic (PA) signal from the multilayered transducer can be
obtained as
where P1, P2, and P3 are the phase of the PA signals which are 1, –1, and 1 in
our case for the 28 µm-, 53 µm-, and 110 µm-thick layer, respectively. Figure 3.28d
shows the calibrated signal and noncalibrated signal. We see that the SNR of the
calibrated signal is improved significantly (by 2.5 times) compared with that without
the calibration.
Figures 3.29b, 3.29c, and 3.29d show the frequency characteristics by fast Fourier
transforming the reflected acoustic waves detected by each of the three layers using
the pulse/echo system (Figure 3.27c). For the 110 µm-, 52 µm-, and 28 µm-thick
single-layer transducer, the central frequency and the bandwidth (-– level) are,
respectively, 5 MHz/85%, 10 MHz/78%, and 15 MHz/59%, while the central fre-
quency of the multilayered transducer is 10 MHz and the –6 dB level bandwidth is
improved to be 140%, as shown in Figure 3.29a.
In vivo animal experiments were performed to evaluate the merits of the improved
bandwidth and sensitivity for photoacoustic imaging. The brain of adult BALB/C mice
(~25g) was imaged. Before imaging, the hair was removed with a hair remover lotion.
The mice were anesthetized by a mixture of ketamine (85 mg/kg) and xylazine during
the imaging, and were sacrificed afterward using the University of Florida Institutional
Animal Care and Use Committee (IACUC)-approved techniques. The laser beam pro-
vided an incident energy density of 9 mJ/cm2 at the skin of the mice, which is much
lower than the safety standard provided by ANSI (20 mJ/cm2 at 532 nm). For each
imaging experiment, we collected PA signals at 120 positions by scanning the trans-
ducer around the mouse brain with a scanning step of 3°. The PA image was recon-
structed by the delay-and-sum algorithm described in Chapter 1.
Figure 3.30a shows a typical signal from in vivo mouse brain measured by the
multilayered transducer without (top panel) and with (bottom panel) calibration.
Without the calibration, the SNR is low and the image formed with such signals has
relatively poor quality. As we see from Figure 3.30b (left), in such case, some blood
vessels inside the brain are not visualized and almost every blood vessel imaged
is not continuous, as compared to the photograph of the brain after removal of the
scalp (Figure 3.30b, right). With the calibration, the SNR is significantly improved as
Image Enhancement Software and Hardware Approaches 89
1 1
Bandwidth Bandwidth
0.8 (78%) 0.8 (59%)
0.6 0.6
13.8 MHz
0.4
0.4
0.2
0.2
0 10 MHz 18.5 MHz
6 MHz
0
0 10 20 30 40 0 10 20 30 40
Frequency (MHz) Frequency (MHz)
(c) (d)
FIGURE 3.29 Frequency response of the multilayered PVDF transducer (a); 110 µm-thick
layer PVDF transducer (b); 52 µm-thick layer PVDF transducer (c); and 28 µm-thick layer
PVDF transducer (d).
shown in the bottom panel of Figure 3.30a. From the image formed with the calibra-
tion (Figure 3.30b, middle), we see that the eyes, blood vessels and other tissues are
identified clearly in comparison with the photograph.
In a separate in vivo experiment, we compared the sensitivity of the multilayered
transducer with the single-layer transducers, and the PA images obtained are pre-
sented in Figure 3.31. We immediately note the clearly higher SNR shown by the
image using the multilayered transducer (Figure 3.31a) compared with that using the
single-layer transducer (Figures 3.31b, 3.31c, and 3.31d). In particular, we can iden-
tify the tissues, organs, and blood vessels inside the brain from Figure 3.31a much
more clearly than that from Figure 3.31b, 3.31c, and 3.31d as indicated by red arrows.
We have shown that the design of variable-thickness multilayered PVDF trans-
ducer provides significant improvement in both the sensitivity and bandwidth com-
pared to the conventional single-layer transducer or commercial hydrophone (e.g.,
from Precision Acoustics, England). However, we note several limitations associated
with the current design, which should be overcome in the future. First, the sensitivity
90 Photoacoustic Tomography
Eye
Max Eye
Min
(b)
FIGURE 3.30 (a) In vivo photoacoustic (PA) signals from the multilayered transducer with
(bottom panel) and without (top panel) calibration. (b) Reconstructed brain images using PA
signals without (left panel) and with (middle panel) calibration. Photograph of the brain after
the removal of the scalp (right panel).
of the current multilayered transducer is still not comparable with that of a commer-
cial PZT unit. This, however, can be overcome by stacking more layers (more than
three) together to make a more sensitive transducer. Second, the PVDF films used
in the current work are commercial units with electrodes on both sides of each film.
After stacking them together, there are two layers of electrodes between two adjacent
films. As a result, the improvement of the sensitivity is not linearly proportional to
the number of layers stacked due to the attenuation from the electrodes. This prob-
lem can be solved by using raw film liquid, which will allow us to use only one much
thinner electrode (just a few µm) between two adjacent films during depositing the
films. Third, no matching layer and electromagnetic shield (EMS) housing were used
in the current design. Hence, we had to average 50 times on the signals collected to
reduce the electromagnetic noise, resulting in a relatively long data acquisition time.
Image Enhancement Software and Hardware Approaches 91
Min
(a) (b)
52 um 28 um
(c) (d)
FIGURE 3.31 In vivo photoacoustic imaging of mouse brain using the multilayered trans-
ducer (a) and single-layer transducer (b: 110 µm thick; c: 52 µm thick; d: 28 µm thick).
Polyimide
film
Upper
electrode
Lower AlN film
electrode
SiO2
Here we describe the fabrication and characterization of the pMUT and its appli-
cation to PAT. As shown in Figure 3.32, the pMUT consists of SiO2, bottom elec-
trode, AlN films, upper electrode, and polyimide (PI). In the microstructures, PI
was fabricated using spin-coating and reactive sputtering technology. PI played two
important roles here: one was to isolate the upper and lower electrodes and to reduce
the possible parasitic capacitance between the two electrodes; the other was to act
as the flexible protective layer on the surface. Compared to SiO2 protective layer, PI
is conducive to the vibration of the films according to the finite element simulation.
In the design of pMUT, AlN-based piezoelectric layer was synthesized through
middle-frequency magnetron reactive sputtering from metallic rectangular Al tar-
gets at room temperature compatible with the standard IC technology. Figure 3.33a
shows a typical field emission scanning electron microscopy (SEM) image of the
cross-sectional structure of the AlN films on the bottom electrode. The SEM image
reveals that the AlN thin film was well-crystallized and comprised well-oriented
columnar grains that were perpendicular to the substrate. The diameter of the colum-
nar grains was approximately 140 nm. Figure 3.33b is the 2θ-ω scan x-ray diffrac-
tion (XRD) spectrum of the AlN thin films, which shows only the (0002) hexagonal
AlN and Pt (111) peaks. The inset shows the rocking curve of the (0002) diffraction
peak for the AlN thin films. The FWHM of the rocking curve is 7.0°.
To increase the receiving response, the pMUT had an area ratio of 0.45 between
the top electrode and vibrating membrane, and the AlN film had only a 300 nm-
thick silicon oxide film and a 200 nm-thick bottom electrode as its supporting layer.
Our experience showed that acrylonitrile-butadiene-styrenecopolymer (ABS) could
protect the superficial silicon oxide in tetramethylammonium hydroxide (TMAH)
solution for about 8 hours when the annealing temperature was 250°C. The rela-
tionship between the protection time and temperature of etching solution is given
in Figure 3.34. Based on this experience, we constructed the pMUT with only 300
nm-thick silicon oxide layer as the device supporting layer.
The electromechanical coupling coefficient of pMUT is defined as (ANSI/IEEE
Std 176-1987):
2
keff fa2 − fr2
2 = (3.34)
1 − keff fr2
Image Enhancement Software and Hardware Approaches 93
1 µm
(a)
X-ray Diffraction Intensity (a.u.)
AlN (0002)
X-ray Diffraction Intensity (a.u.)
Pt (111)
5 10 15 20 25 30
Omega (deg)
20 30 40 50 60
2 Theta-Omega (deg)
(b)
FIGURE 3.33 (a) SEM image of the AlN on bottom electrode. (b) 2θ-ω scan X-ray diffrac-
tion (XRD) spectrum of the AlN thin films.
where fr is the resonance frequency and fa the antiresonance frequency in the imped-
ance frequency spectrum. As shown in Figure 3.35, the impedance and phase frequency
spectrum of the pMUT was characterized using an impedance analyzer (Agilent 4294,
Agilent Technologies, Inc), fa and fr were measured to be 2.885 MHz and 2.93 MHz,
respectively, with 0.005 MHz frequency sweep step. The calculated coupling coefficient
is 2.38% ~ 3.71%, compared to a value of 0.056% for AlN-based pMUT which has a
high parasitic capacitance (Shelton et al. 2009). However, it is generally lower than PZT-
based pMUT (Akasheh et al. 2004, Muralt et al. 2005). In addition, the small phase
change seen from Figure 3.35 means a small value of ke2Q , which might be resulted
from the parasitic capacitance between the lower electrode and upper electrode lead.
94 Photoacoustic Tomography
12
4
70 75 80 85 90
Temperature of TMAH Solution (25%)
FIGURE 3.34 The relationship curve of protection time and temperature of TMAH.
32400 87.6
31200 88.0
Impedance (ohm)
Phase (degree)
30000 88.4
28800 88.8
27600 89.2
Coupler
Nd: YAG
Focusing lens
Optical fiber
PC
2D translation stage
Self-focusing lens
Phantoms
DAQ card pMUT
Amplifier
The self-focusing lens was scanned for 80 steps with a step size of 50µm along
x and y axes. In the experiment, a piece of human hair was embedded in a stan-
dard agar-powder solidified tissue-mimicking phantom, consisting of Intralipid as
the scatterer and India ink as the absorber. The photoacoustic signals transmitted
through the phantom and coupling agent and reached to the device. Figure 3.37a
shows the photoacoustic signal from the human hair, while Figures 3.37b and 3.37c
present the photograph and reconstructed photoacoustic image of the phantom. We
see that the signal from the hair is strong and the image is well recovered. Compared
with other systems, pMUT has a significant impact on photoacoustic imaging
because it has wide operating bandwidth, can be configured as large array, and can
be easily integrated with electronics.
The pixel amplitude profile of the recovered hair (Figure 3.37c) is shown in
Figure 3.38. This allows us to calculate the average FWHM of the profile or the lateral
resolution of the system to be 240 µm. It is, however, possible to obtain higher resolu-
tion by using more tightly focused laser beam with the trade-off of penetration depth.
We have shown the design and fabrication of AlN-based pMUT with a high cou-
pling efficiency, and demonstrated the application of such pMUT for photoacoustic
imaging. We believe significant applications of pMUT to PAT will be quickly identi-
fied, especially when the array of pMUTs and their integration with SOC become
available. While the results presented here are encouraging, we note several limita-
tions associated with the current design, which should be overcome in the future.
First, the sensitivity of the current pMUT is still not comparable with that of a PZT
unit. To improve response of AlN-based pMUT, we can increase the piezoelec-
tric coefficient of thin film by doping scandium inside AlN film (Akiyama et al.
2009). Previous studies showed that the transverse piezoelectric coefficient e31, f of
ScxAl1-xN would be augmented with the increased Sc content (Matloub et al. 2013).
Second, the bandwidth of the pMUT needs to be improved for in vivo photoacoustic
imaging. This can be realized by integrating together several sets of elements with
different central frequencies into one chip.
96 Photoacoustic Tomography
0.2
0
Amplitude (mV)
–0.2
–0.4
–0.6
–0.8
0 500 1000 1500 2000 2500 3000
(a)
1 mm
(b) (c)
FIGURE 3.37 (a) Photoacoustic signal from the hair detected by the pMUT. (b) Photograph
of human hair embedded in the phantom. (c) Recovered photoacoustic image.
1
0.9
0.8
0.7
0.6
Intensity
0.5
0.4
0.3
0.2
0.1
0 10 20 30 40 50 60 70 80
Distance Along Profile
FIGURE 3.38 Intensity profile for the recovered photoacoustic image shown in Figure
3.37(c).
Image Enhancement Software and Hardware Approaches 97
PDMS
Hard resin
(a) Lens mold (b) Filling and curing PDMS in the mold
Needle Liquid
(c) Membrane bonded with lens (d) Liquid filled into the chamber
chamber and two holes punched through the hole with needle
through from sides
Holder
Transducer
FIGURE 3.39 Fabrication process. (a) A lens mold is built by a 3D printer; (b) PDMS is
poured into the mold and cured with 80°C; (c) a piece of membrane (PDMS) is bonded and
seals the lens chamber; (d) liquid is infused into the chamber through the holes to shape the
curved lens interface; (e) a holder, which is also made by a 3D printer, mounts the lens as well
as the transducer.
the true features of the targets, especially when the targets are located far from the
center of the scanning area. Efforts have been made to enlarge the acceptance angle
by gluing a diverging (negative) acrylic lens with a flat transducer (Xia et al. 2011).
However, the problems associated with this method arise: (1) the transducer can
hardly be reused for other purposes because the removal of the acrylic lens may
damage the transducer; (2) the solid-based lens inherently has a high acoustic imped-
ance, which leads to considerable loss of acoustic energy by reflection.
These problems motivated us to propose and test a liquid acoustic lens. The test-
ing of the lens was implemented in three steps: (1) the transducer worked in its trans-
mission mode and its pressure distributions with and without the liquid lens were
mapped and compared; (2) pencil lead, as an ideal target, was used in photoacoustic
tomography to look into how much image deformation would be caused due to the
limited directivity for both cases with and without lens; (3) phantom was used to
mimic tissue and imaged by PAT for both cases with and without lens. In this test,
the transducer (2.25 MHz, V133, Olympus) was in its transmission mode emitting
ultrasound pulses. A holder mounted the transducer as well as the liquid acoustic
lens and fixed their positions. A hydrophone (HNR-0500 ONDA) was employed to
scan laterally and axially while detecting the ultrasound pulses from the transducer
Image Enhancement Software and Hardware Approaches 99
(a)
Hydrophone
Scanning area
Transducer
1
0.9
0.8
0.7
0.6
Without lens
0.5
0.4
0.3
0.2
0.1
With lens
(b) (c)
FIGURE 3.40 (a) Schematic setup for testing the performance of the liquid lens; (b) acous-
tic pressure distribution of the transducer with and without the liquid lens; (c) photograph of
the prototype of the liquid lens mounted on the holder.
–10
–5 8
0 4
5 0
10
0 5 10 15 20 25 30
(mm)
(a)
–10
10
–5
0 5
5 0
10
0 5 10 15 20 25 30
(mm)
(b)
1.5
Intensity Profile
0.5
–0.5
–4 –3 –2 –1 0 1 2 3 4
(mm)
(c)
FIGURE 3.41 PAT of pencil leads. Seven pencil leads (0.7 mm diameter) are aligned along
the radial direction with the right-most one located at the center of the scanning. (a) Imaging
without liquid lens; (b) imaging with liquid lens; (c) intensity profile along the tangential
direction of the pencil lead with black arrow pointed.
Image Enhancement Software and Hardware Approaches 101
from tangential deformation when its distance from the center was about 5 mm. This
effect of tangential deformation became increasingly magnified to the targets located
further from the center. The other observation was that the image contrast dropped with
the increasing distance of the target from the center. The tangential deformation is ubiq-
uitously presented in photoacoustic imaging because the transducer is not an ideal point
detector. Considering the circularly scanning detection method in most PAT systems,
the effect of tangential deformation could be suppressed as long as the target could be
always covered by the FOV (field-of-view) of the detector. However, in reality, only the
target close to the center could be always covered by the scanning detector. Therefore,
the further the target from the center, the worse its image suffered from deformation.
Enlarging the acceptance angle using a diverging lens is an efficient way to sup-
press the tangential deformation. Figure 3.41b shows that the image of the target
(with black arrow pointed), located at about 15 mm away from the center, remained
an approximately similar shape as its true size. On the other hand, the corresponding
image generated without the lens (Figure 3.41a) went through a severe deformation.
The tangential profiles of the images (with and without the lens) were plotted in
Figure 3.41c. The comparison indicates that the diverging liquid acoustic lens can
efficiently suppress the deformation by enlarging the acceptance angle of the detector.
In the phantom experiments, three targets of phantom, which had an µa =
0.049 mm–1 and a µs = 0.1 mm–1, were positioned at the center (No. 1), with a hori-
zontal distance of 11~mm from the center (No. 2), and with a vertical distance of
10 mm from the scanning plane (No. 3), respectively (Figure 3.42). All the targets
had the same diameter of 4 mm. Figures 3.42a and 3.42b show the reconstructed
images of PAT without and with liquid acoustic lens, respectively. Generally, the
image produced with lens (Figure 3.42b) has a better SNR than that produced with-
out lens (Figure 3.42a). This is because the lens can enlarge the acceptance angle and
cover a larger FOV, and thus the signals from the off-center targets could contribute
more in the linear delay-and-sum reconstruction process.
No. 2 target, located 11 mm away from the center, was imaged to have tangen-
tial deformation without the lens. The comparison between the tangential profiles
in Figure 3.42c could apparently illustrate the deformation. On the other hand, the
reconstruction of image over radial direction did not present any deformation under
either condition with or without the lens (Figure 3.42e). Another interesting inspec-
tion is the imaging of the target (No. 3) off the scanning plane. The image produced
without using the lens presented only a fragment of the target, which is the part close
to the central axis (Figure 3.42a). This is because the bare transducer with a lim-
ited directivity can cover only the signal emitted from the part of the target, which
is close to the central axis of the scanning plane, and the signal of the other part,
which is out of the detection cone of the transducer, can hardly be detected at any
scanning points. This signal-blind-zone resulted in a phenomenon that is different
from the tangential deformation of No. 2 target. On the other hand, by enlarging the
acceptance angle using the lens, the detection cone of the transducer can cover the
entire body of No. 3 target. As a result, the image could be correctly reconstructed
(Figure 3.42b). Figures 3.42d and 3.42f show that the image produced with the lens
remains approximately the true size of 4 mm diameter (tangential and radial), but
the image produced without using lens loses its fidelity by shrinking tangentially and
102 Photoacoustic Tomography
–20 –20
6 8
No.1 No. 1
–10 –10 6
No. 2 No.3 4 No. 2 No. 3
4
(mm)
(mm)
0 2 0
2
10 0 10 0
–2 –2
20 20
–20 –10 0 10 20 –20 –10 0 10 20
(mm) (mm)
(a) (b)
2
1.8 No. 2 target No. 3 target
Tangential Profile
Tangential Profile
1
1.4
0
1
With lens –1 With lens
0.6 Without lens Without lens
0 1 2 3 4 5 6 7 0 1 2 3 4 5 6 7
(mm) (mm)
(c) (d)
2
No. 2 target No. 3 target
1
1
Radial Profile
Radial Profile
0
0
With lens With lens
–1 Without lens –1 Without lens
0 1 2 3 4 5 6 7 0 1 2 3 4 5 6
(mm) (mm)
(e) (f )
FIGURE 3.42 Results of phantom experiments. A target (No. 1) is placed close to the center
for reference. No. 2 target is located in the same plane of No. 1 but with a distance of 11 mm
from the center. No. 3 target is lifted up to have a vertical distance of 10mm from the center.
All the targets have the same diameter of 4 mm. (a) Imaging without lens; (b) imaging with
lens; (c) tangential profile of No. 2 target; (d) tangential profile of No. 3 target; (e) radial pro-
file of No. 2 target; (f) radial profile of No. 3 target.
Image Enhancement Software and Hardware Approaches 103
distorting radially. Therefore, enlarging the detection cone using the liquid acoustic
lens could also be promising to improve the SNR and correct imaging deformation
in 3D photoacoustic tomography (Wang et al. 2012, Xiang et al. 2013), which has
been demonstrated as an effective imaging method for the diagnosis of breast cancer
(Xi et al. 2012, Manohar et al. 2004, Ermilov et al. 2009).
In sum, compared with previous solid-based acoustic lens, the liquid lens presents
inherently low acoustic impedance and the convenience to be attached to or detached
from any commercial transducers without damaging the transducers. The material
PDMS that encapsulates the liquid is biocompatible, which makes this lens suitable
for biomedical applications.
Trigger
Nd: YAG
Pre-amplifier
(a)
MEMS MEMS
mirror mirror
Transducer
Fixed
mirror
(b) (c)
1 mm
Actuator
(d) (e)
FIGURE 3.43 (a) Schematic of our photoacoustic imaging system. 3D rendering (b) and
photograph (c) of the scanning probe. (d, e) Photograph of the MEMS mirror.
and the device footprint size is 2 × 2 mm2. When applying a voltage generated by two
function generators to the MEMS mirror, each actuator can move up or down due to
the Joule heating effect, resulting in a tilt to the mirror plate. Two-dimensional scan-
ning can be realized by applying different voltages to the four actuators as shown
in Figure 3.43e. In the system, an output trigger signal is used to synchronize the
data acquisition and function generators (AFG3022B, Tektronics). The ultrasound
signal is amplified by a preamplifier with a gain of 17 dB and further amplified by
an amplifier with a controllable gain from 5 to 20. The data acquisition sampling
rate is 50 MHz and a band-pass filter (Panametrics, Waltham, Massachusetts) from
1 to 5 MHz is used to cut off the low-frequency and electromagnetic noises. In the
Image Enhancement Software and Hardware Approaches 105
experiments, ultrasound gel is applied between the probe and the sample to mini-
mize ultrasound attenuation.
400
1 mm Raw data
300 Hilbert transform
200
100
–100
–200
–300
–400
0 200 400 600 800 1000
(a) (b)
1 mm
(c)
FIGURE 3.44 (a) Photograph of a phantom containing a single object. (b) A typical raw
signal and signal after the Hilbert transform. (c) The recovered 2D image.
106 Photoacoustic Tomography
1 mm 1 mm
(a) (b)
FIGURE 3.45 Photograph of a phantom containing multiple objects (a), and the recovered
2D image (b).
−1
scatterer and India ink as the absorber. The phantom had a = 0.007 mm (absorp-
tion coefficient) and s = 1.0 mm −1 (reduced scattering coefficient). Agar powder
(2%) was used to solidify the Intralipid/ink suspensions. Figure 3.44b presents a typi-
cal A-line signal collected, along with its amplitude after the Hilbert transform, while
Figure 3.44c shows the 2D image of the object. Using the criteria defined by the
FWHM of multiple A-line signals, the size of the recovered object is estimated to
be 0.72 mm compared to the actual object size of 0.70 mm. The SNR of the image
signals was found to be 32 dB, which decreases with increased depth of the target.
Figures 3.45a and 3.45b give the photograph and recovered image of another
phantom where multiple targets were embedded at different positions/depths. From
the image shown in Figure 3.45b, we see that the targets are all detected.
The results given in Figure 3.46 show the 3D imaging ability of our system. In
this experiment, a pencil lead with a diameter of 0.7 mm was embedded in one piece
1 mm
Target
(b) (d)
FIGURE 3.46 (a) Photograph of the chicken with an embedded object. (b)–(d) Coronal,
sagittal, and cross-section views of the recovered 3D image. (e) The 3D rendering of the
recovered image.
Image Enhancement Software and Hardware Approaches 107
1 mm
Discontinued part
(a) (b)
FIGURE 3.47 (a) Photograph of the hand with highlighted imaging area. (b) 3D rendering
of the recovered blood vessels.
109
110 Photoacoustic Tomography
(E) Computer
(D) A/D
(C) Preamplifier
(F) Tank
(A) Laser
(b)
FIGURE 4.1 Photograph of the array-based PAT system. (a) Top view. (b) Front view.
the #1~#64 channel preamplifiers and data acquisitions. Each channel has a sample
rate of 50MHz, controlled by signal CLK50M (frequency variable). The post delay
and the sample waveform length are variable and can be assigned by Labview pro-
gram. EN_CH65-128 enables the #65~#128 channels preamplifiers and data acqui-
sitions. EN_CH129-192 enables the #129~#192 channels preamplifiers and data
acquisitions. This provides the opportunity to read all captured pointers into the
Transducer Array-Based Photoacoustic Tomography 111
…
Fixed Pre-Amp (26 dB)
Fixed Pre-Amp (26 dB) MUX (3 to 1)
…
Fixed Pre-Amp (26 dB)
1-64
8 channel AD board
Data Memory A/D Variable gain Amp (0–80 dB) BP Filter
Acquisition
Control & PC 1-64 Memory A/D Variable gain Amp (0–80 dB) BP Filter
Labview
Memory A/D …
Variable gain Amp (0–80 dB) BP Filter
8 AD board in PC
computer through the local bus after the acquisition and easy readout of the photo-
acoustic data from all the channels.
4.1.1 Laser Source
A Ti:Sapphire (Symphotics TII, LT-2211A) laser optically pumped with a Q-switched
Nd:YAG laser (Symphotics-TII, LS-2137/2) delivers 8 to 25 ns pulses at 10 Hz with
wavelength tunable from 690 to 950 nm. The beam is expanded with a Galilean
telescope assembly and subsequently diverged with either a plano-concave lens or
100 us
100 ms 100 ms
Q-Switch
Laser 10 ns
CLK50M
EN-CH1-64 Post dealy
EN_CH65-128
Sample waveform length
EN-CH129-192
192 transducers
··· ···
MAX4051 MAX4051 MAX4051
··· ···
USB
GAIN AMP GAIN AMP GAIN AMP
1024LS
··· ···
10bitGAIN
A/D AMP 10bitGAIN
A/D AMP 10bitGAIN
A/D AMP
··· ···
GAIN
DSP AMP A/GAIN
10bit PCI AMP
D Bridge 10bit A/GAIN
D AMP
··· ···
··· 10 bit A/D 10 bit
PCIA/D
Bridge 10 bit A/D
Laser
Computer
Q-switch
FIGURE 4.5 Flow diagram of the 192-channel photoacoustic data acquisition system.
+VS C2 MAX4051
RF 10μF
499Ω NO4 1 16 V+
C3
RG 0.1μF
FB NO6 2 15 NO2
26Ω
– +V COM 3 14 NO1
AD8099 VC VOUT
VIN CC RL NO7 4 13 NO0
+ –V 1kΩ
R1 D
NO5 5 12 NO3
26Ω
DISABLE
C5 INH 6 11 ADDA
0.1μF
V– 7 LOGIC 10 ADDB
C4
10μF GND 8 9 ADDC
DIP/SO/QSOP
–VS
(a) (b)
FIGURE 4.6 (a) 26 dB AD8099 circuit design. (b) Design of the MAX4051 as a 3-to-1 switch.
114 Photoacoustic Tomography
PCI bus
will simultaneously convert the analog signals to digital data with user-selected post-
trigger delay and waveform length. Other programmable parameters include sam-
pling rate, trigger source, trigger rate, gain, DC offset, low- and high-pass filters.
Both boards feature user-selectable 10-bit resolution and up to 50 MB samples per
second. With one board set as the master and remaining set to slave mode, all chan-
nels on the slave boards will start taking data upon receiving a trigger signal from
the master board. The jitter between channels is less than 2 ns. PCIAD850 has low-
pass filters with different cutoff frequency: All frequency, 16 MHz, 7.3 MHz, and
4.8 MHz. It also has high-pass filters with different cutoff frequency: 4.8 MHz,
1.6 MHz, 0.6 MHz, and 0.016 MHz.
Labview programming is used to control the entire data acquisition. The software
is written to allow maximum flexibility for various system configurations. Pointers
created during the data acquisition process are used to quickly access the photo-
acoustic data. Data is then saved for offline image reconstructions. Figure 4.8 shows
the front control panel of the Labview software.
transducers over time-of-flight before and after the calibration. Before calibra-
tion, we see that the signal from the point target arrives each transducer at dif-
ferent time indicating positioning error Figure 4.9). By adjusting the position of
each transducer, we obtained the signals from the point target that arrived at the
transducer at the same time point (red curves in Figure 4.9). Figure 4.10 shows the
reconstructed image of three circular targets before and after the calibration. We
Before calibration
After calibration
0 2 4 6 8 10 12 14 16 18 20
Time/μs
FIGURE 4.9 Measured PA signals from six representative transducers before and after the
calibration.
116 Photoacoustic Tomography
153.460000 mm 151.490000 mm
8 8
6 2000 6 3000
4 4 2000
2 1000 2
1000
0 0 0
2 2 0
4 1000 4 1000
6 2000 6 2000
8 8
10 3000 10 3000
4000
5 0 5 10 5 0 5 10
Before calibration After calibration
FIGURE 4.10 Reconstructed photoacoustic images of three circular targets before and after
calibration.
4.1.4.2 Spatial Resolution
To evaluate the system spatial resolution, we constructed a phantom consisting of
two point targets (brass wires; 100 μm in diameter each) suspended in tissue phan-
tom (solidified Intralipid scattering medium). Figure 4.11a shows the reconstructed
image of the two point targets, while Figure 4.11b is the normalized profile of the
reconstructed image along a line y = 10 mm. From the profile, the half-width-at-
half-maximum (AB) and the half-width-at-quarter-maximum (CD) were calculated
to be 0.13 mm. Using the resolution criterion, the spatial resolution of the system was
estimated to be AB+CD–target diameter = 0.13+0.13–0.1 = 0.16 mm.
FIGURE 4.11 (a) Reconstructed image of two point targets. (b) The normalized profile of
the reconstructed image shown in (a) along y = 10 mm.
Transducer Array-Based Photoacoustic Tomography 117
152.330000 mm 151.490000 mm
5 25000 8
4 6 3000
3 20000 4 2000
2 15000 2
1000
1 0
10000 2 0
0
1 5000 4 1000
2 6 2000
0
3 8
4 (a) 5000 10 (b) 3000
5 4000
5 0 5 5 0 5 10
FIGURE 4.12 Reconstructed image of a single target (a) and three targets (b).
4.1.4.3 Phantom Evaluation
Phantom experiments were conducted to evaluate the 2D imaging ability of the
array-based system. Figure 4.12a shows the recovered photoacoustic image of a
0.1 mm-diameter target, while Figure 4.12b gives the reconstructed image of three
targets having a diameter of 4 mm, 1.2 mm and 0.6 mm, respectively. All the images
obtained show that these targets with different sizes can be clearly detected using
this array-based imaging system.
Figure 4.13 presents cross-sectional photoacoustic images of a simulated blood
vasculature located at various depths ranging from 0 to 4.6 cm below the phantom
surface. The cross-sections illustrate the capability of the system to provide high-
resolution and high-contrast images at least at a depth of up to 3.5 cm (Figures 4.13a
and 4.13b show the photograph of the samples).
We also conducted phantom experiments to test the high temporal resolution
ability of the system for real-time tomographic imaging of dynamic fluid flow
(3–10 Hz). Figure 4.14 depicts image frames from a 10 sequence of dynamic ink
flow through a 1.6 mm-diameter polyethylene tube using a manual syringe push.
Laser
Tube
Ink
Experiment diagram
(a)
?? ??
?? ?? ?? ??
?? ??
?? ?? ?? ??
?? ?? ?? ??
?? ?? ?? ??
?? ??
?? ??
?? ??
?? ?? ?? ??
?? ?? ?? ??
?? ?? ?? ??
?? ?? ?? ??
?? ?? ?? ?? ?? ?? ?? ??
?? ??
?? ?? ?? ??
?? ??
?? ?? ?? ??
?? ?? ?? ??
?? ?? ?? ??
?? ??
?? ??
?? ??
?? ?? ??
??
?? ?? ?? ??
?? ?? ?? ??
?? ?? ?? ??
?? ?? ?? ?? ?? ?? ?? ??
?? ??
?? ?? ?? ??
?? ?? ??
?? ?? ??
?? ??
?? ??
?? ?? ??
??
?? ?? ?? ??
?? ?? ??
?? ?? ??
?? ?? ??
??
?? ?? ?? ??
?? ?? ?? ??
?? ?? ?? ?? ?? ?? ?? ??
PA slices during circulation
(b)
0.333000 Second
14 5000
12
10 4000
8 3000
6 2000
4 1000
2
0 0
2 1000
4 2000
6
3000
0 5 10 15
Real-time movie
(c)
FIGURE 4.14 Photoacoustic images of dynamic ink flow through a 1.6-mm-diameter tube.
Transducer Array-Based Photoacoustic Tomography 119
4.2 3D IMAGING
For 3D imaging, scanning in z direction is needed for a circular or linear array of
transducers, which leads to a nonoptimal elevational resolution in z direction. Two-
dimensional planar array of transducers are easy to implement for 3D PAT imag-
ing; however, due to the limited aperture of 2D planar array transducers, features
with high aspect ratio or with orientations oblique to the transducer surface suffer
from distortion, and the azimuthal resolution is reduced. Compared with a 2D planar
array, a spherical array can offer more complete angular views of the object, provid-
ing both high resolution and accurate feature definition regardless of shape or loca-
tion of the object. Here we describe a sparse spherical array based PAT system that,
for example, can be used for 3D imaging of small animal brains.
4.2.1 System Description
Figure 4.15 depicts the block diagram of the real-time 3D PAT system. A Ti:Sapphire
laser optically pumped with a Q-switched Nd:YAG laser sent 8–12 ns pulses at 10
Hz with a wavelength tunable from 690 to 950 nm. The beam was delivered with
an optical fiber through an opening on the top of the transducer array and produced
Pipe to
hold
the rat 8 channel AD board
Data Memory A/D Variable gain Amp (0–60 dB) BP Filter
Acquisition
Control & PC 1–64 Memory A/D Variable gain Amp (0–60 dB) BP Filter
Labview
8 AD boards in PC
FIGURE 4.15 Block diagram of the real-time 3D PAT system. The inset is a photograph of
the close-up view of the chamber holding a rat head.
120 Photoacoustic Tomography
Third 64
Second 64
First 64
(a) (b)
FIGURE 4.16 The spherical transducer array. (a) Photograph of the transducer array. (b) 3D
schematic of the transducer distribution on the interface.
The system allows the selection of transducer positions on the spherical interface.
The total 610 holes formed 11 evenly spaced layers along the vertical direction of
the ball, and the 192 transducer positions were indicated with three different colors
in Figure 4.16b. Three different colors were used to indicate the 3 to 1 multiplexing
from the 192 transducers to the 64-channel data acquisition. This system can also be
used as a real-time 2D system operating at 10 f/s using the 64 transducers arranged
in the vertical center layer (blue). For in vivo experiments, the rat head was elevated
to the center of the spherical interface through a chamber fixed at the tank bottom,
whose top was about 15 mm beneath the interface center, and a transparent plastic
wrap was used to cover the chamber top. For phantom experiments, a homemade
silicone holder was used to hold the phantom.
4.2.2 Phantom Experiments
Three different types of phantoms were used: One containing a point object for
system calibration, one phantom containing three hairs tilted along different ori-
entations for static imaging, and one phantom with ink flowing through a thin tube
embedded in a phantom for real-time 3D imaging.
The point object used for calibration was a small spherical graphite particle
(0.1 mm in diameter) located at the center of the spherical array and ensured an
isotropic acoustic emission profile for all directions. We measured and compensated
the delay of time for all the 192 channels in the radical direction, and reconstructed
this point object after calibration. The hairs-containing phantom was used to demon-
strate the high imaging quality of our system. Finally, we imaged an embedded tube
filled with flowing ink to show the real-time imaging ability of our system. The tube
had a 0.3 mm inner diameter and was horizontally placed in a phantom. No averag-
ing of signals was performed for the phantom experiments except for the point object
experiment where 10 times averaging was applied.
We calibrated the system by recording the emission profiles from the spherical
graphite particle for all the 192 channels, and then measured and compensated the
time delay of each channel. We then evaluated the system resolution by reconstruct-
ing the image of the point object.
Figures 4.17a and 4.17c present the reconstructed x–y and z-x cross-section
images of the point object located at the array center. The quality of these images
is determined by both the distribution and the characteristics of the transduc-
ers. The profiles of the two reconstructed images were also extracted in x and
z directions, as shown in Figures 4.17b and 4.17d, respectively. The FWHM of
the profiles was measured to be 0.19 mm (x direction) for Figure 4.17b, and
0.27 mm (z direction) for Figure 4.17d, compared to the theoretical value of
0.16 mm for the 5 MHz central frequency transducer with an estimated cut-off
frequency of 7 MHz. It is noted that the profile in Figure 4.17d is noisier than
that in Figure 4.17b. This along with a larger FWHM from Figure 4.17d was due
to the asymmetric distribution of the transducers. For targets located away from
the center of the array, the radial resolution will stay nearly the same as that for
a centrally located target, while the lateral resolution will be linearly reduced
with increased distance away from the array center. In our system, the lateral
122 Photoacoustic Tomography
0.5 200
200
100 100
0
0 0
0.5
0.5 0 0.5 0.5 0 0.5
(a) (b)
0 100 100
0.5 0 0
0.5 0 0.5 0.5 0 0.5
(c) (d)
FIGURE 4.17 (a) and (c) x–y and z-x cross-section images through the center plan of the
point object. (b) and (d) The profile extracted in x and z directions from (a) and (c), respec-
tively. Units are in mm.
resolution will be reduced by 0.1mm when the target is located 5mm off the
array center.
Figure 4.18a is the photograph of the phantom containing three hairs tilted along
different orientations, and Figures 4.18b and 4.18c show the reconstructed 3D images
from two different views. The reconstructed volume is 10 × 10 × 10 mm with a 0.1
mm voxel size. The spatial distribution and tails for all the three hairs were clearly
revealed. This result indicates that our system is capable of 3D imaging small objects
of different spatial distribution and orientation in high quality.
30
FIGURE 4.18 (a) photograph of the phantom containing three tiled hairs; (b)–(c) recon-
structed 3D images of the three hairs in two different views. Scale bar represents 5 mm.
Transducer Array-Based Photoacoustic Tomography 123
220
(f) 12.9 S (g) 13.2 S (h) 13.5 S (i) 13.8 S (j) 14.1 S
70
FIGURE 4.19 Reconstructed 3D images of ink flowing through a 0.3 mm tube embed-
ded in a background phantom. (a) Photograph of the phantom containing the tube. (b)–(j)
Reconstructed 3D images at different time points. The time interval is 0.3 s. Scale bar rep-
resents 5 mm.
The reconstructed 3D images of ink flowing through a thin tube are shown
in Figure 4.19. The image domain is 15 × 5 × 10 mm with a 0.1 mm voxel size.
Figure 4.19a is the photograph of the phantom containing a tube filled with ink, and
Figures 4.19b–4.19j are the reconstructed 3D images at different time points. The
time interval between two consecutive images was 0.3 s. These 3D images clearly
tracked the flow through the tube over the course of 2.4 s with high spatial and tem-
poral resolution, and the flowing speed of the ink was measured to be 6 ± 0.9 mm/s
from the reconstructed results with a time interval of 0.3 s.
20
20
FIGURE 4.20 (a) and (c) Photograph of the rat with BMI injection and the control rat after
scalp removed. (b) and (d) 3D PAT images at six time points for (a) and (c), respectively. The
three main blood vessels are indicated by the white arrows, and the seizure focus is indicated
by the circle in (b). The time interval between two successive images is 0.3 s. Scale bar rep-
resents 10 mm.
The corresponding photographs of the two rats with scalp removed right after the
experiments are shown in Figures 4.20a and 4.20c, respectively. The reconstructed
domain for the images shown in both Figures 4.20b and 4.20d is 20 × 20 × 4.5 mm,
with a 0.1mm pixel size. The three main blood vessels on the rat brain are clearly
revealed for both cases, as indicated by the white arrows, and for the rat with BMI
injection a seizure focus can be clearly seen right at the BMI injection position (the
white circle in Figure 4.20b), where strong absorption is observed during seizure
onset. The rapid changes of the absorption both in the main blood vessel and in the
seizure focus were observed from Figure 4.20b, while no such changes were noted
for the control rat (Figure 4.20d). The seizure focus had a diameter of ~3 mm. This
experiment demonstrates that our system can be used to investigate the hemodynamic
changes in small animal brain both spatially and temporally during seizure onset,
although the complex microvasculature cannot be resolved due to the limited number
of transducers used here. The microvasculature can be revealed when the 192-ele-
ment full-ring array is used for 2D tomographic imaging of rat brains (see Chapter 8).
4.3 4-D IMAGING
Real-time 3D or 4-D imaging is necessary in many cases. Real-time 3D imaging
of epilepsy shown in Section 4.2 is one example. Four-dimensional PAT can also
play a significant role in the localized drug delivery using metal needles. Accurate
Transducer Array-Based Photoacoustic Tomography 125
Time
t θ1 t θ2
Space
θ1
z
θ2
θ3
θ y
α
θ4
x
θ5
and updated knowledge of the position and orientation of the needle can increase
the needle guidance and placement within the tissue. Four-dimensional PAT can
be used to guide photothermal therapy for monitoring temperature distribution
during the therapy. In all these cases, real-time 3D photoacoustic imaging is
needed to capture the physiology and pathology dynamically in three dimensions.
In 4-D PAT, the time resolution as the fourth dimension is integrated with the
3D spatial resolution obtained from a series of 2D projections of an object. Here
we demonstrate the methodology in needle based drug delivery guidance, with a
temporal resolution of 330ms, thus enabling the visualization of dynamic biodis-
tribution and pharmacokinetics of indocynine green (ICG). We also demonstrate
4-D PAT for imaging dynamic changes of temperature during thermal therapy of
tumor.
The 4-D PAT imaging system is described in Section 4.2.1. The concept 4-D
PAT is illustrated in Figure 4.21, which depicts the construction of tomograms
from 2D projections at different angles and times. Because of the various dimen-
sions involved, we note that at a given time each 2D projection represents a 3D
frame (including time), whereas a 3D tomogram when constructed from all the 2D
126 Photoacoustic Tomography
FIGURE 4.22 PAT-guided curved needle insertion and drug delivery. From left to right, the
needle was imaged over time from 2.331 to 9.990s.
projections represents a 4-D frame. For the reconstructed images shown in this sec-
tion, z scanning from −15 mm to +15 mm with an increment of 100 µm was used, and
the time scale t ranged from 330 µs to tens of minutes.
A B
y
z
13 243 13 243
9
1
250
2
2 200
3
3 150
100
8
0.2 mm 50
1 2
∆T (°C)
4
0.8 min
150 s 180 s
(b)
5 6
6
Maximum ∆T (°C)
4
7 8
(a)
2
0
0 50 100 150 200 250
Time (s)
(c)
FIGURE 4.24 Tumor margin detection and thermal therapy monitoring. (a) Three-
dimensional stack of consecutive photoacoustic image sections from a breast tumor on
mouse; the 0.2 mm slice corresponds to sections 1–5. Maximum intensity projection of the
entire z stack is shown in 6; 7 is the 3D reconstruction of the entire z-stack. Yellow dashed
circle represents the tumor margin in different slices as in 1–6 which correlated well with
tumor histology in 8. (b) 3D photoacoustic-based thermal images at different time points (90,
120, 150 and 180s) after photothermal therapy. (c) Photoacoustic measurement of temperature
rise during photothermal therapy; a maximum temperature rise of ~6.6ºC within the tumor
is seen. Error bars (±s.d.) were calculated from five consecutive measurements. In this study,
1-month-old BALB/C mice were injected in the right flank with EMTCL2 human cancer cells
(about 53,106 cells) and tumors were allowed to grow for 15 days to a size of 10 mm in diam-
eter. Sliver nanoplates were used for thermal therapy due to their enhanced thermal stability
and enhanced photoacoustic signal response. By precisely controlling the plate diameter and
thickness, the nanoplate’s optical resonance can be tuned to peak at specific wavelengths at
785 nm. The injected volume of nanoparticle solution was 200 mL, and the injected concen-
tration was 0.08 mg/ml. The nanoparticles were intradermally injected at the tumor site. A
CW laser (785 nm, 80 mW, Model DL7140-201S, Sanyo, Japan) was used for photothermal
therapy. Prior to taking experimental measurements, tissue temperatures were measured by a
thermistor to calibrate the relationship between temperature and photoacoustic signal ampli-
tude. During experiments, tissue samples were immersed in a water tank maintained at 37ºC,
and thermistor was inserted into the center of tissue sample to measure the temperature.
Transducer Array-Based Photoacoustic Tomography 129
wavelength of 810 nm were collected. The CW laser beam spot size on the skin of
the mouse was 0.8 mm in diameter. Based on temperature calculation models (Shah
et al. 2008), a temperature distribution was estimated. A maximum temperature
rise of 6.6ºC during therapy was observed by photoacoustic-based thermal imag-
ing (Figure 4.24c). During the first 1 minute 40 seconds, mouse tumor temperature
gradually increased, and then the temperature was relatively constant until the CW
laser was turned off.
5 Photoacoustic
Microscopy and
Photoacoustic
Computed Microscopy
131
132 Photoacoustic Tomography
Transducer
2D linear stage
Water tank
Optical window
Objective lens
Light beam
50 µm
250 µm RBC
(a)
(b)
FIGURE 5.2 (a) Maximum amplitude projection (MAP) photoacoustic images of mouse
ear. (b) Two blood vessels imaged by ORPAM (top panel) in comparison with that by conven-
tional transmission microscope image (bottom panel). Note that the blood vessels cannot be
identified by transmission optical microscope; the two red lines are used to help the visualiza-
tion of the position of the blood vessels.
direct or bright-field illumination without a dark center. Although the lateral resolu-
tion of ARPAM is determined by the bandwidth of the transducer (typically from
50 μm to 1 mm) and lower than ORPAM, it can detect target more deeply located
than that by ORPAM. Because of this, ARPAM has found more applications than
ORPAM (see Chapter 8 for details).
Figure 5.3a shows the schematic of a typical ARPAM system. In this system,
a short-pulsed laser beam of 6 ns duration at 10 Hz repetition rate from a 532 nm
Nd:YAG laser is divided into two beams by a beam splitter and coupled into two
optic fiber bundles. Wideband photoacoustic waves induced as a result of ther-
moelastic expansion of target is collected by a focused high-frequency ultrasound
transducer (50 MHz) with 3 mm aperture and 6 mm focal length. The optical illu-
mination through the optic fiber bundles is adjusted to optimize the signal-to-noise
ratio (SNR) of the system as shown in Figure 5.3b. The laser beams are delivered to
the target through a water tank with a window sealed with an optically and ultra-
sonically transparent membrane. The acoustic transducer and optic fiber bundles are
raster scanned by a 2D moving stage with a scanning step size of 60 μm. The image
formation method is the same as that for ORPAM (see Chapter 1).
For the purpose of demonstration, the vasculature of rat ear is imaged in vivo
using ARPAM. Figure 5.4 presents the maximum amplitude projection (MAP) pho-
toacoustic image projected along the vertical direction (z-axis) to the orthogonal
plane. We see that the vasculature of the rat ear is clearly identified.
134 Photoacoustic Tomography
Beam splitter
(a) Lens
Nd: YAG laser
PC Lens
Fiber bundle II
Amplifier
2D
scanner
(b)
Illumination
Tank pattern
Membrane
Active area of Holder
transducer (3 mm)
FIGURE 5.3 (a) Schematic of an ARPAM system. (b) Illustration of optical illumination
pattern and active acoustic area.
1 mm
PC Lens
Fiber bundle II
Amplifier
(b) Focused
transducer
3D
scanner
Start
2 position
Focal point X Y
4
3
End position Holder Z
FIGURE 5.5 (a) Schematic of the PAM system. (b) C-scan PAM concept. The start and end
positions are typically 0.5 mm away from the focal point.
136 Photoacoustic Tomography
rate. Then the laser beam is split into two subbeams and each of the subbeams is
coupled into an optic fiber bundle. The two optic fiber bundles are fixed in opposite
positions to produce a homogenous volume illumination through the object. Induced
photoacoustic waves are received by a focused high-frequency ultrasound transducer
(50 MHz, 3 mm aperture, and 6 mm focal length). The imaging probe consisting of
the transducer and optic fiber bundles is mounted on a 3D moving stage. This system
can provide a lateral resolution of 61µm on the focal point (Figure 5.5b) and an axial
resolution of 15 µm.
In the experiments, a Drosophila pupa was fixed on a holder with its long side
parallel to the y-axis (Figure 5.5a). The scanning step was set to be 25.4µm along the
x- and y-axis and 50 µm along the z-axis. During the in vivo experiments, low laser
power (0.5 mW) was used to ensure uninterrupted biology development of the pupa.
We noticed that all the pupae imaged developed into fruit flies with this laser power.
j =1
∑ p( j, t − t )
n
0
P(t ) = (5.1)
n
where p(j, t) is the signal received at the j the focal point during the axial scanning,
t0 is the calculated time of acoustic wave propagation for one scanning step along the
z-axis (t0 = 0.03 µs in this study), and n is the total scanning steps used for the axial
scanning.
5.3.1.3 Phantom Preparation
The image reconstruction method described above was validated through phantom
experiments. In these experiments, a human hair was embedded in an agar powder
(2%) solidified tissue mimicking phantom, consisting of Intralipid as the scatterer
and India ink as the absorber. The absorption coefficient of the phantom was 0.007 mm–1
and the reduced scattering coefficient was 1.0 mm–1. Due to the high absorption of
hair, we used a very low power illumination of 0.1mW for these experiments.
from the vial. A small hole was made at the posterior as well as the anterior end of
the pupal case with a fine tungsten needle under a dissection scope. The punctuated
pupae were quickly immersed in 1 ml of 4% paraformaldehyde in PBS (phosphate
buffered saline) in a 1.5 ml eppendorf tube. The tube was let rotate in room tempera-
ture for 24 hours. Certain histological sections were performed by frozen sectioning
machine and imaged by fluorescence optical microscopy system.
T1 1 T2 2 T1
1' 2'
100 µm 5 6 7
T3 3 T4 4 T2
3' 4'
5' 6' 7'
(a) (b)
3
2.5 Line 1 Line 1'
Line 2 2.5 Line 2'
Photoacoustic Signal
0 0
–0.5
100 200 300 400 500 0 150 300 450
x-axis (µm) z-axis (µm)
(c) (d)
FIGURE 5.6 (a) The image of hair by C-scan PAM (T1), and the image of hair located at
different positions by conventional PAM (T2, T3, T4). (b) In vivo pupa images by C-scan
PAM (T2) and by conventional PAM (T1). (c) Lateral profiles for the images shown in Figure
5.6(a). (d) Axial profiles for the images shown in Figure 5.6(a).
138 Photoacoustic Tomography
TABLE 5.1
Comparison of the Target Size Measured by CPAM and Actual Value
Recovered Recovered
Distance from the Actual Size Lateral Size Axial Size
Line # Focal Point (um) (um) (um) (um) Error (lateral)
1 NA 98 105 99 6.1%
2 450 98 170 99 73.5%
3 300 98 135 99 37.6%
4 0 98 99 99 1.0%
Transverse and axial profiles along the dashed lines 1, 1’, 2, 2’, 3, 3’, 4, and 4’ in
Figure 5.6a are given in Figures 5.6c and 5.6d after Hilbert transform of the raw data.
Quantitative information was extracted from the profiles shown in Figures 5.6c and
5.6d and summarized in Table 5.1.
From Table 5.1, we see that T1 image provides accurate quantitatively recon-
structed transverse hair diameter with an error of 6.1% compared to the true value.
T2 and T3 images show a large error of 73.5% and 37.6%. T4 image gives the most
accurate transverse size of target with an error of less than 1% because the hair was
placed right at the focal point of transducer. For axially imaged size, accurate results
with an error of less than 1% were obtained for all four cases since the axial reso-
lution (15 µm) was determined by the frequency and bandwidth of the transducer
regardless of the target positions.
4
2 1 3
1
2
5
b 100 µm
c d
e
2 4
1
2
3
1 5
3 4
1
1
5
2
2
1
3
1 5
(d) (e )
FIGURE 5.7 In vivo CPAM images (top row, b–e), and fluorescence microscopic images of
histologic sections (bottom row, b–e).
resolution. For other DsRed-expressing cells indicated by 6, 6’, 7, and 7’, we also see
that the image quality with C-scan is notably higher.
Figure 5.7a shows the position/section corresponding to the CPAM/FM slices
presented in Figures 5.7b to 5.7e, where the top row shows the in vivo photoacous-
tic images and the bottom row gives fluorescence microscopic images of the cor-
responding histological sections. Major features are clearly identifiable in CPAM
images and agree well with the corresponding histological sections.
To validate if longitudinal observations could be possible, the morphogenetic
movements that occur in the pupae were monitored in vivo by our CPAM as shown
in Figure 5.8. We monitored the same position of one pupa during the first 3 hours
after pupariation. The time-lapse CPAM imaging sequence shows the movements of
DsRed-expressing cells inside the pupa with high resolution.
140 Photoacoustic Tomography
0:00 0:30
100 µm
(a) (b)
1:30 3:00
(c) (d)
Ti:Sapphire laser
Mirror
Fiber bundle I
PC
Coupler
Amplifier
Transducer
Function
generator Fiber bundle II
3D scanner
(b)
Illumination
Tank pattern
Water tank
Membrane
Active area of
transducer (3 mm)
FIGURE 5.9 (a) Schematic of the PAM system. (b) Top view illustration of the optical illu-
mination area.
5.4.1 Methods
5.4.1.1 PAM Imaging System
The PAM imaging system is schematically shown in Figure 5.9a. Short-pulsed laser
beam at 10 Hz repetition rate from a Nd:YAG laser and/or a Ti:Sapphire laser can
be used for imaging. The laser beam from each laser is delivered to the sample via
an optic fiber bundle. Both of the optic fiber bundles are positioned to produce an
optimal illumination at the sample surface as shown in Figure 5.9b. Depending on
the application, either one of the lasers or both of them are used for multispectral
imaging (e.g., 1064 nm from Nd:YAG and 730nm from Ti:Sapphire) where a 50 µs
time delay between the two lasers provided by the function generator is used to avoid
signal overlap. The imaging probe consisting of a focused transducer and the optic
fiber bundles is mounted on a 2D moving stage. The sample is positioned under
an open imaging window at the bottom of the water tank sealed with an ultrasoni-
cally and optically transparent membrane. Ultrasound gel is used between the mem-
brane and the animal tissue/phantom for acoustic transmission. At each imaging
142 Photoacoustic Tomography
5.4.1.2 Phantom Experiments
For these experiments, laser pulses at 730 nm from the Ti:Sapphire laser were used
to image one 0.8 mm-diameter circular target (for cases 1–5) or three circular targets
(0.8 mm in diameter each) (for case 6) embedded in the background, which was a
scattering medium composed of a fat emulsion suspension (Intralipid) with India ink
added as an absorber. The method we used to determine the absorption and reduced
scattering coefficients of the phantoms in this study has been widely adopted in the
field of diffuse optical imaging and spectroscopy (Pogue and Patterson 1994, Hale
and Querry 1973, Madsen et al. 1992, van Staveren et al. 1991). The absorption of the
suspension is essentially due to water24–26, hence the ink provided a controlled way of
achieving a higher level of absorption (Madsen et al. 1992). It is also known that the
added ink should not have a significant impact on the scattering coefficient (Madsen
et al. 1992). The targets were transparent tube containing different concentrations
of Intralipid and India ink, which were used to simulate heterogeneities. For cases
1–3, the single target had an absorption coefficient of 0.07 mm–1 and was located at
a depth of 0.0, 4.8, and 7.6 mm below the surface, respectively. For case 4, the single
target with an absorption coefficient of 0.035 mm –1 was embedded at a depth of
2.1 mm. For case 5, the single target having an absorption coefficient of 0.021 mm–1
was placed at a depth of 1.1mm. For case 6, three targets with different absorption
coefficients (0.07 mm–1, 0.035 mm–1, and 0.021 mm–1, respectively) were embedded
at a depth of 1.9 mm. For all these cases, the absorption coefficient of the background
was 0.007 mm–1, and the reduced scattering coefficient of the background and tar-
gets were 1.0 mm–1.
5.4.1.3 Animal Experiments
Before the experiment of imaging the rat ear, one rat weighing 28 g was anesthetized
using urethane and the hairs of the ear were removed. The laser beam at 532 nm
generated by a Nd:YAG laser was split into two subbeams and coupled into two
optical fiber bundles. A focused ultrasound transducer (50 MHz, 3 mm aperture,
and 6 mm focal length) was used to receive the induced photoacoustic waves. A 2D
moving stage mounted with the optical fiber bundles and transducer carried out a
2D raster scanning. The raster scanning provided 200 A-lines along each direction
with an interval of 60 μm over a distance of 12 mm. For the experiment of imaging
the brain of a rat weighing 25 to 30 g, the brain area of interest was gently depilated,
Photoacoustic Microscopy and Photoacoustic Computed Microscopy 143
and the scalp was removed before the imaging (Figure 5.12a). Pulsed light at 1064 nm
from the Nd:YAG laser and at 730 nm from the Ti:Sapphire laser were used for
multispectral imaging. B-scan photoacoustic signals were recorded by a focused
50 MHz high-frequency transducer. During the imaging, the rat was anesthetized
with urethane (1mg/g) and tightly positioned with a holder to avoid motion effect
due to seizure activity. The body temperature of the animal was maintained at 37°C
with a temperature controlling pad. At the end of study, all rats were sacrificed. All
procedures have been approved by the University of Florida Institutional Animal
Care and Use Committee (IACUC).
5.4.1.4 Image Reconstruction
5.4.1.4.1 Recovery of Absolute Optical Absorption Coefficient
Our PACM approach is based on a strategy that iteratively solves the radiative trans-
fer equation (RTE) and the photoacoustic wave equation (PWE) using finite element
method (FEM). In PACM, we first obtain the distribution of optical fluence, Φ(r )
through the finite element (FE) solution to the RTE from an initial (guess) distribu-
tion of optical absorption coefficient, 0a (r ) . For quantitative imaging of individual
blood vessels, the use of RTE is necessary and most rigorous to model the light prop-
agation in tissue since the absorption coefficient of blood vessels is high, making the
commonly used photon diffusion equation inappropriate for quantitative PAM. The
RTE used is described as follows:
( ⋅ + s + a )φ(r , ) = s
∫ S n −1
φ(r , )Θ( , ) d + q(r , ) (5.2)
where s isthe scattering coefficient; φ(r , ) is the radiance; q(r , ) is the source
term;
and ∈ S n−1 denotes a unit vector in the direction of interest. The kernel,
Θ( , ), is the scattering phase function describing the probability density that a
photon with an initial direction will have a direction after a scattering event.
We assume that the scattering phase function depends only on the angle between the
incoming and outgoing directions; thus
the commonly used Henyey-Greenstein scat-
tering function can be applied: Θ ( , ) =
Θ ( ⋅ ) = (1 − g 2 )/2π(1 + g 2 − 2 g cos γ ) ,
where γ is the angle between and , and −1 < g < 1. The optical fluence is
related to the radiance by Φ(r ) = ∫ S n −1 φ(r , )d .
Next, we computationally obtain the acoustic pressure, pkc ( x k , yk , z , t ) at each
transducer location along the surface of the medium/tissue (rk (xk , yk), k = 1,2,…M
transducer locations) using the following Helmholtz PWE (Yao and Jiang 2009):
where p is the pressure wave; v0 is the speed of acoustic wave in the medium; β is the
thermal expansion coefficient; Cp is the specific heat; and J (t ) = δ(t − t0 ) is assumed
in this study.
144 Photoacoustic Tomography
Finally, using the regularized Newton’s method, the matrix equation for inversion
of the absorption coefficient can be written as
(ℑ ℑ
T
k k + λI ) χ k = ℑTk ( pko − pkc ) , (5.4)
where pko and pkc are the observed and computed complex acoustic field data for k =
1,2,…M transducer locations; Δχk is the update vector for the absorption coefficient;
ℑ is the Jacobian matrix formed by ∂ pk /∂χ k at the boundary measurement sites; λ
is the regularization parameter determined by combined Marquardt and Tikhonov
regularization schemes (Jiang 2010, Jiang et al. 2006) (λ = 0.5 was used in this
study—we have found that the quality of inversion was not sensitive to the choice
of this parameter); and I is the identity matrix. Thus here an A-line image (χ k(z) or
μa,k(z)) is formed by updating absorption coefficient distribution via iterative solution
of Equations (5.2) to (5.4) so that an object function composed of a weighted sum of
the squared difference between computed and measured acoustic data can be mini-
mized. A B-scan or 2D image is obtained by combining all the A-line images and
a 3D image is provided by combining all the B-scan images. In these calculations,
the incident laser source strength and the scattering coefficient μs are estimated in
advance.
5.4.1.4.2 Recovery of Functional/Hemodynamic
Parameters (HbR, HbO2, BF and MRO2)
In most cases for microvasculature imaging, HbO2 and HbR are the two major
absorbers. Using multispectral measurements, the oxy- and deoxy-hemoglobin con-
centrations, [HbO2] and [HbR], can be obtained using the Beer-Lambert law:
(r , λ) = ε HbR (λ) [ HbR ](r ) + ε HbO2 (λ) [ HbO 2 ](r ) (5.5)
a
where ε HbR (λ) and ε HbO2 (λ) are the known molar extinction coefficients of HbR
and HbO2 at wavelength λ. Oxygen saturation can be calculated as sO2 = [HbO2]/
[HbT] where HbT is the total hemoglobin ([HbT] = [HbO2] + [HbR]).
We can further calculate the blood flow (BF) and rate of oxygen metabolism
(MRO2) in blood vessels from the functional parameters obtained above. The
method for such calculation is based on the principle of mass balance to the trans-
port of oxygen in a blood vessel segment (Tsai et al. 2003). To model oxygen trans-
port in a blood vessel by this principle, we consider a 1-D cylindrical vessel (blood
vessel) with Ri and Ro as the inner and outer radii, respectively, surrounded by the
biological tissues. In addition, we assume that all the oxygen (O2) diffusing out the
segment is consumed in a tissue region (Sharan et al. 2008). The law of mass con-
servation stipulates that the amount of O2 lost from a vascular segment must be
equal to the O2 flux diffused to the tissue, determined by the perivascular oxygen
gradients. The oxygen consumed by the tissues (organs) is supplied from three blood
vessels sources: capillaries, arterioles, and venules. If the oxygen supply of tissues
depends on the averaged oxygen saturation at the inlet and outlet of the tissues,
Photoacoustic Microscopy and Photoacoustic Computed Microscopy 145
tissue oxygen saturation should represent the weighted average of the arterial and
venous saturation:
where sO 2,ti and sO 2,to are the averaged oxygen saturation at the inlet (artery) and
outlet (vena) of the tissues, respectively. As such, mass balance for O2 in all tissues
(organs) based on global analysis yields the following estimation of intravascular
flux for a dynamic case, considering expressions of molar amount of oxy-hemoglobin
concentration of tissue and tissue oxygen saturation:
d OC BF sO 2,ti − sO 2 d [ HbT ] sO 2
sO 2 = − + [HbT]blood − , (5.7)
dt 4V [HbT] V [HbT] 1− f dt [HbT]
where BF is the mean blood flow for all the blood vessels inside the tissues and is
specified as the mean blood flow of tissues, OC = MRO2 is the mean oxygen con-
sumption for the whole tissue, [HbT] is the mean total hemoglobin concentration in
the tissues, [HbT]blood is the mean total hemoglobin concentration in the blood circu-
lating through the tissues, and V is the tissue volume and is assumed constant here. In
our calculations, we assumed [HbT]blood = 0.72 mM, f = 0.2, and sO2,ti = 0.98. Further
explanations on the determination of these parameters can be found elsewhere (Carp
et al. 2008).
Equation (5.7) is the mathematical model we have developed for predicting BF and
MRO2 from [HbT] and SO2. It is an ordinary partial differential equation that can be
solved iteratively by Runge-Kutta fourth-order method coupled with the FEM (Press
et al. 1992). Thus, mean BF and MRO2 can be recovered by fitting Equation (5.7) to
time-resolved tissue oxygenation measurements. The fitting method is described as
follows: with any given initial values for OC and BF within the specified range, this
scheme is to optimize the OC and BF parameters based on the solution of Equation
(5.7) to reach the minimized objective function in Equation (5.8):
∑(sO
2
Min: F = m
2i − sOc2i ) (5.8)
i =1
in which sO 2mi is the measured oxygenation parameter from M discrete time points,
and sOc2i is the oxygenation parameter calculated from Equation (5.6) for the same
M time points. Note that the BF and OC are assumed constant during the measure-
ments for the specified time range, due to the need for a sufficient time interval to
obtain stable fitting results.
5.4.2 Results
The PACM reconstruction algorithm (described above) is first validated using
six sets of tissue-mimicking phantoms with known optical properties (Table 5.2).
146 Photoacoustic Tomography
TABLE 5.2
Exact and Recovered Size and Absorption Coefficient Value
of Target for the Phantom Experiments
µa of Target (mm–1) Size of Target (mm)
Depth of the
Case # Target (mm) Exact Recovered Exact Recovered
I 0 0.071 0.90
II 4.8 0.07 0.071 0.90
III 7.6 0.072 0.95
IV 2.1 0.035 0.034 0.95
V 1.1 0.021 0.020 0.8 1.0
VI 1.9 0.07 0.068 0.9
0.035 0.033 1.0
0.021 0.019 1.1
Figures 5.10a to 5.10f show the reconstructed absorption coefficient images from
these phantom experiments. We see that the object(s) in each case are clearly
detected by our PACM approach. In particular, these images are quantitatively accu-
rate in terms of the absorption coefficient of target. This conclusion is further con-
firmed by the quantitative absorption coefficient profiles for these images provided
in Figures 5.10g to 5.10h. We found that the relative error of the recovered absorption
coefficient of target compared to the exact value is less than 4.7% for the single-target
cases 1 to 5, while it ranges from 2.8 to 9.5% for the multitarget case 6 (Table 5.2).
We note that the target having higher contrast (cases 1–3) is better reconstructed than
for the lower contrast cases (case 4).
A rat ear experiment was chosen to further validate the algorithm and show its
possible application to functional imaging based on physiologically specific endog-
enous optical absorption contrasts in biological tissue. In Figure 5.11a we present the
maximum amplitude projection (MAP) images projected along the vertical direction
(z-axis) to the orthogonal plane where seven orders of vessel branching, indicated
by numbers 1 to 7, can be observed in the image. The B-scan image and the recon-
structed absorption coefficient image obtained by our FE-based PACM algorithm
are shown in Figures 5.11b and 5.11c, respectively. These images are in the vertical
plane (x-z plane) at the location indicated by the dashed line in Figure 5.11a. It can
be clearly seen that all of the vessel branching can be reconstructed using both of the
methods. The laser applied in this experiment was at a wavelength of 532 nm, and
from Figure 5.11c we found that at most locations within the blood vessel, the value
of the reconstructed absorption coefficient is in the range of 20~30 mm–1, which is
in good agreement with that of blood vessels at 532 nm reported in the literature
(Barton et al. 1998, Kienle et al. 1996).
(mm–1) (mm–1) (mm–1) (mm–1) (mm–1)
3 1 2
0.07 –3 0.07 –5 0.07 0.0.35
2 0 1 0.02
0.06 0.06 –6 0.06 0.03
1 –4 –1 0
0.05 0.05 –7 0.05 0.025
0 –5 0.04 –2 0.015
0.04 0.04 0.02 –1
0.03 –6 0.03 –8 0.03 –3
–1 y 0.015 –2
0.02 –7 0.02 –9 0.02 0.01
–2 1 mm –4 0.01 –3
0.01 0.01 –10 0.01
z –8 0.005
0 2 4 0 2 4 0 2 4 0 2 4 0 2 4
(a) (b) (c) (d) (e)
(mm–1)
1 0.100 0.100
Case 1 Case 6
0 0.06 Case 2
0.075 Case 3 0.075
0.05 Case 4
–1
0.050 Case 5 0.050
0.04
–2
0.03 0.025 0.025
–3
0.02
–4 0.000 0.000
0.01 0 1 2 3 4 5 6 0 4 8 12
FIGURE 5.10 Reconstructed absorption coefficient images from the phantom experiments. (a) Case 1. (b) Case 2. (c) Case 3. (d) Case 4. (e) Case 5.
Photoacoustic Microscopy and Photoacoustic Computed Microscopy
(f) Case 6. (g) and (h) Quantitative plots: reconstructed absorption coefficient profiles along y = 0.0 mm for case 1, y = –4.8 mm for case 2, y = –7.6 mm
for case 3, y = –2.1 mm for case 4, y = –1.1 mm for case 5, and y = –1.9 mm for case 6.
147
148 Photoacoustic Tomography
1 2 3 4 5 6 7 5 7
6
3
4
1 2
1 mm
(b)
(mm–1)
5 7 60
6
4 40
2 3
1 20
1 mm
(a) (c)
FIGURE 5.11 In vivo imaging of the blood vessels in a rat ear. (a) The MAP image of the
photoacoustic signals projected on the orthogonal plane, with seven small vessels that can be
observed in the image as indicated by numbers 1–7; (b) B-scan image in the vertical plane at
the location indicated by the dashed line in (a); (c) reconstructed absorption coefficient image
in the vertical plane at the location indicated by the dashed line in (a).
We then demonstrate the ability of PACM in in vivo imaging HbO2, HbR, sO2,
cerebral blood flow (CBF), and cerebral rate of oxygen metabolism (CMRO2) using a
rodent model in nondiseased condition. Figure 5.12b shows a typical in vivo B-scan
PAM image of a single blood vessel located 1.5 mm below the surface of the rat
brain with intact skull (along y-z plane) at a selected time point, which was taken at
the location indicated by the dashed line (Figure 5.12a) (a total of 10 B-scan images
were obtained over 60 s duration). Electroencephalographic (EEG) signal from one
electrode is given in Figure 5.12c. For simplicity, the reconstructions of quantitative
absorption coefficient and all the hemodynamic response parameters were conducted
over the square area shown in Figure 5.12b. Using the PACM reconstruction algo-
rithm, we obtained the absorption coefficient images at 730 and 1064 nm over 60 s
duration, and Figures 5.13a to 5.13h show the absorption coefficient images for the two
wavelengths at four selected time points, t1 = 12s, t2 = 24s, t3 = 42s, and t4 = 54s. These
high-resolution images not only allow for the accurate recovery of the size/shape/
location of single blood vessel, but the absolute absorption coefficient itself (indicated
by the gray scale on right). The absorption coefficient images at 730 and 1064 nm
allowed us to obtain HbO2 (Figures 5.14a–5.14d), HbR (Figures 5.14e–5.14h), and
sO2 (Figures 5.14i–5.14l) using Equation (5.5) at the four selected time points as well
as at other time points. It can be seen that these functional parameters can also be
reconstructed quantitatively over time. Based on these results, the reconstructed CBF
and CMRO2 images at four different times are provided in Figures 5.15a–5.15d and
5.15e–5.15h. To closely observe dynamic evolvement of the functional parameters
including HbO2, HbR, sO2, CBF, and CMRO2, we calculated the average value of
Photoacoustic Microscopy and Photoacoustic Computed Microscopy 149
x 0.5
–0.3 Surface
y –0.6
0.4
–0.9 FE reconstruction
area
–1.2
0.3
Z (mm)
–1.5
–1.8 0.2
–2.1
–2.4 0.1
–2.7
–3.0 0
0.3 0.6 0.9 1.2 1.5
1 mm
Y (mm)
(a) (b)
0.04
EEG Intensity (mV)
0.02
0
–0.02
–0.04
10 20 30 40 50 60
t (s)
(c)
FIGURE 5.12 (a) Photograph of the rat brain with intact skull under PAM imaging. The
arrow/dashed line indicates the B-scan path for PAM. (b) Typical B-scan PAM image. Note
that the blood vessel imaged is ~1.5mm below the surface. (c) EEG signal recorded at one
electrode close to the scanned region.
5.4.3 Discussion
The reconstructed results of optical absorption coefficient obtained from the phan-
tom experiments and the rat ear are both qualitatively and quantitatively in terms of
the location, size, shape, and optical property values of the target and background,
which indicates that the PACM method has the capability to provide absolute func-
tional imaging based on physiologically specific endogenous optical absorption coef-
ficient. This capability is confirmed with an animal experiment by using a rodent
model (see Section 5.4.2), but in principle any animal model associated with neural
activation/stimulation could be used.
150 Photoacoustic Tomography
(mm–1) (mm–1)
0 0
–0.1 0.08 –0.1 0.08
–0.2 –0.2
0.06 0.06
–0.3 –0.3
y
–0.4 0.04 –0.4 0.04
–0.5 0.1 mm –0.5
z 0.02 0.02
0 0.2 0.4 0.6 0 0.2 0.4 0.6
(a) (b)
(mm–1) (mm–1)
0 0
0.08
–0.1 –0.1
0.08
–0.2 0.06 –0.2
0.06
–0.3 –0.3
0.04 –0.4
–0.4 0.04
–0.5 –0.5
0.02 0.02
0 0.2 0.4 0.6 0 0.2 0.4 0.6
(c) (d)
(mm–1) (mm–1)
0 0
0.08
–0.1 –0.1 0.08
(mm–1) (mm–1)
0 0
0.08
0.08
–0.1 –0.1
–0.2 0.06 –0.2 0.06
–0.3 –0.3
0.04 0.04
–0.4 –0.4
–0.5 –0.5
0.02 0.02
0 0.2 0.4 0.6 0 0.2 0.4 0.6
(g) (h)
FIGURE 5.13 Reconstructed absorption coefficient images using the in vivo rodent model.
(a)–(d): 730 nm; (e)–(h): 1064 nm at four different time points (a and e: t = 12 s; b and f: t =
24 s; c and g: t = 42 s; d and h: t = 54 s).
Photoacoustic Microscopy and Photoacoustic Computed Microscopy 151
( g/L) ( g/L)
0 0 0
–0.1 6 –0.1 8 –0.1
0.6
–0.2 –0.2 –0.2
–0.3 –0.3 6 –0.3
4 0.4
–0.4 –0.4 4 –0.4
–0.5 2 –0.5 –0.5 0.2
2
0 0.2 0.4 0.6 0 0.2 0.4 0.6 0 0.2 0.4 0.6
(g) (h) (i)
FIGURE 5.14 Reconstructed functional images using the in vivo rodent model. (a)–(d):
HbO2, (e)–(h): 1 HbR, and (i)–(l): sO2 at four different time points (a, e, and i: t = 12 s; b, f,
and j: t = 24 s; c, g, and k: t = 42 s; d, h, and l: t = 54 s).
ml/ml/s ml/ml/s
× 10–3 × 10–3
0 0
8
–0.1 –0.1 8
–0.2 6 –0.2
6
–0.3 –0.3
–0.4 y 4 –0.4 4
–0.5 0.1 mm 2 –0.5
z 2
0 0.2 0.4 0.6 0 0.2 0.4 0.6
(a) (b)
ml/ml/s ml/ml/s
× 10–3 × 10–3
0 0
–0.1 8 –0.1 8
–0.2 –0.2
6 6
–0.3 –0.3
–0.4 4 –0.4 4
–0.5 –0.5
2 2
0 0.2 0.4 0.6 0 0.2 0.4 0.6
(c) (d)
µmol/ml/s µmol/ml/s
× 10–3 × 10–3
0 0
8 8
–0.1 –0.1
–0.2 6 –0.2
6
–0.3 –0.3
–0.4 4 –0.4 4
–0.5 2 –0.5
2
0 0.2 0.4 0.6 0 0.2 0.4 0.6
(e) (f )
µmol/ml/s µmol/ml/s
× 10–3 × 10–3
0 0
–0.1 –0.1 8
8
–0.2 –0.2
6 6
–0.3 –0.3
–0.4 4 –0.4 4
–0.5 –0.5
2 2
0 0.2 0.4 0.6 0 0.2 0.4 0.6
(g) (h)
FIGURE 5.15 Reconstructed CBF (a)–(d), and CMRO2 (e)–(h) images at t = 12 s (a,e), 24 s
(b,f), t = 42 s (c,g) and t = 54 s (d,h). In the calculation, the initial parameters used were
[HbT]blood = 0.72 mM, f = 0.2, and sO2,ti = 0.98 (see Section 5.4.1.4.2 for the explanation of
these initial parameters). In addition, due to the high nonlinear distribution of sO2 over time,
the sO2 distribution curve was first separated into several approximated linear segments to
improve the fitting accuracy of CBF and CMRO2. The mean CBF and CMRO2 were fitted for
each linear segment based on different initial values of [HbT] and sO2.
Photoacoustic Microscopy and Photoacoustic Computed Microscopy 153
40
(b)
HbO2 (g/l)
36
32
28
0 10 20 30 40 50 60
t (s)
11 (c)
HbR (g/l)
9
7
5
0 10 20 30 40 50 60
t (s)
0.88
(d)
0.84
sO2
0.8
0.76
0 10 20 30 40 50 60
t (s)
0.009
(e)
CBF (ml/ml/s)
0.0086
0.0082
0.0078
0 10 20 30 40 50 60
t (s)
0.0092 (f )
CMRO2
0.0088
0.0084
0.008
0 10 20 30 40 50 60
FIGURE 5.16 Comparison of the EEG signal and the average values of the recovered func-
tional parameters of the ROI during the entire time course. (a) Filtered EEG signals; (b) aver-
age HbO2 of the ROI; (c) average HbR of the ROI; (d) average sO2 of the ROI; (e) average CBF
of the ROI; (f) average CMRO2 of the ROI.
154 Photoacoustic Tomography
we find that two significant spikes at 20 and 50 s in the EEG signals can also be
identified in Figures 5.16b and 5.16c.
Using Equations (5.7) and (5.8), we have also calculated CBF and CMRO2 over
the total time course. We can observe significant changes in CBF and CMRO2 in
terms of the shape/size of blood vessel and values of CBF/CMRO2. We also see
from the peak values of CBF shown in Figure 5.15 that the recovered blood flow
values (7.5–8.5 ml/ml/s, or 45–51 ml/100 ml/min) are in good agreement with the
reported CBF of rats (10–120 ml/100 ml/min) and of humans (20–160 ml/100 ml/
min) (Hernandez et al. 1978, Sharples et al. 1995). Average values of CBF and
CMRO2 in the ROI are provided in Figures 5.16e and 5.16f. Interestingly, CBF and
CMRO2 spikes stronger in intensity than that seen in HbR and HbO2 are clearly
notable.
While it is a novel approach to monitor the dynamic response of functional
parameters quantitatively, PACM presented here has some limitations and needs to
be improved in the future. In this study, we estimated a homogenous distribution of
scattering coefficient in advance when the absorption coefficient was recovered. For
the animal experiments the reduced scattering coefficient was assumed as 1.0/mm.
It is well known that the propagation of light in turbid media is strongly character-
ized by optical absorption and scattering; thus this approximation would introduce
a negative impact on the accuracy of the recovery of absorption coefficient. This
impact, however, was small since the contribution of scattering to the photoacoustic
data is much less than that of absorption, if the estimated distribution of scattering
coefficient is in a reasonable range. For example, based on our numerical simula-
tions, when the estimated error of the reduced scattering coefficient is within the
range of ±50% of the actual value, the error of the recovered absorption coefficient
is less than 5%. Recovering absorption and scattering coefficients simultaneously
will improve the accuracy of the reconstructed results, but it will suffer from strong
nonuniqueness for such inversion of multiple parameters. In this study, since we
focus on introducing a new method exploiting the recovery of absorption coefficient
and other associated parameters of biological tissue, this approximation for scatter-
ing coefficient was used for simplicity.
To produce the functional parameters in the blood vessel by PACM, laser beams
at 730 and 1064 nm were used in this study due to the limitation of our current hard-
ware. The contribution of water to the absorption coefficient should be considered,
since the molar extinction coefficient of water at the wavelength 1064 nm cannot be
ignored compared to that of the oxygenated and deoxygenated hemoglobin. In this
study, we made an assumption that the ratio of the concentration of water to the total
hemoglobin in blood was about 55%:45%, based on the fact that the blood plasma
makes up about 55% of total blood volume. Dynamic images (i.e., CBF and CMRO2)
with similar values as the ones obtained under the assumption were obtained if dif-
ferent ratios other than 55% were used in the calculations. The impact of water can
be better considered by using laser beams at three different wavelengths, or at two
wavelengths (for example, 730 and 850 nm), at which the molar extinction coefficient
of water is relatively low.
The model we used to calculate CBF and CMRO2 in blood vessels from the func-
tional parameters is based on the principle of mass balance to the transport of oxygen
Photoacoustic Microscopy and Photoacoustic Computed Microscopy 155
in a blood vessel segment. Some assumptions are made in order to use this model.
For example, oxygen consumption and blood flow are assumed constant during the
measurements, due to the need for a sufficient time interval to obtain stable fitting
results. While this is a very good assumption for oxygen consumption, blood flow
may change during tissue relaxation, indicating that the fitted blood flow value rep-
resents a measure of an average blood flow during the measurement period. The
accuracy of the fitting results can be improved by increasing the temporal resolution
of our imaging system.
In our calculations we assumed [HbT]blood = 0.72 mM, f = 0.2, sO2,ti = 0.98. The
values of sO2,ti and f were taken from the literature (An and Lin 2002, Duong and
Kim 2000). With respect to the choice of [HbT]blood, the normal hemoglobin concen-
tration is in the range of 120–160 g/L for females (Berkow et al. 1997), and since
the hematocrit values of the capillary vessels is known to be two to five times lower
than the systemic hematocrit (Desjardins and Duling 1987). For the purposes of this
study, we assumed a value of 140 g/L for systemic hemoglobin concentration and a
three-times dilution factor in the small blood vessels, resulting in a value of 0.72 mM
for [HbT]blood. We note that the choice of [HbT]blood does influence the flow results,
but it does not affect the calculated oxygen consumption, because the inflow and out-
flow terms appear as part of a product with [HbT]blood in the mass balance equations.
In summary, we have demonstrated that PACM is capable of in vivo imaging
a full set of functional parameters at the small vessel level. The dynamic changes
are comparable with concurrently recorded EEG signals. The spatial resolution of
PACM is the same as PAM, scalable with the central frequency of the focused trans-
ducer, i.e., higher resolution at the single cell level is achievable with the sacrifice of
penetration depth. The imaging speed of the current PACM is relatively slow (6 s), but
it can be improved considerably by using faster lasers. We expect PACM will be a
valuable tool for neuroscience research where hemodynamics associated with micro-
vasculature in response to neural activation/stimulation need to be imaged. PACM
will also be applicable to visualize microvasculature dynamics involved in tumor
angiogenesis and in inflammatory joint diseases.
acoustic lenses have a fixed focal length once the lens is fabricated. Recently the field
of optofluidics has emerged to enlighten the idea that solid-based optical components
could be replaced by liquids or fluids with better performance and novel functional-
ities (Psaltis et al. 2006). Optical liquid or fluidic lenses have been demonstrated to
have an adjustable focal length by either pneumatically control (Zhang et al. 2004) or
flow rate management (Song et al. 2009, 2010). We have described a liquid acoustic
diverging lens that can enlarge the acceptance angle for PAT in Chapter 3. Here, we
demonstrate a liquid acoustic lens with an adjustable focal length and apply it to PAM.
The tunable function of this liquid acoustic lens is realized by pneumatically
controlling the infusion volume of the liquid. Specifically, a syringe pump (KDS
210, KD Scientific) is used to accurately infuse or withdraw liquid into or from the
lens chamber. In this way, the lens interface, which is an elastic membrane, can be
tuned with its curvature. Therefore, the focal length of this acoustic lens can be
adjusted simply by changing the infusion volume of the liquid. The relationship
between the curvature of the interface and the infusion volume of the liquid is char-
acterized and shown in Figure 5.17. Assuming that the lens interface has a spherical
shape, the infusion volume can mathematically determine the radius of the inter-
face curvature (solid line in Figure 5.17) via V = πh2(r–h/3), where V represents the
infusion volume of the liquid and h and r denote the thickness of the lens and radius
of interface, respectively. The experimental results show a good agreement with the
mathematical prediction.
Setting this liquid acoustic lens on its focusing or diverging mode can be real-
ized by purposely choosing the working liquid inside the lens chamber, which by
principle is similar to manipulating optofluidic lens (Song et al. 2011). Here, we
choose silicone oil as the working liquid, which has a relative refractive index of 1.4
0.2
0.18 Theoretical curve
Experimental results
Curvature of the Interface (1/mm)
0.16
0.14
0.12
0.1
0.08
0.06
0.04
0.02
0
0 50 100 150 200 250 300 350
Infusion Volume (µL)
FIGURE 5.17 Curvature of the lens interface as a function of the infusion volume of liquid.
The inset photographs illustrate the lens interface tuned by the infusion volume of the liquid.
Photoacoustic Microscopy and Photoacoustic Computed Microscopy 157
1 mm
(a)
26
Fitting curve
24 Experimental results
Focal Length (mm)
22
20
18
16
14
50 100 150 200 250 300
Infusion Volume (µL)
(b)
FIGURE 5.18 (a) Pressure distribution mappings of the transducer with and without the liq-
uid acoustic lens. The arrow shows the propagation direction of the acoustic beam; (b) focal
length of the liquid acoustic lens as a function of the infusion volume of liquid.
and density of 0.986 g/cm3. The higher refractive index (relative to water) as well
as the convex shape of the lens will enable an ability of focusing acoustic beam.
To characterize the focal length of this liquid acoustic lens, a transducer (30 MHz,
Olympus) was used in its transmission mode for providing ultrasound pulses. The
liquid acoustic lens was attached to the transducer head to focus the acoustic wave.
A hydrophone (HGL-0200, ONDA) was used to scan laterally and axially while
detecting the ultrasound pulses from the transducer (Song et al. 2013).
Figure 5.18a shows the comparison of pressure distributions between the trans-
ducers with and without the liquid acoustic lens. The arrow in the figure shows the
propagation direction of the acoustic beam. The acoustic wave emitted from the
transducer has a slightly focused wavefront (refer to the pressure mapping of trans-
ducer without the lens). When the transducer is working with the lens attached, the
acoustic wave can be focused with variable focal length. Since the infusion volume
of liquid determines the curvature of the lens, increasing the infusion volume by
158 Photoacoustic Tomography
2
1
Liquid acoustic lens
Hairs buried at
Laser pulsed illumination different depths
1 2 3
Translational stage
Transparent glass slide
FIGURE 5.19 Schematic of the experimental setup. Two groups of tests were carried out to
validate the functionality of the adjustable liquid acoustic lens: (1) Three hairs were buried
at different depths within the background phantom with 4mm distance between each hair;
(2) two tumor samples were buried at different depths within the background phantom with a
distance of 1.5 cm between each.
controlling the syringe pump can lead to a shortening of the focal length. Figure 5.18b
shows the focal length as a function of the infusion volume of the liquid. This test
gives a validation that the liquid acoustic lens can effectively focus acoustic wave
with adjustable focal length, and provides a calibration of relationship between the
focal positions and the infusion volume.
The advantage of this liquid acoustic lens with an adjustable focal length lies in
imaging objects at different depths without axial scanning of the detector. We designed
and carried out two groups of tests to demonstrate this advantage: one using human
hair as microscale targets, and the other using excised tumors from a mouse as mac-
roscale targets (Figure 5.19). The targets were buried in a background with an absorp-
tion coefficient of μa= 0.007 mm–1 and a reduced scattering coefficient μ′s = 1 mm–1.
A 30 MHz transducer (Olympus) was used to collect the signal from the hair targets,
and a 10 MHz transducer (V315, Panametrics) was employed for the tumor targets.
The targets were illuminated and scanned by laser pulses with wavelength 532 nm and
pulse duration 6 ns (NL 303HT, EKSPLA) coupled with a translational stage (Zaber
Tech T-LSM200A). Upon the energy deposition, an acoustic wave was generated from
the local thermo-expansion and reflected by a glass slide to the ultrasound detector.
The liquid acoustic lens was attached to the transducer and mounted on a hard-resin
holder. The liquid was injected into the lens chamber through needle and tubing by a
syringe pump (KDS 210, KD Scientific). During the data acquisition, the axial distance
between the targets and the transducer was fixed, and the translational stage provided a
lateral scanning under each condition of infusion volume of the liquid.
For the hair imaging, three hairs were aligned in parallel and buried at different
depths within the background phantom with 4 mm distance between each other. The
Photoacoustic Microscopy and Photoacoustic Computed Microscopy 159
Hair 1
1 mm
Hair 2
Hair 3
FIGURE 5.20 Images of hair by photoacoustic microscopy. Images of the three hairs in
left column were captured by the 30 MHz transducer without the liquid acoustic lens, and
the other three columns of images were captured with the lens under different conditions of
infusion volumes.
photoacoustic signal was firstly collected without using the liquid acoustic lens and
processed with maximum amplitude projection (MAP) method. The images of the
hairs (left column in Figure 5.20) show a broadened profile regarding the thickness
of the hair. This lack of image fidelity is owing to the finite size of the effective
diameter of the transducer. Considering the transducer has an effective diameter
of several millimeters, the hairs were imaged to have approximately the same size
regarding the thickness. After the transducer was equipped with the liquid acoustic
lens, the PAM system managed to obtain sharper images of the hairs (Figure 5.20)
when the focus of the lens was axially adjusted to coincide with the position of the
hair. During the test, the focal length was continuously shortened by increasing the
infusion volume of liquid. Thus, hair 3, which was the farthest from the transducer,
was firstly focused and imaged under a condition of low infusion volume of 10 µL.
With the infusion volume going higher and focal length getting shorter, hair 2 and
hair 1 came to their focus afterward and were sharply imaged under the infusion
conditions of 30 µL and 100 µL, respectively.
For the tumor imaging, two tumor samples with an approximately meniscus shape
(see the photograph in Figure 5.21) were buried in the background with a distance of
1.5 cm between each other. The superficially buried tumor 1 was located 0.5 and 5 cm
away from the surface and transducer, respectively. In this test, a 10MHz transducer
was used to collect the photoacoustic signal. A liquid acoustic lens was attached to
the transducer with its focal length varied by adjusting the infusion volume of the liq-
uid. When the infusion volume went to 50 µL, tumor 2, which was farther away from
the transducer, came into focus. Under this condition, tumor 2 was imaged to have
a meniscus shape that was approximately the same as its real shape, while tumor
160 Photoacoustic Tomography
2
1
FIGURE 5.21 (a) Images of the tumors captured by a 10 MHz flat-head transducer with
liquid acoustic lens attached under different conditions of infusion volumes; (b) photograph
of the tumor samples.
1 was out of focus and had a blurred image (Figure 5.21a). As the infusion volume
was increased to 250 µL leading to a shorter focal length, tumor 1, which was closer
to the transducer, came into focus and was sharply imaged. Figure 5.21b shows the
photograph of the tumor samples for reference. Using the liquid acoustic lens, two
targets with an axial distance of 1.5 cm can be imaged without the implementation
of axial scanning but alternatively by adjusting the focal length of the acoustic lens.
experience based, especially for ORPAM systems. We have described a liquid variable-
focus lens to effectively achieve either acoustic diverging or converging, and evaluated
it using both PAT imaging system and ARPAM imaging system (see Sections 3.8 and
5.5). This liquid lens has several advantages: (1) it has inherent low acoustic impedance
that can effectively reduce the reflective loss of acoustic signals; (2) it has tunable focal
length that enables axial adjustment of acoustic focus without mechanical scanning;
and (3) it is cheap and easily replaceable. A liquid-filled optical lens has been already
widely used and offers the potential to minimize and simplify the optical systems
(Knollman et al. 1970, Sugiura and Morita 1993). In principle, the liquid-filled acoustic
and optical lens performs similar physical functions based on the difference of acoustic
or optical refractive index between the internal and external liquids. Here, we propose
the use of two different types of liquids but with the same relative acoustic and optical
refractive index to achieve coaxial confocal of optical illumination and acoustic detec-
tion. We use the new liquid lens to build a reflection-mode ORPAM and evaluate its
performance with phantom and in vivo animal experiments.
Similar to most ORPAM imaging systems, as shown in Figure 5.22a, the visible
laser beam (532 nm) generated by a Nd:YAG pulsed laser (Surelite I-20, Continuum,
CA) was firstly attenuated, then coupled into a single-mode fiber (P1-460B-FC-1,
Thorlabs) using two neutral density filters and one objective lens. The whole imag-
ing probe consisting of an optical collimator (F260FC-A, Thorlabs), a ring-shaped
5 MHz transducer (external diameter: 11 mm, internal diameter: 5 mm) and a liquid
lens with size of 11.5 mm in diameter was mounted on a 2D scanner. The captured
photoacoustic signals were amplified (5072PR, Olympus), digitalized (NI5124,
National Instruments), and stored in the computer. A beam splitter and an ultrafast
photodiode were used to record the pulse fluctuation that was used to calibrate the
imaging data. Figure 5.22b is the close view of the imaging probe shown in the
dashed rectangle in Figure 5.22a. The laser beam was firstly collimated by the opti-
cal collimator with an output size of 2.8 mm in diameter, then focused by the liquid
lens infused with the cinnamon oil through needle and tubing by a syringe pump
(KDS 210, KD Scientific). The ring-shaped transducer was confocally positioned
with the optical illumination by the same lens. The tank was filled with a mixture
of glycerol and water.
Under the condition of fully infused biconvex lens, both the optical and acoustic
focal length can be formulated as follows (Song et al. 2009, 2010, 2013):
r
f= . (5.9)
2(n − 1)
where r is the radius of the interface curvature, and n is the optical/acoustic relative
refractive index which is calculated by the following equations:
ninternal
noptical = (5.10)
nexternal
Vexternal
nacoustic = (5.11)
Vinternal
162 Photoacoustic Tomography
ND BP Lens
SMF
Nd: YAG
pulsed laser
PD
2D scanner
PC
Pre-amplifier
(a)
Acoustic beam
Collimator
Optical beam
Ring-shape
transducer
Liquid lens
Cinnamon
oil Needle
Glycerol and
water mixture
(b)
FIGURE 5.22 Schematic of the ORPAM imaging system (a) and imaging probe (b). ND:
neutral density filters, PD: photodiode, BP: beam splitter, SMF: single mode fiber.
where ninternal and nexternal represent the optical refractive index of internal and
external liquids, and Vinternal and Vexternal are the sound velocity of internal and exter-
nal liquids. Coaxial confocal of optical illumination and acoustic detection can be
achieved as long as the following conditions are satisfied: (1) the internal liquid has
higher refractive index and lower sound velocity compared with the external liquid
and (2) both the internal and external liquids are optical and acoustic transparent
and nontoxic. Cinnamon oil with impressively high optical refractive index (1.63)
and modest sound velocity (1480 m/s) serves well as the internal liquid in the area of
optical liquid lens and microfluidics. We already described the use of pure glycerol
with higher sound velocity (1920 m/s) for the fabrication of an acoustic diverging
lens (Section 3.8). We note that the pure glycerol has a lower optical refractive index
(1.47) compared with that of cinnamon oil. Hence, we can mix glycerol with water
to make the acoustic and optical relative refractive index identical.
Photoacoustic Microscopy and Photoacoustic Computed Microscopy 163
Acoustic Optical
1.3
1.25
Relative Refractive Index
1.2
1.15
1.1
1.05
1
0 0.2 0.4 0.6 0.8 1
Water Percentage by Weight
(a)
Lens
2 mm
19 mm 19 mm Max
Min
(b)
FIGURE 5.23 (a) Acoustic and optical relative refractive index versus weight percentage of
water in the mixture. (b) Optical rays and acoustic pressure distribution when the lens was
flat and fully infused.
Trenton NJ). To characterize the acoustic converging ability of the lens, the trans-
ducer was driven by a pulser/receiver (5072PR, Olympus) to generate acoustic emis-
sion. A hydrophone (HGL-0200, ONDA) with a circular sensing area of 0.2mm in
diameter was scanned laterally to map the acoustic pressure distribution from the
transducer. Figure 5.23b shows the measured optical rays and acoustic pressure dis-
tributions when the lens was flat and fully infused. We can see that the lens enabled
coaxial acoustic and optical confocal with a focal length of 19mm. As we expected,
the optical and pressure intensities were highly confined when the lens was fully
infused. However, the measured focal length was slightly longer than the theoretical
estimation since the alignment between the collimator/transducer and the lens was
not optimal.
The 1D profile along the dashed white line cross the acoustic focal point shown in
Figure 5.23b is given in Figure 5.24a, and was used to estimate the size of acoustic
250
Acoustic Intensity (a.u.)
205 µm
200
0.7 mm
150
Focal plane
100
50 130 µm
0
8 9 10 11 12 13 14
Distance (mm)
(a) (b)
40 µm 4.8 µm
300
205 µm
Photoacoustic Intensity (a.u.)
250 0 µm
130 µm
200
150
100
50 60 µm
0
100 150 200 250 300
Distance (µm)
(c)
FIGURE 5.24 (a) 1-D profile along the dashed white line crossing the acoustic focal point
shown in Figure 5.23(b). (b) B-scan images of the blade in the focal plane, and 130 μm and
205 μm off the focal plane. (c) 1D profile along the dashed line shown in (b).
Photoacoustic Microscopy and Photoacoustic Computed Microscopy 165
focus. The FWHM was found to be 0.7 mm, which was slightly larger than the
theoretical estimation (0.6 mm) calculated by the following equation, due to some
misalignment of the transducer and the lens:
1.028 × f × c
Resolution ( = 6 dB ) = (5.12)
F×D
where f is the focal length, c is the sound velocity, F is the central frequency, and D
is element diameter of the transducer.
We employed the edge spread function (ESF) to estimate the lateral resolution
of this imaging system. The shape edge of blade (surgical scalpel, blade no. 10) was
imaged. One B-scan image contained 200 A-lines with an interval step of 2.5 μm,
and a total of 100 B-scan images were obtained by performing axial scanning with
a scanning step of 5 μm. Figure 5.24b shows the B-scan images when the blade was
in the focal plane, and 130 μm and 205 μm off the focal plane. From the profiles
given in Figure 5.24c, we found that the resolution in the focal plane is 4.8 μm, and
the resolutions in the out-of-focus planes are much poorer (40 μm in the plane and
130 μm off the focal plane, and 60 μm in the plane and 205 μm off the focal plane).
To evaluate the sensitivity with and without acoustic focused detection, a human
hair was embedded inside a tissue mimicking phantom with an absorption coefficient
of 0.007 mm–1 and a reduced scattering coefficient of 1 mm–1. In the first experi-
ment, the mixture of glycerol and water served as external liquid and enabled acoustic
and optical confocal. In the control experiment, pure water that enabled only opti-
cal converging was used. The MAP images (Figure 5.25a) and quantitative compari-
son (Figure 5.25b) show the significant improvement of sensitivity by using confocal
configuration.
In vivo experiments were carried out to demonstrate the potential biomedical
application of this technique. Rat was kept motionless using a breathing anesthe-
sia system and the body temperature was maintained at 37°C by a temperature-
controlling pad. The rat ear was gently depilated before tightly contacting with
the membrane sealed imaging window in the bottom of the tank. All procedures
were approved by the Institutional Animal Care and Use Committee (IACUC)
at the University of Florida. The total raster scanning points was 200 along each
transverse direction with an interval of 5 μm over a distance of 1 mm. The laser
pulse energy after the lens was measured to be 100 nJ. The experimental time for
a complete volumetric data collection was 40 minutes. After the data acquisition,
the rat fully recovered without any observable laser damage. The microvasculature
was clearly imaged as shown in Figure 5.26a where the top panel shows the MAP
image along axial direction, and the bottom panel gives a selected cross-section
image along the dashed white line in MAP. Figure 5.26b shows the 3D pseudo
color visualization of the vasculature.
We note that the application of this lens is not limited to ORPAM systems. If
we use an optical fiber bundle instead of single-mode optical fiber, we can build
an ARPAM imaging system with weakly confocal of the optical illumination and
166 Photoacoustic Tomography
Min Max
(a)
0.1
0.08
0.06
0.04
0.02
0
0 20 40 60 80 100 120
Distance (um)
(b)
FIGURE 5.25 (a) MAP of human hair with and without acoustic focusing. (b) 1-D plots
along the dashed lines in Figure 5.25(a). Scale bar: 20 μm.
100 um
(a) (b)
FIGURE 5.26 (a) MAP image (top) and selected B-scan image (bottom) of the rat ear.
(b) Volumetric rendering of the vasculature of the imaged rat ear.
Photoacoustic Microscopy and Photoacoustic Computed Microscopy 167
acoustic detection using a single liquid lens. In addition, the variable focal length of
the lens enables us to easily integrate fluorescence confocal microscopy with pho-
toacoustic microscopy. We also recognize that the central frequency of the current
ring-shaped transducer is not high, which led to relatively poor axial resolution and
low sensitivity to capillaries. In addition, the internal diameter of the transducer is
larger than the laser beam. A high-frequency ring-shaped transducer with a small
inner diameter can significantly improve the sensitivity and axial resolution of this
imaging system. Finally, a fast laser can significantly reduce the data acquisition
time.
6 Multimodal Approaches
When two or more than two different imaging modalities are physically combined
appropriately, the combined system can take full advantage of different modalities
to provide complementary information for most accurate clinical decision making.
This is evidenced by the combination of computed tomography (CT) and positron
emission tomography (PET) where CT and PET provide, respectively, structural and
physiological information of common tissue. In this chapter, we present examples of
combining photoacoustic tomography (PAT) with several other modalities includ-
ing diffuse optical tomography (DOT), fluorescence molecular tomography (FMT),
ultrasound imaging, and optical coherent tomography (OCT) to realize various PAT-
based multimodal approaches.
6.1 PAT/DOT
While PAT has been shown so far to be particularly powerful in imaging vascula-
tures and tissue-absorption related parameters such as hemoglobin and oxygenation,
PAT has limitations. For example, PAT can provide high-quality images only for
certain sizes of targets due to the limited detection band of an ultrasound transducer.
It is difficult to accurately detect a relatively large size target (e.g., ≥ 6 mm) using
a typical ≥1 MHz transducer. In addition, the limited directivity of a transducer
may lead to imbalanced image quality for the entire imaging area/volume. Yet, it
still remains a major challenge for PAT to recover tissue-scattering coefficients, an
important parameter for breast cancer detection (Jiang 2010).
DOT is capable of overcoming the above-mentioned limitations associated PAT,
although it has relatively low spatial resolution. Thus, a combination of PAT and DOT
appears to be an ideal approach for tissue imaging. Here we describe a combined
PAT/DOT system and demonstrate its ability using phantom and ex vivo experiments.
169
170 Photoacoustic Tomography
(b)
Positive electrode #1
Optical swith
Positive electrode #2
FIGURE 6.1 (a) Schematic of the PAT/DOT system. (b) Photograph of the interface and
schematic of a single transducer.
Laser 12000
1 MHz
10000
Exam table
8000
1 6000
Prism 2 1.48 MHz
4000
Prism 1
2000
3 4 2 0.38 MHz
Prism 3 0
0 0.5 1 1.5 2 2.5 3 3.5 4
Frequency (MHz)
(a) (b)
Sensitivity
1
1
Directivity 0.9
0.9
0.8
0.8
0.7
Normalized Signal
Normalized Signal
0.7
0.6 0.6
0.5 0.5
0.4 0.4
0.3 0.3
0.2 –30- 30-
0.2
0.1
0.1
0
0
–80 –60 –40 –20 0 20 40 60 80 0 10 20 30 40 50 60
Angle (Degree) Transducer Element
(c) (d)
FIGURE 6.2 (a) 3D schematic of the light delivery system. (b) Frequency response of a trans-
ducer. Dashed line shows the –6 dB level. (c) Directivity of a transducer. Dashed line shows the
–6 dB level. (d) Normalized sensitivity for all the elements in the transducer array.
was delivered from bottom of the exam table to the phantom. During an imaging
experiment, the light beam is scanned in 2D (arrows 1 and 2 in Figure 6.2a) via the
two step motors to realize the light illumination to cover a large area.
where Φ is the photon density, I is the identity matrix, and λ can be a scalar or a
diagonal matrix. q = ( D1 , D2 ,..., DN , a ,1 , a ,2 ,..., a ,N )T is the update vec-
tor for the optical property profiles, where N is the total number of nodes in the finite
element mesh used and D is the diffusion coefficient. Φ( m ) = (Φ1(m ) , Φ(2m ) ,..., Φ(Mm ) )
and Φ(c ) = (Φ1(c ) , Φ(2c ) ,..., Φ(Mc) ), where Φ(i m ) and Φ(i c ), respectively, are measured
and calculated data for i = 1,2,...,M boundary locations. ℑ is the Jacobian matrix
that is formed by ∂Φ/∂D and ∂Φ/∂ a at the boundary measurement sites. In DOT,
the goal is to update the a and D or s distributions through the iterative solu-
tion of Equation (6.1) so that a weighted sum of the squared difference between
computed and measured data can be minimized. A single mesh of 700 nodes was
used, and the images were converged within 15 iterations in a 3 GHz PC with
1 GB memory.
–10 –10
–8 –8
–6 3 –6
–4 –4 2
2
–2 –2
1
0 1 0
2 2
4 4
6 6
8 8
10 10
–10 –5 0 5 10 –10 –8 –6 –4 –2 0 2 4 6 8 10
(a) (b)
FIGURE 6.3 (a) PAT image of three separated metal wires (0.15 mm in diameter each)
embedded in the phantom background. (b) PAT image of two metal wires (0.15 mm in diam-
eter each) placed in the phantom background.
delivery system, and conduct a comparison between PAT and DOT in these areas.
We also test our PAT/DOT system using ex vivo tumor tissue embedded in a tissue-
mimicking phantom.
6.1.2.2 PAT Performance
Figure 6.4a shows the geometry of the tissue-mimicking phantom (Intralipid + India
ink) and the positions of five targets used for four phantom experiments. The param-
eters for the five targets are listed in Table 6.1. These experiments were performed
using the 532 nm Nd:YAG pulsed laser. The light beam was guided and extended
by our light delivery system to be an area source of 3 cm2 and the PAT images were
reconstructed by a delay and sum method. The light energy density at the surface of
the phantom was 20 mJ/cm2.
174 Photoacoustic Tomography
3
4
1 5
2 Phantom
(a)
–20 –20
–15 –15
–10 –10
–5 –5
1
1 0
0
5 5
10 10
15 15
20 20
–20 –15 –10 –5 0 5 10 15 20 –20 –15 –10 –5 0 5 10 15 20
(b) (c)
–40 –40
–30 –30
–20 –20 4
3
–10 –10
0 0
10 10 5
20 20
30 30
2
40 40
–40 –30 –20 –10 0 10 20 30 40 –40 –30 –20 –10 0 10 20 30 40
(d) (e)
FIGURE 6.4 (a) Geometry and positions of targets used for four phantom experiments.
(b) PAT image of target 1 without any depth. (c) PAT image of target 1 positioned at 22 mm
beneath the surface. (d) PAT image of target 3 (30 mm off-center positioned). (e) Fused image
of multiple targets.
Multimodal Approaches 175
TABLE 6.1
Exact Size, Location (Off Center), Depth, and Absorption and Reduced
Scattering Coefficients of the Five Targets
Size (mm) Location (mm) Depth* (mm) μa (mm–1) μ′s (mm–1)
Target 1 2 0 0 (22) 0.028 2
Target 2 2 20 5 (15) 0.028 2
Target 3 3 30 5 (10) 0.028 2
Target 4 3 20 5 (15) 0.028 2
Target 5 1 20 5 (15) 0.028 2
* The maximum experimental imaging depth for certain cases is given in the parentheses.
Figures 6.4b and 6.4c show the PAT images of target 1 located at 0 and 22 mm
below the phantom surface, respectively. While the target is clearly better imaged
with 0 mm depth, the target with 22 mm depth is still detectable. The active imag-
ing area for PAT is validated by the result shown in Figure 6.4d where target 3
(30 mm off-center positioned) is detected. Due to the limited directivity of trans-
ducers, we note that the shape of reconstructed target was distorted and stronger
artifacts are seen around the target. We also tested and found that when a target
was placed at a >30 mm off-center position, the image quality became unaccept-
able. For the single target experiments, the light beam was directly delivered to
the target area without scanning. The image shown in Figure 6.4e demonstrated
the ability of imaging multiple targets using the developed light delivery system.
Three targets (2, 4, and 5) separated with a large distance were imaged by scan-
ning the laser beam nine (3 × 3) times to cover the whole phantom surface. The
nine images obtained from the nine laser beam positions were then fused into a
single image shown in Figure 6.4e.
40 PAT PAT y = 2 mm
Exact
0.02 0.025 Recovered
20
0.02
0.015
µa 0.015
0
0.01
0.01
–20 0.005
0.005
0
–40 8 10 12 14 16 18 20 22 24 26
–40 –20 0 20 40 x (mm)
40 DOT DOT
y = 2 mm
0.018 Exact
Recovered
20 0.016 0.025
0.014
0.02
0 0.012 µa
0.015
0.01
–20 0.008 0.01
0.006
–40 8 10 12 14 16 18 20 22 24 26
–40 –20 0 20 40
x (mm)
1.2
–20 0.7
1
0.8
–40 0.6
–40 –20 0 20 40
8 10 12 14 16 18 20 22 24 26
x (mm)
(a)
FIGURE 6.5 Quantitative PAT and DOT images of target 5 (a), target 2 (b), target 3 (c), and
target 6 (d). (continued)
(Figure 6.5b). In this case, DOT can detect the target from both the absorption and
scattering images; however, the values of both absorption and scattering coefficients
are significantly underestimated. Figure 6.5c shows that target 3 (radius = 3 mm) is
quantitatively recovered by both PAT and DOT while we note an overestimated target
size by DOT.
Interestingly, the largest target 6 (radius = 5 mm) is poorly recovered by PAT
(Figure 6.5d), due to the loss of low-frequency signals given the limited frequency
response of the transducers. Here we see that only the edge of the target is recovered
by PAT and the reconstructed absorption coefficient value is considerably underesti-
mated. DOT in this case provides accurate recovery of the target in terms of its size,
position, and absorption and scattering coefficient values.
Multimodal Approaches 177
x = 3 mm
40 PAT PAT
Exact
30 0.02 0.025
Recovered
20
0.02
10 0.015
0 µa 0.015
0.01
–10 0.01
–20
0.005 0.005
–30
–40 0
–40 –20 0 20 40 –28 –26 –24 –22 –20 –18 –16 –14 –12 –10
y (mm)
10 1.2 1.6
FIGURE 6.5 (continued) Quantitative PAT and DOT images of target 5 (a), target 2 (b),
target 3 (c), and target 6 (d). (continued)
Overall from the results shown in Figure 6.5 and the summary in Table 6.2, PAT
can provide both qualitatively and quantitatively better images than DOT when the
target size is equal to or small than 3 mm in radius, while DOT can offer better
image quality when the target size is larger than 3 mm in radius. We also notice that
the smallest detectable target size is 4 mm for DOT given the experimental condi-
tions used in this study.
4
× 10
40 PAT PAT x = –22 mm
16 Exact
30
14 0.025 Recovered
20
12 0.02
10
10
0 µa 0.015
8
–10
6 0.01
–20 4
–30 0.005
2
–40 0
–40 –20 0 20 40 4 6 8 10 12 14 16 18 20 22
y (mm)
40 DOT
DOT x = –24 mm
30 2 Exact
1.7
Recovered
20 1.8
1.6
10
1.5 1.6
0 µs'
1.4
–10 1.4
1.3
–20 1.2
–30 1.2
1.1 1
–40
–40 –20 0 20 40 4 6 8 10 12 14 16 18 20
y (mm)
(c)
FIGURE 6.5 (continued) Quantitative PAT and DOT images of target 5 (a), target 2 (b),
target 3 (c), and target 6 (d). (continued)
–3
×10 0.03 y = 17 mm
40 PAT PAT Exact
12 Recovered
0.025
20 10
0.02
8
µa 0.015
0
6
0.01
4
–20
0.005
2
–40 0
2 4 6 8 10 12 14 16 18 20 22
–40 –20 0 20 40
x (mm)
40 DOT DOT y = 17 mm
Exact
0.025
0.025 Recovered
20
0.02
0.02
0 µa
0.015 0.015
–20
0.01 0.01
–40
–40 –20 0 20 40 2 4 6 8 10 12 14 16 18 20 22
x (mm)
DOT DOT y = 17 mm
40 4 4 Exact
3.5 3.5 Recovered
20
3 3
µs'
2.5 2.5
0
2
2
–20 1.5
1.5
1
–40 1
2 4 6 8 10 12 14 16 18 20 22
–40 –20 0 20 40
x (mm)
(d)
FIGURE 6.5 (continued) Quantitative PAT and DOT images of target 5 (a), target 2 (b),
target 3 (c), and target 6 (d).
6.2 PAT/FMT
Fluorescence molecular tomography (FMT) has received particular attention in
recent years (Jiang 2010), due to its remarkably high sensitivity as well as the increas-
ing availability of fluorescent dyes and probes, which provide a variety of molecular
signatures. However, the major limitation of FMT is its low spatial resolution, espe-
cially in reflection mode. While PAT has high spatial resolution, it has relatively lower
sensitivity compared with FMT when near-infrared (NIR) fluorescent dyes such as
indocynine green (ICG) are used (see more discussion about molecular or contrast
180 Photoacoustic Tomography
TABLE 6.2
Exact and Reconstructed Target Size and Absorption and
Reduced Scattering Coefficients for the Phantom Experiments
Size (mm) μa(mm–1) μ′s (mm–1)
Case 1 Exact 2 0.028 2
PAT 2 0.026 NA
DOT NA NA NA
Case 2 Exact 4 0.028 2
PAT 4 0.025 NA
DOT 10 0.017 1.1
Case 3 Exact 6 0.028 2
PAT 6 0.024 NA
DOT 10 0.024 1.8
Case 4 Exact 10 0.028 4
PAT 8 0.015 NA
DOT 12 0.028 4
agents based PAT in Chapter 7). While many contrast-agents-based PAT methods
use nanoparticles (Chapter 7), some of them use fluorescent dyes as dyes are usually
strong absorber. In this case, a dual-imaging modality of PAT and FMT can be real-
ized where PAT offers excellent spatial resolution and FMT provides high sensitivity,
making the combination a better molecular imaging approach. Here we describe a
combined 3D PAT and FMT in reflection-mode where the common contrast agent is
ICG dye.
6.2.1 Methods
The PAT system is schematically shown in Figure 6.7a. A tilted fiber bundle deliv-
ers laser light from a Ti:Sapphire tunable laser providing a generally homogenous
illumination. The incident intensity is up to 20 mJ/cm2 on the object surface, which is
100% of the American National Standards Institute limit. The laser source has a rep-
etition rate of 10 Hz, and the pulse duration is about 6ns. Different laser wavelengths
were chosen for the different ICG concentrations in the phantom experiments so
that the absorption peak of ICG could be reached. A 4 MHz immersion ultrasound
transducer was scanned by a linear moving stage along x and y directions with a step
size of 1.27 mm and 1.00 mm, respectively. The generated photoacoustic waves were
averaged 50 times for each position, taking about 40 mins for a complete area scan.
The complex signal was amplified by a commercial preamplifier and acquired by a
high-speed PCI data acquisition board. A LabView program controlled the entire
system for data acquisition. The 3D images were reconstructed using the delay-and-
sum algorithm described in Chapter 1.
Multimodal Approaches 181
x = 1.2 mm
× 10–3
16
40 PAT PAT Recovered
14
0.015
12
20
10
0.01 µa 8
0
6
0.005 4
–20
2
0
–40 0
–40 –20 0 20 40 10 15 20 25
y (mm)
(a)
–40 4 4
–40 –20 0 20 40 5 10 15 20 25
y (mm)
(b)
x = 1.2 mm
40 DOT 1.4
1.4 DOT Recovered
20 1.3 1.3
1.2 1.2
0
µs'
1.1 1.1
–20
1
1
–40 0.9
–40 –20 0 20 40 5 10 15 20 25
y (mm)
(c)
FIGURE 6.6 Reconstructed ex vivo absorption image by PAT (a), and absorption (b) and
scattering (c) images by DOT.
182 Photoacoustic Tomography
Diode laser y
Pulsed laser Transducer x
CCD
y
x
Filter
Acoustic waves
Multiply scattered
Target Target fluorescence light
(a) (b)
FIGURE 6.7 Schematic of the PAT (a) and FMT (b) system.
0 4000
0
4
5 3000
8
X
PAT
2000
Z 10
12
15 16 1000
20
0 5 10 15 20 0 4 8 12 16 20
X Y
(a) (b)
0 0
0.3
4 4 0.25
8 8 0.2
FMT
Z X 0.15
12 12
0.1
16 16
0.05
20 20 0
0 4 8 12 16 20 0 4 8 12 16 20
X Y
(c) (d)
FIGURE 6.8 PAT and FMT images for a 100 µM ICG target at 7 mm depth. (a) and (b) z-x
image, x–y PAT image; (c) and (d) corresponding FMT image. The white rectangle/circle
indicates the exact target location.
184 Photoacoustic Tomography
2 mm 3.5 mm
0 0
350
500
5 5 300
400
10 10 250
300
15 15 200
200
20 20 150
0 10 20 0 10 20
(a) (b)
7 mm
0 350 0 0.2
5 300 5
0.15
10 250 10
0.1
15 200 15
20 150 20 0.05
0 10 20 0 10 20
(c) (d)
0 0.2 0 0.2
5 5
0.15 0.15
10 10
0.1 0.1
15 15
20 0.05 20 0.05
0 10 20 0 10 20
(e) (f )
FIGURE 6.9 Reconstructed images with different target separations. (a)–(c) PAT image;
(d)–(f) FMT image.
Multimodal Approaches 185
separations where the exact target positions are indicated by the black circles. We
see that the two targets at the three separations are clearly reconstructed by both
methods. However, it is noted that the two targets are not fully separated by FMT
when they are only 2 mm apart (Figure 6.9d), but they are clearly separated by PAT
(Figure 6.9a).
6.2.2.3 Comparison of Sensitivity
Nine sets of phantom experiments were conducted to evaluate the sensitivity using
three different ICG concentrations (100 µM, 10 µM, and 2 µM) and three different
target depths. To achieve the maximum absorption at different ICG concentration,
three different wavelengths (698 nm, 780 nm, and 790 nm) were used for PAT. For each
ICG concentration, the targets (2 mm in diameter and 1 mm in height each) were buried
at 7 mm, 12 mm, and 17 mm below the surface, respectively. The targets were 2 mm in
diameter and 1 mm in height. Figure 6.10 shows the reconstructed PAT images along
x-z plane (y = 10 mm), while Figure 6.11 gives the corresponding FMT images. We
immediately note that the PAT images provide much more reliable target position and
size because of the high spatial resolution compared to its FMT counterpart. From
Figure 6.10, it can be seen that as the ICG concentration decreases or the target posi-
tion goes deeper, the reconstructed PAT image has greater artifacts and decreased
image quality. While similar trend is seen from the FMT images, we note that FMT
has clearly greater sensitivity compared to PAT for detection of ICG fluorochromes.
For example, the target with 10 µM ICG at 17 mm depth or 2 µM ICG at 12 mm depth
100 µm 10 µm 2 µm
5 x 1500 5 80 5 50
10 60 40
10 z 1000 10
7 mm
40 30
15 500 15 15 20
20 10
20 20 20
0
0 10 20 0 10 20 0 10 20
5 5 40 5 40
200 10 30
30
12 mm
10 10
15 20 20
15 100 15
10 10
20 20 20
0 0 0
0 10 20 0 10 20 0 10 20
5 140 5 5
120 60
40
17 mm
10 100 10 10
80 40
15 60 15 15 20
40 20
20
20 20 20
0
0 10 20 0 10 20 0 10 20
FIGURE 6.10 PAT images for a target with different ICG concentrations at different depths.
186 Photoacoustic Tomography
100 µm 10 µm 2 µm
0
4
8
7 mm
Z
12
16
20
0 4 8 12 16 20
X
12 mm
17 mm
0.1 0.2 0.3 0 0.1 0.2 0 0.2
FIGURE 6.11 FMT images for a target with different ICG concentrations at different
depths. The black block indicates the true target position.
6.3 PAT/ULTRASOUND
While PAT can provide high-resolution tissue absorption coefficient images and sub-
sequently derived functional parameters such as hemoglobin and oxygenation, it often
lacks certain tissue structural information. One way to compensate this is to combine
ultrasound (US) imaging with PAT. This is actually the most convenient way to realize
a combined PAT and US since both share the common detection channel for ultra-
sound signal collection. We illustrate such a PAT/US combination using intravascular
Multimodal Approaches 187
1 mm
Micro transducer
Lens PA or US signal
(IV) imaging system as an example (see Figure 6.12). In this PAT/US combined system,
light beam from a pulsed Ti:Sapphire (750–1700 nm) laser is reflected by a mirror
and focused into a multimode (MM) optic fiber via a lens. The light is delivered into a
1.5 mm-diameter miniature imaging probe consisting of the MM fiber with angle pol-
ished end and a 20 MHz central frequency microtransducer with a size of 0.7 × 0.5 mm2 (see
the insert photograph in Figure 6.12). The angle polished fiber end ensures the delivery
of light to the wall of blood vessel, and the light-induced ultrasound is collected by the
microtransducer. The photoacoustic (PA) signal is received by the receiver part of the
pulser/receiver and digitized by the data acquisition board/computer. An ultrasound
pulse is sequentially generated by the pulser and delivered into the miniature imaging
probe. In this case, the microtransducer acts as both the excitation source and detector,
and the received ultrasound signal goes through the pulser/receiver and is digitized
by the data acquisition board/computer. A 360º rotation of the imaging probe or the
sample allows a full scan of the blood vessel wall.
Figure 6.13 shows the intravascular PA (IVPA) and ultrasound (IVUS) images
obtained from two ex vivo human blood vessels with plaques (see the photographs;
the plaques are indicated by white arrows). While both IVPA and IVUS can iden-
tify the plaques in both cases, the images generated from the two different contrast
mechanisms certainly complement each other, which can be seen more striking from
the PAT/US fused images. We will have more discussion on IVPA in Chapter 8
where the unique ability of IVPA for quantitatively imaging biochemical composi-
tions of the plaques is demonstrated.
188 Photoacoustic Tomography
FIGURE 6.13 Intravascular photoacoustic (IVPA) and ultrasound (IVUS) images obtained
from two plaque-containing human blood vessels. Arrow indicates the location of plaque.
6.4 OPTICAL-RESOLUTION PAM/OCT
As a final example in this chapter, we present the combination of ORPAM and OCT
based on a miniature probe of only 2.3 mm in diameter, which will be ideal for endo-
scopic and intravascular imaging. The optical paths of both the ORPAM and OCT
are built upon a single-mode fiber, a miniature GRIN lens and two microprisms,
enabling the two modalities to scan the same tissue area. The self-focusing ability of
the GRIN lens results in a highly focused light beam for OCT and ORPAM, yielding
a high lateral resolution of 15 μm for both modalities.
Single-mode fiber
1 mm
Tissue
(a) (b)
FIGURE 6.14 Schematic (a) and photograph (b) of the hybrid probe.
have 0.7 mm in diameter. A stainless steel tube with 1.0 mm in diameter is used
to protect the light path (i.e., the single mode fiber, GRIN lens and cubic prism
pair). The probe is glued to the transducer and protected by another bigger stain-
less steel tube (2.3 mm in diameter).
The probe is mounted on a 3D linear stage (Figure 6.15) and scanned in 2D for 3D
image formation. The light beam for ORPAM is generated from a pulsed Nd:YAG
laser with 532 nm wavelength and a 10 Hz repetition rate. Two neutral density filters
are used to attenuate the light energy, and a small iris is utilized to provide homoge-
neous light distribution. The shaped light beam is focused by a convex lens, which
passes through a 50 μm pinhole for spatial filtering and is then coupled into a 2 × 1
beam coupler. The detected acoustic waves are amplified by two wideband ampli-
fiers and digitized by a data acquisition board (NI5152, National Instrument) at a
sampling rate of 250 MS/s.
Iris
ND L1 L2
L3
Nd: YAG pulsed laser
Pinhole
SLD source
Circulator 2×1
2 × 2 BP
Probe
PD RSOD
2D linear stage
PC
Pre-amplifier
FIGURE 6.15 Schematic of the integrated ORPAM and OCT system. ND: neutral density,
L1: lens 1, L2: lens 2, L3: lens 3, BP: beam splitter, PD: photodiode.
190 Photoacoustic Tomography
70 100
OCT ORPAM
60
80
OCT Profile (a.u.)
50
PA Signal (a.u.)
40 60
30
40
20
10 20
0
0
0.1 0.15 0.2 0.25 0.3 0.35 0.1 0.18 0.27 0.35
x-axis (mm) x-axis (mm)
(a) (b)
FIGURE 6.16 Resolution test for ORPAM and OCT. 1-D profile of OCT (a) and ORPAM
(b) of Group 6, Element 1.
0.4
Without calibration
PA Intensity (Volt)
0.2 PA1 PA2
0
A2 0 1 2 3
z-axis (mm)
A1 (b)
0.4
With calibration
PA Intensity (Volt)
(a)
0.2
0
0 1 2 3
z-axis (mm)
(c)
FIGURE 6.17 Signal calibration. (a) Schematic of the probe (bottom view). (b) and (c):
Typical A-line (Hilbert Transform of raw data) without (b) and with (c) calibration.
the calibration allowed us to add the two signals together, resulting in improved SNR
for image formation.
100 µm
ED
BV
CT D
FIGURE 6.18 In vivo imaging of mouse ear by the integrated probe. MAP image (top) and
cross-section (bottom) of ORPAM (a) and OCT (b). ED: epidermis, CT: cartilage, D: dermis,
BV: blood vessel.
the sebaceous gland with high resolution. The bottom rows (cross-section of tissue)
of Figures 6.18a and 6.18b show the benefits of combining these two modalities more
clearly. The ear’s thickness in the OCT image is from 400 to 600 µm. The dermal
structure and the sebaceous gland are clearly observed. In the cross-sectional OCT,
we can identify epidermis, dermis, and cartilage as indicated with arrows. We have
observed that ORPAM is good at locating micro vessels in the ear with limited sur-
rounding tissue information.
The SNR of the ORPAM is 25dB, which is lower than that of conventional
ORPAM. There are several reasons contributing to the reduced SNR: (1) A 10 MHz
ultrasound transducer was used in this probe, while it is known that the strongest
generated acoustic signal lies between 30 to 70 MHz; (2) the transducer is flat, result-
ing in reduced sensitivity compared with a focused transducer commonly used in
conventional ORPAM; and (3) during the experiments, we used an 8 bit resolution
DAQ card, which can resolve only signals that are larger than 40 mV. As a result, we
lost some signals from small capillaries. These limitations, however, can be easily
overcome using a miniature focused high-frequency transducer (>30 MHz), which
will in turn improve the axial resolution of ORPAM and make the whole probe
smaller as well. Using a high-resolution DAQ card will also help solve the afore-
mentioned problems as well as (4) the actual resolution of a GRIN probe tends to
be worse than this theoretical limit, because the effective NA of GRIN lenses is
degraded owing to the spatial aberrations in the lenses and misalignment between
optical components. For future improvements, we also need a suitable micromotor
to implement an internal scanning mechanism and a faster laser to reduce the scan-
ning time. Finally, we can replace the current slow time-domain (TD) OCT with fast
frequency-domain (FD) OCT. These are necessary steps towards the clinical evalu-
ation of the ORPAM/OCT.
7 Contrast Agents–Based
Molecular Photoacoustic
Tomography
7.1 GOLD NANOPARTICLES
Gold nanoparticles have become a prime candidate for PAT due to their unusual
optical properties and inherent biocompatibility. The intense scattering and absorp-
tion of light that occurs under the plasmon-resonant condition coupled with the abil-
ity to tune the resonance into the NIR by manipulating the aspect ratio make gold
nanoparticles attractive as contrast agents for optical imaging techniques. Further,
gold-protein chemistry is well developed and several bioconjugation protocols are
available in the literature (Grobmyer and Moudgil 2010), which allows the combina-
tion of the targeting functionality of antibodies with such gold nanoparticles. The
inertness and biocompatibility of gold in general hold promise for the use of gold
nanoparticles for in vivo imaging applications.
Figure 7.1 shows the TEM image and optical absorption spectra for 20 nm and/or
50 nm gold nanoparticles. We see that these gold nanoparticles exhibit strong plasma
resonance peak around 530 nm. The molar extinction coefficient of the nanoparticles
was determined to be 1 × 109 M–1cm–1, about three orders of magnitude larger than
that of a typical organic molecule. Here phantom experiments were conducted to
demonstrate gold nanoparticles enhanced PAT using the imaging system described
193
194 Photoacoustic Tomography
0.03
20 nm
0.02
0.015
0.01
0.005
0
400 500 600 700 800
Wavelength (nm)
(a) (b)
FIGURE 7.1 (a) TEM image of gold nanoparticles having an average size of 20nm. (b) Optical
absorption of 20nm (solid line) and 50nm (dashed line) nanoparticles.
30 30 4
(a) 2 (b)
20 20 3.5
3
10 1.5 10
2.5
0 0 2
1
1.5
10 10
0.5 1
20 20
0.5
30 30
30 20 10 0 10 20 30 30 20 10 0 10 20 30
30 30
(c) (d) 1
20 2 20 0.9
0.8
10 1.5 10 0.7
0.6
0 0
1 0.5
0.4
10 10
0.3
0.5 0.2
20 20
0.1
30 30
30 20 10 0 10 20 30 30 20 10 0 10 20 30
FIGURE 7.2 Reconstructed photoacoustic images. (a) A single target containing 0.25 nM
nanoparticles. (b), (c), and (d) Two targets containing 0.5, 0.25, and 0.125 nM nanoparticles in
each target, respectively. The gray scale indicates the relative optical absorption (dimension-
less), while the left and bottom axes refer to spatial coordinates in millimeters.
4.5
4.0 0.50 nM
3.5 0.25 nM
Relative Absorption
0.125 nM
3.0
2.5
2.0
1.5
1.0
0.5
0.0
–10 –8 –6 –4 –2 0 2 4 6 8 10
x or y (mm)
FIGURE 7.3 Optical absorption profiles along a transect through one of the two targets for
the images shown in Figures 7.2b, 7.2c, and 7.2d.
196 Photoacoustic Tomography
15 15
10 0.2 10 0.2
5 5
x (mm)
x (mm)
0.15 0.15
0 0
5 5
0.1 0.1
10 10
15 0.05 15 0.05
10 0 10 10 0 10
Y (mm) Y (mm)
FIGURE 7.4 PAT images of tumor following tail vein injection of gold nanoparticles at 5
minutes following injection (a) and 5 hours following injection (b). The color scale (right)
represents optical absorption of tissue (arbitrary units). Gold nanoparticles were modified
with PEG5000-thiol following the procedure of Bergen et al. (2006). For these experiments,
human breast cancer cells, BT474 (ATCC), were implanted on the lower abdominal wall of
Nu/Nu mice and allowed to grow to ~1.5 cm in size.
permeability and retention” (Maeda and Matsumura 1989, Kommareddy and Amiji
2007). In the present case, the accumulation of gold nanoparticles inside the tumor
can be visualized by PAT and is shown clearly 5 hours following the systemic admin-
istration of gold nanoparticles (Figure 7.4).
7.2 GRAPHENE NANOSHEETS
Inspired by the strong NIR absorption, high photothermal conversion efficiency, and
exceptionally large surface area of graphene, graphene nanosheets have emerged as
a new high-potential nanomaterial for biomedical applications, especially in the area
of photothermal therapy including photothermal-enhanced drug and gene delivery
systems. It would be highly desirable to monitor/image the in vivo distribution of
multifunctional drug delivery systems, evaluate their posttreatment therapeutic out-
comes in situ, and most importantly to track the long-term fate of graphene sheets in
the human body. These capabilities could largely facilitate their application in practi-
cal multifunctional nanomedicine regimes, fighting various diseases.
Here we describe the use of graphene sheets as contrast agents for PAT and dem-
onstrate the potential of graphene-sheets-based PAT for in vivo monitoring the dis-
tribution of multifunctional drug delivery systems. To generate photoacoustic (PA)
signals with NIR light excitation, the following conditions should be satisfied: strong
NIR absorption, nonradiative relaxation, heating, and acoustic wave generation. We
found that the microwave-enabled low-oxygen graphene (ME-LOGr) nanosheets
exhibit strong and wavelength-independent absorption in the visible and NIR regions
(Figure 7.5). Their absorption (with a coefficient of 22.7 L/g cm at 808 nm) exceeds
the best NIR fluorophores (for example, indocyane green has an absorption coeffi-
cient of 13.9 L/g cm at 808 nm) and the endogenous cellular background. The differ-
ence in NIR absorption between the graphene sheets and the background provides
Contrast Agents–Based Molecular Photoacoustic Tomography 197
1.4
5.0 nm ME-LOGr
Nano sheet GO
1.2
1.0
Absorbance (a.u.)
0.8
2.5 nm
0.6
0.4
0.2
250 nm 0.0
0.0 nm
200 400 600 800 1000 1200
Wavelength (nm)
(a) (b)
FIGURE 7.5 (a) AFM images of ME-LOGr nanosheets, (B) UV vs. NIR spectra of
ME-LOGr nanosheets with concentrations of 20 (pink), 10 (olive), 6.7 (blue), 5 (red), and
3.3 mg/L (black), respectively. Inset (b), photograph of an aqueous suspension of ME-LOGr
nanosheets (left) and graphene oxide (GO) nanosheets (right) shows different gray scales,
indicating they are in different oxidation states.
FIGURE 7.6 Photoacoustic images of graphene oxide (GO) and graphene nanosheets of dif-
ferent concentrations, illuminated with 720 and 800 nm pulsed laser coupled with a 1 MHz
central frequency transducer.
198 Photoacoustic Tomography
and NIR illumination, possibly because of their low NIR absorption capability.
Furthermore, the intensity of the PA signals depends on the concentration of the
ME-LOGr nanosheets, suggesting that the ME-LOGr nanosheets can be used as
NIR contrast agent for in vivo NIR PA imaging. Since the strong NIR absorption of
ME-LOGr nanosheets is almost independent of the wavelength in the NIR region,
their NIR PA signal shows a similar trend of wavelength independence. Figure 7.6
also shows that PA signals generated under 800 nm illumination are similar to those
illuminated at 700 nm. This “wavelength-independent” characteristic is very differ-
ent from other PA contrast agents, such as Au nanorods and Ag nanoplates, which
are highly wavelength dependent (De la Zerda et al. 2011).
of uPA cDNA sequence and expressed in E. coli BL21 (Invitrogen, Carlsbad, CA).
ATF peptides were purified from bacterial extracts with Ni2+ nitrilotri-acetic acid
(NTA)-agarose columns (Qiagen, Valencia, CA) using established protocols (Yang
et al. 2009). IR-783 (Sigma-Aldrich, St. Louis, MO) was used to synthesize near-
infrared dye (NIR-830) as described by Lipowska et al. (1993). The schematic and
spectral characterization (excitation wavelength: 800 nm, emission wavelength:
825 nm) of NIR-830 dye were shown in Figures 7.7a and 7.7c. Free thiol groups on
the ATF peptide or control bovine serum albumin protein (BSA) were labeled with
NIR-830 dye and then conjugated to amphiphilic polymer-coated, 10 nm magnetic
iron oxide nanoparticles (IONPs) (Oceannanotech, LLS, Springdale, AR) via cross-
linking of carboxyl groups of the amphilphilic polymer to the animo side groups of
the peptides (Figure 7.7b) using an established protocol. Briefly, cysteine residues of
ATF or BSA were subjected to reduction by TCEP (5 mM at pH 7.4 for 30 mins at
RT). Immediately after reduction, NIR830-maleimide dyes were added and allowed
ATF of uPA
H O
O N
C N
NIR-830
O
S IONP
+
N N
SO–3 SO3Na
(a) (b)
0.15 20
NIR830-ATF
0.12 16
0.09 12
Fluorescence
Absorption
0.06 8
0.03 4
0.00 0
400 500 600 700 800 900
(c)
FIGURE 7.7 Illustration of NIR-830 dye and ATF-conjugated IONP probe. (a) Schematic of
NIR-830 dye. (b) NIR-830 dye is conjugated to mouse ATF peptide through a bond between
maleimide esters and free thiol groups of cystidine residues of the peptide. Dye-labeled
peptides were then conjugated to carboxyl group of the polymer coating on the IONPs.
(c) Resulting optical probe has an Ex 800 nm and Em 825 nm.
200 Photoacoustic Tomography
Beam splitter
(a) Beam expander
Pulsed laser
Lens
PC
Lens Fiber
Pre-amplifier Bundle I
Transducer
2D Scanner
Fiber Bundle IV
Filter
Animal
Holder
7.3.1.6 Image Processing
Photoacoustics tomograms were reconstructed by a program implemented in
MATLAB® 7.0 and merged with Amira 5.3.3. The photoacoustic signals collected
were processed by the Hilbert transform before reconstruction, and the photoacous-
tic signals from each mouse were normalized to the same scale (0~256). Fluorescent
images were collected by RS Image (Roper Scientific, Inc) provided by the manufac-
turer and processed by MATLAB 7.0 and fused through Amira 5.3.3.
7.3.1.7 Histological Analysis
Tumors collected from mice were preserved in 10% neutral buffered formalin for
10 hours at room temperature. Histological sections were stained with Prussian blue
staining and analyzed using standard procedures to confirm the presence of iron
oxide nanoparticles.
202 Photoacoustic Tomography
7.3.1.8 Statistical Analysis
All data obtained from the experiments were summarized using means ± standard
error of the mean (SEM). All data points within a given field-of-view (FOV) (i.e.,
the tumor region in this study) were used to calculate the means/standard errors
presented in this study. For the nontargeted or control tumors, four fiducial markers
were used to indicate the FOV for the analysis.
7.3.2 Results
The absorbance of light by IONPs is much stronger compared with blood at wavelength
longer than 650 nm. The absorbance of IONPs decreases with increasing wavelength
(Figure 7.9b). Although the strongest absorption for IONPs lies near 650 nm, the appropri-
ate wavelength for medical imaging is within the transparent optical window of 700 to
1000 nm, which increases the penetration depth. We measured the photoacoustic signal
of NIR830-ATF-IONP at wavelengths from 730 to 870 nm (Figure 7.9b), and our findings
agreed well with IONP absorption spectra presented by Galanzha et al. (2009). Tumor-
bearing mice were imaged at 24 hours after injection of NIR830-ATF-IONPm and
730 nm was chosen as the best wavelength for in vivo photoacoustic imaging. From the
MAP photoacoustic images shown in Figure 7.9a, NIR830-ATF-IONP-targeted tumors
displayed the highest absorption at 730 nm compared with 800 nm and 850 nm. From the
1
730 nm 800 nm 850 nm
Normalized PA Signal
2 mm
0
(a)
Absorbance/PA Signal (a.u.)
40 NIR830-ATF-IO
IO absorption 4000
Photoacoustic Signal
35 Blood absorption
3000
30
25 2000
20
15 1000
10
0
650 700 750 800 850 900 730 nm 800 nm 870 nm
Wavelength (nm) Wavelength
(b) (c)
FIGURE 7.9 (a) In vivo MAP photoacoustic images of tumors using signals of 730 nm,
800 nm, and 870 nm, 24 hours after injection of NIR830-ATF-IONP. (b) Absorption char-
acteristics of NIR830-ATF-IONP, IONP, and blood. (c) In vivo PA signals using different
wavelength inputs.
Contrast Agents–Based Molecular Photoacoustic Tomography 203
quantitative plot shown in Figure 7.9c, the in vivo photoacoustic signal at 730 nm is 21 and
49% greater, compared with 800 and 870 nm, respectively. Similarly, the absorption of
NIR830-ATF-IONP at 730 nm was 18 and 47% higher, compared with 800 and 870 nm,
respectively. This in vivo data is consistent with in vitro signal enhancement studies.
As shown in Figures 7.10b, 7.10f, and 7.10j, the contrast between tumor and nor-
mal tissue is low before injection. At 24 hours post-NIR830-ATF-IONP injection, the
contrast increased significantly while there was no significant contrast increase using
FL image
24 hrs
0 hr
5 hr
24 hr
FIGURE 7.10 In vivo photoacoustic MAP and fluorescence images before and after injec-
tion. Micrographs were merged with fluorescence images taken 24 hours postinjection with
indicated agent (a, e, i). (b)–(l) Photoacoustic MAP images were merged with images of blood
vessels before injection (b, f, j), and at 5 hours (c, g, k) and 24 (d, h, l) hours postinjection.
204 Photoacoustic Tomography
4000 4000
Photoacoustic Signal
Photoacoustic Signal
3000 3000
2000 2000
1000 1000
0 0
rs
rs
n
l
ol
ol
ol
ro
tio
ou
ou
BS
pm
pm
pm
nt
jec
H
H
Co
50
0
5
24
In
10
17
e-
Pr
(a) (b)
(c)
FIGURE 7.11 Quantitative plot and comparison of photoacoustic signals. (a) Photoacoustic
signals before and after injection of NIR830-ATF-IONP. (b) Comparison of photoacoustic
signals with different concentrations of injected NIR830-ATF-IONP. (c) NIR fluorescence
imaging of resected tumor (left panel), cross-section of tumor along black dashed line (mid-
dle panel), and section stained with Prussian blue (right panel).
Contrast Agents–Based Molecular Photoacoustic Tomography 205
45
2 mm 40
35
SBR (dB)
30
25
20
15
0 10 20 30
Depth (mm)
(a) (b)
PA Signal (a.u.)
0 1 d5 d6
d1 d2 d3 d4
2 mm
31 mm
25 mm
16 mm
12 mm
0 mm 5 mm
(c)
FIGURE 7.12 In vivo photoacoustic images with adding chicken breast between detector
and tumor. (a) MAP image of tumor at 24 hours after injection without adding chicken breast
(left) and photograph of 31mm-thick chicken breast (right). (b) SNR versus depth. (c) B-scan
images by adding different thickness of chicken breast.
is strong and no NIR signals from the surrounding tissue or organs. The right panel of
Figure 7.11c shows the histology section stained with Prussian blue staining. The arrow
indicates the IONP cluster inside the tumor.
To evaluate the clinical utility of this technique, the imaging depth was inves-
tigated by adding biological tissues (chicken breast) to the top of the mice skin.
Figure 7.12a (left) shows the MAP of the tumor in mice that received NIR830-ATF-
IONP at 24 hours postinjection without adding chicken breast. The dashed line repre-
sents the position of B-scan images shown in Figure 7.12c. Even after adding 31 mm
of chicken breast (Figure 7.12a, right), the tumor is clearly seen in Figure 7.12c (d6).
As the imaging depth increased, the signal-to-background ratio (SBR) decreased
from 40 to 18 dB as shown in Figure 7.12b.
7.3.3 Discussion
The photoacoustic signal enhancement for NIR830-ATF-IONP was three times
higher compared to that for nontargeted NIR830-BSA-IONP and 10 times higher
206 Photoacoustic Tomography
TABLE 7.1
Comparison of Nanoparticles Used as Contrast Agent for Photoacoustic
Tomography
Retention
__Nanoparticles Absortion* Size Time** Dose Multimode
Iron oxide Medium 10nm (sphere) >8 hours pmol PAT and
MRI
Gold nanocage Medium 50nm < 5 hours pmol-nmol NA
Gold nanorod High 52nm × 15nm NA nmol NA
Gold nanoshell High 140nm NA pmol-nmol NA
Gold nantotube Medium 100nm × 11nm NA nmol NA
Single-walled
carbon nanotube Low 186nm × 1nm <2 hours μmol NA
* The wavelength range for the comparison is limited to between 680nm and 800nm.
** The retention time is estimated relative to the size of nanoparticles.
than the noninjection controls. We compared IONP with other nanoparticles used as
contrast agents for PAT in Table 7.1. From the detailed comparison, it is noted that
IONP has modest absorption, smaller size (10 nm) and longer retention time, and
can be used with small dose. These advantages make IONP a good contrast agent for
photoacoustic imaging.
The uPAR-targeted IONP nanoparticle probe used in this study addresses some
of the challenges for the use of targeted tumor-imaging agents. Our methodology uti-
lizes stable and high-affinity targeting ligands and produces strong imaging contrast
for multi-image modalities. It has been shown that human breast cancer and tumor
stromal cells have a higher level of uPAR receptor compared with other normal
breast tissues (DelVecchio et al. 1993, Li et al. 1999). In addition, the highest level
of uPAR receptor expression is detected in the invasive edge of the tumor region
usually enriched in blood vessels making it accessible for uPAR receptor-targeted
IONP in this area (Dublin et al. 2000, Hemsen et al. 2003). The high-quality and
uniformly sized IONPs used in this study were coated with a thin ampiphilic copo-
lymer and have a relatively small particle complex (25 nm), which is more suitable
for in vivo delivery of the imaging probe compared with other receptor-targeted or
nontargeted nanoprobes for photoacoustic imaging (Zerda et al. 2008). It also has
been shown that polymer-coated IONPs have more than 8 hours of plasma retention
time (Moore et al. 2000) compared with other molecular imaging agents for PAT,
which are usually less than 2 hours (Zerda et al. 2008). The delivery of NIR830-
ATF-IONP to the tumor was verified by planar NIR fluorescent imaging. This
shows the potential for us to combine fluorescence molecular tomography (FMT)
with photoacoustic tomography (see Section 7.4 below). We have shown in Chapter 6
that while FMT has lower spatial resolution than PAT, it has higher specificity than
Contrast Agents–Based Molecular Photoacoustic Tomography 207
photoacoustic imaging techniques, and the combination of PAT and FMT would
provide complementary information for receptor-targeted molecular imaging.
The demonstrated imaging depth of 31 mm indicates the potential of our method for
tumor imaging in humans. While the photoaocustic system as described here is not yet
suitable for clinical applications, it can be improved to be a clinical prototype using a
commercial ultrasound array and/or a high frame rate of laser pulses with a high-speed
scanning system. By using high-frequency focused ultrasound transducer (>50 MHz), the
microvasculature inside and around the tumor can be imaged as shown in Figure 7.10,
indicating the potential of understanding the interplay between the tumor microvascula-
ture and delivery of targeted contrast agents.
after systemic delivery of a receptor targeted IONPs. Therefore, IONPs have the
potential to be good PAT contrast agents. However, PAT has relatively low sensitiv-
ity compared to pure optical imaging due to the limitations of acoustic transducers
(see Section 6.2). Additionally, the presence of endogenous photoacoustic signals in
some normal organs, such as the kidney, liver, spleen, and heart, may interfere with
specificity of PAT imaging.
Fluorescence molecular tomography (FMT) is an inexpensive and fast 3D optical
imaging modality that has been used for molecular imaging. In contrast to conven-
tional 2D fluorescence molecular imaging, FMT provides improved localization and
quantification in deep tissues (Jiang 2010). In Chapter 6, we have shown that FMT
is more sensitive and specific for the detection of low-concentration contrast agents
in tissues compared with PAT. However, FMT has limited spatial resolution. Hence
the combination of these two modalities with targeted nanoprobes should allow non-
invasive imaging of ovarian tumors with high resolution, specificity, and sensitivity.
Results of a recent study have shown that near-infrared dye labeled ZHER2:342 con-
jugated IONPs (NIR-830-ZHER2:342-IONP) specifically targeted to primary and meta-
static tumors in an orthotopic human ovarian cancer xenograft model and produced
strong imaging signals for 2D-optical and magnetic resonance imaging (Satpathy et
al. 2013). In this study, we wanted to determine the feasibility of application of this
HER-2 targeted IONP as a multimodality imaging probe for the detection of ovarian
cancer using both FMT and PAT.
7.4.1 Methods
7.4.1.1 Cell Lines
Cells from the human ovarian cancer cell line SKOV3, stably expressing a firefly
luciferase gene (SKOV3-luc), were cultured in McCoy’s 5A (Cellgro, Mediatech
Inc. VA, USA) supplemented with 10% fetal bovine serum (Hyclone from Thermo
Scientific) and 1% penicillin streptomycin (Hyclone, Logan, Utah). Cultured cells
were maintained at 37°C in a humidified atmosphere containing 5% CO2.
0.14
Photon Intensity (a.u.)
Absorbance
0.12 Fluorescence
0.1
0.08 HER-2 specific Near Infrared dye
0.06 affibody NIR-830
0.04 IO
0.02
IO NIR-830-Z HER2:342-IONP
0
650 700 750 800 850 900
Wavelength (nm) IO nanoparticle
(a) (b)
IONP
0.25
NIR-830-ZHER2:342-IONP
0.20
PA Signal (v)
NIR-830- NIR-830-
BSA-IONP IONP ZHER2:342-IONP 0.15
0.10
0.05
0.00
(c) 650 700 750 800 850 900
Wavelength (nm)
(d)
NIR-830 (4 picomole/uL)
0.06 NIR-830-ZHER2:342-IONP (0.1 picomole/uL) NIR-830-ZHER2:342-IONP
2.5
Ovarian tumor
2.0
0.04
PA Signal (a.u.)
PA Signal
1.5
0.02 1.0
0.5
0.00 0.0
(e) 0 1 2 3 4 5
Concentration (pmol/μL)
(f )
FIGURE 7.13 Schematic and characteristics of imaging probes. (a) Excitation and emis-
sion spectrum of NIR-830 dye. Excitation wavelength: 797 nm; Emission wavelength: 815 nm.
(b) Schematic representation of NIR-830-ZHER2:342-IONP: NIR-830 dye was conjugated to
a cysteine residue at the C-terminal end of ZHER2:342. The ZHER2:342-NIR-830 complex was
conjugated to the carboxyl groups of amphiphilic polymer-coated IONPs (21.6 nm in diam-
eter). (c) Specific binding and uptake efficiency of the NIR-830-ZHER2:342-IONP, IONP, and
NIR-830-BSA-IONP. HER-2 positive SKOV3 cells treated with NIR-830-ZHER2:342-IONP,
IONP, and NIR-830-BSA-IONP were scraped separately and scanned for absorbance from
500 to 784 nm. Since the cells were stained with nuclear fast red, the peak absorbance for
Prussian blue was seen at 528 nm. Absorbance of cells treated with NIR-830-ZHER2:342-
IONP and NIR-830-BSA-IONP at 528 nm was 0.549 and 0.155. (d) Absorbance spectrum
of IONP and NIR-830-ZHER2:342-IONP within NIR range. (e) Photoacoustic evaluation of
NIR-830 and NIR-830-ZHER2:342-IONP with tumors without any contrast agents. The equiva-
lent concentrations of NIR-830 and NIR-830-ZHER2:342-IONP to normal ovarian tumor were
4 pmol/μL and 0.1 pmol/μL. (f) Photoacoustic intensity from different concentrations of
NIR-830-ZHER2:342-IONP.
210 Photoacoustic Tomography
molar ratio 1:4) overnight at 4°C. Finally, the NIR dye-Affibody conjugated to the
IONPs using ethyl-3-dimethyl amino propyl carbodiimide (EDAC, Sigma-Aldrich)
and N-hydroxy-sulfo-succinimide (sulfo-NHS, Sigma-Aldrich) according to the
carbodiimide method overnight at 4°C (IONP: ZHER-2:342 molar ratio 1:10). Briefly, 100 nmol
of EDAC and 200nmol of sulfo-NHS were added to the aqueous solution of 1 mg of
IONPs, allowing the activation of IONPs for 15 minutes in Borate buffer at pH 5.5.
The reaction was terminated by removing the buffer through a brief centrifugation
using 100 K Nanosep column (Pall Corporation, Port Washington, NY), and then
adding 10mM Borate buffer, pH 8.5. After a brief wash with Borate buffer, pH 8.5,
the reaction of IONPs with ZHER2:342-NIR-830 dye was carried out for 4 to 6 hours
at room temperature. The final product NIR-830-ZHER2:342-IONP was made after
washing the mixture through Nanosep 100 K column three times and conjugate was
stored in 10 mM Borate buffer at pH 8.5.
Beam
Beam splitter
Probe expander
Pulsed laser
Detectors
Sources
CCD Lens
Lens Fiber
Detector PC Pre-amplifier
Bundle I
interface
Source Transducer
Laser Fiber Bundle II
interface
Lens 2D Scanner
Probe
Holder
Animal
Holder
2D linear stage
(a) (b)
FIGURE 7.14 (a) Schematic of the hand-held FMT imaging system. (b) Schematic of the
PAT imaging system.
7.4.1.10 Histology Analysis
Tumors along with normal organs were collected after sacrificing the mice. The tis-
sues were fixed with 10% buffered formalin. Paraffin tissue sections were stained
with Prussian blue or hematoxylin and eosin (H&E). Images were acquired at 200X
magnification using a Zeiss Axioplan 2 upright microscope.
6 ×105
5 ×105
4 ×105
3 ×105
2 ×105
1 ×105
Week 2 Week 3 Week 4 Week 5 Week 6
Counts (p/s)
7.4.1.11 Statistical Analysis
All data used for statistical analysis were summarized using means ± standard error
of the mean (SEM).
7.4.2 Results
Taking advantage of smaller size and highly affinity nature of the Affibody, we have
used a relatively new class of affinity molecule, HER-2 Affibody (ZHER2:342), for conju-
gating to IONPs. Prior to the conjugation, ZHER2:342 was prelabeled with a near-infrared
dye (NIR-830 dye) to the cysteine residue of the Affibody molecule. The biconjugate
NIR-830-ZHER2:342 was finally coupled to the carboxyl group of the amphiphilic poly-
mer coating of IONP. Figure 7.13b depicts the schematic representation of the pro-
duction of the imaging probe, NIR-830-ZHER2:342-IONP. By using Zeta-sizer Nano,
the hydrodynamic size of the IONP was 14.7 ± 3.58nm and increased upon conju-
gating with ZHER2:342 (NIR-830-ZHER2:342-IONP: 21.6 ± 5.61nm) and BSA (NIR-830-
BSA–IONP: 26.12 ± 2.22nm). Specific binding and uptake efficiency of the targeted
nanoparticles (NIR-830-ZHER2:342-IONP) were examined in the HER-2 over expressing
SKOV3 cells as shown in Figure 7.13c. In general, when 100nM of Fe equivalent con-
centration of targeted IONPs were incubated with SKOV3 cells for 5 hours followed
by 1 hour Prussian blue stain, about 60% cells were blue-stained iron-positive cells.
The iron-stained cells increased with higher concentrations of targeted nanoparticles.
However, cellular uptake by nontargeted IONPs was significantly lower when SKOV3
cells were incubated with nontargeted IONPs under similar conditions (Figure 7.13c).
The absorbance spectrum of IONPs and NIR-830-ZHER2:342-IONP used for photo-
acoustic imaging was evaluated within NIR range and shown in Figure 7.13d, where
absorption of IONPs decreased as the wavelength increased. From the spectrum, we
noted that even the absorption of NIR-830-ZHER2:342-IONP had a peak value at 820
nm contributed by the NIR dye, its absorption at 730 nm was still higher than that
at 820nm since IONPs were likely to be the major contributor to the photoacoustic
enhancement due to a low concentration of NIR-830 dye molecules on each IONP
(10 dye molecules per IONP). To verify the assumption, we compared the contribu-
tion to the photoacoustic effect by pure NIR-830 dye and NIR-830-ZHER2:342-IONP.
From the results shown in Figure 7.13e, we noticed that when the concentration of
NIR-830 dye was 40 times higher than the concentration of IONPs in NIR-830-
ZHER2:342-IONP, they contributed equally to the photoacoustic effects. That means
one IONP equals 30 dye molecules in the generation of photoacoustic signals with
consideration of 10 dye molecules per IONP in NIR-830-ZHER2:342-IONP.
To obtain the equivalent concentration of IONPs in NIR-830-ZHER2:342-IONP to nor-
mal ovarian tumors in photoacoustic imaging, we compared the photoacoustic signals
of different concentrations of IONPs in NIR-830-ZHER2:342-IONP with that of the tumors
without any contrast agents. Photoacoustic signals decreased as expected when the
concentration of IONPs in NIR-830-ZHER2:342-IONP decreased. When the concentra-
tion of IONPs reduced to 0.1 pmol/μL, the amplitude of photoacoustic signals becomes
almost the same as that from the tumor (Figure 7.13e). The amplitude change of pho-
toacoustic signals relevant to the concentrations of IONPs in NIR-830-ZHER2:342-IONP
was quantitatively plotted in Figure 7.13e. A linear regression fit of the data yielded an
214 Photoacoustic Tomography
0.9 NIR-830-ZHER2:342-IONPs
0.9 pmol/uL 0.18 pmol/uL
× 104
10 10
0.18 6
5
0 0 × 105
5 6
5
0.09 10 10
4 4
10 0 10 10 0 10
3 2
0.06 0.04 pmol/uL 0.04 pmol/uL
0
2
10 10
5 5
1
0.04 0 0
0 5 5
10 10
10 0 10 10 0 10
0.01 (b)
150 ms
(a)
R2 equaling to 0.99, which was expected given the amplitude of the photoacoustic sig-
nal was proportional to the concentration of IONPs in NIR-830-ZHER2:342-IONP.
To compare the sensitivity of FMT and PAT in imaging when NIR-830-ZHER2:342-
IONP produced contrast, we performed planar fluorescence and FMT phantom
experiments using different concentrations of IONPs in NIR-830-ZHER2:342-IONP. We
found that when the concentration reduced to 0.04 pmol/μL, they were still detect-
able for both planar fluorescence imaging and FMT (Figure 7.16). In the case of
photoacoustic imaging, there was no enhancement when the concentration became
as low as 0.1 pmol/μL. Results of our study suggested that this handheld FMT has
higher sensitivity compared to PAT for imaging NIR-830-ZHER2:342-IONP.
From in vivo planar fluorescence imaging, it showed that the fluorescence inten-
sity reached to the strongest level in the ovarian cancer at 48 hours following sys-
temic delivery (Figure 7.17) when the tumor size was <5 mm (Satpathy et al. 2013).
However, in tumor-bearing mice without receiving injection of the imaging probes
or injected with NIR-830-BSA-IONP, no significant imaging signals were detected
Contrast Agents–Based Molecular Photoacoustic Tomography 215
969
24h 48h
772
Tumor
region
575
377
180
FIGURE 7.17 HER-2 targeted imaging of orthotopic ovarian tumors following systemic
delivery of NIR-830-ZHER2:342-IONP detected by 2D-NIR optical imaging. Optical imaging
was performed 24 and 48 hours postinjection using the Kodak FX in vivo imaging system. The
NIR signal scale was generated using the Kodak molecular imaging software. Anatomical
x-ray images were simultaneously captured to validate tissue position.
in the tumor region. The mean fluorescence intensity (MFI) of the tumor for the
animal groups that received injection of NIR-830-ZHER2:342-IONP or NIR-830-BSA-
IONP 24 and 48 hours postinjection are plotted in Figure 7.18a. The high level of
the MFI found in the tumors of the mice that received NIR-830-ZHER2:342-IONP sug-
gested effective accumulation of the nanoprobes in the tumor at both time points.
Four time points were selected to perform photoacoustic imaging, and quantita-
tive analysis was shown in Figure 7.18b. Photoacoustic contrasts in tumors of mice
before injection of imaging probes were comparable. Twenty-four hours postinjec-
tion, the contrast of tumors of mice that received NIR-830-ZHER2:342-IONP increased
significantly and reached the peak value 48 hours postinjection, which is consistent
with the planar fluorescence imaging. However, no significant contrast enhancement
appeared in nontargeted and noninjection groups.
All three groups of mice were imaged by both PAT and FMT. The imaging area
was guided by both bioluminescence imaging (BLI) (Figure 7.18c) and planar fluo-
rescence imaging (Figure 7.18d). As expected, tumors of the mice that received injec-
tion of NIR-830-ZHER2:342-IONP had the strongest photoacoustic signal (left image
in Figure 7.18e), compared with the tumors of the mice that received injection of
NIR-830-BSA-IONP (middle image in Figure 7.18e) or mice in the no injection con-
trol group (right image in Figure 7.18e). The corresponding FMT results were shown
in Figure 7.18f, which was consistent with the results of planar fluorescence imag-
ing and PAT. Data from multispectral plots shown in Figure 7.18g revealed that the
strongest photoacoustic signal in the tumors of mice that received injection of NIR-
830-ZHER2:342-IONP was at 730nm, which was 500% higher than that of noninjection
controls, and 300% greater than that of the mice injected with NIR-830-BSA-IONP.
After the mice were sacrificed, tumors as well as normal organs such as the liver,
kidney, spleen, and heart in targeted and nontargeted groups were resected and eval-
uated by planar fluorescence imaging, PAT and FMT. In fluorescence imaging, we
found strong optical signals in the kidney and liver but not in other normal organs
(Figures 7.19a and 7.19b). However, in PAT images, the tumor of targeted group
216 Photoacoustic Tomography
2.50 Targeted
N=4 Normal tumor
P<0.005*** P<0.005*** 4
2.00
PA Signal (a.u.)
Non-targeted
1.50 3
MFT
1.00 2
0.50 1
0.00 0
24 h 48 h 24 h 48 h 0 20 40 60
NIR-830-ZHER2:342-IONPNIR-830-BSA-IONP Hours
(a) (b)
Non-targeted Non-targeted
Targeted Control Targeted Control
4
(c) 6 ×10 (d) 6.5 ×104
FMT (a.u.)
PAT (a.u.)
MAP
1 mm
4
targeted injection
3
1
0
nm
nm
nm
0
0
73
80
87
Wavelength (nm)
(g)
FIGURE 7.18 In vivo fluorescence and photoacoustic imaging of tumor-bearing mice and
quantitative analysis. (a) The mean fluorescence intensity plot of tumors in targeted or non-
targeted group 24 and 48 hours postinjection. Fold increases shown in the bar figure were the
mean fluorescence intensity of the tumor area divided by the mean fluorescence intensity of
the body background. Results were from four mice in each group. Student’s t-test was used to
determine the p value. (b) Bioluminescence imaging of the mice showed the position of the
tumor that was used to select the imaging areas of FMT and PAT. (c) Bioluminescence images
of typical mice from targeted (left), nontargeted (middle), and control (right) animal groups.
(d) Planar fluorescence imaging of mice from targeted, nontargeted, and control groups 24
hours postinjection of nanoparticles. (e, f) MAP images and typical cross-sections of PAT
and FMT of mice in the targeted (left), nontargeted (middle), and noninjection (right) animal
groups. Tumors of the mice from the targeted group were clearly detected by both PAT and
FMT, while the imaging contrasts of the tumors of the mice from nontargeted and noninjec-
tion groups were too low to be detected. (g) Quantitative photoacoustic intensity plots of
tumors from the targeted (red), nontargeted (black) and noninjection (blue) animal groups at
730, 800, and 870 nm, respectively.
Contrast Agents–Based Molecular Photoacoustic Tomography 217
1 cm
3000
4000
5000
6000
7000
8000
9000
10000
11000
NIR-830-ZHER2:342-IONP
1 cm
3000
4000
5000
6000
7000
8000
9000
10000
11000
NIR-830-BSA-IONP
Tumor Liver Kidney Heart Spleen 1 × 104
(c) Planar FL
2D FL
1 mm
0
1
PAT: MAP
PAT (a.u.)
0
1
FMT: MAP
FMT (a.u.)
1 mm
0
1
PAT: MAP
PAT (a.u.)
0
1
FMT (a.u.)
FMT: MAP
FIGURE 7.19 Ex vivo evaluation of tumors and normal organs using planar fluorescence
imaging, PAT, and FMT. Planar fluorescence imaging of the tumor, heart, spleen, kidney, and
liver of the mouse in the targeted (a) and nontargeted (b) groups. PAT and FMT evaluation of
the tumor and small samples of normal organs of the mice in targeted animal group (c) and
nontargeted group (d).
218 Photoacoustic Tomography
NIR-830-ZHER2:342-IONP NIR-830-BSA-IONP
(a)
0.8
NIR-830-ZHER2:342-IONP
NIR-830-BSA-IONP
Photoacoustic Signal
0.6
0.4
0.2
0.0
or
ey
rt
n
r
ve
e
ea
m
dn
le
Li
Sp
Tu
Ki
(b)
FIGURE 7.20 Histological and quantitative analysis of tumors and normal organs. (a) Prussian
blue staining of tissue sections showed high levels of iron-positive cells in the tumor of mice
in targeted group and low to intermediate levels of iron-positive cells in the livers and spleens
of mice in both targeted and nontargeted groups. No distinct iron-positive cells were visible in
the tumors of mice in nontargeted group, and in the kidney and heart. PB: Prussian blue, HE:
hematoxylin and eosin. (b) Comparison of photoacoustic intensity of the tumors and normal
organs resected from the mice in targeted and nontargeted groups. The heart and targeted
tumor produced the strongest photoacoustic emission. The spleen generated intermediate
photoacoustic emission.
and hearts emitted strongest photoacoustic signals and the photoacoustic signals of
the spleen were stronger than that of the liver and kidney. From histological sec-
tions and quantitative analysis in Figure 7.20, systemic accumulation of IONPs in the
tumor led to strongest photoacoustic emission, and nonspecific uptake of the IONP
by the reticuloendothelial system (RES) in the liver and spleen resulted in a higher
Contrast Agents–Based Molecular Photoacoustic Tomography 219
photoacoustic emission compared with that of the kidney. A large volume of blood
in the heart generated stronger photoacoustic signals than other normal organs since
the light absorption of blood at 730nm is stronger than that of IONPs. Additionally,
we did see strong optical signals of the kidney but didn’t detect blue iron-stained
cells in the tissue section of the kidney. This may be caused by the renal clearance of
free dye molecules or breakdown products of targeting ligands in the kidney to retain
the nanoparticles in the tissue section (Satpathy et al. 2013).
To evaluate the clinical potential of PAT/FMT technique for intraoperative imag-
ing of ovarian tumors in humans, we examined the ability of imaging of tumors
located in deep tissues by adding different thicknesses of chicken breast tissues on
the top of mouse back. The top row in Figure 7.21a shows the B-scan PAT images
with the different thicknesses of the chicken breast. The bottom row (Figure 7.21a)
shows the corresponding MAP images. As shown, ovarian tumors of the mice that
received injection of NIR-830-ZHER2:342-IONP were clearly imageable even after the
addition of a lay of 19 mm exogenous tissues. Signal-to-background ratio (SBR)
decreased from 38 to 17 dB when imaging depth increased. We also investigated the
imaging depth of FMT (third and fourth rows in Figure 7.21b) and found an imaging
depth of up to 10 mm.
We then compared the spatial resolution of FMT and PAT imaging at different
depths. Representative 3D images of PAT and FMT with different thicknesses of the
chicken breast are shown in Figure 7.22. MAP images and typical cross-sections of
both PAT and FMT areshown in Figure 7.21b. We found that the axial and lateral
resolutions of FMT were relatively poor. Although the position of image center was
in the right depth, the axial and lateral sizes were much larger than the actual sizes.
In contrast, high lateral and axial resolutions of PAT made the reconstructed lateral
and axial sizes very close to the actual dimensions of the tumor. The recovered
lateral and axial sizes of PAT and FMT were plotted (Figure 7.23) and the average
lateral and axial full-width at half-maximum (FWHM) of the tumor in PAT images
at three different depths (3, 6, and 10mm) were 5 and 3 mm, which are comparable
to the size of the resected tumor. Using FMT, the lateral FWHM was 5 to 13 mm and
axial FWHM was 3 to 14 mm, as the depth increased, which were relatively larger
than the actual tumor size.
7.4.3 Discussion
In this section we demonstrated the feasibility of using PAT and FMT to detect
orthotropic ovarian tumors in mice after systemic delivery of HER-2/neu targeted
imaging probes. In Section 7.3, we compared the PAT imaging signals of IONPs
with several other nanoparticle contrasts, such as carbon nanotubes, gold nano-
cages, gold nanorods, and gold nanoshell. Although our results showed that gold
nanoparticles and carbon nanotubes had stronger photoacoustic contrasts than that
of IONPs in animal tumor models, the effects of systemic delivery of those nanopar-
ticles in humans have yet to be determined since the mechanisms of clearance of the
nanoparticles and short- and long-term toxicity are still unclear. In contrast, IONPs
are a class of biodegradable and biocompatible nanoparticles with a low toxicity in
220 Photoacoustic Tomography
0 mm 10 mm 19 mm
Surface Surface
Surface
B-scan
MAP
(a)
3 mm 6 mm 10 mm
PAT: MAP
Surface Surface
Surface
PAT: Cross-
section
FMT: MAP
(b)
FIGURE 7.21 Evaluation of imaging depth and spatial resolution of PAT and FMT.
(a) B-scan (top row) and MAP (bottom row) PA images of a tumor in a representative mouse
that received NIR-830-ZHER2:342-IONP. The back flank of the mouse was overlaid with 0, 10,
and 19 mm-thick layers of chicken breast to mimic the normal tissue in humans. Red arrows
showed the surfaces of the chicken breast. Scale bar: 5 mm. (b) Dual modality imaging of the
same tumor located at different depth (3, 6, and 10 mm). The dashed white lines indicate the
position of the selected cross-sections of PAT and FMT, and arrows showed the positions of
the imaging surfaces. Scale bar: 5 mm. From the MAP and cross-sections of PAT and FMT,
we noticed that the lateral and axial resolutions of PAT were higher than that of FMT.
Contrast Agents–Based Molecular Photoacoustic Tomography 221
3 mm 6 mm 10 mm
PAT (3D)
z
y
x
FMT (3D)
z
y
x
FIGURE 7.22 Dual-modality 3D imaging of the same actively targeted tumor located in
different depth. The back flank of the mouse was overlaid with 3, 6, and 10 mm-thick layers of
chicken breast to mimic the thick normal tissue in humans. The white arrow showed the sur-
faces of the chicken breast tissue in the PAT images. The top surfaces of the boundary boxes
in FMT represented the surfaces of the chicken breast tissue. From the results, we noticed
that the reconstructed lateral and axial sizes of FMT were overestimated when imaging depth
increased, while the reconstructed lateral and axial sizes of PAT were within the range of the
measured size of the tumor.
humans and have already been used in human patients for MRI detection of liver
cancer and lymph node metastasis after systemic delivery (Harisinghani et al. 2003).
Based on our in vitro test result, we found that the photoacoustic signal was gen-
erated primarily from the IONPs as the concentration of NIR-830 dye conjugated
to the IONPs is too low to show a marked signal enhancement effect. However, it is
feasible to enhance the PAT signal by modifying our nanoparticle design to incorpo-
rate large amount of NIR-830-dye molecules (over 1000 molecules per IONP) into
the polymer-coating of the IONPs. Increased concentration of NIR dye in NIR-830-
ZHER2:342-IONP will further enhance the PAT signal.
Planar fluorescence imaging has been commonly used in molecular imaging for
monitoring the delivery of nanoprobes. However, it lacks the ability to map tumors
with the imaging probes that localize deep inside the body because of a poor spatial
resolution and lack of depth information. In this study, the handheld FMT system
showed the potential to noninvasively map ovarian tumors in 3D at depths up to
10 mm. The imaging procedure is fast, taking less than 2 minutes to cover a
20 × 20 mm2 imaging area. Additionally, FMT can specifically detect tumors with
a high sensitivity because of inherent characteristic of fluorescent imaging modality.
However, relatively low axial and lateral resolutions affect the imaging accuracy. As
a complementary method to FMT, PAT was employed to detect ovarian tumors with
contrast enhancements from IONPs. We anticipate that in a clinical setting, the fast
222 Photoacoustic Tomography
3 mm 3 mm
1 6 mm 1 6 mm
Photoacoustic Signal (a.u.)
0.6 0.6
0.4 0.4
0.2 0.2
0 0
0 3 6 9 12 15 18 0 3 6 9 12 15 18
x-axis (mm) x-axis (mm)
(a) (b)
3 mm 3 mm
1 6 mm 1 6 mm
Photoacoustic Signal (a.u.)
0.6 0.6
0.4 0.4
0.2 0.2
0 0
0 3 6 9 12 15 18 0 3 6 9 12 15 18
z-axis (mm) z-axial (mm)
(c) (d)
FIGURE 7.23 Quantitative comparison of spatial resolutions between PAT and FMT with
increased imaging depth. Lateral profiles of the recovered tumor by PAT (a) and FMT
(b) along the dashed white lines in Figure 7.21b. Axial profiles of the recovered tumor by PAT
(c) and FMT (d).
hand-held FMT system could be used to mark the possible tumor-containing areas,
and then PAT could be used to image the marked area to accurately identify tumor
margins and disseminated small tumor lesions in the peritoneal cavity. This combi-
nation imaging approach takes the advantage of both FMT and PAT to complement
each single modality.
8 Clinical Applications
and Animal Studies
In this chapter, we discuss the applications of photoacoustic tomography (PAT) to
several clinically significant areas and demonstrate its validity in humans or in ani-
mals. As described in Chapter 2, quantitative PAT (qPAT) can provide high-resolu-
tion functional images including hemoglobin and oxygenation, which promises its
potential in breast cancer detection. Quantitative PAT is also uniquely suited for
imaging joint diseases (e.g., osteoarthritis). The possibility of miniaturizing a PAT
imaging probe renders it an excellent modality for intravascular, endoscopic, or
intraoperative imaging. The high spatial and temporal resolutions of PAT in microm-
eter and millisecond scales offer its bright future in brain imaging, as it will play an
unprecedented role in noninvasively mapping brain activity and connectivity, one of
the hottest topics in today’s neuroscience and neurology.
8.1 JOINT IMAGING
Osteoarthritis (OA), or degenerative joint disease, is the most common form of joint dis-
ease. It is a slowly progressive disease and a major cause of morbidity in the population
over age 50, affecting more than 21 million Americans. It also imposes considerable
expense on the health care system. Although a number of factors contribute to its devel-
opment, including obesity, trauma, and genetic predisposition, the hallmark of OA is
progressive damage to articular cartilage. As damage progresses, fissuring of the articu-
lar cartilage can result in fragmentation and deposition of small loose bodies within the
joint space, causing a low-grade inflammatory reaction and increased turbidity in the
synovial fluid, with a decrease in viscoelastic and lubricating properties. Subchondral
bone also becomes exposed and results in sclerosis. While there is currently no cure for
this disease, numerous studies have shown that the progression of articular damage may
be modified by medical or surgical intervention if the disease is detected early (Itay et
al. 1987, Howell and Altman 1993, Brittberg et al. 1994). These studies, coupled with
recent developments in gene therapy and anticipated novel therapeutic approaches, have
generated substantial demand for noninvasive techniques for detecting early changes in
the joints, when intervention is likely to have its greatest effect.
Several noninvasive imaging modalities have been applied to detect OA, includ-
ing X-ray radiography, computed tomography (CT), ultrasound (US), and magnetic
resonance imaging (MRI) (Brandt 1997, Chan et al. 1991, Myers et al. 1995, Tyler
et al. 1995). Radiography and CT are excellent techniques to image hard tissues and
are primarily used for advanced OA stages when bone damage has occurred (Brandt
1997, Chan et al. 1991). It is difficult for these techniques to detect changes in carti-
lage or the early inflammatory process in synovial membrane/fluid. Ultrasound has
223
224 Photoacoustic Tomography
limited utility in evaluation of the early stages of OA and is very operator depen-
dent, making it difficult to be used as a quantitative tool. MRI represents a power-
ful modality for evaluating joint abnormalities, particularly when a surface coil or
contrast agent is used (Tyler et al. 1995, Ostergaard et al. 1997). However, MRI is
expensive and may not be suitable for long-term, routine monitoring of OA.
It has been recently recognized that optical contrast is highly sensitive to structural
and metabolic changes associated with an OA joint (Yuan et al. 2008, Jiang 2010) due
to a strong correlation between optical changes and the diseased joint. Collagen fibril
network deterioration has been observed when OA was present in the cartilage area
(Panula et al. 1998). This deterioration decreased the collagen-induced optical path
difference by 19% to 71%. The variation in fibrils together with the loss of proteogly-
cans in OA should result in significant changes in both the scattering and absorption
coefficients of the cartilage. In addition, it is known that with the onset of OA, the
synovial membrane/fluid in articular cavity becomes increasingly turbid (Beuthan
et al. 1996, Prapavat et al. 1997). The increased turbidity would accompany increased
scattering and absorption coefficients in the diseased synovial membrane/fluid. In
fact, this optical increase can be as large as 100% at certain wavelengths in the near-
infrared (NIR) region (Beuthan et al. 1996, Prapavat et al. 1997), which would provide
good optical contrast for imaging purposes. Finally, there is increasing evidence that
OA is a disease involving a metabolic dysfunction of bone (Mansell 2007, Knott and
Bailey 1998). It is likely that this metabolic dysfunction of bone, often associated with
high metabolism of subchondral bone, will cause changes in its tissue oxygenation,
Hb, HbO2, and water content. These metabolic and functional parameters should also
be measurable by multispectral optical/photoacoustic imaging.
The demonstrated optical detection of OA, however, was based on diffuse optical
tomography (DOT) (Jiang 2010), a pure optical imaging method that has relatively low
spatial resolution (2~4 mm) due to strong tissue scattering. While it is known that an
adult articular cartilage has a typical thickness of from 2 to 4 mm, diseased cartilage
typically has a thickness of 1 mm or smaller (Yuan et al. 2008). Thus, to correctly image
an OA joint, DOT needs to be combined with a high-resolution modality such as X-ray
so that the high-resolution X-ray images can be used as a priori structural guidance
for DOT reconstruction (Yuan et al. 2008). Laser-induced photoacoustic tomography
(PAT) alone can achieve the same as the combined X-ray/DOT does, since it retains
the desired high optical contrast while providing much better spatial resolution than
pure optical tomographic methods by detecting much less-scattering ultrasonic waves.
In addition, PAT is free of the speckle artifacts present in pulse-echo ultrasonography.
Here we demonstrate the application of single- and multispectral quantitative
PAT for imaging OA in the finger joints in both 2D and 3D.
(a) (b)
T7 T6
T8
T5
T4
T3
T2
T1
Section
line
FIGURE 8.1 (a) Photograph of the transducer array/finger interface of the photoacous-
tic tomography system used for the current study. S: Laser source; T1-T8: Transducers.
(b) Schematic of the coronal section/plane for the transducer scanning.
laser (LOTIS, Minsk, Belarus) with a repetition rate of 10 Hz and a pulse width
of <10 ns, illuminates the distal interphalangeal (DIP) joint of interest. The light
beam is guided via an optical fiber and the laser energy at the DIP joint is con-
trolled around 10 mJ/cm 2, which is far below the safety standard of 22 mJ/cm 2.
The ultrasound detection array (Figure 8.1a) is composed of eight 1 MHz trans-
ducers (Valpey Fisher, Hopkinton, MA) arranged equally spaced along a 210° arc
arm. The position and performance of each transducer in the ultrasound array was
calibrated carefully with controlled tissue phantom experiments. During an exam,
the palmar side of the DIP joint faced up, allowing the DIP joint to be illuminated
from the dorsal side of the finger. Our experience indicates that this way of light
illumination can give us maximized tissue penetration. To secure the position of
the finger, rubber bands were applied to the distal tip and the proximal end of the
finger. Both the ultrasound array and the examined finger were immersed in a
water tank for minimized ultrasound attenuation. The detected acoustic signals
were fed into preamplifiers and converted to digital signals by a multichannel A/D
board (PREAMP2-D and PCIAD1650, US Ultratek, Concord, CA). Labview pro-
gramming was used to control the entire examination procedure. With the ultra-
sound array, the photoacoustic exam took just about 30 s, allowing the collection of
acoustic signals at 40 detection positions (5 array positions) along a coronal plane
of the DIP joint (Figure 8.1b).
The qPAT reconstruction method detailed in Section 2.1 was used to quantita-
tively recover the optical absorption coefficient images of the DIP joints.
8.1.1.1.2 Patient Examination
Six subjects (two OA patients and four healthy controls) were enrolled in the study.
All participants (white females; mean age 61 years, range 45 to 71 years) provided
226 Photoacoustic Tomography
informed consent as part of the protocol approved by the Institutional Review Board
of University of Florida (UF). Participants were recruited from the Rheumatology
Clinic at the Shands Health Center of UF. One DIP finger joint, joint II of the left
hand, from each subject was examined clinically and photoacoustically. The DIP
joint is mostly vulnerable to OA disease and easily accessible with our current pho-
toacoustic scanning system.
Clinical examination of each patient was performed independently by a single
rheumatologist. Patients with OA were identified by clinical history and main clini-
cal features, including symptoms (predominantly pain and stiffness), functional
impairment and signs (joint enlargement and redness). The healthy controls had no
known OA or other joint diseases.
5
5 5
0
0 0
–5 –5 –5
0.035
–5 0 5 –5 0 5 –5 0 5 0.03
(a) (b) (c) 0.025
0.02
5 0.015
5 6
0.01
4
0.005
2 0
0
0
–2
–5 –4 –5
–6
–5 0 5 –4 –2 0 2 4 –8 –6 –4 –2 0 2
(d) (e) (f )
FIGURE 8.2 Recovered absorption coefficient images for the six subjects examined. (a)–(d)
Normal joints. (e)–(f): OA joints. The color scale indicates the absorption coefficient (mm–1).
Clinical Applications and Animal Studies 227
with highest absorption) are clearly delineated from the adjacent tissues (cartilage
and fluid) in the joint cavity for all the cases. While each joint has different shape
and size due to their positions relative to the scanning plan, the joint tissue/space
(indicated by the arrow) is readily identifiable. Compared with the healthy joints
(Figures 8.2a to 8.2d), we note that the value of absorption coefficient in the joint
cavity is elevated for the OA joints (Figures 8.2e and 8.2f). It is also notable that the
joint space narrowing appears to be evident for the OA joints.
The average absorption coefficient of joint tissues (cartilage, fluid, and bone) for
both the normal and OA joints were calculated and presented in Figures 8.3a and
8.3b. We can immediately tell from Figure 8.3a that both the diseased cartilage and
synovial fluid have significantly increased absorption coefficient compared with the
0.04
Cartilage
Fluid
0.03
Average µa (mm–1)
0.02
0.01
0.00
H1 H2 H3 H4 OA1 OA2
Case Number
(a)
1.0
µ(c)
a /µa
(b)
(f )
µa /µa(b)
Ratio of Absorption Coefficient
0.8
0.6
0.4
0.2
0.0
H1 H2 H3 H4 OA1 OA2
Case Number
(b)
FIGURE 8.3 (a) Average absorption coefficient of cartilage (light gray) and fluid (dark gray)
for the healthy (H1–H4) and osteoarthritic (OA1–OA2) joints. (b) Ratio of absorption coeffi-
cient of cartilage to that of bone (red), or ratio of absorption coefficient of fluid to that of bone
(blue) for the healthy and OA joints.
228 Photoacoustic Tomography
normal cartilage and fluid. While we note that the value of absorption coefficient of
bone for the OA joints is just slightly larger than that for the normal joints, the ratio
of the absorption coefficient of the cartilage or synovial fluid to that of the bone is
striking, as can be seen from Figure 8.3b. These quantitative optical changes asso-
ciated with the diseased joints are certainly attributed to the increased turbidity of
synovial fluid, collagen fibril network deterioration, and metabolic dysfunction of
bone, as elaborated previously.
12
4.5
10 10
4 10
5 3.5 5
dp 8
3 dp
jt 2.5 ee jt vp
0 vp 0 su 6
ee mp 2 mp
–5 1.5 –5 4
1
2
–10 0.5 –10
–10 –5 0 5 10 –10 –5 0 5 10
(a) (b)
40
10 10 35
20
30
5 5
dp 15 dp 25
ee jt vp ee jt
0 0 vp 20
su
mp vp 10 mp
15
–5 –5
5 10
–10 –10 5
–10 –5 0 5 10 –10 –5 0 5 10
(c) (d)
FIGURE 8.4 Recovered Hb (a, b) and HbO2 (c, d) images for typical normal (a, c) and OA
(b, d) joints. The color scale indicates the Hb or HbO2 (μM). dp: distal phalanx; mp: middle
phalanx; jt: joint cavity; vp: volar plate; ee: extensor expansion; su: skin and subcutaneous
soft tissues.
Qualitatively, the structural differences between OA and normal joints are not
apparent from Figures 8.4 and 8.5; however, quantitative differences in functional
parameters between OA and normal joints are striking from the extracted aver-
age oxygen saturation and water content of the joint and phalanx bones given in
Figures 8.7 and 8.8. We can immediately tell from Figure 8.7 that both the diseased
joint and bones have significantly decreased oxygen saturation compared with the
normal ones. In particular, the oxygen saturation of the OA joints is smaller than
72%, while it is larger than 79% for the normal joints. Figure 8.8 shows that the
OA joints have elevated water content especially for the joint cavity. The average
acoustic velocity of the joint and bones is given in Figure 8.9, where we can see that
the acoustic velocity of the joint tissues is notably larger for the OA joints than that
for the normal joints, while the value of this quantity for bones is indistinguishable
among the OA and normal joints.
Changes in cartilage, underlying subchondral bone, and synovial fluid are the
tissues most affected by OA (these three types of tissues are the major components
230 Photoacoustic Tomography
60
10 40 10
50
35
5 5
d 30 40
ee vp
d
jt 25 ee
0 0 jt
30
m vp 20
vp su
m
–5 15 –5 20
10
–10 10
–10 5
–10 –5 0 5 10 –10 –5 0 5 10
(a) (b)
× 106 × 106
2
10 10 2
n & sus 1.9
n & sus 1.9
5 dp 1.8 5
ee 1.8
ee dp
jt 1.7
0 0 jt 1.7
mp tdpt mp
1.6 1.6
–5 su –5 tdpt
1.5 su 1.5
–10 1.4 –10 1.4
–10 –5 0 5 10 –10 –5 0 5 10
(c) (d)
FIGURE 8.5 Recovered H2O (a, b) and acoustic velocy (c, d) images for a typpical normal (a, c)
and OA (b, d) joints. The color scale indicates the H2O (%) or acoustic velocity (m/s). dp: distal
phalanx; mp: middle phalanx; jt: joint cavity; ee: extensor expansion; su: skin and subcutaneous
soft tissues; fdpt: flexor digitorum profoundus tendon; n and sus: nail and subungual space.
n
sus
sus
dp fd pt dp
vp fdpt
vp
ee
fdpt
FIGURE 8.6 Sagittal MRI of a model normal DIP joint. dp: distal phalanx; mp: middle
phalanx; jt: joint cavity; vp: volar plate; ee: extensor expansion; su: skin & subcutaneous
soft tissues; fdpt: flexor digitorum profoundus tendon; n and sus: nail and subungual space.
Clinical Applications and Animal Studies 231
85
Joint
Phalanx bones
H: Healthy Subject
OA: Osteoarthritic Subject
80
Oxygen Saturation (%)
75
70
65
H1 H2 H3 OA1 OA2
Case Number
FIGURE 8.7 Average oxygen saturation of the joint (light gray) and phalanx bones (dark
gray) for the healthy (H1–H4) and osteoarthritic (OA1–OA2) joints.
80
Joint
Phalanx bones
70
Water Content (%)
60
50
40
30
H1 H2 H3 OA1 OA2
Case Number
FIGURE 8.8 Average water content of the joint (light gray) and phalanx bones (dark gray)
for the healthy (H1–H4) and osteoarthritic (OA1–OA2) joints.
232 Photoacoustic Tomography
2.4
Joint
2.2 Phalanx bones
Velocity (×106 mms–1)
2.0
1.8
1.6
1.4
H1 H2 H3 OA1 OA2
Case Number
FIGURE 8.9 Average acoustic velocity of the joint (light gray) and phalanx bones (dark
gray) for the healthy (H1–H4) and osteoarthritic (OA1–OA2) joints.
of the joint or joint cavity defined in this paper) (Gerwin et al. 2006, Blanco et al.
2004). Bone is frequently remodeled in OA, with new bone formed either as osteo-
phytes at the margins of the articular cartilageor as a change in subchondral bone
density (Gerwin et al. 2006, Blanco et al. 2004). These changes in bone structure can
allow blood flow to more easily penetrate the subchodral bone plate and the calcified
cartilage that lies between subchondral bone and cartilage (Gerwin et al. 2006, Blanco
et al. 2004). There is now cumulative scientific evidence suggesting that the trigger
for many of these changes is the low oxygen levels in the diseased tissues includ-
ing the cartilage of an OA joint (Blanco et al. 2004; Henrotin et al. 2005). These
low oxygen levels will cause the formation of new blood vessels, allowing the dis-
eased synovium to invade the surrounding tissues (Blanco et al. 2004). It seems these
changes correlate well with the decreased oxygen saturation seen in the joint cavity
of OA joints in our study. The mild inflammatory changes frequently seen in the
synovial fluid of patients with OA would also produce high water content in the joint
cavity, a feature that is also seen in our measurements (Figure 8.8). In addition, nor-
mal cartilage is tough, elastic, and very durable and comprises mainly collagen (20%
to 30%) and water molecules (67% to 80%). Early changes in OA cartilage include
proteoglycan loss, with a resultant increase in hydration of the articular cartilage.
These changes also likely contribute to the increase in water content and decrease in
oxygen saturation seen in the joint cavity of OA joints. While the changes in water
content and hemoglobin concentration in the joint cavity may all contribute to the
change in acoustic velocity associated with an OA joint, a full understanding on the
mechanism behind this change warrants future studies.
Clinical Applications and Animal Studies 233
8.1.3.1.2 Patient Examination
The same six subjects described in Section 8.2.1 were used for this 3D PAT study.
Y (mm)
CL
–5 CL
0.05
IP IP IP
IP IP IP
–10
–10 –5 0 –10 –5 0 –10 –5 0 –10 –5 0 –10 –5 0 –10 –5 0 0.04
X (mm) X (mm) X (mm) X (mm) X (mm) X (mm)
(a) Coronal Section (dorsal palm)
0.03
X = –5.5 mm X = –5 mm X = –4.5 mm X = –4 mm X = –3.5 mm X = –3 mm
Clinical Applications and Animal Studies
10
DP DP DP DP DP DP 0.02
SF SF SF
SF SF SF
5
0.01
DIP DIP DIP
0
Y (mm)
CL DIP DIP CL CL
CL CL DIP CL 0
–5
IP IP IP IP IP
IP
0 5 10 0 5 10 0 5 10 0 5 10 0 5 10 0 5 10
Z (mm) Z (mm) Z (mm) Z (mm) Z (mm) Z (mm)
FIGURE 8.10 Selected coronal (a) and sagittal (b) planes of the recovered 3D absorption coefficient image for a typical normal joint. DP: distal pha-
235
lanx; IP: intermediate phalanx; DIP: distal interphalangeal joint; CL: cartilage; SF: synovial fluid.
Z = –9 mm Z = –8.5 mm Z = –8 mm Z = –7.5 mm Z = –7 mm Z = –6.5 mm
10
236
DP DP DP CL DP DP 0.07
DP
SF
SF
5
DIP 0.06
Y (mm)
DIP DIP
0 DIP DIP CL
DIP
0.05
–5 IP
IP IP IP IP IP
–5 0 5 –5 0 5 –5 0 5 –5 0 5 –5 0 5 –5 0 5 0.04
X (mm) X (mm) X (mm) X (mm) X (mm) X (mm)
Y (mm)
0 0
TE
–5
IP IP IP IP IP IP
–10 –5 0 –10 –5 0 –10 –5 0 –10 –5 0 –10 –5 0 –10 –5 0
Z (mm) Z (mm) Z (mm) Z (mm) Z (mm) Z (mm)
(b) Sagittal section (Medial Lateral)
FIGURE 8.11 Selected coronal (a) and sagittal (b) planes of the recovered 3D absorption coefficient image for a typical OA joint. DP: distal phalanx;
Photoacoustic Tomography
IP: intermediate phalanx; DIP: distal interphalangeal joint; CL: cartilage; SF: synovial fluid; TE: tendon.
Clinical Applications and Animal Studies 237
TABLE 8.1
Averaged Absorption Coefficient and Size of Joint Tissues for 2 OA and 4
Normal Joints
Joint
Joint Mean Thickness µ (ac ) µ (af ) µa
µ (mm )
a
a
–1
µ (ab ) µ (ab ) µ (ab )
No. Tissues μa(mm–1) (mm)
Healthy 1 Bone 0.035 0.004 1.7 0.43 0.25 0.11
cartilage 0.015
fluid 0.008
2 Bone 0.039 0.010 2.1 0.61 0.45 0.26
cartilage 0.024
fluid 0.017
3 Bone 0.034 0.009 2.5 0.59 0.45 0.26
cartilage 0.020
fluid 0.0 IS
4 Bone 0.037 0.008 1.8 0.61 0.46 0.22
cartilage 0.023
fluid 0.017
5 Bone 0.036 0.018 1.5 0.76 0.63 0.50
cartilage 0.028
fluid 0.023
6 Bone 0.037 0.014 1.1 0.72 0.58 0.38
cartilage 0.026
fluid 0.021
a
a is the lowest sectional optical absorption coefficient in the joint cavity.
with a spherical mesh of 7519 finite element nodes and 41,323 tetrahedral elements.
Figures 8.12a and 8.12b are the recovered optical absorption maps in coronal section
and sagittal section, respectively, of an OA finger joint at wavelengths from 730 to
880 nm. In Figures 8.12a and 8.12b, the joint tissue/space is differentiated from the
adjacent bones in the coronal and/or sagittal sections at several optical wavelengths
(760, 805, 850, and 880 nm). Relative structural consistency of the recovered optical
absorption images are observed between the wavelengths of 760, 805, and 850 nm,
which are selected to further quantitatively resolve the tissue chromophore concen-
trations. The reason that the joint tissue/space is missing or fused with the adjacent
bones at some optical wavelengths (such as 730 and 825 nm) is most likely because of
the motion error during the 30-minute examination procedure, despite an optimized
wavelength order is followed in our 3D multispectral PAT scanning. We believe that
the motion error and the structural inconsistency of the recovered optical absorption
maps at some wavelengths can be significantly reduced by using an ultrasound array
with larger number of ultrasound detectors (e.g., see the 192-element array system
described in Chapter 4).
The quantitatively resolved concentrations of HbO2 and Hb of the same finger
joint are displayed in Figure 8.13. The concentrations of HbO2 (Figure 8.13a) in dif-
ferent joint tissues are clearly differentiated in high resolution both in coronal section
and sagittal section, and it is relatively higher in the bones than in the joint cavity.
Figure 8.13b shows the concentrations of Hb both in coronal section and sagittal
section, where the joint cavity is differentiated from the adjacent phalanges in the
sagittal section and is barely identifiable in the coronal section. We believe that the
relatively poor quality of the resolved Hb concentration images in coronal section
is most likely due to (1) the low-quality image at the wavelength 760 nm, where the
absorption spectrum of Hb has a high peak, and (2) the potential motion error during
the 3D multispectral PAT scanning.
Figure 8.14 shows the quantitatively resolved concentrations of HbO2 and Hb
from a typical healthy finger joint. Again, the structure of joint space is clearly iden-
tified in the images of each resolved chromophore, and the concentrations of HbO2
and Hb in the joint cavity are found to be relatively lower than that in the adjacent
phalanges. In comparison with the OA finger joint shown in Figure 8.13, the joint
cavity in healthy subjects seems more distinctive.
For all four examined in vivo finger joints, the average concentrations of oxy-
hemoglobin (HbO2), total hemoglobin (Total-Hb), and the oxygen saturation (SO2)
in the joint cavity and the adjacent bones are calculated and presented in Figure 8.15.
Figure 8.15a shows that the average concentrations of HbO2 and Total-Hb in the
finger phalanges are 44~58 μM, and 68~96 μM, respectively. The quantitatively
resolved concentration levels of HbO2 and Total-Hb are in agreement with the
measurements by diffuse optical tomography (Jiang 2010). Further observations
indicate a difference in the levels of total-Hb between finger phalanges with OA
disease and healthy phalanges, which are 86~96 μM and 68~69 μM, respectively.
The finding of enhanced hemoglobin levels in OA joints has the pathologic roots
revealed by previous studies, where angiogenesis has been observed in diseased
Clinical Applications and Animal Studies 239
Y (mm)
Y (mm)
5 0.05 5
0.04
0.04 0.03
0 0.03 0 0 0.03
0.02
0.02 0.02
–5 0.01 –5 0.01 –5 0.01
0 0
–5 0 5 –5 0 5 –5 0 5
X (mm) X (mm) X (mm)
825 nm 850 nm 880 nm
10 10 10 0.06
0.04 0.05
0.05
5 5 0.04 5
0.03
Y (mm)
Y (mm)
Y (mm)
0.04
0.03
0.03
0 0.02 0 0
0.02 0.02
0.01
–5 –5 0.01 –5 0.01
0 0
–5 0 5 –5 0 5 –5 0 5
X (mm) X (mm) X (mm)
(a)
730 nm 760 nm 805 nm
10 10 10 0.05
0.06
0.04
0.05 0.04
5 5 5
Y (mm)
Y (mm)
Y (mm)
0.04 0.03
0.03
0.03 0.02
0 0 0 0.02
0.02
0.01 0.01
–5 0.01 –5 –5
0 0 0
–10 –5 0 –10 –5 0 –10 –5 0
Z (mm) Z (mm) Z (mm)
825 nm 850 nm 880 nm
10 0.035 10 10 0.035
0.04
0.03 0.03
5 0.025 5 0.03 5 0.025
Y (mm)
Y (mm)
Y (mm)
0.02 0.02
0 0.015 0 0.02 0 0.015
0.01 0.01
0.01
–5 0.005 –5 –5 0.005
FIGURE 8.12 Recovered optical absorption images in coronal section (a) at z = –8 mm and
sagittal section (b) at x = –1 mm of an in vivo finger joint for light source in six wavelengths.
240 Photoacoustic Tomography
Z= –8 mm X= –1 mm
10 10 70
80 60
5 5 50
60
Y (mm)
Y (mm)
40
0 40 0 30
20
20
–5 –5 10
0 0
–5 0 5 –10 –5 0
X (mm) Z (mm)
(a)
10 10
50 50
40 40
5 5
Y (mm)
Y (mm)
30 30
0 0
20 20
10 10
–5 –5
0 0
–5 0 5 –10 –5 0
X (mm) Z (mm)
(b)
tissues. The oxygen saturations of the four in vivo finger joints are observed from
58 to 66% in the finger phalanges, while finger phalanges with OA disease seems to
have lower oxygen saturations levels (55~60%) than healthy phalanges (65~66%).
Hypoxia in osteoarthritic phalanges has been revealed and seemed to appear in
prearthritic stage by previous studies of finger joints diseases (Jeon et al. 2008).
Our results are in good agreement with the previous findings of hypoxia in osteo-
arthritic phalanges.
As shown in Figure 8.15b, the average concentrations of HbO2 and Total-Hb
in the finger joint cavity are 16~29 μM, and 27~53 μM, respectively. An obvious
Clinical Applications and Animal Studies 241
Z = 2 mm X = –0.5 mm
60
10 10 60
50
50
5 40 5
40
Y (mm)
Y (mm)
30 30
0 0
20 20
–5 10 –5 10
0 0
–5 0 5 –5 0 5
X (mm) Z (mm)
(a)
35
35
10 10
30
30
25
5 25
5
20
Y (mm)
Y (mm)
20
15 0 15
0
10 10
–5 5 –5 5
0 0
–5 0 5 –5 0 5
X (mm) Z (mm)
(b)
difference in the level of total-Hb between joint cavities with OA disease and
healthy joint cavities can be observed in Figure 8.15b, which are 46~53 μM and
27~42 μM, respectively. The oxygen saturations in the joint cavities are observed
to be 55~69%, and osteoarthritic fingers seem to have lower oxygen saturations
levels (55~59%) than healthy phalanges (59~69%) in the joint cavities. Again, the
enhanced hemoglobin levels and dropped oxygen saturations in OA joint cavities
are in agreement with the pathologic angiogenesis and hypoxia related with finger
joint diseases.
242 Photoacoustic Tomography
100 100
100 100
Concentrations of HbO2 and Total-Hb (μm)
HbO2
90 Total-Hb 90
SO2
80 80
8.2 INTRAOPERATIVE IMAGING
Breast cancer remains a major public health problem affecting one in four women in
the United States (Jemal et al. 2008). As a result of population-based screening pro-
grams for breast cancer, most women are currently diagnosed with early stage breast
cancer. In a lumpectomy, margin is typically examined using pathological methods
to determine if there is at least 2 mm of surrounding normal breast tissue beyond
the tumor border; this is considered to be a negative margin. However, if the tumor
cells come to the edge of the resected tissue, it is considered to be a positive margin
(Taghian et al. 2005, Assersohn et al. 1999).
Clinical Applications and Animal Studies 243
Aperture Footprint
Fixed Mirror
Actuator
Transducer
Reflection
Mirror
MEMS Mirror
Plastic Film Electrodes
(a) (b)
Light
22.5°
Fixed Mirror
Transducer 2
MEMS Mirror
3 4
Tissue 1
(c) (d)
FIGURE 8.16 Schematic representation of the MEMS probe. (a) 3D rendering of the probe.
(b) Photograph of the MEMS mirror with four actuators and five pads bonded with gold wire.
(c) Schematic of the miniaturized probe. A wedge-shaped mirror plated with aluminum is
fixed in position to redirect the light beam to the MEMS mirror. (d) The MEMS mirror is
fixed on a ramp with an angle of 22.5°.
One set of data measured from a cylindrical phantom with ink covering the
imaged surface was used to calibrate all the experimental data used in this study
to reduce the effect of focal signal from the center area from the ring-shaped trans-
ducer. All slices were processed and displayed by normalizing the images to the
same scale (0–256) with a threshold (–6dB level of the normalized peak photo-
acoustic signal).
1
Simulation (Transverse)
0.9
Transverse
0.8 Axial
Resolution (mm)
0.7
0.6
0.5
0.4
0.3
0.2
0 0.5 1 1.5 2 2.5
Distance (mm)
(a)
SNR
40
30
SNR (dB)
20
10
0
0 0.5 1 1.5 2 2.5
Distance (mm)
(b)
FIGURE 8.17 Performance of this system. (a) Axial and transverse resolutions versus target
depth. (b) Signal-to-noise ratio (SNR) versus target depth.
target position. In Figure 8.17a, the system’s axial and transverse resolutions are plot-
ted versus the target’s position. The experimental axial resolution (black crosses),
determined by the central frequency of transducer, varied from 0.36 to 0.4 mm, which
agrees well with the theoretical value of 0.3 mm. The experimental transverse reso-
lution, based on the size of light spot, decreased from 0.24 to 0.96 mm when the
depth increased. Again this is consistent with the theoretical depth-dependent trans-
verse resolution of 0.21~0.95 mm. The SNR measured in turbid media is shown in
Figure 8.17b, decreasing from 32 to 18 dB within 2.3 mm.
The volumetric shape, size, and position of the tumor are shown in Figure 8.18b.
The surgeon removed the tumor marked with T in Figure 8.18b. In these experi-
ments, the line of surgical resection of tumor was intentionally carried out beyond the
margin of the detectable tumor, consistent with clinical practice. After the surgical
246 Photoacoustic Tomography
y (c) y
(a)
x x
(e)
y (d) y
(b)
x z x z
T
10 mm 10 mm
10 mm 7 mm
10 mm 7 mm
FIGURE 8.18 In vivo 3D tumor mapping in a mouse model. (a) Photograph of the mouse
with tumor implanted in abdomen before surgery. (b) In vivo 3D photoacoustic image. The
distance shown along z-axis includes the tumor depth and the distance between the surface
of transducer and mouse skin. (c) Photograph of the mouse after tumor resection. (d) 3D
photoacoustic image of the tumor cavity after surgery to examine the completeness of the
tumor resection. (e) Photograph of the tumor resection guided by 3D photoacoustic image
shown in (b).
procedure, the surgical area shown in Figure 8.18c was inspected again by the same
imaging method to ensure the complete resection of tumor. The image shown in
Figure 8.18d confirmed the completed resection of tumor. The signal (indicated by
the arrow in Figure 8.18d) was from the inner surface of the ring-shaped transducer.
This signal disappeared in Figure 8.18b due to strong signals from the tumor, and
normalized image procedure was used.
Following tumor removal, the tumor size as determined by iPAT was compared
with the actual tumor size as measured histologically. Hematoxylin and eosin (H&E)
stained sections along the x-z plane were obtained after the photoacoustic experi-
ments. A 2D PAT slice along the red dashed line in Figure 8.18b was selected and
compared with the corresponding H&E stained section (the black dashed line in
Figure 8.18a) as shown in Figures 8.19a and 8.19b. Figure 8.19c shows the photo-
graph of the tumor in comparison with the H&E and PAT slices. Also, the red dashed
line along the top margin of the tumor shown in Figure 8.19a matches well with the
top margin (black dashed line in Figure 8.19b) of the H&E section and photograph
(red dashed line in Figure 8.19c). The size along two typical directions (Lines 1 and
2 in Figure 8.19a) was estimated to be 2.1 and 1.2 mm, respectively, in excellent
agreement with the actual dimensions of 2.2 and 1.1 mm measured from the histol-
ogy slice (Lines 1′ and 2′ in Figure 8.19b). Such comparisons were applied to other
two H&E staining sections along the x-z plane for this mouse, and we found that the
measured error was less than 8.5% for all three slices.
Clinical Applications and Animal Studies 247
1'
1 2'
2 x
z
1'
1 2'
2 x
y
FIGURE 8.19 Quantitative analysis of the photoacoustic slices and H&E stained sections.
(a) Photoacoustic slice (along the red dashed line in Figure 8.18b). Lines 1 and line 2 indicate
the imaged dimensions along two different directions. (b) H&E stained section in the same
position. Lines 1′ and line 2′ denote the measured dimensions in the H&E stained section.
(c) Photograph of the tumor. All the lines indicated by arrows in (a), (b), and (c) show the top
margins of the tumor. (d) Transverse photoacoustic slice from another mouse. Lines 1 and
line 2 indicate the imaged dimensions along two different directions. (e) H&E stained section
in the same position. Lines 1′ and line 2′ present the actual dimensions in the H&E stained
section. (f) Photograph of the tumor. All the lines indicated by arrows in (d), (e), and (f) pres-
ent the margins of the tumor in transverse plane. Scale bar, 500µm.
(d) (e) (f )
FIGURE 8.20 Quantitative analysis of photoacoustic images in correlation with H&E sec-
tions with increasing image depth. (a, b) PAT slices and H&E sections for a tumor beneath
the skin of mouse (0.3 mm depth). (c, d) PAT slices and H&E sections for a tumor beneath
the mouse skin and a 1 mm-thick chicken breast. (e, f) PAT slices and H&E sections for a
tumor beneath the mouse skin and a 1.8-mm-thick chicken breast. White dashed lines show
the surface of chicken breast. Scale bar, 500 μm.
8.3 BRAIN IMAGING
Epilepsy is a common, chronic, neurological disorder characterized by seizures.
Three percent of people will be diagnosed with epilepsy at some time in their lives
(Hauser et al. 1996). Indeed, approximately 50 million people worldwide have
epilepsy, and 20% to 30% of these patients are refractory to all forms of medical
treatment (Hauser 1992). Seizures are transient signs and symptoms of abnormal,
excessive, or hypersynchronous neuronal activity in the brain (Fisher et al. 2005). In
most cases, seizures are controlled, although not cured, with anticonvulsant medi-
cation. Seizure types are classified according to whether the source of the seizure
is localized (partial or focal onset seizures) or distributed (generalized seizures)
(Greenfield et al. 2011). Localization-related epilepsies arise from an epileptic focus.
A partial seizure may spread within the brain, a process known as secondary gen-
eralization. Generalized epilepsies, in contrast, arise from many independent foci
(multifocal epilepsies) or from epileptic circuits that involve the whole brain. In epi-
lepsies of unknown localization, it remains unclear as to whether they arise from a
portion of the brain or from more widespread circuits.
For those patients with medically intractable focal epilepsy, the best treatment
option is resective brain surgery (Berg et al. 2007). Although several factors can
impact the success of epilepsy surgery, the primary reason of failure is the incomplete
Clinical Applications and Animal Studies 249
mapping of the local epilepsy network, which results in incomplete resection of epi-
leptogenic foci (Engel 2004). Much of the attention on epilepsy surgery has been
directed at identifying single neuronal populations. This approach has, in many
cases, led to failed surgical outcomes, because seizures typically involve groups of
neurons interacting both locally and across several cortical and subcortical brain
regions. A better understanding of the regional interactions occurring at the site of
seizure onset and spread may provide important insights about the pathophysiology
of seizures and aid with accurate brain mapping and resection of the epileptic focus.
In theory, removing the focus should result in a patient’s seizures being cured.
However, there is much evidence to suggest that the focus is more of a region of
seizure onset with a number of sites that can act independently to initiate seizures
(Thom et al. 2010). Seizures in animal models and in people often have a multifocal
or broadly synchronized onset. The best evidence for multifocality within the sei-
zure onset zone comes from surgical experience with intracranial monitoring. The
challenge of mapping the epileptic focus stems from the observation that the pathol-
ogy associated with focal epilepsy is often distributed across a number of brain sites
(Bertram 2009) and that current diagnostics methods frequently fall short of iden-
tifying such sites. Animal studies indicate that the neurons involved in the epileptic
circuitry have enhanced excitability throughout (Bertram et al. 1998, Mangan et
al. 2000). The implication of these observations is that each of the sites could act
independently to initiate a seizure or, potentially, to drive another site into a seizure.
Thus, in focal epilepsy, one may view a cortical region as a broad seizure onset zone,
with the potential that multiple foci can act as a seizure focus for any given seizure.
Much of our understanding about focal seizure circuitry comes from electro-
physiological recording methods. Although electrophysiology is currently the gold
standard in mapping the epileptic focus, it is often inadequate to define the boundary
of the epilepsy circuitry due to spatial sampling limitations and volume conduction.
What we truly desire is a brain-mapping modality that could give a high-resolution
real-time spatial and temporal “read out” of the dynamics of cortical processing and
seizures. It has been well established that optical contrast is highly sensitive to neu-
ronal activity (Hill and Keynes 1949, Grinvald et al. 1988). The high optical contrast
is largely due to the changes in blood volume and blood oxygenation, both of which
substantially increase during seizures, and increase in oxygenation metabolic rate
results in increased demands on autoregulatory mechanisms. Taking advantage of
the oxygenation dependence in the optical absorption spectrum of hemoglobin, opti-
cal imaging or intrinsic optical signal (IOS) methodologies have provided excellent
surface maps of epileptic focus (Haglund et al. 1992, Haglund and Hochman 2004).
Near-infrared spectroscopy (NIRS) has also shown to be able to detect seizure
(Steinhoff et al. 1996, Sokol et al. 2000, Watanabe et al. 2000). However, the major
limitation of NIRS and IOS is that each provides only surface depth information.
Greater depth information can be obtained by using tomographic reconstruction
methods such as diffuse optical tomography (Jiang 2010). Whereas diffuse optical
tomography has low spatial resolution, laser-induced PAT has both superior spatial
and temporal resolution. PAT detects absorbed photons ultrasonically by employing
the photoacoustic effect. It combines both high contrast and spectroscopic specific-
ity based on the optical absorption of both oxy- and deoxy-hemoglobin with high
250 Photoacoustic Tomography
8.3.1 Methods
8.3.1.1 Animals
Male Sprague-Dawley rats (Harlan Labs, Indianapolis, IN) weighing 50g to 60g
on arrival were allowed one week to acclimate to the 12-h light/dark cycle and
given food and water ad libitum. All procedures were approved by the University
of Florida Animal Care and Use Committee and conducted in accordance with the
National Institutes of Health Guide for the Care and Use of Experimental Animals.
8.3.1.2 PAT Imaging
The 192-element array based system described in Section 4.1 was used for the in vivo
study presented in this section.
8.3.1.4 Induction of Seizures
Rats (n = 10) received 10 μl of 1.9 mM bicuculline methiodide and 10 μl normal
saline into the left and right parietal cortex, respectively. The infusion was performed
through the previously implanted electrode sites at a rate of 0.3 µL/min. The infu-
sion system consisted of a 100 µL gas-tight syringe (Hamilton, Reno, NV) driven by
a syringe pump (Cole-Parmer, Vernon Hills, IL) connected to polyaryletheretherk-
etone (PEEK) tubing (ID = 0.381 mm, OD = 0.794 mm, length = 0.5 m, Upchurch
Scientific, Oak Harbor, WA). The PEEK tubing was coupled to a silica cannula (ID
= 50 µm, OD = 147 µm, Polymicro Technologies, Phoenix, AZ) via a microfluidic
connector. Cortical local field potentials were recorded 5 min before each injection
and continued for up to 30 min thereafter.
8.3.1.5 Image Analysis
A custom software utility was written and incorporated into the computer software pack-
age MATLAB® (MathWorks, Massachusetts, USA) to analyze and display recorded
data. This software enabled the reconstruction of the PAT images and the determination
of the blood vessel diameter. Amira (version 5.3.3, TGS Template Graphics Software)
was used for 3D reconstruction of the seizure foci. We used the frequency-domain
Granger causality methodology to evaluate the dynamic interactions within the seizure
circuitry and the associated brain networks (Brovelli et al. 2004). The time-series data
were selected from 200 sets of PAT images sampled at 3 Hz within the seizure onset
period, and at each time point we chose nine regions of interest (ROI), R1–R9, and then
the PA signal was averaged within each ROI, as shown in Figure 8.24b. Changes in pho-
toacoustic signals were quantified as –ΔA/A in Figure 8.24b, right. The PA data shown
in Figures 8.22–8.24 were not averaged. The image color scale was determined by the
photoacoustic signal intensity in arbitrary units (between 0 and 1).
RH
1 4 LH RH LH
1 4
2
5
2 5
3
3
ROB LOB
ROB LOB
2 mm
0 0.3 0.6 1
FIGURE 8.21 (a) Noninvasive PAT imaging of a rat brain in vivo with the skin and skull
intact. MCA, middle cerebral artery; RH, right hemispheres; LH, left hemispheres; LOB,
left olfactory bulbs; ROB, right olfactory bulbs. (b) Open-skull photograph of the rat brain
surface acquired after the PAT experiment. Numbers 1–5 indicate the corresponding blood
vessels in the PAT image and rat brain photograph.
was observed in the bicuculline methiodide cortical injection site and surrounding
region (Figure 8.22c, left), but not in the saline injection site or surrounding region
(Figure 8.22c, right). PAT scanning for subcortical changes was also performed.
Slices 5, 7, and 9 (Figure 8.22d) are the images obtained 3, 5, and 7 mm below the
scalp, respectively. Figure 8.22e is the 3D rendering of the epileptic foci obtained
from different tomographic layers. Each image exhibited the variable patterns of
seizure onset and propagation both at the cortical and subcortical regions. Seizures
were confirmed by concomitant time-locked PAT/video-electroencephalography
(Figure 8.22a). Experiments were repeated for each animal at 2-hour intervals.
200 μV
100 s
(a)
Max
Optical Absorption
B S BV BV
B S
2 mm
Min
(b) (c)
Max
Optical Absorption
500 μm
Min
Slice 5 Slice 7 Slice 9
(d)
x
y
z
3 mm
3 mm
(e)
FIGURE 8.22 Noninvasive epileptic foci localization. (a) EEG recordings of seizure onset
after bicuculline methiodide injection. (b) Schematic showing the location of BMI injection
and saline injection (control). B, BMI injection; S, saline injection. (c) Reconstructed PAT
image before (left) and after (right) the bicuculline methiodide injection. The BMI and saline
were injected into the left and right parietal neocortex, respectively. A significant increase
of optical absorption is seen in the region of the bicuculline methiodide injection, while no
absorption contrast is observed in the region of the saline injection relative to its surround-
ings. (d) Series of PAT images from three representative transverse planes parallel to the skin
surface with 755-nm wavelength. Slices 5, 7, and 9 are the images obtained 3, 5, and 7 mm
under the skin, respectively. The images show different spatial patterns at the seizure foci
at different tomographic layers. (e) 3D rendering of the epileptic foci from different tomo-
graphic layers.
254 Photoacoustic Tomography
ROI
2 mm
(a)
200 μV
20 s
(b)
65.934 Sec 1 min 6.267 Sec 1 min 6.600 Sec 1 min 6.933 Sec
0 mm2 0.2053 mm2 0.2342 mm2 0.2670 mm2
1000
500
500
1 min 7.266 Sec 1 min 7.599 Sec 1 min 7.932 Sec 1 min 8.265 Sec
0.3877 mm2 0.3986 mm2 0.4234 mm2 0.5004 mm2
500 μm
(c)
FIGURE 8.23 Real-time monitoring of ictal onset. (a) A PAT image showing the cerebral vas-
culature and the position of BMI injection. ROI, region of interest; Scale bar: 2 mm. (b) EEG
recordings showing the seizure onset about 1 min after BMI injection. (c) PAT images recording
the ictal onset in real time. At 1 min 6.267 s the area of the focus was 0.2053 mm2. The area of
the focus then increased in size over the next 2 s to 0.5004 mm2, corresponding to an increase in
the amplitude of the EEG spikes. The area of the seizure focus was derived from the PAT images
by thresholding to a pixel value one standard deviation above the pixel values from the area of
the focus during the control conditions. Scale bar: 500 μm.
Clinical Applications and Animal Studies 255
1 2 3 4 1200
1000
800
600
400
200
0
200
2 mm
2 mm 400
(a)
100
50
0
50
100 R1 (focus)
R4
150
0 1 2 3
Time (s)
(b)
(c)
FIGURE 8.24 In vivo maps of contralateral homotopic seizure foci. (a) Left, the PAT image
of the rat brain; the yellow dotted rectangular shows the region of interest for analysis; Right,
noninvasive imaging of the primary and mirror foci in contralateral homotopic cortex. A1–4
shows how secondary epileptic foci were generated and diminished. The PA signal in the
secondary foci was smaller in magnitude and delayed in time compared to the signal recorded
from the primary foci. (b) Left, Granger analysis of the image around the foci and its immedi-
ate secondary foci; right, PA signal from the foci (red) and secondary area (blue) during an
ictal event. (c) Pairwise Granger analysis of the seizure foci, contralateral homotopic foci and
middle cerebral artery. C1, PAT image showing the 9 selected regions of interest; C2, arrows
indicate Granger connectivity maps between the primary and secondary foci; C3, connectiv-
ity maps between the primary and secondary foci, and the middle cerebral artery.
256 Photoacoustic Tomography
foci was smaller in magnitude and delayed in time compared to the signal recorded
from the primary seizure foci. We also observed a region of inverted optical signal
immediately surrounding the seizure onset zone (Figure 8.24b2-3). We performed
analysis of the optical signal at a distance of at least 2 mm away from the edge of the
focus. Our results demonstrate that the photoacoustic signal was inversely related to
the optical signal recorded from the focus (Figure 8.24b3).
To evaluate the dynamic interactions within the seizure circuitry and capture
the associated networks we used the frequency-domain Granger causality (Brovelli
et al. 2004). PAT time series data were selected from 200 sets of PAT images sam-
pled at 3 Hz within the seizure onset period. At each time point we chose 9 ROIs
(Figure 8.24b) and used the averaged optical absorption within each region for the
network analysis. Nine sets of time series analysis were classified into three groups
including the primary ictal onset area and corresponding four surrounding ROIs
including the primary or initiating seizure focus, contralateral homotopic foci, and
two ROIs surrounding the primary focus in the contralateral cortex. The middle
cerebral artery, primary seizure focus, and secondary homotopic seizure focus were
also analyzed. Results from group 1 analysis demonstrate the causal influence from
the primary focus to the surrounding regions of interest (Figure 8.24b1). The negative
zero-lag correlation (–0.68) between both the primary and secondary foci showed
that neural activities within these two regions changed in opposite direction over
time. Figure 8.24b4 shows the Granger causality analysis for group 2 data, where we
see that the primary and secondary contralateral brain foci influenced each other
mutually with a stronger influence of the primary focus relative to the contralateral
focus. Moreover, we suggest that the primary seizure focus may influence the con-
tralateral foci as previously suggested using optical intrinsic imaging in epilepsy
(Khalilov et al. 2003) and microelectrode recordings of hippocampus local field
potentials (Zhou et al. 2009). Finally, from group 3 analyses (Figure 8.24b5) we
found that the primary focus strongly influenced the hemodynamic change in the
middle cerebral artery, which in turn showed influence on the contralateral homo-
topic foci.
MV
2 mm
(a)
Amplitude (A.U.)
0.2
PA Signal
0.1
0
0S
0.1 1.6 S
3.2 S
0.2 4.4 S
6.4 S
0.3
0 20 40 60 80 100 120 140 160 180
Diameter (um)
(b)
50 μY
1s
(c)
36
34
Diameter (μm)
32
30
28
26
24
22
5 10 15 20 25 30
Time (s)
(d)
FIGURE 8.25 Changes of blood vessel diameter during interictal onset. (a) PA image of
the rat cortex vasculature with the intact scalp and skull. The arrow indicates the microvas-
cularity (MV) along the dashed line direction for a cross-sectional scanning using a 50 MHz
ultrasound transducer. (b) Typical photoacoustic signals from the targeted vessel at different
times. (c) EEG recordings show the interictal spikes. (d) Change of the vessel size captured by
PAT. There was a clear correlation between the interictal spikes and the changes of the vessel
size. Error bars (±s.d.) were calculated from 20 consecutive spikes.
258 Photoacoustic Tomography
8.4.1 Methods
8.4.1.1 Photoacoustic Imaging System
In this study, a photoacoustic microscopy (PAM) system was used to monitor
vasculature change in breast cancer xenograft models. The system is the same as
shown in Figure 5.3. In this application, a focused ultrasound transducer (50 MHz,
3 mm aperture, and 6 mm focal length) was used to receive induced photoacous-
tic waves. PA signals amplified by two different amplifiers (one was 17 dB from
100 kHZ to 1 GHz and the other was 20 dB from 20 MHz to 3 GHz) were digi-
tized by an 8-bit data acquisition board (NI5152, National Instrument, Austin,
TX) at a sample rate of 250 MS/s. The 2D transverse scanning combined with the
depth-resolved ultrasonic detection generated 3D PA images displayed in maxi-
mum amplitude projection (MAP). Three-dimensional PA signals were processed
by Hilbert transform, normalized to the same scale (0–256) and applied with a
threshold (–6 dB level). The volumes of the blood vessels were calculated by inte-
grating the corresponding image voxels (1 for blood vessel and background being
set to 0). Entropy was calculated from normalized MAP of each PA image at the
same scale (0–256).
nude mice (nu/nu) at 5 to 8 weeks were purchased from Charles River Laboratories
(Charles River Laboratories, Inc., Wilmington, MA) and caged in groups of five or
fewer. Mice breast cancer xenografts were established by subcutaneous injection
of 1 × 105 NeuT (a mouse breast cancer cell line) or NeuT EMTCL2 (a malignant
variant of NeuT) cells into the mammary fat pads of the mice. Tumor volume was
calculated from caliper measurements of the large (a) and smallest (b) diameters of
each tumor using formula a × b2 × 0.4. Three days after inoculation, most tumors
had grown to ~30 mm3. All mice were euthanized when the tumor volume in the
nontreated group reached ~1000 mm3.
For in vivo siRNA (a class of antiangiogenic drugs) knockdown of the unfolded
protein response (UPR) proteins, mice with similarly sized tumors were divided into
two groups (siRNA treatment and scrambled control). Mice were anesthetized with
a mixture of ketamine (85 mg/kg)/xylazine (4 mg/kg) and were intratumoral injected
(10 μl) with scAAV2 sept-mut vectors encoding siRNAs against IRE1α or XBP-1 or
ATF6 or scrambled siRNA at a titer of 2 × 1013 genome copies per ml.
NeuT
NeuT EMTCL2
(a)
9 100
NeuT NeuT METCL2 NeuT NeuT METCL2
Normalized Volume
*p<0.05 vs NeuT *p<0.05 vs NeuT
8 80 * *
* * *
* *
Entropy
*
7 60
6 40
5
3
9
ay
ay
ay
ay
ay
ay
ay
ay
D
D
(b) (c)
300
NeuT NeuT
NeuT EMTCL2
Tumor Volume (mm3)
200
0
0 3 5 7 9 11
Time (days)
(d)
FIGURE 8.26 In vivo serial photoacoustic imaging of developing tumor vasculature and
quantitative analysis. (a) Representative serial PA images of NeuT model (top panel) and
NeuT EMTCL2 model (bottom panel). (b) Entropy extraction for change in the vessel den-
sity over different time points as indicated. (c) Comparative analysis of normalized vessel
volumes at different time points as indicated. (d) Tumor volume was calculated from daily
caliper measurements of the large A and smallest B diameters of each tumor using formula
a × b2 × 0.4. Representative images of H&E staining for breast cancer tissue sections of NeuT
(top) and NeuT EMTCL2 (bottom) xenograft models.
Clinical Applications and Animal Studies 261
scr
IRE1
siRNA
XBP-1
ATF6
(a)
* *
7 * * 60 *
* ** * * ** *
*
* **
*
6 ** 40 *
5 20
3
1
1
1
ay
ay
ay
ay
ay
ay
ay
ay
ay
ay
D
D
D
(b) (c)
300
scr
IRE1 siRNA
Tumor Volume (mm3)
XBP-1 siRNA
200
ATF6 siRNA
100
0
0 3 5 7 9 11
Time (days)
(c)
FIGURE 8.27 Knockdown of the siRNAs against the UPR protein resulted in decreased
tumor growth and tumor vasculature in mouse breast cancer xenografts. Mice breast can-
cer xenografts received intratumoral scAAV2 sept-mutant vector-delivered siRNAs against
IRE1α or XBP-1 or ATF6. PA imaging was performed on the same tumor inoculation site on
day 3, 5, 7, and 9 post tumor inoculations. (a) Representative PA images of different treatments:
the scrambled siRNA (top panel), IRE1α siRNA (second panel), XBP-1 siRNA (third panel)
and ATF6 siRNA (bottom panel). (b) Entropy extraction for change in the vessel density over
different time points as indicated. (c) Comparative analysis of normalized vessel volumes at
different time points as indicated. (d) Tumor volume was calculated from daily caliper mea-
surements of the large A and smallest B diameters of each tumor using formula a × b2 × 0.4.
262 Photoacoustic Tomography
tumor volume reached ~250 mm3 after 11 day of NeuT EMTCL2 inoculation in the
scrambled siRNAs treatment group (Figure 8.27d). Figure 8.27d also shows that
the knockdown of IRE1α and XBP-1 both exhibited significant decreased tumor
growth to a similar extent (~45% on day 11) and the ATF6 siRNA was even more
effective on inhibition of tumor growth (~54% by day 11). Serial PA imaging was
employed to monitor and evaluate the development of tumor vasculature in the
NeuT EMTCL2 xenograft models on day 3 after NeuT EMTCL2 inoculation fol-
lowed on days 5, 7, 9, and 11. Entropy method delineated a significant decrease in
vessel density (25%) by day 9 and sustained at that level thereafter in the IRE1α
siRNA treatment group compared with the scrambled group (Figure 8.27b). The
NeuT EMTCL2 xenograft models treated with the siRNAs against XBP-1 or ATF6
evidenced a steady decrease in vessel density with statistic significant in com-
parison with the scramble group (Figure 8.27b). All three siRNAs exhibited sig-
nificantly steady decrease in vessel volume compared with the scrambled siRNA
treatment (Figure 8.27c).
8.5 INTRAVASCULAR IMAGING
In the United States, the leading cause of death is acute cardiovascular events, and
the majority of them are due to the rupture of an unstable coronary atherosclerotic
plaque (Kolodgie et al. 2001, Schaar et al. 2004). The demand for imaging/diagnos-
tic technologies that can assess the vulnerability of these plaques is great. During the
past decades, there has been considerable progress in the development of new imag-
ing techniques for high-risk coronary atherosclerotic plaque detection. Compared
to the noninvasive screening modalities such as MRI, CT, and ultrasound, the cath-
eter-based intravascular imaging techniques have the advantages of providing 3D
information of structural, compositional, biochemical, and molecular features of
coronary lesions and play an increasing significant role in the detection and assess-
ment of coronary atherosclerotic plaques (Honda and Fitzgerald 2008).
Catheter-based X-ray angiography can provide real-time images of the stenosis
and is the most widely used intravascular imaging modality (Rodgers et al. 1994).
Intravascular ultrasound (IVUS) imaging offers a relatively high-resolution and
deep penetration depth cross-section image of the blood vessel (Yock et al. 1988).
Optical coherence tomography (OCT) provides very high spatial resolution images
(Toutouzas et al. 2009). Near-infrared spectroscopy (NIRS) opens up the possibil-
ity of identifying absorption properties of tissue within the artery wall (Jaffer et al.
2009). However, these techniques still suffer from various limitations when applied
to intravascular imaging. For example, X-ray angiography has the risk of unreliable
estimation of the vessel lumen narrowing when projecting the 3D arterial network to
2D silhouettes and offers no compositional information; IVUS has limited capacity
in characterizing the component of atherosclerotic plaques. OCT is limited by its 1
to 2-mm penetration depth as light loses its coherence in deeper tissue. NIRS offers
little depth/structural information of the plaque.
Clinical Applications and Animal Studies 263
According to autopsy studies, researchers found that most unstable plaques are
featured a thin fibrous cap, a large lipid-rich necrotic core, and activated macrophage
near the fibrous cap (Virmani et al. 2000, Schroeder et al. 1995). In addition, there is
a strong association between atherosclerotic plaque stability and the components of
the plaques (Virmani et al. 2000, Schroeder et al. 1995). Limitations of intravascular
imaging modalities prevent them from achieving the goal of complete evaluation of
atherosclerotic plaque, which requires the 3D structural and compositional informa-
tion with deep penetration depth and high resolution. Thus, a new imaging modality/
technology that can provide deep depth, high-resolution 3D structural and composi-
tional information of the atherosclerotic plaque is highly desirable for assessment of
the vulnerability of coronary plaques.
Intravascular photoacoustic imaging (IVPA) is a catheter-based optical-induced
ultrasound imaging modality and has the potential to offer both structural and com-
positional information of the vessel wall/plaque with deep penetration depth (~1cm)
and high spatial resolution (~tens of microns). The atherosclerotic plaque is com-
posed of different chemical components that possess certain optical absorption coef-
ficients that respond to certain wavelengths of incident light. These absorbers can
be identified with the information from detected photoacoustic signals and incident
light as the ultrasound signal is proportional to the local fluence and corresponding
absorption coefficient. Specifically, by comparing the change in the photoacoustic
signals with the absorption coefficient spectra or using model-based quantitative
reconstruction algorithms (see Chapter 2), the atherosclerotic plaque components
can be characterized.
Using the PAT system described in Chapter 6 (Figure 6.12) and quantitative
PAT methods discussed in Chapter 2, experiments using ex vivo blood vessels
were conducted to demonstrate quantitative intravascular photoacoustic imaging
(IVPA). For the ex vivo experiments, IVPA measurements at multiple wavelengths
(710, 800, 900, 1100, and 1150 nm) were performed to extract the composition
of plaques. Figure 8.28 shows the reconstructed images by the delay-and-sum
(Figure 8.28c) and by the FE-based method (Figure 8.28d) for one set of repre-
sentative ex vivo atherosclerotic blood vessel (Figure 8.28a gives the photograph
of the sample). Comparing these results with the artery sample, we can see that
the plaque shows up in each image at all wavelengths and in the right position
while the shape and PA signal or absorption coefficient change in response to the
wavelength.
Using the absorption coefficient images at the five wavelengths coupled with the
Beer-Lambert law (Figure 8.28b), we can obtain the concentrations of the four main
components of blood vessels, which are lipid, elastin, collagen, and water. Based
on these compositional images shown in Figure 8.29, the wet weight percentages of
the components in this atherosclerotic artery were estimated as 15, 28, 32, and 25%
for lipid, elastin, collagen, and water, respectively. Comparing with normal tissue
which has 24% elastin, 37% collagen, and 1.6% lipid (Black and Hastings 1998), our
reconstructed results show a higher lipid percentage, which means that a plaque with
a lipid core existed in the imaging section.
264 Photoacoustic Tomography
15
800 1000 1200 10 0 10
Wavelength
(b)
15 0.05 15
10 0.04
0.04 10
5 5 0.03
0.03
0 0
0.02 0.02
5 5
10 0.01 10 0.01
15 15
10 0 10 10 0 10
(d) 15 15
10 0.025 10 0.02
5 0.02 5
0.015
0 0.015 0
5 5 0.01
0.01
10 10 0.005
0.005
15 15
10 0 10 10 0 10
FIGURE 8.28 (a) Photograph of a human atherosclerotic artery sample; (b) optical absorp-
tion coefficient spectra of various components within the artery; (c) PA images recovered by
the delay-and-sum method at multiple wavelengths; (d) Absorption coefficient images recov-
ered by the FE-based method at multiple wavelengths.
8.6 BREAST IMAGING
Breast cancer is a leading cause of death for women in the United States. The best
way of combating the increased incidence of breast cancer is early detection. While
X-ray mammography has long been accepted as the most effective method to detect
breast cancer prior to outward signs and symptoms, it has unacceptable false nega-
tive rates for patients with radiodense breast tissue. For example, the sensitivity of
screening mammography decreases from a high of 88% in fatty breast tissue to 62%
in dense breast tissue (Carney et al. 2003). Patients with dense breasts represent the
general population of premenopausal women as well as those with fibrocystic tissue
Clinical Applications and Animal Studies 265
15 15
1
10 0.3 10
0.8
5 5
0.2 0.6
0 0
5 5 0.4
0.1
10 10 0.2
15 15
10 0 10 10 0 10
Lipid 15% Elastin 28%
15 15
1.2
10 10 0.8
1
5 5
0.8 0.6
0 0
0.6 0.4
5 0.4 5
10 10 0.2
0.2
15 15
10 0 10 10 0 10
Collagen 32% Water 25%
disease. Yet, cancer in younger women tends to be more virulent and grow faster.
Another concern is that the positive predictive value of X-ray mammography is quite
low. This has numerous ramifications both medical and economic, in terms of the
number of necessary biopsies. Consequently, there is a critical need to develop breast
cancer detection methods that could serve either a complementary or competitive
role with respect to conventional X-ray mammography.
Several other conventional techniques are currently under investigation for breast
cancer detection, including ultrasound (US), MRI, CT, positron emission mammog-
raphy (PEM), and breast-specific gamma imaging (BSGI). Among these conventional
alternatives or supplements, MRI, PEM, and BSGI are the most promising solutions to
dense breast imaging. Bluemke and colleagues showed a sensitivity of 88% and cor-
responding specificity of 68% regardless of breast density from an MR study of 821
women (Bluemke et al. 2004). Similar high sensitivities of MR for breast cancer have
also been found in other studies involving targeted imaging of high-risk populations
(Warner et al. 2004). While MR appears to offer high sensitivity in the dense breast,
a major disadvantage of MRI is the limited specificity resulting from its enhancement
of benign breast lesions (Kuhl 2007). Recent emerging nuclear imaging techniques
like PEM and BSGI provide molecular information of tumor tissue. Carmen and col-
leagues showed 88% sensitivity and specificity from PEM of 808 subjects (Carmen et
al. 2005). However, the high cost and the use of exogenous contrast agents may render
these techniques impractical for frequent monitoring and screening.
The need for a nonionizing, noninvasive imaging technique that can provide
high-contrast images at microscale resolution with functional information for breast
266 Photoacoustic Tomography
cancer can be fulfilled with PAT. As described in Chapter 2, PAT can obtain infor-
mation on hemoglobin and oxygen saturation at high resolution and contrast, without
the use of exogenous contrast agents, which is a significant advantage when com-
pared with other tumor hypoxia imaging techniques (e.g., blood oxygen level depen-
dent-MRI and PET).
Another unique advantage of PAT for breast imaging is to facilitate monitoring
treatment for breast cancer. At the time of diagnosis of breast cancer, more than 30%
of cases are locally advanced breast cancer (LABC). For these women, lumpectomy,
or curative surgery, is often an option if preoperative (neoadjuvant) chemotherapy
(NAC) is given to shrink the tumor to allow for optimal surgical therapy. While NAC
is emerging as a promising treatment strategy for locally advanced breast cancer,
deploying monitoring methods during the course of therapy is important for mak-
ing further advances. MR and fluorodeoxyglucose (FDG) PET have been evaluated
in several clinical trials and have proven useful in this setting, but both come at
considerable cost and inconvenience (Partridge et al. 2005, Rousseau et al. 2006).
Assessing tumor shrinkage by clinical breast examination is possible as well but
is poorly correlated with treatment responses. Nonionizing, noninvasive PAT with
high resolution has shown its promise in NAC monitoring (see Figures 8.33 and
8.34). Significant changes in absolute total hemoglobin (HbT) and oxygen saturation
(O2%) of tumor regions have been observed during NAC from patient data. Our ini-
tial results have shown that PAT is very promising and invaluable in assessing and
predicting tumor response to NAC of breast cancer.
(a)
Data
Breast Amplifier Acquisition
Boadrs
(b) Computer
Light Delivery
System
Pulsed
Mirror Laser
(c)
FIGURE 8.30 (a) Photograph of breast/transducer interface. (b) Photograph of PAT clinical
exam table. (c) Schematic of PAT system.
Clinical Applications and Animal Studies 267
light delivery system was built to illuminate a large area of breast tissue for opti-
mized tissue penetration. Frequency response of the transducers is from 380 kHz
to 1.48 MHz and maximum frequency response is up to 2 MHz. The PAT system
offers a spatial resolution of 0.5 mm. A depiction of the breast/transducer interface
and the system in a clinical setting is shown in Figures 8.30a and 8.30b, respectively.
During the exam, the patient was in a prone position with her breast pendant through
an aperture. The breast was slightly compressed between a PETG plate at the cranial
plane and the ultrasound detector array at the caudal plane surrounding the breast.
Cross-section images of breast tissue can be captured by a ring-shaped interface. The
breast was illuminated through the PETG plate with light from a pulped Ti:Sapphire
laser optically pumped with a Q-switched Nd:YAG laser.
8.6.2 Clinical Experiments
Here we demonstrate two pilot clinical studies with our prototype system shown
in Figure 8.30. Two studies including breast cancer detection and monitoring NAC
treatment for breast cancer are demonstrated below. The first study involved breasts
with different types of lesions, diagnostic imaging (mammogram, MRI, and/or
ultrasonography), and healthy controls. In the second part, a case study is shown to
monitor NAC response using PAT technique on a patient (47-year-old female) who
underwent NAC prior to surgery. Maps of absolute total hemoglobin (HbT = HbR
+ HbO2) and oxygen saturation (O2%) of coronal plane were calculated from two
absorption coefficient images reconstructed at 775 and 808nm, respectively, for both
studies. This is the first attempt to use functional PAT methods in clinical breast can-
cer diagnosis and monitoring NAC. Protocols for both studies were approved by the
institutional review board and conducted in full compliance with the accepted stan-
dards for research involving humans. Written informed consent was obtained from
all the participants. Both African American and Caucasian patients were included
in the study.
20 0.4
0.2
10
0
10 mm 50 mm
FIGURE 8.31 (a) Ultrasound image of lesion area. (b) HbT and O2% maps of coronal plane
from PAT.
2008). The finding of increased oxygen saturation in part of the tumor region is con-
firmed with other optical methods (Srinivasan et al. 2003).
Case 2 is a PAT study with a malignant lesion in the left breast while no abnormal-
ity in the right breast from pathology report and diagnostic imaging (mammogram,
ultrasound, and/or MRI). The left breast lesion is high-grade DCIS and invasive
mammary carcinoma with apocrine features and squamous metaplasia (Nottingham
Grade 7). Contrast MRI examination with gadolinium was taken one week before
PAT examination. Three-dimensional MRI images of both breasts shown in
Figures 8.32c and 8.32d were constructed with 2D slices obtained from the MRI
(a) (b)
Right Breast (Healthy) Left Breast (Abnormal)
(µM) (%)
1
40
0.8
PAT 30 0.6
20 0.4
10 0.2
0
50 mm HbT O2% HbT O2 % HbT O2
(c) R L (d)
MRI 50
mm
FIGURE 8.32 (a) MRI image of healthy breast. (b) MRI image of abnormal breast.(c) HbT
and O2% maps of coronal plane from PAT of healthy breast. (d) HbT and O2% maps of coronal
plane from PAT of abnormal breast.
Clinical Applications and Animal Studies 269
scans. It showed benign MRI in right breast and abnormal left breast—an irregular
area of suspicious non-masslike enhancement with plateau kinetics that extends 5 cm
in AP dimension and 2.6 cm in craniocaudal dimension, and the anterior margin of
the suspicious area of enhancement begins 5.6 cm posterior to the nipple. HbT and
O2% maps of coronal plane taken from PAT are shown in Figures 8.32a and 8.32b
for the right and left breast, respectively. We can see that our multiwavelength PAT
system successfully detected a lesion area at 5.6 cm depth from nipple. Increased
HbT concentration is detected with diameter 1.9 × 2.7 cm (maximum diameter in
each axis), which is compatible with the MRI findings. Decreased O2% concentra-
tion circling an increased concentration is shown in O2% map (Figure 8.32b), which
may relate with blood flow and necrosis of tumor region. From the right breast PAT
images shown in Figure 8.32a, we can see that while some nonhomogeneous pattern
exists in HbT and O2% maps, the contrast of these benign patterns is less than 1.2
for both HbT and O2%, which is much smaller than the contrast of the lesion region
compared to normal tissue (which is higher than 1.6). Similar findings are confirmed
with other in vivo cases examined by this PAT system. These findings indicate that
PAT has a great potential for breast cancer diagnosis, and its further clinical explora-
tion is very promising.
1
0.8
O2% 0.6
0.4
0.2
0
20 mm 20 mm
MRI US US MRI
(b) (c)
FIGURE 8.33 (a) HbT and O2% maps of an invasive mammary carcinoma with DCIS patient
obtained at baseline, each month during treatment and after treatment. (b) Reconstructed cor-
onal plane (5.6cm from nipple) from MRI and US image of lesion area before NAC.
(c) Reconstructed coronal plane (5.6cm from nipple) from MRI and US image of lesion area
after NAC. Note that all the PAT, MRI, and US images displayed in the same scale.
2.5
200
2
150
1.5 HbT contrast
100 O2% contrast
1
Area(mm2)
50
0.5
0 0
B NAC 1M 2M 3M A NAC
FIGURE 8.34 Quantitative analysis of size of lesion area, contrast of HbT and O2% maps of
tumor and surrounding normal tissue from PAT exams before NAC, 1 month, 2 months, and
3 months after first NAC treatment till NAC treatment was completed before surgery.
Clinical Applications and Animal Studies 271
273
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Electrical Engineering
Photoacoustic Tomography
"… a very comprehensive collection of photoacoustic imaging methods, covering
everything from the basics of photoacoustic generation and image reconstruction
methods to the various implementations, including numerous applications. This is
the first time the topic of photoacoustic imaging has been covered in this detail
by a single author."
—Günther Paltauf, Karl-Franzens-Universität Graz, Austria
The concept of photoacoustic tomography (PAT) emerged in the mid-1990s,
and the field of PAT is now rapidly moving forward. Presenting the research of
a well-respected pioneer and leading expert, Photoacoustic Tomography
is a first-of-its-kind book covering the underlying principles and practical
applications of PAT in a systematic manner. Written in a tutorial format,
the text:
K24289