University of Cyprus

Biomedical Imaging and Applied Optics

Tissue Optical Properties

Introduction
Interaction between
Light and Tissue

Reflection
Refraction
Absorption
Fluorescence
Scattering

Light
Source

Optical
Signal
Tissue

Depends on
Constituents of tissue
Optical properties of
tissue
Propagation of light
2

Absorption
Extraction of energy from light by a molecular species
Diagnostic applications: Transitions between two energy
levels of a molecule that are well defined at specific
p
wavelengths could serve as spectral fingerprint of the
molecule
Various types of Chromophores (light absorbers) in Tissue
Wavelength-dependent absorption
Tumor detection and other physiological assessments (e.g. pulseo
oximetry)
et y)

Therapeutic applications: Absorption of energy is the primary

mechanism that allows light
g form a source (laser)
(
) to produce
p
physical effects on tissue for treatment purpose
Lasik (Laser Assisted in situ Keratomileusis) Eye Surgery, Tatoo
Removal, PDT
3

Absorption
Absorption occurs when the photon frequency matches the
frequency associated with the molecule
molecules
s energy transition
frequency
Electrons absorb the energy of the light and transform it into vibrational
motion
The
Th absorption
b
ti off a photon
h t results
lt in:
i

quantized change in charge separation

quantized excitation of vibrational modes

Electrons interact with neighboring atoms convert vibrational energy
into thermal energy

 Each electronic energy

gy
levels is associated with
many vibrational energy
levels
Absorption of UV and
visible
i ibl light
li ht promotes
t
transition between
electronic energy levels

Potentia
al Energy

Absorption

Vibrational Energy Level

S1
Electronic
E
Energy
Level

Absorption of infrared
light promotes
transitions between
vibrational energy
gy levels
5

S0

Absorption
Absorption
p
Cross-section,,
[m2]
Consider a chromophore
id li d as a sphere
idealized
h
with
ith a
particular geometrical size.
Consider that this sphere
p
blocks incident light and casts
a shadow, which constitutes
absorption.
absorption
The size of absorption shadow
= absorption cross-section
Qa: absorption efficiency

a = Qa A

Absorption
Pabs = Ioa

Pin =IoA

Pout = Io(A-a)

Outgoing Beam

Incident Beam
Area =A

Area = a

Pout = Io(A-a)
area = A - a

Pabs
a =
Io
7

Absorption
Assumptions
Cross section is independent of relative orientation of the impinging
light and absorber uniform distribution of Na (molecules/cm3) identical
absorbing particles

Absorption Coefficient, ma [1/m]

a = N a a
Absorption
p
cross-sectional area p
per unit volume of medium

Absorption mean free path, la [m]

la =

Represents the average distance a photon travels before being

absorbed
8

Absorption
Transmission and Absorbance ((macroscopic view))

Transmission

I
T=
Io
Absorbance (attenuation, or optical density)

Io
A = log(T ) = log
I
9

Absorption
Lambert Beer Law:
The linear relationship between absorbance and
concentration of an absorbing
g species.
p

Relates , transmission, and absorbance

I = Ioe

a b

a = N a a
Pabs
a =
IO

= absorption cross-sectional area [cm2]

IO = The intensity entering the sample at z = 0 [w/cm2]
I = The intensity of light leaving the sample [w/cm2]
b = pathlength traveled in the sample [cm]
10

Absorption
Absorbers in Tissue

NIR

NIR
Hemoglobin
Lipids
Water

VISIBLE

UV

UV-VIS

DNA
Hemoglobin
Lipids
Structural protein*
protein
Electron carriers*
Amino acids*

* Absorbers that fluoresce when excited in the UV-VIS

11

Absorption
UV Absorption
Protein
Protein, amino acid,
acid fatty acid
and DNA absorption dominate
UV absorption
Protein
Dominant non-water
constituent of all soft tissue, ~
30%
Absorption properties
determined by peptide bonds
and amino acid residues
Peptide excitation about =
190 nm
Amino acids absorption at =
210 - 220 nm and 260 280 nm

DNA

Amino Acid

Peptide

Absorbs radiation for 320 nm

Large water absorption < 180

nm
12

Absorption
Infrared Absorption
Protein IR absorption peaks at
6.1, 6.45, and 8.3 m due to
amide excitation
Absorption depth 10 m in
= 6-7 m region

Water absorption peak at

0.96, 1.44, 1.95, 2.94 and
6.1 m
Absorption depth
~ 500 mm at = 800 nm
<1 m at =2.94 m
20 m throughout 6 m

13

Extinction Coe
eff (1/cm M)

Absorption
10

10

10

Main Absorbers at visible and NIR

Hemoglobin
Lipid
p
Hb

10

10

Hemoglobin

HbO2
400

500

600

700

800

900

WAVELENGTH (NM)

1000

Each hemoglobin has 4 heme

(Fe2+) sites to bind O2
Responsible for oxygen transport
HbO2 and Hb
oxygen saturation is an indicator
of oxygen delivery and utilization
as wellll as metabolic
b li activity
i i

14

Hemoglobin
Responsible for oxygen transport

HbO2 and Hb
oxygen saturation is an indicator of
o gen delivery
oxygen
deli er and utilization
tili ation as well
ell
as metabolic activity

Deoxyhemoglobin has lower

absorption than oxyhemoglobin in
the blue and green
Bright red arterial blood
Bluish venous blood

Ex
xtinction Coe
eff (1/cm M)

Absorption
10

10

10

Hb
10

HbO2
10

400

500

600

700

800

900

WAVELENGTH (NM)

Absorption peaks for HbO2

418, 542, 577, and 925 nm

Absorption peaks for Hb

550, 758, 910 nm

Isosbestic points

547, 569, 586, and 798 nm

15

1000

Absorption
Lipid (Fat)

Monitoring of physiological
changes in female breast tissue
Tissue layer model

HEMOGLO
OBIN (1/mm mM
M)

Site-specific measurements of
body composition

3.0

0.06

Water

25
2.5

0.04

2.0

HB

0.03

1.5

0.02

1.0
0.5

0.05

Lipid
HbO2

0.01
0.00

0.0
0
0
600

700

800

900

1000

WAVELENGTH (nm)

16

WATER & FA
AT (1/ mm mM)

Important energy store in the

body

Scattering
Change
g of direction of propagation
p p g
and/or energy
gy of
light by a molecular species
Diagnostic applications: Scattering depends on the
size, morphology, and structure of the components
in tissues (e.g. lipid membrane, collagen fibers,
nuclei).
l i)
Variations in these components due to disease would
affect scattering properties
properties, thus providing a means for
diagnostic purpose

Therapeutic applications: Scattering signals can be

used to determine optimal light dosimetry and
provide useful feedback during
p
g therapy
py
17

Scattering
Purely absorbing

With Scattering

Photon pathlength = L

Photon pathlength >> L

Lambert- Beer Law does not apply here!!!

Need to calculate true pathlength of light
18

Scattering
Why is the sky blue, clouds
white and sunsets red?
white,
Blue skies are produced due to
scattering at shorter wavelengths
Visible light (violet & blue) are
selectively
l ti l scattered
tt d b
by O2 and
d N2
much smaller than wavelengths of the
light
violet and blue light has been
g
scattered over and over again

When light encounters larger

particles (cloud, fog), Mie scattering
occurs
Mie scattering is not wavelength
dependent appears white
Cigarette smoke, too

At sunset
The light must travel over a longer
path in the atmosphere
Blue/green is scattered away and only
red/orange (scattered less) reaches
your eyes

19

Scattering
Mechanism for Light
g Scattering
g
Light scattering arises from the presence of
heterogeneities within a bulk medium
Physical inclusions
Fluctuations in dielectric constant from random
thermal motion

Heterogeneity/fluctuations non-uniform
temporal/spatial distribution of refractive index
in the medium
Passage of an incident EM wave sets electric
charges into oscillatory motion and can excite
vibrational modes
Scattered light is re-radiated by acceleration of
these charges and/or relaxation of vibrational
transition

20

Scattering
Elastic scattering:
g no energy
gy change
g
Frequency of the scattered wave = frequency of incident
wave
Probes static structure of material
Rayleigh and Mie scattering

Inelastic scattering: energy change

Frequency
q
y of the scattered wave frequency
q
y of incident
wave
Internal energy levels of atoms and molecules are excited
Probes vibrational bonds of the molecule
Raman scattering (stokes and anti-stokes )

21

Scattering
Elastic Scattering
The light scattered by a system
has interacted with the
inhomogeneities
g
of the system
y
Photons are mostly scattered
by the structure whose size
matches the wavelength
Principal parameters that
affect scattering

Wavelength,
W
l
th
Relative refractive index
Particle radius
Sh
Shape
and
d orientation
i t ti

Two types of scattering:

y g and Mie
Rayleigh
22

Scattering
Rayleigh
y g Scattering
g

Light
Source

Detector

Scattering from very small particles /10

Rayleigh scattering is inversely related to fourth power of the
wavelength of the incident light

1
I 4

is the wavelength of the incident light

I is the intensity of the scattered light
23

Scattering
Mie Scattering
g

For scattering of particles comparable or larger than the

wavelength, Mie scattering predominates
Because of the relative particle size
size, Mie scattering is not
strongly wavelength dependent
Forward directional scattering
24

Scattering
Pin =IoA

Pscatt = Ios Pout = Io(A-s)

Incident Beam

Outgoing Beam

Scattering Cross Section, scatt [m2]

area of an index-matched, perfectly absorbing disc necessary to produce

The measured reduction of light

scatt = Qs*As
Qs: Scattering efficiency (calculated by Mie theory); defined as the ratio of the
scattering section to the projected area of the particle on the detector
As: Area of Scatterer [m2]
25

Scattering
Scattering
g Coefficient, s [[1/m]]
s =Nss ,
Ns = the number density of scatterers
s = scattering
tt i efficiency
ffi i

Cross-sectional area for scattering per unit volume of medium

Scattering Mean Free Path

Path, ls
Average distance a photon travels between scattering events

1
ls =
s

26

Scattering
Anisotropy, g
d
scattered
Imagine that a photon is
photon S
scattered by a particle so
hv
that its trajectory is
Scatterer
d fl t d b
deflected
by an angle,
l
Scattering
Then, component of a new
hv
Angle ()
S
trajectory aligned forward Incidenet
Photon
direction is cos()
cos ()
Photon
Anisotropy is a measure of
trajectory
forward direction retained
Scattering
after a single scattering
event
event, < cos()>
totally
y backward
bac a d scattering
scatte g
11 tota

g = 0 isotropic scattering
1 totally forward scattering

Biological Tissues:
0.65 < g <0.95

27

Scattering
Reduced Scattering
C ffi i t s [1/m]
Coefficient,
[1/ ]
s incorporates the
scattering
g coefficient,,
s
and the anisotropy factor, g

s ' = (1 g) s
s can be regarded as an
effective isotropic
scattering coefficient that
represent the
h cumulative
l i
effect of several forwardscattering events
Special significant with
respect to photon diffusion
theory

26o
g = cos = 0.90

s ' = (1 g ) s = 0.10
0 10 s
mpf = 1/ s
mpf ' = 1/ s ' = 10mpf = 10 / s
28

Scattering
Scattering
g in Tissue
Tissue is composed of a mixture
of Rayleigh and Mie scattering
10 m

cells
nuclei

1 m

Mie Scattering
0 1 m
0.1

mitochondria
lysosomes, vesicles
striations in collagen fibrils
macromolecular aggreagates

Rayleigh Scattering
0.01 m

membranes
29

Scattering
Scattering in Tissue
Refractive
R f ti index
i d mismatch
i
t h
between lipid and
surrounding aqueous
medium
ed u

Soft tissues are dominated by

lipid contents
Celluar membranes, membrane
folds,, and membraneous
structure

Mitochondria, ~ 1m

Intracelluar organelle
composed of many folded
membrane, cristae

Collegan fibers, 2 ~ 3m

Collegan fibrils, 0.3 m

Periodic
P i di fl
fluctuation
t ti iin collegan
ll
ultrastructure source of
Rayleigh scattering in UV and
Visible range

Cells

30

Light Transport in Tissue

Scattering and absorption
occur simultaneously and
are wavelength dependent

t = s' + a
Scattering monotonically
decreases with
wavelength
Absorption is large in UV,
near visible,
visible and IR
Absorption is low in red
and NIR Therapeutic
window (600 1000 nm)

s ' = A

s ' ~ 0.5 4
31

Light Transport in Tissue

Modeling of light transport in
tissues are often governed by
the relative magnitudes of
optical
p
absorption
p
and
scattering
a >> s : Lambert-Beer Law
( 300nm;2000nm)
s >> a : Diffusion
Approximation (600nm ~
1000nm)
s ~ a : Equation of Radiative
Transfer, Monte Carlo (300nm
~ 600 nm; 1000nm ~ 2000nm)

Use Monte Carlo, Transport

Theory,
y, or Diffusion Theory
y

Physical Pathlength:
Optical Pathlength:

Lp
Lo

Biological Tissue
Lo/Lp = 4 or
32

Light Transport in Tissue

Modeling Photon Propagation

a, s, g, phase function S
Stochastic Description
p
33

Light Transport in Tissue

Radiative Transport
p
Theory
y
The direct application of EM theory is complicated
RTT deals with the transport of light energy
RTT ignores wave phenomena (polarization, interference) of EMT
ds

y State Radiative Transport

p
Equation
q
Steady

Loss due to
G 
G 
L r ,
scatt and abs
= ( a + s )L( r , s )
s
G  
G 
 
+ s p(s , s ') L( r , s ) d s ' + S( r , s )

( )

Overall Energy
balance at position
r and direction s

dA

Source
gain due to scattering
term
from s to s at r
L = radiance [W/m2 sr], propagation of photon power
P(s, s) = phase (scattering) function
s, s = directional vectors of photon propagation

34

Light Transport in Tissue

Diffusion Approximation
Simplified
S
f
form
f
off RTT at diffusion
ff
limit
s >> a
the number of photon undergoing the random walk is large

G
j( r , t ) / t << c( a + s ') = c t '

G 
1 G
3 G G

L( r , s , t )
( r , t ) +
j( r , t ) s
4
4
Isotropic source beyond 1/t

~10/t (~ 1mm in biological tissue)

far
f from
f
sources & boundaries
assume tissue is macroscopically homogeneous

G
1 ( r , t )
G
G
G
G
D(r ) ( r ) a ( r , t ) = S ( r , t )
c t
G
G
G
where D(r ) = 1/ 3[ a (r ) + s (r )]
35

Tissue Optical Properties

Measurement Strategies
g
Black Box
Optical
Source
input

TISSUE
H(a,
(
s))

Detector
output
p

H: System Function
Goal: To find out H(a, s)
Requires Non-Static
Non Static system Perturbations in either optical source
or tissue

36

Tissue Optical Properties

Measurement Schemes
CW (Continuous Wave) Measurement

Simplest
p
form of measurement
Static, continuous wave input
requires dynamic tissue property changes
E.g. pulse oximetery

Time-Resolved Measurements
Temporal changes in optical sources
Time Domain Photon Migration (TDPM)
Frequency Domain Photon Migration (FDPM)

Spatially-Resolved Measurement
Spatial changes in optical path
37

attenuation t-total

Tissue Optical Properties

arterial

t-oxy
pulsatile

venous (Hb-O2)
t-background
ti
tissue

time
t =

non-pulsatile

CW
(continuous
wave)
pulse
l oximetry
i t
locks into
pulse
healthy adult
calibration
accounts for
tissue scatter
(ms)
typically
t i ll att 2
wavelengths
(660, 940 nm)

a + s
38

Tissue Optical Properties

Time Domain Photon Migration
(TDPM)

Impulse
Function,

Directly measure ma and ms from

TPSF using Diffusion Equation
Complicated and expensive
detection system
rather low SNR
Temporal Point
Spread Function

(TPSF)

39

Tissue Optical Properties

Frequency Domain Photon Migration (FDPM)
SOURCE

TISSUE

DETECTED

stuff
happens

AMPLIT
TUDE

ACsrc AC
SRC

ACdet
ACDET

DCsrc
DC
SRC
DC
DCdet
DET

TIME

~ TIME
M = AC/DC

40