Origin of signals in tissue imaging

and spectroscopy

Andrew J. Berger
The Institute of Optics
University of Rochester
Rochester, NY 14627
A very brief outline

• Absorption
• Emission
• Scattering
Who are you? Why are you here?
(with apologies to Admiral Stockdale)

• experienced in some branch of optics

• biomedical not your main shtick
• interested in survey of fundamentals
• want introduction to applications
• interested in following the later talks
• want pointers to the literature
 Fred the photon
photons

absorption events

I (λ )
I 0 (λ )
Absorption = molecular transition between states

• electronic
• vibrational
• rotational
• (translational)
 Electronic transitions

What's quantized:
angular momentum = n ⋅ 
Consequently: energy 4
3
me 4  1 1 
∆E = ⋅ 2 − 2 
8ε 0 h 2 n n  2
 i f 

13.7 eV = 91 nm outer shell: n>1

1
Biologically: typically UV or blue
 Vibrational transitions

r
energy

What's quantized:

r − r0 ≈ 0.5A oscillator levels : ∆En→n +1 = ω
Representative values:
U ≈ 12 k ( r − r0 ) → k ~ 6 ×10 2 J/m 2
2
U ≈ 5 eV
µ = 6 amu (2 carbon nuclei)

r → ω = k µ = 2.5 × 1014 rad/sec

r0
λ = 6 µm
mid-IR
Rotational transitions

What's quantized:
angular momentum L2 = J ( J + 1)  2
Consequently:
2
∆E J → J +1 = 2 ⋅ J
µr
Representative values:
µ = 6 amu
r =1A 
≈ 2 × 10 −3 eV

λ0→1 ≈ 0.5 mm microwave regime

 How to talk about absorption
I0

L
I −ε cL −µa L
= 10 =e
I0

molar
µ a ≡ ln 10 ⋅ εc
extinction "absorption coefficient" [1/length]
concentration
 What's absorbing?

DNA

biological
window

rotational
vibrational
electronic

courtesy V. Venugopalan, http://www.osa.org/meetings/archives/2004/BIOMED/program/#educ

 Hemoglobin

courtesy V. Venugopalan, http://www.osa.org/meetings/archives/2004/BIOMED/program/#educ

 Typical tissue absorption!
adipose tissue ~ 1% blood
by volume

blood = 45% red

blood cells by volume red blood cell =
1/3 hemoglobin
by weight

Hemoglobin molecular weight

= 65,000 mg/mmole

Hb concentration = 23 µ M
 Hemoglobin

at isosbestic point,
µ a = 0.023 mM ⋅ 0.09 mm -1 / mM = 0.002 mm -1
Mean free absorption pathlength = 500 mm (!)
 Hemodynamics calculations
single µ a = ln 10 ⋅ εc
absorber :

 µ a1  ε1Hb
ε 1
 Hb
HbO2

 µ  = ln 10 ⋅ ε Hb  c 
two
absorbers :  a2   2 ε 2
HbO2
 c HbO2 
    
    
measure the look up the molar extinction calculate the
absorption coefficients (e.g. concentrations
coefficients http:/omlc.ogi.edu)

[ HbO2 ]
parameters
of interest :
oxygen saturation:
[ Hb] + [ HbO2 ] theory works
for N>2
total hemoglobin [ Hb] + [ HbO2 ] chromophores,
too!
Further adventures of Fred the photon

absorption
photons

fluorescence
Fluorescence: level diagram
• absorption: fsec
• internal conversion: fsec
• upper state lifetime: psec-nsec
• emission: fsec

shift is to the RED (Stokes) of the excitation light

r
r0
 Fluorescence Spectroscopy
Major biological fluorophores:
10

• structural proteins: collagen Tryptophan B

Fluorescence Intensity [a.u.]

Porphyrins (Hp)
and elastin crosslinks 8
Pyridoxine

• coenzymes for cellular energy

metabolism (electron 6 Collagen
acceptors):
• flavin adenine dinucleotide Elastin
NADH
Flavins
4
(FAD)
• nicotinamide adenine
dinucleotide, reduced form 2

(NADH)
• aromatic amino acids: side 0
groups on proteins 300 350 400 450 500 550 600 650 700

• porphyrins: precursors to Fluorescence emission wavelength [nm]

heme

Ref. Mycek and Pogue, Handbook of Biomedical Fluorescence courtesy M.-A. Mycek
 A fluorescence scenario

cellular epithelium
thickening

collagen support

healthy trending towards cancer

• increased FAD fluorescence

• reduced collagen fluorescence
(farther from surface)
• polyp formation → neovasculature;
increased absorption & decreased
fluorescence
 The time dimension
• absorption: fsec
• internal conversion: fsec
• upper state lifetime: psec-nsec
• emission: fsec

• radiative decay rate: kr

• nonradiative loss rate: knr

• knr varies with environment

• fluorescence decay lifetime
 1 
varies, too: τ =  
r  ∑k 
r0  
not intensity-based!

combined spectral and temporal fluorescence measurements:

Pitts and Mycek, Rev. Sci. Inst. 72:7, 3061-3072 (2001).
 More introductions to fluorescence

R. Redmond, "Introduction to fluorescence and

photophysics," in Handbook of Biomedical
Fluorescence (ed. Mycek and Pogue).
N. Ramanujam, "Fluorescence spectroscopy of
neoplastic and non-neoplastic tissues," Neoplasia,
2:1, 89-117 (2000).
 Yet more adventures for Fred

scattering
photons

Stokes
Anti-Stokes

Raman scattering
 Level diagram for Raman

incident photon has

energy

energy E

molecule gains
energy ∆ E

scattered photon has

energy E -∆ E
r0 r

excitation usually in near-IR or <300 nm UV to avoid visible fluorescence

 Basic mechanism of Raman scattering
cos ωt
cos Ωt

induced
dipole moment  ∂α 
: p = αE =  α 0 + ∆r0 cos Ωt  E0 cos ωt
 ∂r 0 

2 cos Ωt cos ωt = cos( ω − Ω ) t + cos( ω + Ω ) t

product
term :
STOKES ANTI-STOKES
 intensity (arb. units)

phenylalanine 619
guanine 667
adenine 720

RNA bases
cytosine, uracil 783
813
tyrosine 853
Typical

902

aromatic amino acids

phenylalanine 1005

1092
C-N, C-C str. 1127
Typical spectrum

1211
amide III 1259

1340

Raman shift (cm-1 )

spectrum (oral

C-H 2 def. 1457

1580

amide I 1651
(oral bacteria)
bacteria)
 Applications for Raman
• Chemical analysis of tissue, in vitro or in vivo (breast,
artery, blood)
• Disease classification
topical review: Hanlon et al., “Prospects for in vivo Raman
spectroscopy,” Phys. Med. Biol. 45, R1-R59 (2000)
(or just talk to me!)

• High-resolution, molecularly specific microscopy

go to: FWN4, “CARS microscopy: coming of age,” Sunney

Xie, 2:45-3:15.

FWN5, “Interferometric contrast between resonant

CARS and nonresonant four-wave mixing,” Daniel
Marks, 3:15-3:30.
Fred keeps going, and going, and...

scattering
photons

elastic scattering
 Elastic scattering
• caused by variations in refractive index
component typical n in the vis/NIR
extracellular fluid 1.35 – 1.36
cytoplasm 1.36 – 1.375
nucleus 1.38 – 1.41
mitochondria 1.38 – 1.41
water 1.33
Drezek et al., Appl. Opt. 38:16, 3651-3661 (1999).

• various approaches to modeling:

full rigor Maxwell’s equations (e.g. Drezek above)
Mie theory plane wave on homogeneous sphere
(e.g., code at philiplaven.com)
van de Hulst three-term approximation to Mie (larger spheres
and modest n values)
Rayleigh scattering very small particles (compared to λ)
 Wavelength dependence varies w/ scatterer size
Polystyrene Spheres of Varying Diameters in Water
0
10

)
-1
Mie Theory Scattering Coefficient (mm

2000 nm
-1
10 1000 nm
200 nm
100 nm
20 nm
-4
λ

500 600 700 800 900 1000 1100

Wavelength (nm)

courtesy Edward Hull, Rochester summer school lecture notes

 A summary of scattering scales

Figure by Steve Jacques,

Oregon Medical Laser Center
http://www.omlc.ogi.edu/classroom

go to: FTuL1, “On the microscopic origin of light scattering

in tissue,” Peter Kaplan, 2:00-2:30.
 Spectral dependence of scattering

sin ( 2δ λ )  sin ( δ λ ) 
2

n2 ~ 1− + 
δ λ  δ λ 
van de Hulst
n1 approximation to
sphere Mie theory
δ ≡ πd ( n2 − n1 )
d

n2 − n1
F sin 2 ( δ λ )
eva w e nal p t nedi c ni

~
1 + F sin 2 ( δ λ )
d/2 (F = cavity finesse)
etalon
 Spectral dependence of scattering

1-D etalon

• d=5 microns

• n1 = 1.36

• n2/n1 = 1.06
3-D sphere

wavelength / nm
 Scattering spectroscopy

δ1

 sin ( 2δ λ )  sin ( δ λ )  2  δ 2 > δ1

~ d 2 1 − +  
δ λ δ λ more rapid
   
oscillations

• spacing of peaks: size of scatterer

• depth of modulation: number of such scatterers

superposition of
mixture spectra
 broadband Scattering spectroscopy
polarized
illumination
polarization-
resolved detection
normal colon cells

cancerous cells

Perelman et al., Phys Rev Lett 80:627 (1998) and following.

 Angularly-resolved scattering
d
n1
n2

angular distribution
has interferometric
(oscillatory) behavior
as well

go to: FTuR1, “Real-time angle-resolved low-coherence

interferometry for detecting pre-cancerous cells,”
Adam Wax, 4:15-4:45.

FTuL4, “Elastic-scattering spectroscopy for cancer

detection: What have we learned from preliminary
clinical studies?” Irving Bigio, 3:00-3:30.
 Bulk tissue interrogation

µ s' µa
reduced scattering
coefficient [1/length]

• determine the absorption coefficient (spectroscopy)

• identify and characterize heterogeneities (functional imaging)
• note: scattering enables absorption studies in backscattering
geometry!
 Absolutely basic photon migration
in the
signal at detector decays
limit of:
according to
Detector
no
− µ a ct
scattering
e absorption

RMS distance from origin

(“random walk”)
increases according to
no
absorption 1
ct = Dct
pulse 3( µ s + µ a )
'

diffusion
scattering coefficient
[m2/sec]
 The real deal: diffusion theory
scattering and different source-detector separations
absorption
µ a = 0.001 mm-1
35 mm
µ s' = 1 mm-1
n = 1.4
25 mm

pulse r = 15 mm
 What are the diffusion measurements?
source(s) detector(s)

• time domain: intensity vs. time

• frequency domain (amplitude-modulation):
modulation depth and/or phase vs. distance or frequency
• steady state: intensity vs. distance

go to: FTuK1, “Multidimensional diffuse optical imaging in

breast cancer detection,” Brian Pogue, 2:00-2:30.

FTuK5, “Functional imaging by optical topography,”

Randall Barbour, 3:15-3:45.
 Still hungry?
• fluorescence: multiphoton-excited microscopy
• second-harmonic: ditto
• elastic scattering: optical coherence tomography,
laser scanning confocal microscopy
• polarization: surface-sensitive imaging, intrinsic
birefringence
• instrumentation: Raman fiber probes,
fluorescence excitation-emission matrices

Thanks to: Mary-Ann Mycek, Vasan Venugopalan, Edward Hull

Have a great rest of the conference!