TISSUE OPTICS

Outline

What is Tissue Optics

Absorption in Tissue

Absorption Process and Parameters

Typical absorbers in Tissue

Scattering in Tissue

Scattering Process and Parameters

Typical scatterers in Tissue

What is Tissue Optics?

Definitions from dictionary

Optics:

a science that deals with

genesis and propagation of light
changes that it undergoes and produces

Tissue:

an aggregate of cells
usually of a particular kind together with their
intercellular substance that form one of the
structural materials of a plant or an animal

What is Tissue Optics?

Tissue
Subject of Interest

Optics

= Tissue Optics

Investigating Principle

We are interested in

Constituents of Tissue
Optical Properties of Tissue
Propagation of Light
Interaction between Light and Tissue
Diagnostic and Therapeutic Implications

Is Tissue Optics Special?

Three Major Components
Optical Source Sun
Medium - Atmosphere
Detector Human Eyes
Interaction between light and particles in atmosphere
- Absorption
- Scattering (Rayleigh & Mie)
Propagation of Light through Atmosphere

Optical Signal from Tissue

Large number of Biological Molecules

Functional and Structural information

Noninvasive

Near real time

Objectives of Tissue Optics

Key Question: How many photons per
second will reach the tissue chromophore
and be absorbed?

Absorption is important because it transfers

energy to tissue

1.

To find the light energy per unit area per unit time
that reaches a target chromophore at some
position

2.

To develop methods by the absorption and

scattering properties of tissue can be measured

Ultimate Goal of Tissue Optics

Assess all optical properties

noninvasive in living tissue (in vivo)
Difficulties

Tissue is a complicated heterogeneous

system
Requires noninvasive in vivo information
for meaningful diagnostic and therapeutic
purposes

ABSORPTION
in
TISSUE

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
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. pulseoximetry)

Therapeutic applications: Absorption of energy is the

primary mechanism that allows light form a source (laser) to
produce physical effects on tissue for treatment purpose

Lasik (Laser Assisted in situ Keratomileusis) Eye Surgery, Tatoo

Removal, PDT

Absorption (quantum view)

Absorption occurs when the photon frequency matches the

frequency associated with the molecules energy transition

The absorption of a photon results in:

quantized change in charge separation
quantized excitation of vibrational modes

Metrics for Absorption

Absorption Cross-section, s [m2]

Consider a chromophore idealized as a sphere with a

particular geometrical size. Consider that this sphere blocks
incident light and casts a shadow, which constitutes
absorption.
The size of absorption shadow = absorption cross-section

sa Qa A

Qa: absorption
efficiency

Metrics for Absorption

Pabs = Iosa

Pin =IoA

Pout = Io(A-sa)

Outgoing Beam

Incident Beam
Area =A

Area = sa

Pabs
sa
Io

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

Metrics for Absorption

Assumptions

Cross section is independent of relative orientation of the

impinging light and absorber
uniform distribution of identical absorbing particles

Absorption Coefficient, ma [1/m]

m a N a sa
Absorption cross-sectional area per unit volume of
medium
Absorption mean free path, la [m]

1
la
ma

Represents the average distance a photon travels before

being absorbed

Absorption Fundamentals

Transmission and Absorbance (macroscopic view)

I
T
Io

Transmission

Absorbance (attenuation, or optical density)

Io
A log( T) log
I

Connection between T/A and ma

Now, absorbing medium is characterized by

ma, transmission, and absorbance. Are they
related?

Lambert Beer Law: the linear relationship

between absorbance and concentration of an
absorbing species.

Lambert-Beer Law

s= absorption cross-sectional area = s a

Pabs
[cm2]
IO

IO = The intensity entering the sample at z = 0 [w/cm2]

I = The intensity of light leaving the sample
IZ = The intensity entering the infinitesimal slab at Z
dI = the intensity absorbed in the slab

Lambert-Beer Law
Total opaque area on the slab
due to absorbers

s a N a A dz

Number of absorbers
in the slab volume

Loss of intensity

dI
sa N a dz

Fraction of photons
absorbed

Io

b
dI
s a N a dz
0
I( z )

I
ln s a N a b
Io

Lambert-Beer Law
I
ln s a N a b
Io

Since
and

3
1mol
molec
1000cm
mol

3
23

liter
cm 6.023x10 molec liter
log( x )
ln( x )
2.303 log( x )
log( e)

I
I 6.023x10 20
1
A log
ln
s a c b c b
I
2
.
303
I
2
.
303
o
o

, Molar Extinction Coefficient [cm-1M-1]

Measure of Absorbing Power of species

I
cb
A
T 10
10
Io

Lambert-Beer Law
I
I
1
c b log
ln
2.303 I o
IO
1

s a Na b
2.303

s a N a 2.303 c

ma = 2.303c
(1) By measuring Transmission or Absorbance for given M, we can obtain
usually ex vivo
(2) With knowledge of , if we can measure ma in vivo, we can quantify
concentration of chromophores

Absorbers in Tissue

VISIBLE

NIR

UV-VIS

NIR

UV

Hemoglobin
Lipids
Water

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

* Absorbers that fluoresce when excited in the UV-VIS

UV Absorption

Protein, amino acid, fatty acid

and DNA absorption
dominate UV absorption

Protein is dominant non-water

constituent of all soft tissue, ~
30%
Absorption properties
determined by peptide bonds
and amino acid residues
Peptide excitation about l =
190 nm
amino acids absorption at l =
210 - 220 nm and 260 280
nm
DNA absorbs radiation for l
320 nm

Large water absorption l < 180 nm

Amino Acid

Peptide

Infrared Absorption

Protein IR absorption
peaks at 6.1, 6.45, and 8.3
mm due to amide excitation

Absorption depth 10 mm in
l 6 7 mm region

Water absorption peak at

0.96, 1.44, 1.95, 2.94 and
6.1mm

Absorption depth
~ 500 mm at l = 800 nm,
<1 mm at l=2.94 mm
20 mm throughout l 6
mm

Respiratory Enzymes: NADH, FAD,

Cytochrome a3
These enzymes play a key role in providing the proton-motive force
necessary for oxidative phosphorylation
If tissue is oxygen starved, [NADH] and [FADH2] will be enhanced
Reduced NADH conc. is indicative of high oxygen consumption and is
characteristic of tumor tissue
NADH

FAD

NADH (FAD) strongly fluoresce while NAD+ (FADH2) does not

Cytochrome a3 has a prominent absorption peak at l = 840 nm

Visible and NIR Absorption

Main Absorbers at visible and NIR
Hemoglobin
Lipid

Extinction Coeff (1/cm M)

10

10

10

Hb

Hemoglobin

10

HbO2
2

10

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 well as metabolic activity

Hemoglobin

Deoxyhemoglobin has lower absorption than

oxyhemoglobin in the blue and green

Absorption peaks for HbO2

418, 542, 577, and 925 nm

Absorption peaks for Hb

Bright red arterial blood (blushing)

Bluish venous blood (cold pale face)

550, 758, 910 nm

Isosbestic points

547, 569, 586, and 798 nm

Lipid (Fat)

3.0

0.06

Water

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
600

700

800

900

WAVELENGTH (nm)

1000

WATER & FAT (1/ mm mM)

Important energy store in the

body
Site-specific measurements
of body composition
Monitoring of physiological
changes in female breast
tissue
Tissue layer model

HEMOGLOBIN (1/mm mM)

Summary - Absorber

UV

Protein
Amino acid
Fatty Acid
Peptide
DNA
NADH
FAD
Water

Visible & NIR

Hemoglobin
Lipid
Cytochrome a3
Therapeutic Window
600 nm ~ 1000 nm

IR
Water
Protein
Glucose

SCATTERING
in
TISSUE

Scattering in Tissue

Much more complicated than absorption

Light is hardly observed from the source, but
reaches our eyes indirectly through scattering
Inhomogeneity causes scattering; cloud,
raindrop, etc
Elastic (Rayleigh, Mie) or inelastic (raman)

Scattering in Tissue

Diagnostic applications: Scattering depends on the size,

morphology, and structure of the components in tissues (e.g.
lipid membrane, collagen fibers, nuclei). Variations in these
components due to disease would affect scattering
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 therapy

Scattering - Example
Purely absorbing
Photon pathlength = L

With Scattering
Photon pathlength >> L

Lambert- Beer Law does not apply here!!!

Need to calculate true pathlength of light

Scattering Blue sky revisited

Blue skies are produced due to scattering
at shorter wavelengths
Visible light (violet & blue) are selectively
scattered by O2 and N2 much smaller
than wavelengths of the light
violet and blue light has been scattered
over and over again

When light encounters larger particles (cloud, fog), Mie

scattering occurs
Mie scattering is not wavelength dependent appears
white

Mechanism for Light Scattering

Light scattering arises from the presence of heterogeneities

within a bulk medium

Physical inclusions
Fluctuations in dielectric constant from random thermal motion

Heterogeneity/fluctuations result in 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

Elastic vs. Inelastic Scattering

Elastic scattering: no energy change

Frequency of the scattered wave = frequency of

incident wave
Probes static structure of material
Rayleigh and Mie scattering

Inelastic scattering: energy change

Frequency of the scattered wave frequency of

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

Elastic Scattering

The light scattered by a system has

interacted with the inhomogeneities of the
system
Photons are mostly scattered by the structure
whose size matches the wavelength
Principal parameters that affect scattering

Wavelength, l
Relative refractive index
Particle radius
Shape and orientation

Two types of scattering: Rayleigh and Mie

Rayleigh Scattering

Light
Source

Detector

Scattering from very small particles l/10

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

1
I 4
l

l is the wavelength of the incident light

I is the intensity of the scattered light

Mie Scattering

For scattering of particles comparable or

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

Scattering in Tissue (I)

Tissue is composed of a mixture of Rayleigh

and Mie scattering
Hierarchy of ultrastructure
10 mm

cells

nuclei

* TiO2 : 0.2 ~ 2 mm
1 mm

Mie Scattering
0.1 mm

mitochondria
lysosomes, vesicles

striations in collagen fibrils

macromolecular aggregates

Rayleigh Scattering
0.01 mm

membranes

Source of Scattering in Tissue

Refractive index mismatch between lipid and surrounding

aqueous medium

Mitochondria, ~ 1mm

Intracelluar organelle composed of many folded membrane, cristae

Collegan fibers, 2 ~ 3mm

Soft tissues are dominated by lipid contents

Celluar membranes, membrane folds, and membraneous structure

Collegan fibrils, 0.3 mm

Periodic fluctuation in collegan ultrastructure source of Rayleigh
scattering in UV and Visible range

Cells

Metrics for Optical Scattering

Pin =IoA

Pscatt = Ioss

Outgoing Beam

Incident Beam
Scattering Cross Section,

Pout = Io(A-ss)

scatt

[m2]

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

The measured reduction of light

sscatt = 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]

Metrics of Optical Scattering

Scattering Coefficient, ms [1/m]

ms =Nsss ,

Ns = the number density of scatterers

ss = scattering efficiency
Cross-sectional area for scattering per unit volume of
medium

Scattering Mean Free Path, ls

Average distance a photon travels between scattering

events

1
ls
ms

Anisotropy, g

Imagine that a photon is

scattered by a particle so
that its trajectory is
deflected by an angle, q
Then, component of a
new trajectory aligned
forward direction is
Incidenet
cos(q)
Photon
Anisotropy is a measure
of forward direction
retained after a single
scattering event, <
cos(q)>

scattered
photon

Scatterer

hv

Photon
trajectory

dW
S

hv
Scattering
Angle (q)

cos (q)
Scattering
event

Anisotropy factor, g
1

g0
1

totally backward scattering

isotropic scattering
totally forward scattering

Biological Tissues, 0.65 < g <0.95

Reduced Scattering Coefficient, ms [1/m]
ms incorporates the scattering coefficient, ms and the anisotropy
factor, g

ms ' (1 g)ms

ms can be regarded as an effective isotropic scattering

coefficient that represent the cumulative effect of several forwardscattering events
Special significant with respect to photon diffusion theory

Reduced Scattering Coefficient

g cos q 0.90
Useful for description of
photon propagation in
diffuse Regime

q 26 o
m s ' (1 g )m s 0.10m s
mpf 1 / m s
mpf ' 1 / m s '

1 iso-scatt step
=1/(1-g)
aniso-scatt.
steps

Each step involves isotropic

scattering.
Such a description is equivalent to
mpf ' 1 / m s ' description of photon movement
using many small steps 1/s that
each involve only a partial
deflection angle

Light Transport in Tissue

Scattering and absorption occur simultaneously and are wavelength

dependent
m s ' A l b
Scattering monotonically decreases with wavelength
m s ' ~ l0.5 l4
Absorption is large in UV, near visible, and IR
Absorption is low in red and NIR Therapeutic window (600 1000 nm)

Light Transport in Tissue

Modeling of light transport in tissues are often governed by

the relative magnitudes of optical absorption and scattering

ma >> ms : Lambert-Beer Law (l 300nm;l2000nm)

ms >> ma : Diffusion Approximation (600nm ~ 1000nm)
ms ~ ma : Equation of Radiative Transfer, Monte Carlo (300nm ~ 600
nm; 1000nm ~ 2000nm)

Propagation of Photons through Tissue

Scattering and absorbing tissue

Absorption Coefficient: ma
Scattering Coefficient: ms
Physical Pathlength: Lp
Optical Pathlength:
Lo

Biological Tissue
Lo/Lp = 4 or
Use Monte Carlo, Transport Theory, or Diffusion Theory !!!!

Modeling Photon Propagation

ma, ms, g, phase function S

Stochastic Description

Radiative Transport Theory

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

Steady State Radiative Transport Equation

Loss due to


L r , W
scatt and abs
m a m s Lr , s
s



m s ps , s ' Lr , s d s ' Sr , s

Overall Energy
balance at position
r and direction s

dA
S

gain due to scattering

from s to s at r

Source
term

L = radiance [W/m2 sr], propagation of photon power

P(s, s) = phase (scattering) function
s, s = directional vectors of photon propagation

Diffusion Approximation

Simplified form of RTT at diffusion limit

ms >> ma
the number of photon undergoing the random walk is large

j( r , t ) / t cma ms ' cm t '

(~ 200 GHz)

1
3

L( r , s , t )
( r , t )
j( r , t ) s
4
4

Isotropic source beyond 1/mt

~10/mt (~ 1mm in biological tissue)
far from sources & boundaries
assume tissue is macroscopically homogeneous

1 r , t
D( r )r m a r , t Sr , t
c t

where D( r ) 1 / 3m a ( r ) m s ( r )

Measurement Strategies
Black Box

Optical
Source
input

TISSUE
H(ma, ms)

Detector
output

H: System Function

Goal: To find out H(ma, ms)

Requires Non-Static system
Perturbations in either optical source or
tissue

Measurement Schemes

CW (Continuous Wave) Measurement

Simplest form of measurement

Static, continuous wave input
requires dynamic tissue property changes
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

TDPM (II)
Directly measure ma and ms from TPSF using
Diffusion Equation
Complicated and expensive detection system
rather low SNR
Temporal Point Spread Function

(TPSF)

FDPM
SOURCE

TISSUE

DETECTED

stuff happens

AMPLITUDE

ACsrc ACSRC

ACdet
ACDET

DCSRC
DC
src
DC
DC det

DET

TIME

~ TIME
M = AC/DC