osa2004tutorial

8/8/2019 OSA2004tutorial

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Origin of signals in tissue imaging

and spectroscopy

Andrew J. Berger

The Institute of Optics

University of RochesterRochester, NY 14627


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A very brief outline

Absorption

Emission

Scattering


3/38

Who are you? Why are you here?

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

(with apologies to Admiral Stockdale)


4/38

Fred the photon

absorption events

photons

)(0 I)(I

Absorption = molecular transition between states

electronic

vibrational

rotational

(translational)


5/38

Electronic transitions

energy

1

4

3

2

What's quantized: = nmomentumangularConsequently:

= 2220

4 11

8 fi nnh

meE

Biologically: typically UV or blue

13.7 eV = 91 nm outer shell: n>1


6/38

Vibrational transitions

en

er

gy

What's quantized:

= + :levelsoscillator 1nnE

r

0rr

A5.00 rr

eV5U

rad/sec105.2 14== k

Representative values:

22 J/m106~ k( ) 2021 rrkU

m6=

nuclei)carbon(2amu6=

mid-IR


7/38

Rotational transitions

What's quantized:

( ) 22 1momentumangular += JJLConsequently:

Jr

E JJ = + 22

1

A1

=r

eV102 3

amu6=

microwave regime

Representative values:

mm5.010


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How to talk about absorption

LcL aeI

I == 0

10

0I

I

L

molarextinction

concentration

ca 10ln"absorption coefficient" [1/length]


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What's absorbing?

DNA

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

electronic

vibrational

rotational

biologi c

al

window
http://www.osa.org/meetings/archives/2004/BIOMED/program/http://www.osa.org/meetings/archives/2004/BIOMED/program/


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Hemoglobin

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


11/38

Typical tissue absorption!

red blood cell =

1/3 hemoglobinby weight

adipose tissue ~ 1% bloodby volume

blood = 45% redblood cells by volume

Hemoglobin molecular weight= 65,000 mg/mmole

Hb concentration = 23 M


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Hemoglobin

at isosbestic point,-1-1 mm0.002mM/mm.090mM023.0 ==a

Mean free absorption pathlength = 500 mm (!)


13/38

Hemodynamics calculations

ca = 10ln

=

2

2

2

22

11

2

1

10lnHbO

HbHbOHb

HbOHb

a

a

c

c

measure theabsorptioncoefficients

look up the molar extinctioncoefficients (e.g.

http:/omlc.ogi.edu)

calculate theconcentrations

oxygen saturation:

total hemoglobin

[ ][ ] [ ]2

2

HbOHb

HbO

+

[ ] [ ]2HbOHb +

singleabsorber :

two

absorbers :

parametersof interest : theory works

for N>2chromophores,

too!


14/38

Further adventures of Fred the photon

photons

fluorescence

absorption


15/38

Fluorescence: level diagram

0rr

absorption: fsec internal conversion: fsec upper state lifetime: psec-nsec emission: fsec

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


16/38

Ref. Mycek and Pogue, Handbook of Biomedical Fluorescence

Fluorescence Spectroscopy

0

2

4

6

8

10

300 350 400 450 500 550 600 650 70

Fluorescenc

e

Intensity

[a.u.

]

Fluorescence emission wavelength [nm]

Porphyrins (Hp)

FlavinsElastin

Collagen

NADH

Tryptophan

Pyridoxine

B

courtesy M.-A. Mycek

Major biological fluorophores:

structural proteins:collagenand elastin crosslinks

coenzymes for cellular energymetabolism (electronacceptors):

flavin adenine dinucleotide(FAD) nicotinamide adeninedinucleotide, reduced form(NADH)

aromatic amino acids: side

groups on proteins porphyrins: precursors to

heme


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A fluorescence scenario

cellular epithelium

collagen support

healthy trending towards cancer

thickening

increased FAD fluorescence

reduced collagen fluorescence(farther from surface)

polyp formation neovasculature;increased absorption & decreasedfluorescence


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The time dimension

0rr

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

varies, too:

= k1

not intensity-based!

combined spectral and temporal fluorescence measurements:

Pitts and Mycek, Rev. Sci. Inst.72

:7, 3061-3072 (2001).


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More introductions to fluorescence

R. Redmond, "Introduction to fluorescence andphotophysics," in Handbook of BiomedicalFluorescence (ed. Mycek and Pogue).

N. Ramanujam, "Fluorescence spectroscopy ofneoplastic and non-neoplastic tissues," Neoplasia,2:1, 89-117 (2000).


20/38

Yet more adventures for Fred

photons

Raman scattering

scattering

Stokes

Anti-Stokes


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Level diagram for Raman

en

er

gy

0rr

molecule gains

energy E

scattered photon hasenergy E - E

incident photon hasenergy E

excitation usually in near-IR or


22/38

Basic mechanism of Raman scattering

tEtrr

Ep

coscos 000

0

+==

tcostcos

induceddipole moment:

( ) ( )tttt ++= coscoscoscos2product

term :

STOKES ANTI-STOKES


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Typical spectrum (oral bacteria)Typical spectrum (oral bacteria)

783 1

005

1457

1651

1092

1340

1259

1211

902

853

813

720

6676

19

1

580

1127

phenylalanine

guanine

adenine

c

ytosine

,uracil

p

henyla

lanine

C-H

2

def.

amideI

amideI I

I

C-N,C- C

str.

tyrosine

Raman shift (cm-1)

intensity

(arb.unit

s)

aromatic amino acids

RNA bases


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Applications for Raman

Chemical analysis of tissue, in vitro or in vivo (breast,artery, blood)

Disease classification

High-resolution, molecularly specific microscopy

topical review: Hanlon et al., Prospects for in vivo Ramanspectroscopy, Phys. Med. Biol. 45, R1-R59 (2000)

(or just talk to me!)

go to: FWN4,CARS microscopy: coming of age,SunneyXie, 2:45-3:15.

FWN5,Interferometric contrast between resonantCARS and nonresonant four-wave mixing,DanielMarks, 3:15-3:30.


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Fred keeps going, and going, and...

photons

elastic scattering

scattering


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Elastic scattering

caused by variations in refractive indexcomponent 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 Maxwells 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 )


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Polystyrene Spheres of Varying Diameters in Water

500 600 700 800 900 1000 1100

10-1

10 0

Wavelength (nm)

MieT

heoryScattering

Coefficien

t(mm

-1)

2000 nm

1000 nm

200 nm

100 nm

20 nm

-4

Wavelength dependence varies w/ scatterer sizeWavelength dependence varies w/ scatterer size

courtesy Edward Hull, Rochester summer school lecture notes


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A summary of scattering scales

igure by Steve Jacques,regon Medical Laser Centerttp://www.omlc.ogi.edu/classroom

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

in tissue,Peter Kaplan, 2:00-2:30.
http://www.omlc.ogi.edu/classroomhttp://omlc.ogi.edu/classroom/ece532/class3/gifs/fibrils.gifhttp://omlc.ogi.edu/classroom/ece532/class3/gifs/mitodrawing.gifhttp://www.omlc.ogi.edu/classroom


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Spectral dependence of scattering

( )12 nnd

( ) ( ) 2sin2sin1~

+

sphere

2n

1nvan de Hulst

approximation to

Mie theory

incid

entp

lanewave

etalon

( )( )22

sin1sin~F

F+

12 nn

(F = cavity finesse)d/2

d


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d=5 microns

n1 = 1.36

n2/n1 = 1.06

1-D etalon

3-D sphere

Spectral dependence of scattering

wavelength / nm


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Scattering spectroscopy

( ) ( )

+

2

2 sin2sin1~

d

spacing of peaks: size of scatterer depth of modulation: number of such scatterers

1

12 >more rapidoscillations

mixturesuperposition of

spectra


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Scattering spectroscopy

Perelman et al., Phys Rev Lett80:627 (1998) and following.

normal colon cells

cancerous cells

broadbandpolarized

illumination

polarization-resolved detection


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Angularly-resolved scattering

d

2n

1n

angular distributionhas interferometric(oscillatory) behavioras 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 cancerdetection: What have we learned from preliminaryclinical studies?Irving Bigio, 3:00-3:30.


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Bulk tissue interrogation

'

s a

determine the absorption coefficient (spectroscopy)

identify and characterize heterogeneities (functional imaging)

note: scattering enables absorption studies in backscattering

geometry!

reduced scatteringcoefficient [1/length]


35/38

Absolutely basic photon migration

ctae

Detector

signal at detector decaysaccording to

noscattering

in thelimit of:

absorption

( )Dctct

as

=+'31

pulse

RMS distance from origin(random walk)

increases according tono

absorption

scatteringdiffusion

coefficient

[m2/sec]


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The real deal: diffusion theory

different source-detector separations

r = 15 mm

25 mm

35 mm a = 0.001 mm

-1

s' = 1 mm-1

n = 1.4

pulse

scattering and

absorption


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What are the diffusion measurements?

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 inbreast cancer detection,Brian Pogue, 2:00-2:30.

FTuK5,Functional imaging by optical topography,

Randall Barbour, 3:15-3:45.

source(s) detector(s)


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Still hungry? fluorescence: multiphoton-excited microscopy

second-harmonic: ditto

elastic scattering: optical coherence tomography,laser scanning confocal microscopy

polarization: surface-sensitive imaging, intrinsicbirefringence

instrumentation: Raman fiber probes,fluorescence excitation-emission matrices

Have a great rest of the conference!

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

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