Lighting up Human Tissue to Detect Tumors

Asima Pradhan

Dept. Of Physics

& Centre for Laser Technology

Indian Institute of Technology, Kanpur REACH 2010gy, p REACH 2010IIT Kanpur10.10.10

Working of biological system is complicated

That the human body works with such precision as it does amazes scientistsThat the human body works with such precision as it does, amazes scientists even today when technology has advanced so much.

The precision lies at both macro-structural and micro-biochemical levels. At the micro level, the proteins occupy an important place in maintaining the functioning of cells and thus the entire system.

The basis of our research lies in extracting the molecular and subtle morphological characteristics of cancer.

Diagnosis of disease is increasingly becoming a technological task.

Cli i i ’ lClinician’s goal:

To access structural and functional changes in

diseased tissue.

Infer the identity and stage of disease.

Ultimate prognosis.

Some relevant statistics on cancer in India

Cancer cases: >25 lakh per year

Death rate 4 lakhs per year

#2 largest cause of mortality

Increasing at the rate of 11% peryear

Every day 2000 people gets affectedby mouth cancery

Highest cases:

Lung cancer in meng

Breast cancer in female

40% of total cancer deaths in

3

women: Breast and Cervical

X-ray computed tomographyIonizing radiation: harmful if used too often for routine screening.

Ultrasound

Lacks resolution to detect objects in sub millimeter scale. May not provide information about tissue chemistryinformation about tissue chemistry

• MRI: Sub millimeter spatial resolutionAbility to detect specific chemicalsCost of equipment makes its operation expensiveCost of equipment makes its operation expensive.

(Adverse effect of strong magnetic field on biological systems is still a topic of debate. People use 1.5 T to 3T)

•Pathology

This high power microscopic view demonstrates intraductal carcinoma. Neoplastic cells are still within the ductules and have not broken through into the

stroma. Note that the two large lobules in the center contain microcalcifications. Such microcalcifications can appear on mammography.

Pathology

provides the most widely used clinical method to obtain chemical information from diseased tissues.

Hi t l ( di i l h i ) b i i l l i d• Histology (traditional technique) probe microscopic structural alterations due to disease

Major drawback

• Can only be applied in-vitro and takes time.

Necessitates removal of tissue/biopsy hence a limited to probing small areas only.

OPTICAL SPECTROSCOPY AND IMAGING

• A non-invasive, • safe, ,• inexpensive, • compact techniquep q

capable of extracting diagnostically relevant structural and biochemical information is the need of hour!!!

• Diagnostics techniques based on optical spectroscopy offer such possibilities.p

• Fluorescence• Scattering

Ab i• Absorption

Essential to Study Essential to Study

Light Tissue InteractionLight Tissue Interaction

TISSUE OPTICSTISSUE OPTICS

Light-Tissue Interaction

Ab ti & S tt i

Elastic scattering Inelastic scattering

Absorption & Scattering

Tissue

Diagnostics

Imaging

Fluorescence & Raman

Diagnostics

Transmitted Light

7

Basic Aim: Detect Early Stages of Diseasei ti l tin particular, tumors

Increase in size of nucleus Changes in elastic scatteringg gCells packed

Increase in metabolic activity Biochemical changesIncrease in metabolic activity Biochemical changesIncrease in concentration of related bio-moleculesBond breaking (cross links) Fluorescence yield changes

Bonds change Raman scattering changes

Blood concentration changes Absorption changesGrowth of blood vessels

Wavelength of the Light Source

Penetration depth of laser light in soft non-colored tissue as a function of wavelength

KrF XeCl Dye Argon Diode Nd:YAG Tm:YAG Ho:YAG Er:YAG CO2248 308 465 514.5 830 1064 2010 2100 2940 10600

5 µm 40

20 µm

1 µm

µµm

250 µm

150 µm

1300 µm 300-400

µm

1400 µm 1400

µm

El ti tt i t d b

SCATTERING IN TISSUES

Elastic scattering events caused by

random spatial variations in density

refractive indexµs = 20-200 mm-1

refractive index

of extra cellular, cellular and sub cellular components.

Described by Mie theory: size ~ wavelength

Tissue scattering decreases monotonically with increasing wavelength

Change in scattering coefficient implies change in size/concentration.

Using LSS: Only morphological information

Absorption in Tissues

•Strongly wavelength dependent

• Tends to generally decrease with wavelength.

• Typical values of µa :

0 1 to 10 000 cm-1 from NIR to UV0.1 to 10,000 cm 1 from NIR to UV.

• Absorption in tissue in UV, visible and IR

Therapeutic Window

700-1000nmregions primarily from

proteins, hemoglobin, water

Coenzymes NADH, FAD and FMN:

Biochemical Aspects (Fluorescence)Fluorophores in Tissue

300-700nmCoenzymes NADH, FAD and FMN:

metabolic activity

Malignancy: Impaired mitochondrial metabolic • Hemoglobin

• Beta Caroteneg y pactivity.

Change in concentration

• Beta-Carotene

• Structural Proteins

CollagenResult:

Change in measured fluorescence from tissue.

C b t b li t t f ti

Collagen

Elastin

• Electron CarriersCan probe metabolic status of tissue

Structural proteins: Collagen , Elastin

Carriers

NADH

FAD

Malignancy : cross-links broken fluorescence quenched

• Amino acids

• Tryptophan

• Tyrosine

Can probe early stages of cancer

Fluorescence Spectra of Endogenous Tissue Fluorophores

Reproduced from Stephen Webb, PhD thesis, University of London, 2003

Developments

Late 1980’s Alfano et.al., Feld et.al pioneered in the field of cancer diagnosis

1990’s First Generation task: In vitro experiments

Diff t h b d ti l i i lDifferent approaches based on optical principles:

Fluorescence Spectroscopy

Elastic Scattering SpectroscopyElastic Scattering Spectroscopy

Raman Spectroscopy

Absorption Spectroscopyp p py

Optical Coherence Tomography(OCT)

Developments….

2000… Second Generation task: bring technology to clinics

Evaluate potential for in vivo diagnosis

Novel approaches:

Two photon fluorescence

Photoacoustic Spectroscopy

M ll I iMueller Imaging

Confocal Imaging

Current Issues Improve diagnostic capabilities

Gain biochemical information

Nano Bio Photonics

Fluorescence from Human Breast Tissue

200 normal

pericanal mal FAD Benign Normal

.u)

120

160

p

intracanal

mixed

others

malig

INTE

NSI

TY (a

80

120

FWH

M(n

m)

500 550 600 650 7000

Fluorescence with point measurements: Average conribution from bulk tissue. Thus, multiple scattering effects play major role.

0 10 20 30 40 500

40

500 550 600 650 700

WAVELENGTH (nm) NO.OF SAMPLES

Sensitivity 95%

Specificity 92%

Differences in width of spectra:

Role of two fluorophores

Various Approaches for Analysis

Large Amount of spectral data and large number of samplesrequired a proper statistics based algorithm for analysis .

Advantage: Entire spectral information content may be exploited.

Common Technique: Principal Component Analysis (PCA)

Recent Advancement: Wavelet analysis

Obstacles in using Static Auto-Fluorescence Spectra for Diagnosis of Cancerg

Modulation of Fluorescence by wavelength

dependent absorption & scatteringdependent absorption & scattering

properties of tissue

Biochemical information masked

Large site to site variation

•Valuable biochemical information on tissuefluorophores may not be retrieved

I t i i fl h tIntrinsic fluorescence has tobe extracted!!

Approach used by us for extraction of Intrinsic Fluorescence

A. Polarized Fluorescence & polarized elastic scattering measurement based approach

A purely experimental approach p y p pp

B. Spatially resolved fluorescence measurementp y f

Depth information of inhomogeneityDepth information of inhomogeneity

Light Propagation in Tissueg p g

Ballistic

SnakeSnake

Underlying PrincipleUnderlying PrincipleDominant Depolarizationcontribution:

Incident polarized lightCo-polarized backscattered lightReduced comp. Cross-polarized contribution:

Multiple scattering of lightbackscattered light

Diffuse comp.I I⊥

Polarized component of thedetected fluorescence

[I- I⊥ ]f f

Extracts contributions whichhave not undergone significantg gscattering in tissue

Therefore originates fromTherefore originates fromsuperficial layer of tissue

B.V. Laxmi et al, Lasers in the Life Sci., 9, 229-243, (2001)

Underlying Principle (Cont…)U de y g c p e (Co …)

• Fluorescence detected in d l i d

Cross-polarized backscattered light

Incident polarized light

Co polarized backscattered lightcrossed polarized channel

(I )

gDiffuse comp.

Co-polarized backscattered lightReduced comp.

I I⊥

(I⊥)

• Contribution of moreContribution of more multiply scattered photons

• Hence originates from Biswal et al, Optics Express, 11, 3320 –3321 (2003)Nidhi Agarwal et al IEEE JSTQE (2003)deeper tissue layer Nidhi Agarwal,et al, IEEE JSTQE (2003)Sharad Gupta et al, JBO (2005)Anita et al, JBO (2008)

(a) Co state. (b) Cross state. (c) Co-Cross state.

PRE 2005JOSA A 2007

Underlying Principle (Cont…)U de y g c p e (Co …)

• Remove scattering effects:g ffdifference in co and cross polarized light

R b ti ff t• Remove absorption effects: fluorescent light /elastically scattered light

• Assumption:

Wavelength dependent scattering & absorption :Similar effects on polarized component of fluorescence & polarized component of elastic scattering spectrapolarized component of elastic scattering spectra

Optics Express, 2003.

Example :

Scheme

Assumption:Wavelength dependent scattering & absorption : similar effects on polarized component of fluorescence & polarized component of elastic scattering spectra

Modification is made so as to eliminate modulation due to blood

A systematic study on phantoms was also performed to validate this scheme

Where is this scheme applicable?Where is this scheme applicable?

• Layered Tissuey• Superficial tumors

Densely packedDensely packed epithelium, NADH increase

C llBasal layer

Basal layer

Collagen cross-links break in stroma

(a)Microscopic images of epithelial layer of (a) normal and (b) dysplastic state of cervix tissue

Intrinsic Fluorescence from Human Cervical Tissue

0 8

1.0

ores

cenc

e

Normal0.8

1.0

uore

scen

ce

NormalI t i i

0 2

0.4

0.6

0.8

mal

ized

ave

rage

flur

oin

tens

ity (a

.u.)

Dysplasia

Co-polarized0.2

0.4

0.6

mal

ized

ave

rage

flu

inte

nsity

(a.u

.)

DysplasiaIntrinsic

350 400 450 500 550 600 650

0.0

0.2

Peak

Nor

m

λ (nm)350 400 450 500 550 600 650

0.0

Peak

nor

m

λ (nm)

Dysplastic tissues : Increase in NADH fluorescenceSensitivity 74%Sensitivity 74%

Intrinsic fluorescence : enhanced discrimination between normaland dysplastic tissues as compared to the co polarized caseand dysplastic tissues as compared to the co-polarized case.

NADH : more reliable discriminating parameter …SPIE 2008

COVARIANCE MATRIXusing Principal Component Analysis

S i h λ 350

Data = Normalized Spectra( i (i) (V l (i) M l )

Spectra with λexc = 350nm

(variance (i)= (Value(i) – Mean value);Covariance Matrix = (DataT Data)/(n-1)

Fluorescence Imaging

Mueller matrix imaging in human cervical tissues

Mueller matrix imaging in human cervical tissueElastic Scattering based measurements

Mueller Matrix M11 M12 M13 M14M11 M12 M13 M14S

1/

S /

S1S

S /S /

Elastic Scattering based measurements

Contains complete polarizationinformation of the medium

It is a blue print for scattering

M21 M22 M23 M24

M31 M32 M33 M34

M41 M42 M43 M44

M21 M22 M23 M24

M31 M32 M33 M34

M41 M42 M43 M44

S2

/

S3

/

S4

/

S2

S3

S4

=S

2 /

S3

/

S4

/

S2

/

S3

/

S4

/

It is a blue print for scatteringmedia

Emerging Stoke’s vector

Incident Stoke’s vectorMueller Matrix

Emerging Stoke’s vector

1200µ1200µ

Mueller image M00 microscopic image

Polar Decomposition of Mueller Matrix

M = M∆MRMDM = M∆MRMD∆ R D

DiattenuationDepolarization

∆ R D

DiattenuationDepolarization RetardanceDepolarization

•Multiplescattering •Differential attenuation

RetardanceDepolarization

•Multiplescattering •Differential attenuation Multiple scattering•Linear & Circularretardance

ff(absorption & scattering)

Multiple scattering•Linear & Circularretardance

ff(absorption & scattering)

1200µ 1200µ1200µ

r

20

25

0.6

0.7

er

20

250.6

0.7

Normal epithelium

Dysplastic epithelium of cervixP

ixel

num

be10

15

0 3

0.4

0.5

Pix

el n

umb

10

15

0.3

0.4

0.5

pof cervix

Pixel number

20 40 60 80

50.2

0.3

Pixel number

20 40 60 80

50.2180µ180µ40µ 40µ

(a (b

Depolarization power images

Pixel number e u be()

()

Normal epithelium

Dysplastic epithelium f i

1000µ

pof cervix

of cervix

Basal B l l

40µ

40µ

Microscope images

layer Basal layer180µ 180µ

er

60

80

0.14

0.16

r

60

80

0.14

0.16

Normal Dysplastic t f

Pix

el n

umbe

20

40

0.08

0.1

0.12

Pie

l num

be

20

40

0.08

0.1

0.12

stroma of cervix

stroma of cervix

Pixel number

20 40 60 80

20

0.04

0.06

Pixel number

20 40 60 80

20

0.04

0.06180µ 180µ

Pixel number e u be

Retardance images

180µ180µ 180µ180µ

Microscope images Optics Express, Vol.17,(3) 2009

(a) (b)

Depolarization power: sensitive to morphological changes during progressionfrom normal to dysplastic state.

Retardance reveals the morphological changes around the stromal region.

Current Status: Automating and increasing data bank for use asCurrent Status: Automating and increasing data bank for use as supplementary tool in clinics

ConclusionsF t f i t i i fl t di ti t l ll tFuture of intrinsic fluorescence spectroscopy as a diagnostic tool as well as to extract biochemical information looks bright. Some refinement is to be done before this technique may be used to extract quantitative information.

Several other light-based tools such as LSS, Raman spectroscopy & OCTwith their individual strengths and weaknesses relative to fluorescence are being used at pre-clinical/clinical stages

Such approaches may not be competing but complementary tools and most importantly, concurrence with histopathological results are important

Mueller Imaging technique can be used as a supplementary technique to the ‘gold standard’ histopathology.

Great deal of clinical / pre-clinical research remains to be done to move these techniques into routine clinical practice

Lastly, focusing on the goal to use optical biopsy in-situ should not detract researchers from trying to understand the biochemical basis of disease through such optical means…

Acknowledgments

• Department of Information Technology (Photonics) [MCIT] Dr Sharad Gupta(Photonics) [MCIT]

• CSIR

• BRNS

Dr. Sharad Gupta

Dr. Maya Nair

Dr.Prashant Shukla

• IIT Kanpur

• Dr.Asha Agarwal (Professor in Pathology, GSVM Medical College)

Jaidip Jagtap

Sridhar RajaGSV edical College)

• Dr. Kiran Pandey (Professor and Head of Gyn and Obstr, GSVM Medical College)

• Dr Nirmalya Ghosh (Univ of Toronto)

Rajbeer Singh

Dharitri Rath

Krishna Kumar Tomar• Dr. Nirmalya Ghosh (Univ. of Toronto) Krishna Kumar Tomar

Meghdoot Mazumdar

Physical Modeling

Takes into account spectral characteristics of contributing fluorophores

ys ca ode g

Takes into account spectral characteristics of contributing fluorophores such as

•intensity,

•peakshift,

•bandwidth

for discriminationfor discrimination

Advantage: Offers insight into biochemical aspects of tissue.

Our study

Flavins

PorphyrinsTo differentiate spectra were fitted to Voigt function.

27 September 2010 Last updated at 00:37 GMT Share this page

Painless laser device could spot early signs of diseasePainless laser device could spot early signs of disease

By Katia Moskvitch

Michael Morris, a chemistry professor at the University of Michigan, US, hasat the University of Michigan, US, has

been using Raman for the past few years to study human bones

Reason for the experimental observation

Normalization of unpolarized fluorescence by unpolarized elastic scattering spectra cannot recover intrinsic fluorescence intensity information

fPropagation path of elasticallyscattered photon

Propagation path of fluorescence photon

λEM λEX λEM

λEX λEX λEMλEMEMλEMEM

Major difference in the survival of the long path photons

Results in the differential effect of absorption & scattering on unpolarizedResults in the differential effect of absorption & scattering on unpolarizedfluorescence spectra and elastic scattering spectra

British researchers at the Rutherford Appleton Laboratory in Didcot and at the Gloucestershire Royal Hospital have dco d e G ouces e s e oy osp ve

been using Raman to analyse calcifications in breast tissue that might be early signs of cancer.

We could target those calcifications and make a decision about whether

they're benign or malignant," Nicholas Stone, head of the

biophotonics research unit at the Gloucestershire Royal Hospital told

the magazine Chemical and Engineering News

IncidentInelastic scattering (Fluorescence, Raman)

I t iti d d O ti l ti f ti h fl ti it tt iElastic scattering Intensities depend on Optical properties of tissue such as reflectivity, scatteringcoefficient, particle size, optical homogeneity, absorption coefficient etc.

Absorbed portion of light produces:

Transmitted

Absorbed portion of light produces:

· Photochemical effect

· Thermal effect

· Inelastic scattering (Fluorescence, Raman)

Depending on λ and nature of tissue

IR and visible lasers generally produce only thermal effects

UV laser: both thermal and photochemical

The extracted polarization parametersThe extracted polarization parameters

Fibrous structure & their orientation in tissue

Linear retardance

Circular retardance (Optical rotation)Dichroic absorption (orscattering) due to presence of

Concentration of chiral substances like glucose in

tissue

Total retardance R

Depolarization ∆

g) poriented structures Conjugate effect of linear &

circular retardance

Multiple scattering effects( i i f i

Diattenuation D(concentration, size, refractiveindices of scatterers present intissue)

Decomposition Procedure

STEP I: Diattenuation Vector

M11 M12 M13 M14

Experimental Mueller matrix

D = (M12 M13 M14 )T / M11

1 DT

D mMD =

M21 M22 M23 M24

M31 M32 M33 M34

M41 M42 M43 M44 (1-D2)1/2 I + (1-(1-D2)1/2DDT∧∧

D mD

STEP II :

D ={1 / M11} × [M122 + M13

2 + M142]1/2

M/= M MD-1Diattenuation M/=M∆ MR

Diattenuation Parameter

M M MDfree matrix∆ R

1 0P m

1 00 mR

1 0P∆ m/1 0 0 0

M∆

Ci l

Linear

P∆ m∆0 mR

(P-mD)/(1-D2)

P∆ m

m∆= ±[m’(m’)T +((λ1λ2)1/2 + (λ2λ3)1/2+ (λ3λ1)1/2)I]-1

P∆(1) ∆1 0 0

P∆(2) 0 ∆2 0

P∆(3) 0 0 ∆3

Circular

∆ [ ( ) (( 1 2) ( 2 3) ( 3 1) ) ]

× [(λ11/2+ λ2

1/2 +λ31/2)m’(m’)T +(λ1λ2λ3)1/2I]

∆ = 1- {tr (M∆) - 1/ 3}Net Depolarization Index

Major absorbers in tissue

WaterW e

In the UV, the absorption increases with shorter wavelength due to protein, DNA and othermolecules.

In the visible, the major absorber is different forms of hemoglobin present in tissue

In the IR, the absorption increases with longer wavelengths due to tissue water content. Scaling thep re ater absorption b 75% mimics a t pical tiss e ith 75% ater contentpure water absorption by 75% mimics a typical tissue with 75% water content.

In the red to near-infrared (NIR), absorption is minimal. This region is called the diagnostic andtherapeutic window

Mueller MatrixMueller Matrix

−−+−−+−−+−

−−−

4RHLVRVLH

4MHPVMVPH

4VHHVVVHH

2OVOH

2LORO

2MOPO

2VOHOOO

−−+−−+−−+−

−−+−−+−−+−

4LRRLLLRR

4MLPRMRPL

4VLHRVRHL

2OLOR

4RPLMRMLP

4MPPMMMPP

4VPHMVMHP

2OMOP

4442

Stokes Vectors

+

VHI

++≥

−−−

=

111111

VUQI,

LRMPVH

VUQ

2222

−

=

=

−=

=

−

=

=

1001

S,

1001

S,

01

01

S,

0101

S,

00

11

S,

0011

S LCPRCPMPVH

STEP III: MR = M∆-1 M/

Linear Retardance

OpticalRotation

1 0 0 00 cos2(2θ)+sin2(2θ)cos(δ) sin(2θ)cos(2θ)(1 − cos(δ)) -sin(2θ)sin(δ)0 sin(2θ)cos(2θ)(1 − cos(δ)) sin2(2θ)+cos2(2θ)cos(δ) cos(2θ)sin(δ)

θ δ θ δ δ

1 0 0 00 cos(2ψ) sin(2ψ) 0 0 -sin(2ψ) cos(2ψ) 0 0 0 0 1

×

Linear retarder with retardance (δ) & orientation angle (θ) Circular retarder with optical rotation (ψ)

0 sin(2θ)sin(δ) -cos(2θ)sin(δ) cos(δ) 0 0 0 1

δ = cos -1 [{(MR (2,2) + MR(3,3))2 + (MR(3,2)-MR(2,3))2}1/2 -1]Linear Retardance

ψ = tan -1 [{MR (3,2)-MR(2,3)} / {MR (2,2) + MR(3,3)}]Optical Rotation

1 ( )cos 12

Rtr MR − = −

Total Retardance 1 2 2cos 2cos ( ) cos ( ) 12

R δψ− = −

H2C CH

OH

C

OH

CH

OH

H2C O P

O

OH

N

N

CNH

CN

OO

O

PO OH

CH 3

CH 3 N

NH 2

H2C CH

OH

C

OH

CH

OH

CH2OH

O O

H2C

O

NN

CH

N

NH

CN

O

2 H H

CH3

OH OH OH

H2

O

FAD

N CNH

O

CH3

NC

NO

H2C CH

C CH

H2C

CH3

O P OH

OH

Rib fl i

N CNH

O

CH3

FMN

Riboflavin

O

Typical fluorescence spectra at various i i l hexcitation wavelengths

325nm

0.09

0.10

0.11

0.12325nm 350nm 370nm

0.05

0.06

0.07

0.08

nsity

(a.u

.)

0 01

0.02

0.03

0.04

0.05

IF In

te

350 400 450 500 550 600 650 700-0.01

0.00

0.01

Wavelength (nm)g ( )

Fluorescence spectra of cervical tissue with 370nm excitation

1500000

2000000

)

Nor Can

co-polarized 200000

250000

300000

.)

Nor Can

cross-polarized

500000

1000000

Inte

nsity

(a.u

.)

50000

100000

150000

Inte

nsity

(a.u

.

400 450 500 550 600 650 700

0

Wavelength (nm)400 450 500 550 600 650 700

0

Wavelength(nm)

0.20

0.25

u.)

Nor Can

Intrinsic

0.05

0.10

0.15

Inte

nsity

(a.u

400 450 500 550 600 650 700

0.00

Wavelength (nm)

Ratio of Intrinsic Fluorescence for Cervical Tissue at 370nm Excitation

Sensitivity 74%

4

5

6

7

8

IF 370nmo

Can

cer

0

1

2

3

4

atio

of N

orm

al to

0 10 20 30 40 50-2

-1Ra

No. Patients

RESULTS

16.8mm16.8mm

40mmGroove to hold optical fiber

FLAVIN PHANTOM: The peak maximum was found to be 522.2 nm.

Optical Coherence Tomographyp g p y

Ex vivo arthroscopic OCT of an embeddedcartilage tear (a) 2-D OCT clearly delineateda minute cartilage tear that was less than 0.2mm thick but embedded 0.6 mm below thecartilage surface. (b) Green fluorescent dye-stained histology from a parallel cross-section of the dashed area in (a). Image size:roughly 6 mm wide and 2 mm deep for (a);3 6 mm wide and 1 mm deep for (b) The3.6 mm wide and 1 mm deep for (b). Thewhite arrows in both images indicate theembedded tears

OCTOCT