Dept. Of Physics & Centre for Laser Technology Indian Institute of Technology, Kanpur Dept. Of Physics & Centre for Laser Technology Indian Institute of Technology, Kanpur Lighting up Human Tissue to Detect Tumors Asima Pradhan REACH 2010 IIT Kanpur

Working of biological system is complicated  That 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. Clinician’s goal:  To access structural and functional changes in diseased tissue.  Infer the identity and stage of disease.  Ultimate prognosis.

3 Cancer cases: >25 lakh per year Death rate 4 lakhs per year #2 largest cause of mortality Increasing at the rate of 11% per year Every day 2000 people gets affected by mouth cancer Highest cases: Lung cancer in men Breast cancer in female 40% of total cancer deaths in women: Breast and Cervical Some relevant statistics on cancer in India

 X-ray computed tomography Ionizing 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 chemistry MRI: Sub millimeter spatial resolution Ability to detect specific chemicals Cost 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 provides the most widely used clinical method to obtain chemical information from diseased tissues. 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. 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.

OPTICAL SPECTROSCOPY AND IMAGING A non-invasive, safe, inexpensive, compact technique capable of extracting diagnostically relevant structural and biochemical information is the need of hour!!! Diagnostics techniques based on optical spectroscopy offer such possibilities. Fluorescence Scattering Absorption

Essential to Study Light Tissue Interaction TISSUE OPTICS

Reflection, Raman, Fluorescent Light Transmitted Light Tissue 7 Light-Tissue Interaction Absorption Incident Light Elastic scattering Diagnostics Imaging Inelastic scattering Fluorescence & Raman Diagnostics Absorption & Scattering

Basic Aim: Detect Early Stages of Disease in particular, tumors Increase in size of nucleus Changes in elastic scattering Cells packed Increase in metabolic activity Biochemical changes Increase in concentration of related bio-molecules Bond breaking (cross links) Fluorescence yield changes Bonds changeRaman scattering changes Blood concentration changesAbsorption changes Growth of blood vessels

KrF XeCl Dye Argon Diode Nd:YAG Tm:YAG Ho:YAG Er:YAG CO m5m 40  m 250  m 1300  m 1400  m 20  m 1m1m  m 150  m 1400  m Penetration depth of laser light in soft non-colored tissue as a function of wavelength Wavelength of the Light Source

 Elastic scattering events caused by random spatial variations in density 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 SCATTERING IN TISSUES  μs = mm -1

Absorption in Tissues Strongly wavelength dependent Tends to generally decrease with wavelength. Typical values of  a : 0.1 to 10,000 cm -1 from NIR to UV. Absorption in tissue in UV, visible and IR regions primarily from proteins, hemoglobin, water Therapeutic Window nm

Coenzymes NADH, FAD and FMN:  metabolic activity Malignancy: Impaired mitochondrial metabolic activity. Change in concentration Result: Change in measured fluorescence from tissue. Can probe metabolic status of tissue Structural proteins:Collagen, Elastin Malignancy : cross-links broken fluorescence quenched Can probe early stages of cancer Biochemical Aspects (Fluorescence) Fluorophores in Tissue nm Hemoglobin Beta-Carotene Structural Proteins Collagen Elastin Electron Carriers NADH FAD Amino acids Tryptophan Tyrosine

Fluorescence Spectra of Endogenous Tissue Fluorophores Reproduced from Stephen Webb, PhD thesis, University of London, 2003

Late 1980’s Alfano et.al., Feld et.alpioneered in the field of cancer diagnosis 1990’s First Generation task: In vitro experiments  Different approaches based on optical principles: Fluorescence Spectroscopy Elastic Scattering Spectroscopy Raman Spectroscopy Absorption Spectroscopy 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 Mueller Imaging Confocal Imaging Current IssuesImprove diagnostic capabilities Gain biochemical information Nano Bio Photonics Developments….

Fluorescence from Human Breast Tissue Sensitivity 95% Specificity 92% Fluorescence with point measurements: Average conribution from bulk tissue. Thus, multiple scattering effects play major role. Differences in width of spectra: Role of two fluorophores

Various Approaches for Analysis Large Amount of spectral data and large number of samples required 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 Cancer  Biochemical information masked  Large site to site variation Valuable biochemical information on tissue fluorophores may not be retrieved Intrinsic fluorescence has to be extracted!! Modulation of Fluorescence by wavelength dependent absorption & scattering properties of tissue

Approach used by us for extraction of Intrinsic Fluorescence A. Polarized Fluorescence & polarized elastic scattering measurement based approach A purely experimental approach B. Spatially resolved fluorescence measurement Depth information of inhomogeneity

Ballistic Snake Diffuse Light Propagation in Tissue

Underlying Principle Dominant Depolarization contribution:  Multiple scattering of light Polarized component of the detected fluorescence  [I  - I  ] f Extracts contributions which have not undergone significant scattering in tissue  Therefore originates from superficial layer of tissue Incident polarized light Co-polarized backscattered light Reduced comp. Cross-polarized backscattered light Diffuse comp. I  II B.V. Laxmi et al, Lasers in the Life Sci., 9, , (2001)

Underlying Principle (Cont…) Fluorescence detected in crossed polarized channel (I  ) Contribution of more multiply scattered photons Hence originates from deeper tissue layer Cross-polarized backscattered light Diffuse comp. Incident polarized light Co-polarized backscattered light Reduced comp. I  II Biswal et al, Optics Express, 11, 3320 –3321 (2003) 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 2005 JOSA A 2007

Underlying Principle (Cont…) Remove scattering effects: difference in co and cross polarized light 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 spectra Optics Express, 2003.

Example :

Scheme Modification is made so as to eliminate modulation due to blood A systematic study on phantoms was also performed to validate this scheme Assumption: Wavelength dependent scattering & absorption : similar effects on polarized component of fluorescence & polarized component of elastic scattering spectra

Where is this scheme applicable? Layered Tissue Superficial tumors Basal layer (a) Microscopic images of epithelial layer of (a) normal and (b) dysplastic state of cervix tissue Densely packed epithelium, NADH increase Collagen cross- links break in stroma

Co-polarized Intrinsic  Dysplastic tissues : Increase in NADH fluorescence Sensitivity 74% Intrinsic fluorescence : enhanced discrimination between normal and dysplastic tissues as compared to the co-polarized case. NADH : more reliable discriminating parameter Intrinsic Fluorescence from Human Cervical Tissue …SPIE 2008

COVARIANCE MATRIX using Principal Component Analysis Data = Normalized Spectra (variance (i)= (Value(i) – Mean value); Covariance Matrix = (Data T Data)/(n-1) Spectra with λexc = 350nm

Fluorescence Imaging

Mueller matrix imaging in human cervical tissues

Mueller Matrix Contains complete polarization information of the medium It is a blue print for scattering media Emerging Stoke’s vector S 1 / S 2 / S 3 / S 4 / S 1 S 2 S 3 S 4 = Incident Stoke’s vector Mueller Matrix Emerging Stoke’s vector S S 2 / S 3 / S 4 / S 2 / S 3 / S 4 / Mueller matrix imaging in human cervical tissue Elastic Scattering based measurements 1200µ Mueller image M 00 microscopic image

Polar Decomposition of Mueller Matrix 1200µ

Depolarization power images Microscope images Normal epithelium of cervix Dysplastic epithelium of cervix Normal epithelium of cervix Dysplastic epithelium of cervix Basal layer 1000µ 180µ 40µ 180µ 40µ (a ) (b )

Normal stroma of cervix Dysplastic stroma of cervix 180µ Microscope images Optics Express, Vol.17,(3) 2009 Retardance images

Depolarization power: sensitive to morphological changes during progression from normal to dysplastic state. Retardance reveals the morphological changes around the stromal region. Current Status: Automating and increasing data bank for use as supplementary tool in clinics (a)(b)

Conclusions Future 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 & OCT with 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 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… Mueller Imaging technique can be used as a supplementary technique to the ‘gold standard’ histopathology.

Acknowledgments Department of Information Technology (Photonics) [MCIT] CSIR BRNS IIT Kanpur Dr.Asha Agarwal (Professor in Pathology, GSVM Medical College) Dr. Kiran Pandey (Professor and Head of Gyn and Obstr, GSVM Medical College) Dr. Nirmalya Ghosh (Univ. of Toronto) Dr. Sharad Gupta Dr. Maya Nair Dr.Prashant Shukla Jaidip Jagtap Sridhar Raja Rajbeer Singh Dharitri Rath Krishna Kumar Tomar Meghdoot Mazumdar

Takes into account spectral characteristics of contributing fluorophores such as intensity, peakshift, bandwidth for discrimination Advantage: Offers insight into biochemical aspects of tissue. Our study Flavins Porphyrins To differentiate spectra were fitted to Voigt function. Physical Modeling

27 September 2010 Last updated at 00:37 GMT Share this page Painless laser device could spot early signs of disease By Katia Moskvitch Michael Morris, a chemistry professor at 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 can not recover intrinsic fluorescence intensity information Propagation path of elastically scattered photon Propagation path of fluorescence photon EM EM EX EM EX EM EX EM  Major difference in the survival of the long path photons Results in the differential effect of absorption & scattering on unpolarized fluorescence spectra and elastic scattering spectra

British researchers at the Rutherford Appleton Laboratory in Didcot and at the Gloucestershire Royal Hospital have 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

Incident Transmitted Inelastic scattering (Fluorescence, Raman) Elastic scattering Intensities depend on Optical properties of tissue such as reflectivity, scattering coefficient, particle size, optical homogeneity, absorption coefficient etc. Absorbed portion of light produces: ·Photochemical effect ·Thermal effect ·Inelastic scattering (Fluorescence, Raman) Depending on and nature of tissue IR and visible lasers generally produce only thermal effects UV laser: both thermal and photochemical

Linear retardance Circular retardance (Optical rotation) Total retardance R Depolarization  Diattenuation D The extracted polarization parameters Dichroic absorption (or scattering) due to presence of oriented structures Fibrous structure & their orientation in tissue Concentration of chiral substances like glucose in tissue Conjugate effect of linear & circular retardance Multiple scattering effects (concentration, size, refractive indices of scatterers present in tissue)

Decomposition Procedure STEP I: M 11 M 12 M 13 M 14 M 21 M 22 M 23 M 24 M 31 M 32 M 33 M 34 M 41 M 42 M 43 M 44 Experimental Mueller matrix Diattenuation Vector D = (M 12 M 13 M 14 ) T / M 11 (1-D 2 ) 1/2 I + (1-(1-D 2 ) 1/2 DD T  1 D T D m D M D = STEP II : D ={1 / M 11 }  [M M M 14 2 ] 1/2 M / = M M D -1 Diattenuation free matrix M / =M  M R 1 0 P  m  m R (P-mD)/(1-D 2 ) 1 0 P   m / m  =  [m’(m’) T +(( 1 2 ) 1/2 + ( 2 3 ) 1/2 + ( 3 1 ) 1/2 )I] -1  [( 1 1/ / /2 )m’(m’) T +( 1 2 3) 1/2 I] Diattenuation Parameter  = 1- {  tr (M  ) - 1  / 3} Net Depolarization Index P  (1)  P  (2) 0  2 0 P  (3) 0 0  3 MM Circular Linear

In the UV, the absorption increases with shorter wavelength due to protein, DNA and other molecules. 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 the pure 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 and therapeutic window Water Major absorbers in tissue

Mueller Matrix Stokes Vectors

STEP III: M R = M  -1 M / Linear Retardance Optical Rotation cos 2 (2  )+sin 2 (2  )cos(  ) sin(2  )cos(2  )(1 − cos(  )) -sin(2  )sin(  ) 0 sin(2  )cos(2  )(1 − cos(  )) sin 2 (2  )+cos 2 (2  )cos(  ) cos(2  )sin(  ) 0 sin(2  )sin(  ) -cos(2  )sin(  ) cos(  ) cos(2  ) sin(2  ) 0 0 -sin(2  ) cos(2  )  Linear retarder with retardance (  ) & orientation angle (  )Circular retarder with optical rotation (  )  = cos -1 [{(M R (2,2) + M R (3,3)) 2 + (M R (3,2)-M R (2,3)) 2 } 1/2 -1] Linear Retardance  = tan -1 [{M R (3,2)-M R (2,3)} / {M R (2,2) + M R (3,3)}] Optical Rotation Total Retardance

FMN Riboflavin FAD

Typical fluorescence spectra at various excitation wavelengths

Fluorescence spectra of cervical tissue with 370nm excitation co-polarized Intrinsic cross-polarized

Ratio of Intrinsic Fluorescence for Cervical Tissue at 370nm Excitation Sensitivity 74%

RESULTS FLAVIN PHANTOM: The peak maximum was found to be nm. 16.8mm 40mm 16.8mm Groove to hold optical fiber

Optical Coherence Tomography Ex vivo arthroscopic OCT of an embedded cartilage tear (a) 2-D OCT clearly delineated a minute cartilage tear that was less than 0.2 mm thick but embedded 0.6 mm below the cartilage 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). The white arrows in both images indicate the embedded tears

OCT