This PDF file contains the front matter associated with SPIE Proceedings Volume 8940, including the Title Page, Copyright Information, Table of Contents, and the Conference Committee listing.

Due to its high sensitivity to subtle alterations in medium morphology the vector light beams have recently gained much attention in the area of photonics. This leads to development of a new non-invasive optical technique for tissue diagnostics. Conceptual design of the particular experimental systems requires careful selection of various technical parameters, including beam structure, polarization, coherence, wavelength of incident optical radiation, as well as an estimation of how the spatial and temporal structural alterations in biological tissues can be distinguished by variations of these parameters. Therefore, an accurate realistic description of vector light beams propagation within tissue-like media is required. To simulate and mimic the propagation of vector light beams within the turbid scattering media the stochastic Monte Carlo (MC) technique has been used. In current report we present the developed MC model and the results of simulation of different vector light beams propagation in turbid tissue-like scattering media. The developed MC model takes into account the coherent properties of light, the influence of reflection and refraction at the medium boundary, helicity flip of vortexes and their mutual interference. Finally, similar to the concept of higher order Poincar´e sphere (HOPS), to link the spatial distribution of the intensity of the backscattered vector light beam and its state of polarization on the medium surface we introduced the color-coded HOPS.

We exploit the directional awareness of circularly and/or elliptically polarized light propagating within media which exhibit high numbers of scattering events. By tracking the Stokes vector of the detected light on the Poincar´e sphere, we demonstrate its applicability for characterization of anisotropy of scattering. A phenomenological model is shown to have an excellent agreement with the experimental data and with the results obtained by the polarization tracking Monte Carlo model, developed in house. By analogy to diffusing-wave spectroscopy we call this approach diffusing-wave polarimetry, and illustrate its utility in probing cancerous and non-cancerous tissue samplesin vitro for diagnostic purposes.

A computer algorithm was developed to automatically identify and count melanocytes and keratinocytes in 3D reflectance confocal microscopy (RCM) images of the skin. Computerized pathology increases our understanding and enables prevention of superficial spreading melanoma (SSM). Machine learning involved looking at the images to measure the size of cells through a 2-D Fourier transform and developing an appropriate mask with the erf() function to model the cells. Implementation involved processing the images to identify cells whose image segments provided the least difference when subtracted from the mask. With further simplification of the algorithm, the program may be directly implemented on the RCM images to indicate the presence of keratinocytes in seconds and to quantify the keratinocytes size in the en face plane as a function of depth. Using this system, the algorithm can identify any irregularities in maturation and differentiation of keratinocytes, thereby signaling the possible presence of cancer.

Resonant Raman (RR) spectroscopy provides an effective way to enhance Raman signal from particular bonds associated with key molecules due to changes on a molecular level. In this study, RR is used for detection of human brain metastases of five kinds of primary organs of lung, breast, kidney, rectal and orbital in ex-vivo. The RR spectra of brain metastases cancerous tissues were measured and compared with those of normal brain tissues and the corresponding primary cancer tissues. The differences of five types of brain metastases tissues in key bio-components of carotene, tryptophan, lactate, alanine and methyl/methylene group were investigated. The SVM-KNN classifier was used to categorize a set of RR spectra data of brain metastasis of lung cancerous tissues from normal brain tissue, yielding diagnostic sensitivity and specificity at 100% and 75%, respectively. The RR spectroscopy may provide new moleculebased optical probe tools for diagnosis and classification of brain metastatic of cancers.

Oral cancer is the most common cancer among Indian males, with 5-year- survival-rates of less than 50%. Efficacy of Raman spectroscopic methods in non-invasive and objective diagnosis of oral cancers and confounding factors has already been demonstrated. The present Raman microspectroscopic study was undertaken for in-depth and site-specific analysis of normal and tumor tissues. 10 normal and 10 tumors unstained sections from 20 tissues were accrued. Raman data of 160 x 60 μm and 140 x 140 μm in normal and tumor sections, respectively, were acquired using WITec alpha 300R equipped with 532 nm laser, 50X objective and 600 gr/mm grating. Spectral data were corrected for CCDresponse, background. First-derivitized and vector-normalized data were then subjected to K-mean cluster analysis to generate Raman maps and correlated with their respective histopathology. In normal sections, stratification among epithelial layers i.e. basal, intermediate, superficial was observed. Tumor, stromal and inflammatory regions were identified in case of tumor section. Extracted spectra of the pathologically annotated regions were subjected to Principal component analysis. Findings suggest that all three layers of normal epithelium can be differentiated against tumor cells. In epithelium, basal and superficial layers can be separated while intermediate layer show misclassifications. In tumors, discrimination of inflammatory regions from tumor cells and tumor-stroma regions were observed. Finding of the study indicate Raman mapping can lead to molecular level insights of normal and pathological states.

Cervix-cancer is the third most common female cancer worldwide. It is the leading cancer among Indian females with more than million new diagnosed cases and 50% mortality, annually. The high mortality rates can be attributed to late diagnosis. Efficacy of Raman spectroscopy in classification of normal and pathological conditions in cervix cancers on diverse populations has already been demonstrated. Our earlier ex vivo studies have shown the feasibility of classifying normal and cancer cervix tissues as well as responders/non-responders to Concurrent chemoradiotherapy (CCRT). The present study was carried out to explore feasibility of in vivo Raman spectroscopic methods in classifying normal and cancerous conditions in Indian population. A total of 182 normal and 132 tumor in vivo Raman spectra, from 63 subjects, were recorded using a fiberoptic probe coupled HE-785 spectrometer, under clinical supervision. Spectra were acquired for 5 s and averaged over 3 times at 80 mW laser power. Spectra of normal conditions suggest strong collagenous features and abundance of non-collagenous proteins and DNA in case of tumors. Preprocessed spectra were subjected to Principal Component-Linear Discrimination Analysis (PCLDA) followed by leave-one-out-cross-validation. Classification efficiency of ~96.7% and 100% for normal and cancerous conditions respectively, were observed. Findings of the study corroborates earlier studies and suggest applicability of Raman spectroscopic methods in combination with appropriate multivariate tool for objective, noninvasive and rapid diagnosis of cervical cancers in Indian population. In view of encouraging results, extensive validation studies will be undertaken to confirm the findings.

Degradation of articular cartilage extracellular matrix (ECM) by proteolytic enzyme is the hallmark of arthritis that leads to joint destruction. Detection of early biochemical changes in cartilage before irreversible structural damages become apparent is highly desirable. Here we report that the autofluorescence decay profile of cartilage is significantly affected by proteolytic degradation of cartilage ECM and can be characterised by measurements of the autofluorescence lifetime (AFL). A multidimensional fluorometer utilizing ultraviolet excitation at 355 nm or 375 nm coupled to a fibreoptic probe was developed for single point time-resolved AFL measurements of porcine articular cartilage explants treated with different proteinases. Degradation of cartilage matrix components by treating with bacterial collagenase, matrix metalloproteinase 1, or trypsin resulted in significant reduction of AFL of the cartilage in both a dose and time dependent manner. Differences in cartilage AFL were also confirmed by fluorescence lifetime imaging microscopy (FLIM). Our data suggest that AFL of cartilage tissue is a potential non-invasive readout to monitor cartilage matrix integrity that may be utilized for diagnosis of arthritis as well as monitoring the efficacy of anti-arthritic therapeutic agents.

The objective of this study is to evaluate the diagnostic potential of stokes shift (SS) spectroscopy (S3) for normal, precancer and cancerous oral lesions in vivo. The SS spectra were recorded in the 250 – 650 nm spectral range by simultaneously scanning both the excitation and emission wavelengths while keeping a fixed wavelength interval Δλ=20 nm between them. Characteristic, highly resolved peaks and significant spectral differences between normal and different pathological oral lesions observed around 300, 355, 395, and 420 nm which are attributed to tryptophan, collagen, and NADH respectively. Using S3 technique one can obtain the key fluorophores in a single scan and hence they can be targeted as a tumor markers in this study. In order to quantify the altered spectral differences between normal and different pathological oral lesions are verified by different ratio parameters.

In order to identify the optimal imaging conditions for the highest contrast in biological tissue, we explored the optical contrast of a phantom as a function of depth and wavelengths of excitation. Our customized optical hardware featured a scanning microscope, and imaging spectrographs equipped with silicon and InGaAs CCD diode array detectors allowed directed comparison of the intensity of NIR (650-900 nm) and exNIR (1000-1600 nm) light transmitted through a phantom (milk). We demonstrated that the contrast depends on the phantom thickness and the wavelength. At low depths (less than 3 mm) NIR light provides the best contrast while exNIR light shows significantly higher contrast in phantoms thicker than 4.5 mm. Our results suggest that distinguishing biological features in deep tissue may benefit from the application of the exNIR for in vivo.

Treating cancer is one of the major challenges of modern medicine. There is great interest in assessing tumor development in in vivo animal and human models, as well as in in vitro experiments. Existing methods are either limited by cost and availability or by their low accuracy and reproducibility. Thermography holds the potential of being a noninvasive, low-cost, irradiative and easy-to-use method for tumor monitoring. Tumors can be detected in thermal images due to their relatively higher or lower temperature compared to the temperature of the healthy skin surrounding them. Extensive research is performed to show the validity of thermography as an efficient method for tumor detection and the possibility of extracting tumor properties from thermal images, showing promising results. However, deducing from one type of experiment to others is difficult due to the differences in tumor properties, especially between different types of tumors or different species. There is a need in a research linking different types of tumor experiments. In this research, parametric analysis of possible contributors to tumor thermal profiles was performed. The effect of tumor geometric, physical and thermal properties was studied, both independently and together, in phantom model experiments and computer simulations. Theoretical and experimental results were cross-correlated to validate the models used and increase the accuracy of simulated complex tumor models. The contribution of different parameters in various tumor scenarios was estimated and the implication of these differences on the observed thermal profiles was studied. The correlation between animal and human models is discussed.

Quantitative photoacoustics is emerging as a new hybrid modality to investigate diseases and cells in human pathology and cytology studies. Optical absorption of light is the predominant mechanism behind the photoacoustic effect. Therefore, a need exits to characterize the optical properties of specimens and to identify the relevant operating wavelengths for photoacoustic imaging. We have developed a custom low-cost spectrophotometer to measure the optical properties of human axillary lymph nodes dissected for breast-cancer staging. Optical extinction curves of positive and negative nodes were determined in the spectral range of 400 to 1000 nm. We have developed a model to estimate tissue optical properties, taking into account the role of fat and saline. Our results enabled us to select the optimal optical wavelengths for maximizing the imaging contrast between metastatic and noncancerous tissue in axillary lymph nodes.

Despite best efforts, bile duct injury during laparoscopic cholecystectomy is a major potential complication. Precise detection method of extrahepatic bile duct during laparoscopic procedures would minimize the risk of injury. Towards this goal, we have developed a compact imaging instrumentation designed to enable simultaneous acquisition of conventional white color and NIR fluorescence endoscopic/laparoscopic imaging using ICG as contrast agent. The capabilities of this system, which offers optimized sensitivity and functionality, are demonstrated for the detection of the bile duct in an animal model. This design could also provide a low-cost real-time surgical navigation capability to enhance the efficacy of a variety of other image-guided minimally invasive procedures.

The pathogenic process of Alzheimer’s Disease (AD), characterized by amyloid plaques and neurofibrillary tangles in the brain, begins years before the clinical diagnosis. Here, we suggest a novel method which may detect AD up to nine years earlier than current exams, minimally invasive, with minimal risk, pain and side effects. The method is based on previous reports which relate the concentrations of biomarkers in the Cerebrospinal Fluid (CSF) (Aβ and Tau proteins) to the future development of AD in mild cognitive impairment patients. Our method, which uses fluorescence measurements of the relative concentrations of the CSF biomarkers, replaces the lumbar puncture process required for CSF drawing. The process uses a miniature needle coupled trough an optical fiber to a laser source and a detector. The laser radiation excites fluorescent probes which were prior injected and bond to the CSF biomarkers. Using the ratio between the fluorescence intensities emitted from the two biomarkers, which is correlated to their concentration ratio, the patient’s risk of developing AD is estimated. A theoretical model was developed and validated using Monte Carlo simulations, demonstrating the relation between fluorescence emission and biomarker concentration. The method was tested using multi-layered tissue phantoms simulating the epidural fat, the CSF in the sub-arachnoid space and the bone. These phantoms were prepared with different scattering and absorption coefficients, thicknesses and fluorescence concentrations in order to simulate variations in human anatomy and in the needle location. The theoretical and in-vitro results are compared and the method’s accuracy is discussed.

Light at wavelengths in the visible and near-infrared (NIR) regions (650 nm – 1,350 nm) is used in numerous medical applications. The NIR region between 650 nm and 950 nm, known as the first optical window, allows for deeper depth penetration in tissue than in the visible region due to a reduction in absorption. There also exists a second NIR optical window with wavelengths from 1,100 nm to 1,350 nm. Longer wavelengths above 1,350 nm were ignored due to major water absorption and lack of 2D photodetectors. In this study, a new therapeutic spectral window with wavelengths between 1,600 nm and 1,870 nm is reported. This third optical window can be used for imaging more deeply into tissue due to a reduction in scattering. In this paper, light attenuation from 400 nm to 2,000 nm, including all three optical windows, was measured. 200 micron slices of normal and benign prostate and breast tissues were studied. The total attenuation lengths (l_t) of light were obtained. The attenuation length of malignant and normal tissue in the third optical window was larger than in the first and second therapeutic windows. Optical images of chicken tissue over three black wires were also obtained using the third optical window.

The first discovery and mechanism of super continuum generation with ultrashort pulses in solids (glasses and crystals) and rare gas media will be presented. How the observation of the white light over 6000cm-1 was unraveled for the first time with excitation of ultrashort pulses 45 years ago.

Spectral broadening and the generation of new frequencies were initially observed in pulsed laser systems in the mid-1960s as an inherent feature of the uncontrollable nonlinear process such as self-focussing and self-phase modulation occurring primarily in the gain media and were looked upon as deleterious rather than a resource. With the advent of mode locked lasers to generate picosecond pulses new effects were observed. Developed by the Alfano group in bulk media external to the laser in the 1970s the supercontinuum or “white light” source has now evolved into a commercially successful and highly compact source that can readily extend over more than three octaves with spectral power densities exceeding 100mW/nm. In this presentation I will describe this remarkable evolution.

A microstructured optical fiber was first used in 2000 for supercontinuum generation. Since then, enormous progress has been made in understanding, controlling, and marketing fiber-based supercontinuum sources. In particular, biomedical applications of such sources are revolutionizing the field of medical imaging. In this talk I review the recent progress in this area and describe how a supercontinuum can be employed for biomedical imaging using the techniques known as coherent anti-Stokes Raman scattering, stimulated emission-depletion microscopy, and optical coherence tomography.

This talk will give an overview of the unique properties of supercontinuum generation (SCG) in all-normal dispersion (ANDi) fibers pumped by ultrashort pulses and the possibilities they offer for ultrafast photonics applications. In contrast to their anomalously pumped counterparts, the SCG process in ANDi fibers conserves a single ultrashort pulse in the time domain, completely suppresses soliton formation and decay, and avoids noise-amplifying nonlinear dynamics. The resulting spectra combine the best of both worlds – the broad, more than octave-spanning bandwidths usually associated with anomalous dispersion pumping with the high temporal coherence, pulse-to-pulse stability and well-defined temporal pulse characteristics known from the normal dispersion regime. These characteristics are ideally suited for ultrafast photonics, and I will present application examples including the generation of high quality single-cycle pulses and their amplification, as well as ultrafast spectroscopy. This talk will also explore the exciting new possibilities enabled by extending this approach into the mid-IR spectral region using novel soft glass fiber designs.

The ability to generate a supercontinuum in microstructure fiber using only the unamplified pulses from a mode-locked oscillator was critical to the development of optical frequency combs. I will briefly introduce the key concepts for stabilizing the comb spectrum of a mode-locked laser and how it relies on continuum generation.

The history of super continuum generation with ultrashort pulses in bulk media will be reviewed. In particular, a description on how the self-focusing dynamics leads to shock formation and the generation of extremely broad spectra when an ultrashort pulse travels through a transparent gas, liquid, or solid.

First observations of cross-phase modulation effects were reported in mid-1980's. This nonlinear interaction between ultrashort pulses is inherent to nonlinear generation processes. It can also be used for all-optical control of spectral, temporal, and spatial properties of ultrashort pulses. This paper reviews the main discoveries that were reported by Alfano and al. at City College of New York.

The aim of this study is to use the Resonance Raman (RR) and fluorescence spectroscopic technique for tumor margin detection with high accuracy based on native molecular fingerprints of breast and gastrointestinal (GI) tissues. This tumor margins detection method utilizes advantages of RR spectroscopic technique in situ and in real-time to diagnose tumor changes providing powerful tools for clinical guiding intraoperative margin assessments and postoperative treatments. The tumor margin detection procedures by RR spectroscopy were taken by scanning lesion from center or around tumor region in ex-vivo to find the changes in cancerous tissues with the rim of normal tissues using the native molecular fingerprints. The specimens used to analyze tumor margins include breast and GI carcinoma and normal tissues. The sharp margin of the tumor was found by the changes of RR spectral peaks within 2 mm distance. The result was verified using fluorescence spectra with 300 nm, 320 nm and 340 nm excitation, in a typical specimen of gastric cancerous tissue within a positive margin in comparison with normal gastric tissues. This study demonstrates the potential of RR and fluorescence spectroscopy as new approaches with labeling free to determine the intraoperative margin assessment.

This study shows tryptophan as the key native marker in cells to determine the level of aggressive cancer in breast cell lines using native fluorescence spectroscopy. An algorithm based on the ratio of tryptophan fluorescence intensity at 340 nm to intensity at 460 nm is associated with aggressiveness of the cancer cells. The higher the ratio is, the more aggressive the tumor towards metastasis.

It is important to detect cervical dysplasia, Cervical Intraepithelial Neoplasia (CIN). CIN is the potentially premalignant and abnormal squamous cells on surface of cervix. In this study, the spatial frequency spectra of pre-cancer cervical tissues are used to detect differences among different grades of human cervical tissues. Seven sets of thick tissue sections of human cervix of normal, CIN 1, CIN 2, and CIN 3 tissues are studied. The confocal microscope images of the stromal region of normal and CIN human tissues were analyzed using Fast Fourier Transform (FFT) to generate the spatial spectra. It is observed that higher frequency components exist in CIN tissues than those in normal tissue, as well as those in higher grade CIN tissue than those in lower grade CIN tissue. The width of the spatial frequency of different types of tissues is used to create a criterion for CIN grading by training a support vector machine (SVM) classifier. The results show that the randomness of tissue structures from normal to different stages of precancer in cervical tissue can be recognized by fingerprints of the spatial frequency. The efficacy of spatial frequency analysis for CIN grading is evaluated as excellent since high AUC (area under the ROC curve), sensitivity and specificity are obtained by the statistics study. This works lays the foundation of using spatial frequency spectra for a histology evaluation.

Increasing the depth to image inside tissue is critical in biomedicine. Two-photon (2P) excitation of the second singlet (S₂) state of a group of fluorescent agents with near infrared emission, Chlorophyll a (Chl a) and Indocyanine green (ICG), is used to extend the optical imaging regime of 2PM into “tissue optical window” for deep tissue penetration. The fast nonradiative from S₂ to S₁ yields both emission and absorption wavelengths in the therapeutic window. The salient feature is to place both the 2P excitation and emission wavelengths of the imaging agents falling into the “tissue optical window”. As a first step to achieve deeper optical imaging, Chl a and ICG are investigated and demonstrated as imaging agents for 2P S₂ excitation microscope image.

In his talk, "Diffuse Optical Methods for Assessing Breast Cancer Chemotherapy," SPIE Fellow Bruce Tromberg (Beckman Laser Institute and Medical Clinic) describes a method combining frequency domain photon migration, essentially a method of tracking photon motion in tissue, with a NIR spectroscopy technique using 850nm LEDs. The result is a scatter corrected absorption spectra. The technique takes advantage of elevated blood and water levels and decreased lipid levels in the presence of tumors to provide a more accurate mapping of the breast, allowing more effective treatment. Tromberg's team recently completed their first full mapping of the breast and have taken the instrument from a standalone unit to a portable one suitable for travel. In addition to providing feedback to enhance breast cancer treatment, Tromberg expects that this technique will be applicable in treating other forms of cancer as well.