The presented work is part of a study investigating the surgical diagnostic potential of Raman spectroscopy for the malignant brain tumor, glioblastoma multiforme.

We have used resonance Raman scattering as a non-invasive optical technique to measure the concentration of carotenoid pigments in the human retina. The technique is highly sensitive and specific strength scales linearly with actual macular carotenoid content as measured by high-pressure liquid chromatography. This will facilitate studies of carotenoid distributions and their role in degenerative diseases of the eye and may allow for the rapid screening of carotenoid levels in large populations at risk for vision loss from age-related macular degeneration, the leading cause of blindness in the elderly in the US.

The ordinary Raman scattering (ORS) spectra of virion protein of Granulosis Virus of Cabbage Butterfly Preris rapae (PrGV-VP) and their surface-enhanced Raman scattering (SERS) spectra adsorbed on the surface of silver colloid particles have been found that the Raman signal in the SERS spectra was enhanced by a factor of about 8 X 10². In ORS spectra of PrGV-VP, it indicates that PrGV-VP has a predominantly random-coil secondary structures. All tyrosine residues are exposed. And the PrGV-VP has a gauche-gauche- gauche configuration of the C-C-S-S-C-C linkage. In SERS spectra of PrGV-VP, the intensities of bands related to COO group and NH₂ group vibrations are relatively strong. We interpret that the PrGV-VP are adsorbed on the Ag surface through both the carboxylate groups and the amino groups. The efficient enhancement of amide I and amide II vibrations are impeded, because the peptide groups of PrGV-VP are screened by the side-chains amino acid residues. The Raman enhancement of PrGV-VP has a short-range mechanism. The chemisorption is a main mechanism on Ag hydrosols of PrGV- VP.

The aim of this work is to investigate light scattering properties of tumorigenic and nontumorigenic and nontumorigenic cells. Specifically, both wavelength dependent and polarization dependent light scattering properties of cells from a multi-step carcinogenesis model have been measure.d Evidence for differences in the light scattering properties of the cells is presented.

Oblique incidence reflectometry is a simple and accurate method for measuring the absorption and reduced scattering coefficients of turbid media. We used this technique to deduce absorption and reduced scattering spectra form wavelength-resolved measurements of the relative diffuse reflectance profile of white light as a function of source- detector distance. In this study we measured the absorption and reduced scattering coefficients of chicken breast tissue in the visible range with the oblique incidence probe oriented at 0 to 90 degrees relative to the muscle fibers. We found that the deduced optical properties varied with the probe orientation. This experiment demonstrated the application of oblique-incidence, fiber-optic reflectometry to measurements on biological tissues and the effect of tissue structural anisotropy on optical properties.

The light scattering is the main reason to degrade the imaging resolution of optical tomography. In order to improve the imaging resolution, polarization and scattering angle differential technique was developed to suppress the effect of scattering in turbid medium. An explicit theory for the technique was presented and a transillumination imaging system under the technique was set up. Using this system, the feasibility of this technique was demonstrated through a serial of experimental measurements in model phantom and higher resolution was obtained than that maintained by conventional method. The technique shows promise in getting the high resolution imaging in conjunction with other optical tomography methods.

In recent years, in-vivo optical spectroscopy has become widely used during many routine endoscopic procedures for purposes of biomedical science and practical medicine by virtue of its non-invasive effect and real time remote detection convenience. Fiber optic probes are an important unit of spectroscopic equipment for providing effective excitation and light gathering on site. Probes should be small enough to be easy introduced into the instrumentation channel of standard flexible or rigid endoscopes. Several commercial and custom types of fiber tips for applications in diagnostics have been examined. Single- and multifiber delivery schemes are also discussed. To provide comparative analyses of probes' optical properties, sensor optical efficiency and tip coupling efficiency coefficients have been proposed. These coefficients are a quantitative measure of probe optical efficiency and have been calculated by means of ray tracing for both fiber-tissue and tissue-fiber traces. These results could be a helpful tool for designers of fiber probes for Raman, laser-induced fluorescence and elastic-scattered spectroscopy of internal human organs.

A real time fluorescence ratio mapping a system for evaluating the surface of tissue can detect changes in tissue state including precancerous conditions. This system, using a personal computer and video camera, functions in near real-time, displaying pseudo color fluorescence intensity ratios maps at 15 frames per second. A fluorescence ratio mapping system functioning at close to video rates can be integrated with an endoscope to give the physician additional information during a patient exam about the state of a tissue. Preliminary measurements on ex vivo samples differentiated tumor from normal regions of tissues. Fluorescence spectral ratio maps give more structural detail of tissue than direct white light viewing. Tissue structure was observed in the fluorescence images. These results were correlated with histology.

Fluorescence, excitation, diffuse reflectance and FT-IR Raman spectral scans of squamous cell carcinoma of the head and neck have been studied. Eleven specimens of squamous cell carcinoma in larynx, lymph node, throat, grand, soft palate and epiglotts and nine corresponding normal specimens was tested. The excitation spectral differences between squamous cell carcinoma and normal were investigated in the protein spectral range 260 nm to 300 nm. The ratios intensity at various excitation wavelengths were used as criterion to separate carcinoma and normal tissues. The t- test of these parameter have been made. The P values of all these three parameters are less than 0.005. The statistical results of sensitivity and specificity of using three parameters compared to pathological results are about 90 percent. The Raman spectroscopy was measured. Characteristics vibrational modes were used to separate out carcinoma and correlated with excitation spectroscopy data.

Spectral and temporal profiles together with polarization for far-red to NIR emission wing beyond 650 nm were measured form adipose and tissues. The characteristics of the emission arising form adipose and tissues are shown to be different under 532 nm and 632 nm laser excitation indicating a different origin of the emission. The difference in the spectral intensity of the far-red to NIR emission under 632 nm laser excitation was used to image and distinguish adipose from tissue.

Fluorescence spectrum is known to be a sensitive tool for detecting microscopic changes in the environment of fluorophores. Steady state fluorescence spectroscopy has been utilized to probe the environment of these molecules in normal and diseased skin human tissue. For fluorescence polarization spectroscopy of human skin tissues, the sample were excited with a plane polarized 50mW argon ion laser operated at 488 nm and the luminescence spectra were recorded after passing through an analyzer. The spectra were recorded with SPEX 1877E triplemate attached with a cooled PMT and DM3000R data acquisition system. The polarized fluorescence spectra of cancerous human skin tissue is different from normal human tissue. The maxima of the spectra of normal and cancerous human tissue are located around 540 nm and 530 nm respectively. The main feature of the computed anisotropy at different wavelengths is the higher value of degree of anisotropy of cancerous tissue compared to the normal counterpart. Higher degree of anisotropy of cancerous tissue may imply that the fluorophores are more tightly bound to proteins or are in a more viscous environment.

Gallstones have been classified as being cholesterol type and pigment type. The classification is important for diet control of the patient to avoid recurrence of the stone. Spectroscopy is a sensitive technique to determine the composition of the gallstone both in-vitro and in-vivo. this work deals with the fluorescence spectroscopy of gallstone. For fluorescence spectroscopic studies of gallstone, samples were excited with 5 mw of 488 nm line of argon-ion laser and spectra were recorded with a SPEX 1877E triplemate attached with a cooled PMT and DM3000R data acquisition system. Fluorescence spectra from pure cholesterol and bilirubin were also recorded for comparison. Different types of gallstones: mixed, cholesterol, pigment type were studied. All spectra exhibited a very broad band, 500 to 800 nm and sometimes two bands, depending on type of stone. Pure cholesterol shows three prominent fluorescence peaks at 513, 550, 583 nm along with two peaks at approximately 568 and 586 nm. Pure bilirubin shows prominent peak at 628 nm, without any Raman line. From fluorescence spectra different types of stones are identified. Different gallstones studied show a mixture of cholesterol and bilirubin types and the ratio of the two varies from one sample type to another.

Surface enhanced FTIR spectroscopy with polarized light can be used to investigate the structure of functionalized membranes. We have used this method to characterize lipid membranes containing integral membrane proteins. The Ge ATR crystal was coated by a 9 nm layer of Ag clusters before adsorbing the membrane. The dipoles of the clusters get excited by the evanescent field. The observed spectra show an enhanced absorption of different functional groups. The enhancement factor depends upon the polarization of the incident light and depends upon the orientation of this groups towards the cluster surface. This allows the detection of the structure and orientation of the membrane fragments at the interface of the clusters.

The quality of platelets transfused is vital to the effectiveness of the transfusion. Freshly prepared, discoid platelets are the most effective treatment for preventing spontaneous hemorrhage or for stopping an abnormal bleeding event. Current methodology for the routine testing of platelet quality involves random pH testing of platelet rich plasma and visual inspection of platelet rich plasma for a swirling pattern indicative of the discoid shape of the cells. The drawback to these methods is that they do not provide a quantitative and objective assay for platelet functionality that can be used on each platelet unit prior to transfusion. As part of a larger project aimed at characterizing whole blood and blood components with multiwavelength UV/vis spectroscopy, isolated platelets and platelet in platelet rich plasma have been investigated. Models based on Mie theory have been developed which allow for the extraction of quantitative information on platelet size, number and quality from multi-wavelength UV/vis spectra. These models have been used to quantify changes in platelet rich plasma during storage. The overall goal of this work is to develop a simple, rapid quantitative assay for platelet quality that can be used prior to platelet transfusion to ensure the effectiveness of the treatment. As a result of this work, the optical properties for isolated platelets, platelet rich plasma and leukodepleted platelet rich plasma have been determined.

The characterization of biological fluids for diagnostic purposes implies the estimation of the particle size distribution, the chemical composition, the shape distribution and the charge of the particles suspended in the fluid. These particle properties are defined as the joint particle property distribution. The shape distribution is especially important for the identification of healthy cells, sickle cells, viruses and bacteria. platelet viability can be determined from cell shape, discoid versus spheroid. Recent theoretical and simulations of the scattering behavior of particle dispersions using Mie and Rayleigh-Gans-Debye scattering models have demonstrated that complementary information on the joint property distribution is available from angular measurements of combined absorption and scattering spectra. A new multiangle- multiwavelength (MAMW) spectrophotometer was designed and developed using state-of-the-art fiber optic technology for the characterization of concentrated and dilute dispersion of biological fluids, primarily for whole blood and platelets. Miniature fiber optic UV/vis spectrometers were used to create a portable and robust characterization system; the spectrometer allows for the application of fields and easy adaptation for on-line analysis. This instrumentation was successfully tested for scattering measurement sensitivity and reproducibility and is capable of simultaneous absorption and scattering measurements at several angles and over a wide wavelength range. The MAMW spectrophotometer has been sued to analyze sizes of macromolecules and particle standards. Experimental results demonstrate that this technology can be used for the characterization of the joint property distribution of dilute dispersion of platelets, other biological particles and fluids.

In this investigation an attempt is made to find the effects of controlled breathing on brain with the help of optical sensor mounted on the left and right temples of a subject. It has already been established that the brain activity can be monitored in terms of arterial blood volumetric changes to the left and right hemispheres of the brain recorded with the help of optical sensors. To investigate the influence of controlled breathing, an expert in controlled breathing is chosen as the subject. Pranayama is believed to be the controlled intake and outflow of breath in a firmly established posture. Some types of pranayama are believed to relieve mental stress. While the subject is practicing one such type of breath control, arterial blood volume changes in the brain are recorded using optical sensor mounted on the left and right temples of the subject. From these measurements at the beginning and end of the pranayama exercise, it could be noticed that the subject could induce changes in the cardiac and respiratory rhythms by controlled breathing. Rhythmic phenomena in the skin perfusion in the vicinity of the brian are also studied when the subject is holding his breath. The arterial blood volume changes to the left and right hemispheres of the brian, as monitored by the optical sensors during this period, exhibit asymmetric reaction when the subject is holding his breath. An attempt is made to understand whether these changes induced by stoppage of breathing are 'chaotic' or 'adaptive' in nature.

Human skin is a formidible light barrier to non-invasive, transcutaneous optical diagnosis of deep-seated lesions. Human skin is composed to two anatomically and physiologically distinct layers, the superficial epidermis and the deeper dermis. Each contains optically active tissue components including scatters, absorber and fluorophores. Beneath the skin is the subcutaneous fibroadipose tissue that varies in thickness and composition depending on the anatomic site and the person's habitus. Delivery of interrogation light and capture of reflected or emitted diagnostic light from deep-seated lesions will be greatly influenced by the skin and subcutaneous tissue. Diagnostic strategies will have to include careful analysis of anatomically distinct 'normal tissues' before 'abnormal lesions' can be distinguished.

We report the development of a fluorescence lifetime imaging (FLIM) system based on a time-gated image intensifier and solid-state laser amplifier with a system response of < 100 ps. We have sued this system to image lifetimes as short as 100 ps and to image changes in the environment of a fluorescent phantom, causing lifetime differences less than 10 ps. The versatility of this FLIM system has been demonstrated by measuring both the temporal and spectral profiles of multiple fluorescent samples in a single acquisition. Fluorescence lifetime imaging of tissue constituents has also been carried out and first results suggest that this technique can provide a means of distinguishing between different tissue constituents.

The goal of this study is to define the spectro-temporal fluorescence emission of the main types of lipoid that accumulate in atherosclerotic lesions cholesterol esters: cholesteryl linoleate (CL) and cholesteryl oleate (CO). CO is commonly found in fatty-streaks whereas CL and FC predominate in advanced lesions and their necrotic core. Fluorescence emission of the purified components was induced with Nitrogen laser pulses and detected with a MCP-PMT connected to a digital oscilloscope. Using a Laguerre expansion of kernels algorithm the intrinsic fluorescence pulses were deconvolved from the measured transient pulses. A double exponential function depicted the fluorescence decay characteristics.

The optical characterization of scattering media and biological samples is a very important topic due to the interest in using optical techniques as medical diagnostic tool. In this paper we reported experimental results concerning absorption and reduced scattering coefficients obtained in different scattering thick samples like latex and intralipid solutions, bovine muscle, chicken and turkey breast using a tunable time-resolved laser transmittance set-up. Our experimental data have been compared with the result of other different techniques and with the predictions of Mie theory indicating advantages and limits of time-resolved transmittance spectroscopy.

The time-dependent decay of arterial wall fluorescence remains relatively unexplored. We measured the timeresolved fluorescence emission (337 urn excitation) of 37 excised samples of human aorta classified histologically as normal or Type I- Type Va atherosclerotic lesion (Stary's classification). Time-resolved spectra (370-510 nm, 0.5 ns temporal resolution) were constructed by deconvoluting the emission waveform using the discrete-time Laguerre basis for expansion of the decay. The spectra changed markedly with tissue type, being spectrally broad for normal samples and peaked for atherosclerotic samples. In the time dimension, the emission lasted longer for the atherosclerotic samples. Biexponential approximation of the fluorescence time-dependent decay (390 nm) showed that the long decay constant for Type II lesions (5.95±0.16 ns) was larger than the corresponding parameter for normal arterial wall (5.42±0.17 ns) and smaller than the decay constant for intermediate (Type III) and advanced (Types P1-Va) lesions (6.88±0.32 ns, 7.38±0.51 ns). Discriminant analysis using the time constants at 390 nm and the intensity ratio I 39011 470 as predictor variables classified near-normal (normal-Type I) and diseased samples (Types ffi-V) with <90% sensitivity. 75% of Type II lesions were correctly classified. These results suggest that atherosclerotic lesions can be accurately classified based on their time-resolved fluorescence spectra. Keywords: time-resolved fluorescence, atherosclerosis, nitrogen laser, arterial wall, classification of lesions

A new method to produce an interference depth-lateral image of skin tissue without axial-lateral scanning with a 20 ms image acquisition time using grating-generated interference microscopy is demonstrated. The depth scan of 1.5 mm and the transversal coordinate image about 0.3 mm of the skin tissue with a depth resolution of 17 micrometers , and a sensitivity of up to -100 dB are achieved using 30 mw from mode locked Ti:sapphire laser at (lambda) equals 800 nm.

A fast grating-generated scan heterodyne interferometric tomography is proposed. Heterodyne detection is performed by use of the Doppler frequency shift that result from moving the reflection diffraction grating with constant speed. Biological tissue and blood flow imaging are considered as applications of this method.

We present a new microscopy system based on the OCT principle, that uses a multiplexed lock-in detection scheme to generate a 2D head-on image in parallel without lateral scanning. Our 'full-field optical coherence microscopy' comprises a Michelson interferometer built with a polarizing beam splitter, and uses a photoelastic birefringence modulator to modulate the optical path difference between the two orthogonal polarizations. A novel signal processing method is used to achieve a demodulation in parallel on every pixel of a 256 X 256 CCD camera. A 840 nm electroluminescent diode with 20 micrometers coherence length is used to illuminate the field of view through the microscope objective lens. In-depth exploration of the sample is realized by changing the plane of focus. The lateral resolution of the images is limited by the camera pixel size and is 2 micrometers . The axial sectioning ability is approximately 8 micrometers . Having validated our setup on model samples, we now evaluate its performance on biological structures. As an example, images of onion cells from 50-400 micrometers below the surface are obtained in 1 s with 100 dB sensitivity.

Second harmonic and two photon fluorescence generated by focused 10 fs ultrashort laser pulses were used to image subsurface structures of highly scattering biological tissues. By scanning the focal point of the incident laser beam, 2D sectional images of the tissues subsurface structure properties, such as symmetry and molecular compositions are obtained.

The purpose of this study is to perform a preliminary evaluation of the diagnostic potential of laser-induced autofluorescence spectroscopy for colonic tumors using fluorescence intensity ratios at specific wavelengths. Measurements were made on normal mucosa and adenocarcinoma of human colon tissues. Each tissue section was examined using an optical probe consisting of a central fiber for delivering the excitation laser and a fiber bundle for detection of the fluorescence. Using different wavelengths of a CW mixed-gas laser, we measured and analyzed the LIAF spectra of tissues through a polychromator coupled with a CCD detector. It can be found that in the range of 520-620 nm, the spectral shapes of tumor tissues are very similar to the normal tissue spectra, and the autofluorescence intensities of normal tissues are about threefold higher than those of tumor tissues. However, in the range of 625- 720nm, the autofluorescence intensities of tumor tissues are higher than those of normal tissue. The spectral characteristics peaks arising from endogenous porphyrins can be observed in some tumor tissues. The preliminary results show that the autofluorescence intensity ratio of I₅₈₀/I₆₃₀ or I₅₈₀/I₆₈₀ can be used to distinguish colonic tumors from normal tissues with good accuracy.

Fourier transform IR fiberoptic evanescent wave spectroscopy has been suggested as a powerful tool for clinical diagnostics of normal, precancerous, and cancerous breast tissue ex vivo and in vitro in the middle IR region of the spectrum. This method is suitable for noninvasive and direct measurements of the spectra of normal and pathological tissues in vitro, ex vivo and in vivo. For the first time this method has been used nondestructively, rapidly, fast and remotely in the operating rooms of clinics. The aim of our studies has been the express testing of various tumor tissues during surgery to optimize and localize an area of removing tissue. We measured very small samples of normal and malignant breast tissue ex vivo with a fiber bent to a special angle, forming a special tip probe configuration. We also suggest her the minimally invasive FTIR-FEW diagnosis tool using 'needle' changeable tip probe for biopsy applications. The spectral histopathology branching could be developed with help of this technique in vitro. The breast tissue at different stages of tumor or cancer has been distinguished very clearly in spectra of amide, cyclic and noncyclic hydrogen bonded fragments, phosphate groups and sugars.