This PDF file contains the front matter associated with SPIE Proceedings Volume 8230, including the Title Page, Copyright information, Table of Contents, and the Conference Committee listing.

We present, for the first time an in vivo implementation of dynamic light scattering (DLS) adapted to optical coherence tomography (OCT). Human bladder carcinoma tumors were grown in dorsal skin-fold window chambers fitted to male nude mice and imaged at a rate of 200 Hz using OCT. Maps of speckle decorrelation times (DT) were generated for regions of skin from individual mice as well as for regions containing tumor tissue before and after treatment with chemotherapy. Variations in DT were found between individual mice exhibiting different skin anatomy (primarily due to deterioration from the window chamber implantation). A significant difference in DT was also observed between tumor regions and surrounding normal tissue. Finally, maps of DT generated for tumor tissue treated with chemotherapy indicated a drop in DT at 24 and 48 hours after treatment. These preliminary results suggest the feasibility of using DLSOCT to measure intracellular motion as an endogenous contrast mechanism in vivo.

We have been investigating the quantification of skin surface roughness by polychromatic speckle contrast. Speckle contrast, being a measure of light coherence, decreases as coherence decays when low coherent light is reflected from a rough surface. The main constraint of applying the technique to skin is the presence of bulk scattering along with surface reflection. Bulk scattering also decays coherence and is a source of noise. To examine the effect of bulk contribution, we studied speckle patterns generated by silicone phantoms with controllable roughness and optical parameters in the range of human skin. We discovered that using the theoretical curve plotting speckle contrast vs. surface roughness as a calibration curve overestimates the phantom surface roughness. We propose to use the effective calibration curve for the proper skin roughness measurements. The effective calibration curve was obtained experimentally taking the advantage of its weak dependence on phantom's attenuation coefficients.

The detection of cellular mitosis inside three-dimensional living tissue at depths up to 1 mm has been beyond the detection limits of conventional microscopies. In this paper, we demonstrate the use of motility contrast imaging and fluctuation spectroscopy to detect motional signatures that we attribute to mitotic events within groups of 100 cells in multicellular tumor spheroids. Motility contrast imaging is a coherence-domain speckle-imaging technique that uses low-coherence off-axis holography as a coherence gate to localize dynamic light scattering from selected depths inside tissue. Fluctuation spectroscopy is performed on a pervoxel basis to generate micro-spectrograms that display frequency content vs. time. Mitosis, especially in Telophase and Cytokinesis, is a relatively fast and high-amplitude phenomenon that should display energetic features within the micro-spectrograms. By choosing an appropriate frequency range and threshold, we detect energetic events with a density and rate that are comparable to the expected mitotic fraction in the UMR cell line. By studying these mitotic events in tumors of two different sizes, we show that micro-spectrograms contain characteristically different information content than macro-spectrograms (averaged over many voxels) in which the mitotic signatures (which are overall a low-probability event) are averaged out. The detection of mitotic fraction in thick living tissue has important consequences for the use of tissue-based assays for drug discovery.

Since random lasers were the objective of fundamental investigation, they have awaited utilities in biomedical applications. We investigate the feasibility that random lasing modes in disordered media can be used for singlenanoparticle quantitation. Our numerical experiments reveal that minute alterations in a single nanoparticle can induce dramatic changes in the eigenmodes of the system, which are self-formed in the disordered media, and that the multiple modes can serve as a "fine fishnet" to capture any perturbations. Combining its simplicity of realization and its sensitivity, random lasers could potentially serve a new biosensing mechanism.

The Radial Angular Filter Array (RAFA) is a novel optical filter consisting of a radially-distributed series of micromachined channels, which converge upon a focal point several millimeters away from the edge of the device. It is designed to measure the angular distribution of light emitted from an object located at the focal point and is capable of selecting ballistic and quasi-ballistic photons at specific angles out of strong background noise due to scattering. We hypothesized that the device might be useful for examining optically absorbing features below the surface of a turbid medium via a depth mapping approach. In order to validate this concept, experiments were performed with a focused laser beam, a series of Intralipid^TM solutions (0.1 wt% to 1.0 wt%), a 0.5 mm diameter graphite rod (absorber), and a RAFA optically coupled to a line camera. By scanning the position of the rod and comparing the light scattering profiles obtained by the RAFA at each scanning step, the location and the depth of the rod were successfully identified. Future work will be directed toward studying the performance of the technique with a collimated broadband illumination beam for spectroscopic applications.

We demonstrate the use of a low-coherence spectral-domain phase microscopy (SDPM) system for accurate quantitative phase measurements in red blood cells (RBCs) for the prognosis and monitoring of disease conditions that affect the visco-elastic properties of RBCs. Using the system, we performed time-recordings of cell membrane fluctuations, and compared the nano-scale fluctuation dynamics of healthy and glutaraldehyde-treated RBCs. Glutaraldehyde-treated RBCs possess a lower amplitude of fluctuations reflecting an increased membrane stiffness. To demonstrate the ability of our system to measure fluctuations of lower amplitudes than those measured by the commonly used holographic phase microscopy techniques, we also constructed a wide-field digital interferometric microscope and compared the performances of the two systems. Due to its common-path geometry, the optical-path-delay stability of SDPM was found to be less than 0.3nm in liquid environment, at least three times better than in holographic phase microscopy under the same conditions. In addition, due to the compactness of SDPM and its inexpensive and robust design, the system possesses a high potential for clinical applications.

We describe the design and performance of a Hyperspectral Imaging System (HSI) for label-free characterization of human metaphase chromosomes. Chromosomes consist of a DNA-protein complex that is organized in sub-structures and can be described by an array of "particles" with different size and refractive indices. This locally resolved stray light pattern can be used to visualize and characterize unstained chromosomes. The paper describes an imaging system where stray light spectra of chromosomes are detected using a Pushbroom Imaging device attached to a standard microscope in combination with multivariate data analysis. To proof the concept, single particle spectra as well as particle array spectra are analyzed and explained by means of Mie scattering theory and the results are confirmed with FDTD (Finite Difference Time Domain) simulations. This label free signature is due to the superposition of the interference pattern of the different layer thicknesses, the spectral interference of the band pattern, changes in refractive indices along the chromosome axis as well as the absorption of chromophores in different spectral regions of the chromatin condensation. This complex spectral signature can be analyzed by means of a principal component analysis (PCA) and classified in a multidimensional PCA space.

The use of empirical models for the extraction of optical and physiological properties from reflectance spectra is a relatively new approach, as compared to other techniques such as those based on the diffusion theory and inverse Monte Carlo algorithms. Empirical models are appealing for their ease of implementation and applicability to conditions for which analytical models are limited. Thus far, however, empirical models have been limited to only a handful of specific probe geometries. In this work, the relationship between reflectance and optical property values is explored for a wide range of geometry and tissue conditions. The influence of variation in scattering phase function, and numerical aperture of the optical fibers are investigated and incorporated into the empirical relationships for the first time. Reflectance data used in this work was simulated using an improved Monte Carlo model designed to run on a graphics processing unit (GPU). Improvements include a Modified Henyey-Greenstein and a Mie theory-based phase function in place of the conventional Henyey-Greenstein phase function, and assignment of probe-specific reflectivity conditions to better model the tissue-probe tip interface. These improvements are particularly important for probe geometries with small sourcedetector separations. Probe geometries that offer the most stable relationships between reflectance and optical property values, and hence, the best accuracy and reliability in extraction of physiological properties from tissue, are presented.

We report an approach for determination of the scattering coefficient, the reduced scattering coefficient, and the anisotropy factor from the quantitative phase map measured by differential interference contrast microscopy based on the scatteringphase theorem. The approach is first validated by showing the excellent agreement between the retrieved optical properties of polystyrene spheres and Intralipid-20% suspension and their known values. The scattering properties of unstained pathological prostate cancer slides and fresh cancerous and normal colon tissue samples are then investigated. A clear trend with cancer in the reduced scattering coefficient and the anisotropy factor is shown. The potential of the approach for tissue diagnosis is discussed at the end.

Brain glial tumors have peculiar features of the perifocal region extension, characterized by its indistinct area, which complicates determination of the borders for tissue resection. In the present study filter-reduced back-scattered laser light signals, compared to the data from mathematical modeling, were used for description of the brain white matter. The simulations of the scattered light distributions were performed in a Monte Carlo program using scattering and absorption parameters of the different grades of the brain glial tumors. The parameters were obtained by the Mie calculations for three main types of scatterers: myelinated axon fibers, cell nuclei and mitochondria. It was revealed that diffuse-reflected light, measured at the perifocal areas of the glial brain tumors, shows a significant difference relative to the signal, measured at the normal tissue, which signifies the possibility to provide diagnostically useful information on the tissue state, and to determine the borders of the tumor, thus to reduce the recurrence appearance. Differences in the values of ratios of diffuse reflectance from active growth parts of tumors and normal white matter can be useful for determination of the degree of tumor progress during the spectroscopic analysis.

Quantitative morphological assessment of biological cells and their subcellular environment is important to characterize cellular state in normal and diseased tissue and cellular response to various experimental treatments. Recently, we showed that optical Gabor-like filtering of light scattered by spheres yields an optical measurement which varies linearly with diameter. In addition, the sensitivity to changes in size was superior to post-processing of digital images. Here, we extend our previous results by showing that the linear relationship between Gabor filter period and particle size holds over a size range from 100nm to 2000nm. We also show that this relationship is independent of the particle's or medium's refractive index. Using simulations, we provide a theoretical basis for our findings. Unlike previous methods, this technique does not require the presence of single isolated particles and thus may be used to directly extract the characteristic size associated with the local texture of heterogeneous objects. We therefore discuss this applicability of our method in heterogeneous samples consisting of collagen and living cells.

Traditional light scattering analysis of cells relies mostly on the one-dimensional distribution of the light scattering intensity, where only the cell size and some limited information regarding the internal structure can be obtained. More recent studies have attempted to analyze the two-dimensional diffraction images of cells using standard texture analysis techniques to extract additional intracellular information. We recently compared the effectiveness of several major methods that are often used in image texture analysis and found that the Gabor filter approach is more effective than most methods and is capable of providing information regarding the major structural features and mitochondrial properties of the cell. In this report we further our investigation by utilizing a Gabor filter technique to analyze light scattering patterns of cells and to correlate their changes to that of the mitochondrial properties of the cells. Numerical simulations of light scattering are performed using the discrete dipole approximation on analytically generated biological cell models with various mitochondrial characteristics. A set of two-dimensional scattering images is produced corresponding to systematic variations in the size, shape, and distribution of mitochondria and is processed with a bank of Gabor filters. Selected mean values of the Gabor-filtered images are displayed in scatter plots, providing a novel approach to grouping the cell models according to mitochondria size, shape, and distribution.

Because of its high spatial resolution and noninvasive imaging capabilities, optical coherence tomography has been used to characterize the morphological details of various biological tissues including urinary bladder and to diagnose their alternations (e.g., cancers). In addition to static morphology, the dynamic features of tissue morphology can provide important information that can be used to diagnose the physiological and functional characteristics of biological tissues. Here, we present the imaging studies based on optical coherence tomography to characterize motion related physiology and functions of rat bladder detrusor muscles and compared the results with traditional biomechanical measurements. Our results suggest that optical coherence tomography is capable of providing quantitative evaluation of contractile functions of intact bladder (without removing bladder epithelium and connective tissue), which is potentially of more clinical relevance for future clinical diagnosis - if incorporated with cystoscopic optical coherence tomography.

A spectral analysis technique to enhance tumor contrast during breast conserving surgery is proposed. A set of 29 surgically-excised breast tissues have been imaged in local reflectance geometry. Measures of broadband reflectance are directly analyzed using Principle Component Analysis (PCA), on a per sample basis, to extract areas of maximal spectral variation. A dynamic selection threshold has been applied to obtain the final number of principal components, accounting for inter-patient variability. A blind separation technique based on Independent Component Analysis (ICA) is then applied to extract diagnostically powerful results. ICA application reveals that the behavior of one independent component highly correlates with the pathologic diagnosis and it surpasses the contrast obtained using empirical models. Moreover, blind detection characteristics (no training, no comparisons with training reference data) and no need for parameterization makes the automated diagnosis simple and time efficient, favoring its translation to the clinical practice. Correlation coefficient with model-based results up to 0.91 has been achieved.

In clinical diagnostic procedures, gingival inflammation is considered as the initial stage of periodontal breakdown. This is often detected clinically by bleeding on probing as it is an objective measure of inflammation. Since conventional diagnostic procedures have several inherent drawbacks, development of novel non-invasive diagnostic techniques assumes significance. This clinical study was carried out in 15 healthy volunteers and 25 patients to demonstrate the applicability of diffuse reflectance (DR) spectroscopy for quantification and discrimination of various stages of inflammatory conditions in periodontal disease. The DR spectra of diseased lesions recorded using a point monitoring system consisting of a tungsten halogen lamp and a fiber-optic spectrometer showed oxygenated hemoglobin absorption dips at 545 and 575 nm. Mean DR spectra on normalization shows marked differences between healthy and different stages of gingival inflammation. Among the various DR intensity ratios investigated, involving oxy Hb absorption peaks, the R620/R575 ratio was found to be a good parameter of gingival inflammation. In order to screen the entire diseased area and its surroundings instantaneously, DR images were recorded with an EMCCD camera at 620 and 575 nm. We have observed that using the DR image intensity ratio R620/R575 mild inflammatory tissues could be discriminated from healthy with a sensitivity of 92% and specificity of 93%, and from moderate with a sensitivity of 83% and specificity of 96%. The sensitivity and specificity obtained between moderate and severe inflammation are 82% and 76% respectively.

A modified polarized Monte Carlo code is developed that allows heterogeneous structure to be modeled. The code is validated with existent polarized Monte Carlo code. Heterogeneous structure simulating colon tissue is simulated to understand the difference between simulations of homogeneous vs heterogeneous tissue structure. Reflectance measurements from simulations containing increased blood vessel size and increased blood volume fraction, both markers for potential cancerous tissue, are studied in order to better interpret reflectance measurement from diagnostic probes.

A blind separation technique based on Independent Component Analysis (ICA) is proposed for breast tumor delineation and pathologic diagnosis. Tissue morphology is determined by fitting local measures of tissue reflectance to a Mie theory approximation, parameterizing the scattering power, scattering amplitude and average scattering irradiance. ICA is applied on the scattering parameters by spatial analysis using the Fast ICA method to extract more determinant features for an accurate diagnostic. Neither training, nor comparisons with reference parameters are required. Tissue diagnosis is provided directly following ICA application to the scattering parameter images. Surgically resected breast tissues were imaged and identified by a pathologist. Three different tissue pathologies were identified in 29 samples and classified as not-malignant, malignant and adipose. Scatter plot analysis of both ICA results and optical parameters where obtained. ICA subtle ameliorates those cases where optical parameter's scatter plots were not linearly separable. Furthermore, observing the mixing matrix of the ICA, it can be decided when the optical parameters themselves are diagnostically powerful. Moreover, contrast maps provided by ICA correlate with the pathologic diagnosis. The time response of the diagnostic strategy is therefore enhanced comparing with complex classifiers, enabling near real-time assessment of pathology during breast-conserving surgery.

We obtain absorption and scattering reconstructed images by incorporating a priori information of target location obtained from fluorescence diffuse optical tomography (FDOT) into the diffuse optical tomography (DOT). The main disadvantage of DOT lies in the low spatial resolution resulting from highly scattering nature of tissue in the near-infrared (NIR), but one can use it to monitor hemoglobin concentration and oxygen saturation simultaneously, as well as several other cheomphores such as water, lipids, and cytochrome-c-oxidase. Up to date, extensive effort has been made to integrate DOT with other imaging modalities such as MRI, CT, to obtain accurate optical property maps of the tissue. However, the experimental apparatus is intricate. In this study, DOT image reconstruction algorithm that incorporates a prior structural information provided by FDOT is investigated in an attempt to optimize recovery of a simulated optical property distribution. By use of a specifically designed multi-channel time-correlated single photon counting system, the proposed scheme in a transmission mode is experimentally validated to achieve simultaneous reconstruction of the fluorescent yield, lifetime, absorption and scattering coefficient. The experimental results demonstrate that the quantitative recovery of the tumor optical properties has doubled and the spatial resolution improves as well by applying the new improved method.

We present a novel spectral Mueller matrix measurement system for both elastic and inelastic scattering (fluorescence) polarimetric measurements. The system comprises of a Xenon lamp as excitation source, a polarization state generator (PSG) and a polarization state analyzer (PSA) unit to generate and analyze polarization states required for 4 x 4 sample Mueller matrix measurements, coupled to a spectrometer for spectrally resolved (λ ~ 400 - 800 nm) signal detection. The PSG unit comprises of a fixed linear polarizer (polarization axis oriented at horizontal position) followed by a rotatable broadband quarter wave plate. The sample-scattered light is collected and collimated using an assembly of lenses, then passes through the PSA unit, and is finally recorded using the spectrometer. The PSA unit essentially consists of a similar arrangement as that of the PSG, but positioned in reverse order, and with the axis of the linear polarizer oriented at vertical position. A sequence of sixteen measurements are performed by changing the orientation of the fast axis of the quarter wave plates of the PSG unit (for generating the four required elliptical polarization states) and that of the PSA unit (for analyzing the corresponding polarization states). The orientation angles (35°, 70°, 105° and 140°) were chosen based on optimization of the PSG and PSA matrices to yield most stable system Mueller matrices. The performance of the polarimeter was calibrated using Eigenvalue calibration method which also yielded the actual values of the system PSG and PSA matrices at each wavelength. The system has been automated and is capable of Mueller matrix measurement with high accuracy over the entire spectral range 400 - 800 nm (elemental error < 0.01). For recording the elastic scattering Mueller matrix of sample, the PSG and PSA matrices for each wavelength are used, while for fluorescence Mueller matrix measurements, the PSG for the excitation wavelength (chosen to be 405 nm) and PSA for varying emission wavelengths (450 - 800 nm) are used. The developed spectral Mueller matrix system has been initially used to record both elastic scattering and fluorescence Mueller matrices from normal and cancerous cervical tissues.

This research proposes a new imaging technique for near real time multispectral acquisition of the so called "Degree Of Polarization" (DOP) in polarimetry for future clinical investigation. The aim of exploiting the DOP as the contrast element is to demonstrate that the elliptical DOP provides more information characterizing complex medium than the more traditional linear and circular ones. The system considers an incoherent input white light beam and opportunely calibrated nematic crystals, so no mechanical tools are necessary. The biomedical application of this method suggests a simple, direct, fast and also easily exploitable future employment, as a desirable mean for clinical investigation. Moreover new elements to improve the model of light scattering will be acquired.

To reduce the cost of near-infrared endoscopic image equipment and the reconstruction time, a measurement method based on the effective detection area is proposed and the corresponding algorithm which simultaneously reconstructs the absorption coefficient and the reduced scattering coefficient is developed. First, the effective detection area is investigated with the Monte Carlo simulation. Secondly, the image reconstruction algorithm based on the effective detection area is studied. The Jacobin matrix is built by combining the adjoint method with the modified Generalized Pulse Spectrum Technique and calibrated by the maximum of its absolute value. The Generalized Minimal Residual Krylov method is used to obtain the iterative update factor. Finally, the impact of the number of measured points in the effective detection area on the reconstructed results is discussed, and the robustness of the algorithm to noise and cross-talk are verified by the simulated test data. The results show that the reconstructed algorithm based on the effective detection area has equivalent accuracy to the traditional ones. The fidelity of reconstructed absorption and reduced scattering coefficients can be 80%, respectively. The scales and positions of the reconstructed lesions are both correspond to the true and the reconstruction time is reduced by half. The optimal number of sources and detectors is 16 depending on the scale of the simulation model. The detection using the effective detection area and the developed reconstruction algorithm will promote the development of diffuse optical tomography which is applied to cervical and other tubular organs.

Diffuse scattering measurements to determine the changes of glucose concentration in a highly scattering medium are conducted by using an optical heterodyne technique. The heterodyne technique can increase the signal-to-noise ratio (SNR) of the detection amplitude and phase by coherence gating and narrow detection bandwidth. The experimental results showed that a good sensitivity of 0.1% scattering change per mM is observed.

Cell density is an important parameter in the question of bio-variation and the studying of cell scattering could be a viable tool. The development of spatially resolved optical fiber probe would enable the characterization of optical scattering from cells within a colony. Single mode fiber probe would be budget friendly as compared to a 50-nm sub-cellular fiber probe. This project develops a calibration procedure to correlate the optical scattering measured by a single mode fiber probe to that of a 50-nm sub-cellular fiber probe in the context of cell density variation. The Fourier transform of intensity angular transmission would give correlation information in the Efield in the spatial coordinate. Monte Carlo simulation could be used to constrain the input intensity function spatial content resembling microscopy. The use of a 50-nm sub-cellular fiber probe for detailed study of biological samples would give sub-micron scale length information.