This PDF file contains the front matter associated with SPIE Proceedings Volume 6446, including the Title Page, Copyright information, Table of Contents, Introduction, and the Conference Committee listing.

We describe path length resolved Doppler measurements of the multiply scattered light in turbid media using phase modulated low coherence Mach-Zehnder interferometer, with separate fibers for illumination and detection. A Doppler broadened phase modulation interference peak observed at the modulation frequency shows an increase in the average Doppler shift with optical path length. The path length dependent Doppler broadening of scattered light due to the detection of multiple scattered light is measured from the Lorentzian linewidth and the results are compared with the predictions of Diffusive Wave Spectroscopy. For particles with small scattering anisotropy, the diffusion approximation shows good agreement with our experimental results. For anisotropic scatterers, the experimental results show deviations from the Diffusion theory. The optical path lengths are determined experimentally from the Zero order moment of the phase modulation peak around the modulation frequency and the results are validated with the Monte Carlo technique.

Laser speckle contrast imaging (LSCI) is a well established technique for imaging blood flow in the brain. Microfluidic devices were designed and fabricated in PDMS and TiO₂ to mimic the capillary network in the brain, with different channel sizes (10microns to 150microns). The flow of intralipid through the channels was imaged with LSCI. The microfluidic devices were used as a tissue phantom to perform controlled experiments to investigate the effect of factors that influence speckle contrast like speed, concentration of intralipid, depth of channels and exposure time. A speckle imaging instrument that allows image acquisition over a wide range of exposure times is presented. Speckle contrast was found to be a function of speed and exposure time, with concentration and channel depth serving to improve signal strength. Exposure time is shown to influence the sensitivity of speckle contrast to speed. It is also shown that speckle contrast as a function of exposure time can potentially be used a method to obtain qualitative measurements of speed.

In the study of epithelial tissues and superficial cancers, it is often important to determine the optical properties of small volumes of tissue, and at varying depths. A model that relates the reflectance spectrum to the optical properties of a turbid medium at small source-detector separations is developed based on Monte Carlo simulations and experiments in tissue phantoms. Four fiber probes are analyzed, for which each fiber probe presents a different tilt angle. Preliminary results show good correlation between known optical properties in tissue phantoms, and the measured optical properties.

The phenomenon of enhanced backscattering (EBS) of light, also known as coherent backscattering (CBS) of light, is a spectacular manifestation of self-interference effects in elastic light scattering, which gives rise to an enhanced scattered intensity in the backward direction. Although EBS has been the object of intensive investigation in non-biological media over the last two decades, there have been only a few attempts to explore EBS for tissue characterization and diagnosis. We have recently made progress in the EBS measurements of biological tissue by taking advantage of lowcoherence (or partially coherent) illumination, which is referred to as low-coherence EBS (LEBS) of light. LEBS possess novel and intriguing properties such as speckle reduction, self-averaging effect, broadening of the EBS width, depth-selectivity, double scattering, and circular polarization memory effect. After we review the current state of research on LEBS, we discuss how these characteristics apply for early cancer detection, especially in colorectal cancer (CRC), which is the second leading cause of cancer mortality in the United States. Although colonoscopy remains the gold standard for CRC screening, resource constraints and potential complications make it impractical to perform colonoscopy on the entire population at risk (age > 50). Thus, identifying patients who are most likely to benefit from colonoscopy is of paramount importance. We demonstrate that LEBS measurements in easily accessible colonoscopically normal mucosa (e.g., in the rectum of the colon) can be used for predicting the risk of CRC, and thus LEBS has the potential to serve as accurate markers of the risk of neoplasia elsewhere in the colon.

Improved methods for detecting dysplasia, or pre-cancerous growth, are a current clinical need. Random biopsy and subsequent diagnosis through histological analysis is the current gold standard in endoscopic surveillance for dysplasia. However, this approach only allows limited examination of the at-risk tissue and has the drawback of a long delay in time-to-diagnosis. In contrast, optical scattering spectroscopy methods offer the potential to assess cellular structure and organization in vivo, thus allowing for instantaneous diagnosis and increased coverage of the at-risk tissue. Angle-resolved low coherence interferometry (a/LCI), a novel scattering spectroscopy technique, combines the ability of low-coherence interferometry to isolate scattered light from sub-surface tissue layers with the ability of light scattering spectroscopy to obtain structural information on sub-wavelength scales, specifically by analyzing the angular distribution of the backscattered light. In application to examining tissue, a/LCI enables depthresolved quantitative measurements of changes in the size and texture of cell nuclei, which are characteristic biomarkers of dysplasia. The capabilities of a/LCI were demonstrated initially by detecting pre-cancerous changes in epithelial cells within intact, unprocessed, animal tissues. Recently, we have developed a new frequency-domain a/LCI system, with sub-second acquisition time and a novel fiber optic probe. Preliminary results using the fa/LCI system to examine human esophageal tissue in Barrett's esophagus patients demonstrate the clinical viability of the approach. In this paper, we present a new portable system which improves upon the design of the fa/LCI system to allow for higher quality data to be collected in the clinic. Accurate sizing of polystyrene microspheres and cell nuclei from ex vivo human esophageal tissue is presented. These results demonstrate the promise of a/LCI as a clinically viable diagnostic tool.

We have developed and implemented a system which can acquire, process, and display the inverse scattering solution for optical coherence tomography (OCT) in real-time at frame rates of 2.25 fps for 512 X 1024 images. Frames which previously required 60 s, now take under 500 ms, an improvement in processing speed by a factor of over 120 times. An efficient routine was designed which requires two interpolations of the columns, one one-dimensional real-to-complex fast Fourier transform (FFT) of the columns, and two two-dimensional FFTs. The limits to speed are now reliant on the parallelizability of the processing hardware. Our system provides quantitatively meaningful structural information from previously indistinguishable scattering intensities and provides proof of feasibility for future real-time systems.

Using phase-dispersion spectra measured with optical coherence tomography (OCT) in the frequency domain, we demonstrated the quantitative sizing of multiple spherical scatterers on a surface. We modeled the light scattering as a slab-mode resonance and determined the size of the scatterers from a Fourier transform of the measured phasedispersion spectra. Using a swept-source OCT system, we mapped the detected size of the scatters to the intensity of a two-dimensional surface image. The image was formed by raster-scanning a collimated beam of 200 μm diameter across a sample with distinct size domains. The image shows a clear distinction between deposited polystyrene microspheres of 26 and 15 μm average sizes. In a separate experiment, we demonstrated tissue-relevant sizing of scatters as small as 5 μm with a Fourier domain OCT system that utilized 280 nm of bandwidth from a super-continuum source. Our previous studies have demonstrated that the light scattered from a single sphere is, in general, nonminimum- phase; therefore, phase spectra can provide unique information about scattered light not available from intensity spectra alone. Also, measurements of phase spectra do not require background normalization to correct for the spectral shape of light sources or the spectral absorption of specimens. The results we report here continue our efforts towards combining intensity and phase spectra to enable improved quantitative analysis of complex tissue structures.

Intrinsic optical properties of biological tissue can be modulated with specific genetic alterations, and used as a phenotypic response to probe specific signaling pathways. Utilizing this approach, we used optical scatter imaging to probe the effect of BCL-x_L on subcellular particle size distribution within monolayers of living CSM14.1 and iBMK cells. Expression of YFP-Bcl-x_L shifted the center of the subcellular particle diameter distribution from 1.5μm to 2μm in CSM 14.1 cells and from 1.5 to 1.8 μm in iBMK cells. This shift was also observed in cells expressing YFP-TM, in which YFP is directly fused to the C-terminal transmembrane (TM) domain of BCL-x_L, but not in cells expressing YFP or YFP-BCL-x_L-ΔTM, which lack the TM domain. YFP and YFP-BCL-x_L-ΔTM were diffusely distributed in the cytoplasm, while YFP-TM and YFP-BCL-x_L were localized on the mitochondria. The measured particle sizes, combined with the localization of the TM domain to the mitochondria, suggest that morphological disturbances of the mitochondrial membrane effected by the TM domain of Bcl-x_L, underlie the measured optical scatter changes. We have also found that expression of BCL-2, another anti-apoptotic protein, in iBMK cells, results in a subcellular particle diameter increase similar to that induced by BCL-xL. However, BCl-xL-ΔTM induced as much apoptosis resistance as BCL-x_L. Thus, mitochondrial morphology changes are not required for apoptosis resistance. Nonetheless, expression of YFP-TM also conferred a moderate level of apoptosis resistance, while apoptosis resistant iBMK cells lacking Bax and Bak, showed an increase in the light scattering contribution of particles less than 1.5 μm in diameter. Our results suggest a possible secondary role of the BCL-xL TM domain in apoptosis resistance. However, the functional relationship between mitochondrial morphology and apoptosis resistance remains to be fully elucidated.

Traditional biological and chemical methods for pathogen identification require complicated sample preparation for reliable results. Optical scattering technology has been used for identification of bacterial cells in suspension, but with only limited success. Our published reports have demonstrated that scattered light based identification of Listeria colonies growing on solid surfaces is feasible with proper pattern recognition tools. Recently we have extended this technique to classification of other bacterial genera including, Salmonella, Bacillus, and Vibrio. Our approach may be highly applicable to early detection and classification of pathogens in food-processing industry and in healthcare. The unique scattering patterns formed by colonies of different species are created through differences in colony microstructure (on the order of wavelength used), bulk optical properties, and the macroscopic morphology. While it is difficult to model the effect on scatter-signal patterns owing to the microstructural changes, the influence of bulk optical properties and overall shape of colonies can be modeled using geometrical optics. Our latest research shows that it is possible to model the scatter pattern of bacterial colonies using solid-element optical modeling software (TracePro), and theoretically assess changes in macro structure and bulk refractive indices. This study allows predicting the theoretical limits of resolution and sensitivity of our detection and classification methods. Moreover, quantification of changes in macro morphology and bulk refractive index provides an opportunity to study the response of colonies to various reagents and antibiotics.

Light scattering from cells originates from sub-cellular organelles. Our measurements of angularly resolved light scattering have demonstrated that at 633 nm, the dominant scattering centers within EMT6 cells are mitochondria and lysosomes. To assess their specific contributions, we have used photodynamic therapy (PDT) to induce organelle-specific perturbations within intact cells. We have developed a coated sphere scattering model for mitochondrial swelling in response to ALA- and Pc 4-PDT, and in the case of Pc 4-PDT we have used this model to map the scattering responses into clonogenic cell survival. More recently, we demonstrated the ability to measure the size, scattering contribution, and refractive index of lysosomes within cells by exploiting the localization and high extinction of the photosensitizer LS11 and an absorbing sphere scattering model. Here we report on time- and fluence-dependant scattering measurements from cells treated with LS11-PDT. LS11-PDT causes rapid lysosomal disruption, as quantified by uptake of acridine orange, and can induce downstream effects including release of mitochondrial cytochrome c preceding the loss of mitochondrial membrane potential (Reiners et al., Cell Death Differ. 9:934, 2002). Using scattering and these various methods of analysis, we observed that the induction of lysosomal morphology changes requires a fluence significantly higher than that reported for cell killing. At lower fluences, we observe that at 1 h after irradiation there is significant mitochondrial swelling, consistent with the onset of cytochrome c-induced cell death, while the morphology of lysosomes remains unchanged. We also expand on the ideas of lysosomal staining to demonstrate the sensitivity of scattering measurements at different wavelengths to different organelle populations.

The changes in light scattering induced by acetic acid in cervical cancer cell suspensions and the attached monolayer cells were studied using elastic light scattering spectroscopy. The results show that Mie scattering is dominant in small forward scattering angles (<10.0 degrees). However Mie fitting was found not to be able to provide a satisfactory interpretation of the scattering spectral signals in the large scattering angles. This creates challenge to extract accurate information on the refractive index of cellular organelles. The internal structures in the cells do not make appreciable contribution to light scattering in the small scattering angles while these structures show up and dominate the light scattering in larger angles. The fractal mechanism captures these internal structures. After applying acetic acid solution to the cells, it was found that the volume fraction of the small size scatterers increases and the largest scatterer size decreases. Meanwhile, the fluctuation amplitude of intracellular refractive index increases. Overall, the results provide the evidence that small-sized organelles are the major contributors to the acetowhitening effect.

We recently developed a novel optical method for observing submicron intracellular structures in living cells which is called confocal light absorption and scattering spectroscopic (CLASS) microscopy. It combines confocal microscopy, a well-established high-resolution microscopic technique, with light scattering spectroscopy (LSS). CLASS microscopy requires no exogenous labels and is capable of imaging and continuously monitoring individual viable cells, enabling the observation of cell and organelle functioning at scales on the order of 100 nm with 10 nm accuracy. To demonstrate the ability of the CLASS microscope to monitor unstained living cells on submicrometer scale we studied human bronchial epithelial cells undergoing apoptosis. Fluorescence microscopy of living cells requires application of molecular markers which can affect normal cell functioning. CLASS microscopy is not affected by this avoiding potential interference of fluorescence molecular markers with cell processes. In addition, it provides not only size information but also information about the biochemical and physical properties of the cell. CLASS microscopy can provide unique capabilities for the study of cell interactions with the environment, cell reproduction and growth and other functions of viable cells, which are inaccessible by other techniques.

In determining true clinical utility and patient benefits of a new diagnostic technique, a variety of issues become important, which are often not considered during the proof-of-concept stage. These relate to the overall cost to the health-care system, user friendliness and clinical practicality, likelihood of adoption, the appropriate statistics for demonstration clinical value, and the relevance of the technology to the specific disease.

Fourier domain low coherence interferometry (fLCI) is an optical technique which combines the depth resolution of low coherence interferometry with the sensitivity of light scattering spectroscopy. The fLCI system uses a white light source in a modified Michelson interferometer with a spectrograph for detection of the mixed signal and reference fields. Depth-resolved structural information is recovered by performing a short-time Fourier transform on the detected spectrum, similar to spectroscopic optical coherence tomography, and analyzing the wavelength dependent variations in scattered light as a function of depth. fLCI has been demonstrated as an excellent technique for probing the nuclear morphology of a monolayer of in vitro cancer cells. We have built a new fLCI optical system which implements an imaging spectrograph for detection and a 4- F interferometer which uses a 4-F imaging system to re-image light scattered from the experimental sample onto the slit of the imaging spectrograph. The new system has allowed us to measure light scattered from the deepest layers of thick scattering samples, such as tissue phantoms and thick animal tissues, for the first time. We now take the first steps to quantitatively determine the diameter of scatterers within a thick experimental sample using the new fLCI system along with the fLCI data processing technique.

The optical properties of a tissue can be specified by the depth dependence of a reflectance-mode confocal measurement, as the focus is scanned down into a tissue. Reflectance-mode confocal scanning laser microscopy (rCSLM) and optical coherence tomography in focus tracking mode (OCT) are two examples of such confocal measurements. The measurement of reflected signal as a function of the depth of focus, R(z), is expressed as ρe^-μz, where ρ [dimensionless] is the local reflectivity from the focus within a tissue and μ [cm^-1] is the attenuation of signal as a function of z. The reflectivity of a mirror defines ρ = 1. This paper describes how the experimental ρ and μ map into the optical properties of scattering coefficient, μs [cm^-1], and anisotropy of scattering, g [dimensionless]. Preliminary results on tissue for the rCSLM and OCT systems are reported.

In apoptotic cells, mitochondria have been shown to undergo morphological and structural changes caused by intra-membrane biochemical events. These subcellular changes are dynamic and progress over time via distinct morphological stages observable using electron microscopy (EM). To investigate whether changes in the morphology of the mitochondrial matrix could be detected and monitored by non-invasive light scattering methods, we performed simulations of light scattering by synthetic three-dimensional mitochondrial matrix models using the finite difference time domain (FDTD) technique. Our models consisted of spherical or ellipsoidal particles (matrix region) contained within a spherical or ellipsoidal mitochondrion. Within the mitochondrion model, the small particles' refractive index was taken to be 1.4, while the refractive index of the surrounding volume was taken to be 1.35, and equal to that of the cytoplasm surrounding the mitochondrion. Depending on the matrix volume ratio, particles within the mitochondrion can be either overlapping or non-overlapping. Our results suggest that measurable changes in light scattering by mitochondria can be detected in central dark-field microscopy. By analyzing the angular dependence of light scattered within the numerical aperture of a standard 63X objective (~67° solid angle), the simulations suggest that matrix regions on the order of 100nm can be detected. Light scattering by mitochondria could be altered both by the shape of these features, and by the matrix volume fraction within the mitochondrion. Our data suggest that optical scatter microscopy could be used to guide EM studies by rapidly assessing relative changes in mitochondrial morphology in living cells, and defining important time points to be further analyzed by EM.

We investigated a custom Monte Carlo (MC) platform in the generation of opto-physiological models of motion artefact and perfusion in pulse oximetry. With the growing availability and accuracy of tissue optical properties in literatures, MC simulation of light-tissue interaction is providing increasingly valuable information for optical bio-monitoring research. Motion-induced artefact and loss of signal quality during low perfusion are currently the primary limitations in pulse oximetry. While most attempts to circumvent these issues have focused on signal post-processing techniques, we propose the development of improved opto-physiological models to include the characterisation of motion artefact and low perfusion. In this stage of the research, a custom MC platform is being developed for its use in determining the effects of perfusion, haemodynamics and tissue-probe optical coupling on transillumination at different positions of the human finger. The results of MC simulations indicate a useful and predictable output from the platform.

Endothelin-1 (ET-1) is a potent vasoconstrictor sometimes used in studies of cerebral ischemia. Its ability to create ischemic regions of various sizes with little additional damage has made it a popular tool in evaluating anti-stroke drugs. Despite its emergence in stroke models, it remains poorly characterized. Attempts to do this with Laser Doppler Flowmetry (LDF) or a histological analysis provide good temporal resolution or good spatial resolution respectively, but not both. An imaging modality that provides both temporal and spatial resolution would be able to better characterize the acute and chronic effects of ET-1 on cerebral blood flow. We have used laser speckle contrast imaging to study the effects of ET-1 after topical application on rats. We observed an immediate decrease in blood flow corresponding to the amount of ET-1 used. After the initial decrease, the blood flow slowly increases towards the baseline value with occasional vasospastic responses observed. Future studies involving multi-spectral reflectance imaging combined with the laser speckle contrast analysis would lead to a better understanding of the hemodynamic effects of ET-1.

We present spectroscopic swept-source optical coherence tomography (OCT) measurements of the phase-dispersion of cell samples. We have previously demonstrated that the phase of the scattered field is, in general, independent of the intensity, and both must be measured for a complete characterization of the sample. In this paper, we show that, in addition to providing a measurement of the size of the cell nuclei, the phase spectrum provides a very sensitive indication of the separations between the cells. Epithelial cancers are characterized by many factors, including enlarged nuclei and a significant loss in the architectural orientation of the cells. Therefore, an in vivo diagnostic tool that analyzes multiple properties of the sample instead of focusing on cellular nuclei sizes alone could provide a better assessment of tissue health. We show that the phase spectrum of the scattered light appears to be more sensitive to cell spacing than the intensity spectrum. It is possible to determine simultaneously the cell nuclei sizes from the intensity spectrum and the nuclei spacing from the phase spectrum. We measure cell monolayer samples with high and low cell density and compare measured results with histograms of the cell separations calculated from microscope images of the samples. We show qualitative agreement between the predicted histograms and the interferometric results.

High resolution 2D side scatter patterns from polystyrene beads were obtained by using an integrated microfluidic waveguide cytometer. A He-Ne laser beam was prism-coupled into a microfluidic chip, and waveguide modes were excited to illuminate a single scatterer. While immobilizing a single scatterer on chip in the observation window area, high resolution 2D scatter patterns were obtained by using a CCD array located beneath the microchip. This cytometer is sensitive to variations in both the refractive index and the size of a single scatterer. Fourier transforms of Mie simulation results from a single scatterer show that forward scattered light at large angles is optimal for micro-size differentiation. While side scatter light was reported to contain rich information about organelles in a single cell, we show here that side scatter light can be used to perform fast micro-size differentiation and cellular analysis. A cross section scan of the experimental scatter pattern gives an oscillation distribution of the scattered intensity. This oscillation has a frequency that is typical for a given micro-size scatterer. A Fourier method for quick micro-size differentiation is reported, based on the comparisons between the Mie simulations and the experimental results. Finite-difference time-domain (FDTD) simulations of single white blood cells in the waveguide cytometer are studied, which allows extraction of microstructural and nano-structural information from single cells.

A unified theory for light scattering by biological cells is presented. It is shown that Mie scattering from the bare cell and the nucleus dominates cell light scattering in the forward directions. The random fluctuation of the background refractive index within the cell, behaving as a fractal random continuous medium, dominates light scattering by cells in other angles. The theory is validated by experimental angular light scattering spectra of epithelial cells for scattering angles from 1.25 to 173.8 degrees and in the spectral range from 400nm to 700nm.