This PDF file contains the front matter associated with SPIE Proceedings Volume 7187, including the Title Page, Copyright information, Table of Contents, Introduction (if any), and the Conference Committee listing

Currently the only method for positively identifying malignant melanoma involves invasive and often undesirable biopsy procedures. Available ex-vivo data indicates increased vascularization in the lower regions of excised melanoma, as compared to dysplastic nevi. The ability to interrogate this region of tissue in-vivo could lead to useful diagnostic information. Using a newly developed fiber based superficial probe in conjunction with a steady-state frequency-domain photon migration (SSFDPM) system, we can probe the upper 1-2 mm of tissue, extracting functional information in the near infrared (650-1000 nm) range. To test the resolution and detection range of the superficial probe in this context, deformable silicone phantoms have been fabricated that simulate normal skin with melanocytic lesions. These phantoms consist of a two-layered matrix with the optical properties of normal light skin, containing several cylindrical inclusions that simulate highly absorbing pigmented lesions such as melanoma. These inclusions are varied in depth, diameter, and optical properties in order to fully test the probe's detection capabilities. It was found that, depending on absorption, we can typically probe to a depth of 1.0-1.5 mm in an inclusion, likely reaching the site of angiogenesis in an early-stage melanoma. Additionally, we can successfully interrogate normal tissue below lesions 1.5mm deep when absorption is about 0.4/mm or less. This data indicates that the superficial probe shows great promise for non-invasive diagnosis of pigmented lesions.

Functional optical characterization of disease progression and response to therapy suffers from loss of spatial resolution and imaging depth due to scattering, impacting the ability of researchers to localize and quantify molecular processes. Here we report on the ability of dimethyl sulfoxide (DMSO) to reduce temporarily the optical scattering of skin. Data collected from in vitro phantom images and in vivo fluorescence images demonstrate the potential of this simple method to mitigate the blurring effects of scattering with topical application, which we expect will improve the accuracy and localization of in vivo molecular imaging studies.

The readings in laser Doppler perfusion monitoring are affected by the optical properties of the tissue in which the microvasculature is embedded, through their effect on the optical path lengths. Thus for a constant perfusion, the LDF output signal is affected by the variance in individual photon path lengths due to the changes in tissue optical properties and probe geometry. We will present efforts to render blood flow measurements independent of the tissue optical properties by using low coherence interferometry. We will give evidence of the improvement in quantification of our approach. In particular we show that low coherence interferometry can measure dynamic properties of particles in Brownian motion, independent of optical properties of the surrounding tissue matrices. Furthermore, demonstration is given of the applicability of the method in vivo.

The propagation of light through complex structures, such as biological tissue, is a poorly understood phenomenon. Current practice typically ignores the coherence of the optical field. Propagation is treated by Monte Carlo implementation of the radiative transport equation, in which the field is taken to be incoherent and is described solely by the first-order statistical moment of the intensity. Although recent Monte Carlo studies have explored the evolution of polarization using a Stokes vector description, these efforts, too are single-point statistical characterizations and thus ignore the wave nature of light. As a result, the manner in which propagation affects coherence and polarization cannot be predicted. In this paper, we demonstrate a Monte Carlo approach for propagating partially coherent fields through complicated deterministic optical systems. Random sources with arbitrary spatial coherence properties are generated using a Gaussian copula. Physical optics and Monte Carlo predictions of the first and second order statistics of the field are shown for coherent and partially coherent sources for a variety of imaging and non-imaging configurations. Excellent agreement between the physical optics and Monte Carlo predictions is demonstrated in all cases. Finally, we discuss convergence criteria for judging the quality of the Monte Carlo predictions. Ultimately, this formalism will be utilized to determine certain properties of a given optical system from measurements of the spatial coherence of the field at an output plane. Although our specific interests lie in biomedical imaging applications, it is expected that this work will find application to important radiometric problems as well.

Pancreatic adenocarcinoma has a five-year survival rate of only 4%, largely because an effective procedure for early detection has not been developed. In this study, mathematical modeling of reflectance and fluorescence spectra was utilized to quantitatively characterize differences between normal pancreatic tissue, pancreatitis, and pancreatic adenocarcinoma. Initial attempts at separating the spectra of different tissue types involved dividing fluorescence by reflectance, and removing absorption artifacts by applying a "reverse Beer-Lambert factor" when the absorption coefficient was modeled as a linear combination of the extinction coefficients of oxy- and deoxy-hemoglobin. These procedures demonstrated the need for a more complete mathematical model to quantitatively describe fluorescence and reflectance for minimally-invasive fiber-based optical diagnostics in the pancreas.

The finite difference time domain method was used to compute scattering of a focused optical beam by multiple heterogeneous biological cells. A perfectly matched layer boundary condition and the scattered-field-only method were utilized in the simulation to increase accuracy and computational efficiency. A fifth-order approximation to the focused Gaussian beam was used for the incident field. A parametric study was performed to determine scattering effects of varying cellular fine structure, such as nuclear refractive index, organelle volume density, cellular shape and the cell membrane on the point spread function of the beam. It was found that two-photon PSF is largely unaffected by increasing numbers of scatterers within cells, while two-photon excitation signal strength is dependent on both beam focal depth and the density of scatterers in tissue.

Image analysis and pattern recognition are key elements of many biomedical analysis schemes. In this work, we show the use of pattern recognition and classification for the study of an interesting biomedical problem- the prediction of organelle arrangement within a cell based on wide-angle light scattering patterns. Organelle distribution is known to relate to disease and drug resistance. However, up until this point it has been unclear how changes to organelle distribution relate to the composition of wide-angle light scattering patterns. As such, we use a rapid new scattering simulation method and standard pattern analysis techniques to demonstrate clear correlations between scattering pattern composition and organelle distribution. The texture of scattering images-specifically the spot and edge content of samples is found to directly relate to the type and size of organelle distributions within a cell. These relationships are used to quickly classify organelle distributions to a high degree of accuracy.

Extraction / unique interpretation of the intrinsic polarization parameters in optically thick turbid media such as tissues is complex due to multiple scattering effects and due to simultaneous occurrences of many polarization effects (the most common polarimetry effects in tissues are depolarization, linear birefringence and optical activity). Each of these polarimetry characteristics, if separately extracted, holds promise as a useful biological metric. We have recently investigated the use of an expanded Mueller matrix decomposition method to tackle this problem, with early indications showing promise. However, for further insight and for practical realization of this approach, it is essential to have quantitative understanding of the confounding effects of scattering, the propagation path of multiply scattered photons and detection geometry on the Mueller matrix-derived polarization parameters (parameters of particular biomedical importance are linear retardance, optical rotation and depolarization). The effect of the ordering of the individual matrices in the decomposition analysis on the derived polarization parameters also needs to be studied. We have therefore investigated these issues by decomposing the Mueller matrices generated with a polarization sensitive Monte Carlo model, capable of simulating all the simultaneous optical (scattering and polarization) effects. The results show that with appropriate choice of detection position, indeed the inverse decomposition analysis enables one to decouple and quantify the individual intrinsic polarimetry characteristics despite their simultaneous occurrence, even in the presence of the numerous complexities due to multiple scattering. The details of these results are presented and the implications of these in diagnostic photomedicine are discussed.

Inverse light scattering methods have been applied by several groups as a means to probe cellular structure in both clinical and scientific applications with sub-wavelength accuracy. These methods determine the geometric properties of tissue scatterers based on far field scattering patterns. Generally, structure is determined by measuring scattering over some range of angles, wavelengths, or polarizations and then fitting the observed data to a database of simulated scattering selected from a range of probable geometries. We have developed new light scattering software based on the T-matrix method that creates databases of scattering from spheroidal objects, representing a substantial improvement over Mie theory, a method limited to simulating scattering from spheres. The computational cost of the T-matrix method is addressed through a simple but massively parallel program that concurrently simulates scattering across hundreds of PCs. We are exploring the use of these T-matrix databases in inverting interferometric measurements of angle-resolved scattering from spheroidal cell nuclei using a technique called angle-resolved low coherence interferometry (a/LCI). With a/LCI, we have previously distinguished between healthy and dysplastic tissue in both cell cultures and in ex vivo rat and hamster tissue using Mie theory to measure nuclear diameter. We now present nuclear volume and spheroidal aspect ratio measurements of unstained, living MCF7 cells using the improved T-matrix database to analyze a/LCI data. We achieve measurement accuracy equivalent to conventional image analysis of stained samples. We will further validate the approach by comparing experimental measurements of scattering from polystyrene microspheroids, and show that the T-matrix is a suitable replacement for Mie theory in ex vivo tissue samples.

Our goal is to demonstrate the use of in vitro elastic scattering spectroscopy (ESS) to monitor changes in light-scattering properties of cells due to apoptotic micro-morphology changes. We developed an instrument capable of wavelength-resolved ESS measurements from cell cultures in the backward direction along with an algorithm to extract the size distribution of scatterers in the sample using Mie theory. CHO cells were cultured to confluence on plates and were rendered apoptotic with 2μM staurosporine. Backscattering measurements were performed on pairs of treated and control samples at times up to 6 hours post treatment. Initial results indicate that ESS is capable of discriminating between treated and control samples as early as 15 minutes post treatment. Extracted size distributions from treated and control samples show an increase in the number of small particles with a corresponding decrease in larger particles after treatment. This is consistent with expected morphological changes during apoptosis, and work continues to correlate these size distributions with underlying morphology.

Using a reflectance mode confocal scanning laser microscope (rCSLM), a noninvasive technique to monitor epidermal thickness in vivo is presented. The modality is characterized by the collection of the reflectance profile from cutaneous tissues, resolved in transverse (x, y) directions at each depth (z) increment. Due to the different light scattering properties of the skin layers, each layer can be identified. The depth of each layer can then be inferred from the axial reflectance profile along the z direction. In pilot experiments an agent that induced epidermal proliferation, 12-O-tetradecanoylphorbol-13-acetate (TPA), was applied topically to the ear of a mouse. Peak-to-valley analysis of the rCSLM A-scans showed the epidermal thickness increasing from an initial 5.4 [μm] to 12.4 [μm] after 24 [hr]. Peak-to-peak analysis showed an increase from 9.1 [μm] to 21.2 [μm]. These results suggest that rCSLM imaging provides a means to study morphologic changes in the epithelium arising from inflammatory response and cell proliferation in vivo without recourse to biopsy or sacrifice of animals.

Fourier Domain Low Coherence Interferometry (fLCI) is an optical technique that recovers depth-resolved spectroscopic information about scatterers. The current fLCI system utilizes a white light Xe arc lamp source, a 4-f interferometer, and an imaging spectrometer at the detection plane to acquire spectra from 256 adjacent spatial points. This configuration permits the acquisition of ultrahigh depth resolution Fourier domain OCT images without the need for any beam scanning. fLCI has traditionally obtained depth-resolved spectral information by performing a short-time Fourier transform (STFT) on the detected spectra, similar to the processing techniques of spectroscopic OCT. We now employ a dual Gaussian window processing method which simultaneously obtains high spectral and temporal resolution, thus avoiding the resolution trade-off normally associated with the STFT. Wavelength dependent variations in scattering intensity are analyzed as a function of depth to obtain structural information about the probed scatterers. We now verify fLCI's ability to distinguish between normal and dysplastic epithelial tissue using the hamster cheek pouch model. Thirty hamsters will have one cheek pouch treated with the known carcinogen DMBA. At the conclusion of the 24 week treatment period the animals will be anesthetized and the cheek pouches will be extracted. We will use the fLCI optical system to measure the neoplastic transformation of the in situ subsurface tissue layers in both the normal and DMBA-treated cheek pouches. Traditional histological analysis will be used to verify the fLCI measurements. Our results will further establish fLCI as an effective method for distinguishing between normal and dysplastic epithelial tissues.

Measurement of intrinsic optical signals (IOSs) is attractive for noninvasive, real-time monitoring of tissue viability in brains. We previously performed measurement of IOSs for a rat global ischemic brain model that was made by rapidly removing blood by saline infusion, and observed that after an induction of ischemia, a unique triphasic change in light scattering occurred. This scattering change preceded the reduction of CuA in cytochrome c oxidase which has been shown to correlate with cerebral ATP decrease. In the present study, we examined whether such triphasic scattering change can be observed in the presence of blood in vivo. Transcranial measurement of diffuse reflectance was performed using a broadband tungsten lamp for a rat brain during hypoxia that was induced by N2 inhalation. The reflectance spectral changes in the visible (500-600 nm) and near-infrared (NIR) (650-850 nm) regions were analyzed to monitor changes in hemodynamics and light scattering, respectively. After starting N2 inhalation, reflectance signals in the visible region showed an increase in deoxy-hemoglobin concentration, and about 80 s after full deoxygenation of hemoglobins, reflectance signals in the NIR region showed a similar triphasic change, which was attributable to change in light scattering. Simultaneous measurement of cerebral EEG showed that neuronal activity ceased about 50 s before this triphasic scattering change. These results show that light scattering will become an important indicator of loss of tissue viability in brain; brain tissue can probably be saved if reoxygenation is achieved before starting this scattering change.

Methods for the optimization of a/LCI for clinical use are presented. First, the use of the T-matrix light scattering model to simulate scattering from spheroidal particles is presented as a more appropriate simulation of cell nuclei scattering than the previously used Mie theory. In addition, the use of a broadband light source with a bandwidth greater than 50nm similar to those utilized in OCT applications is demonstrated. Accurate sizing of scatterers in tissue phantoms containing stretched and unstretched polystyrene microspheres along with measurements of unstretched polystyrene microspheres in solution are presented, demonstrating advances in system performance and design. In addition, preliminary human in vivo esophageal tissue data are presented.

The primary goals of this study are to improve the accuracy of noninvasive diagnosis of early cervical cancer. In this study, a novel 3-D optical imaging system based on active stereo vision and motion tracking is developed to track the motion of patient and to register the time-sequenced images of cervix recorded during the examination of colposcopy. This technology can quantify the acetic acid induced optical signals associated with early cancer development at cervix. The results of a preliminary clinical study of 65 patients demonstrate that the accuracy to differentiate pre-cancerous cervical tissue from normal tissue can be significantly increased.

Infiltrating neoplastic epithelia induce ultra-structure changes in tissue providing an intrinsic contrast in terms of their local light scattering response. Imaging systems that can enhance this contrast allow for better visualization of tumor boundaries and thus have enormous potential in guiding complex surgical procedures like breast lumpectomy. Highly localized reflectance measurement probes can quantify scattering changes in tissues in situ, but in order to be useful in surgical settings these techniques require an extension to imaging. A novel microsampling reflectance imaging system has been developed to allow rapid quantitative imaging of ultra-structure associated scattering changes in tissues in situ. The imaging system is described in terms of its design, construction and testing for multi-wavelength, telecentric, darkfield illumination and confocal spectroscopic detection, with imaging fields of up to 1.5 cm × 1.5 cm at 100 microns resolution. Spatial confinement of the incident and detected light allows for direct sampling of the scattering spectrum in tissues in situ and the telecentric design ensures consistent sampling of the scattering phase function throughout the entire imaging field. The imaging system was modeled and optimized using the ZEMAX optical design software. Description of the design and results from the optimization process are presented.

Different techniques have been developed to determine the optical properties of turbid media, which include collimated transmission, diffuse reflectance, adding-doubling and goniometry. While goniometry can be used to determine the anisotropy of scattering (g), other techniques are used to measure the absorption coefficient and reduced scattering coefficient (μ_s(1-g)). But separating scattering coefficient (μ_s) and anisotropy of scattering from reduced scattering coefficient has been tricky. We developed an algorithm to determine anisotropy of scattering from the depth dependent decay of reflectance-mode confocal scanning laser microscopy (rCSLM) data. This report presents the testing of the algorithm on tissue phantoms with different anisotropies (g = 0.127 to 0.868, at 488nm wavelength). Tissue phantoms were made from polystyrene microspheres (6 sizes 0.1-0.36 μm dia.) dispersed in both aqueous solutions. Three dimensional images were captured. The rCSLM-signal followed an exponential decay as a function of depth of the focal volume, R(z) = ρexp(-μz) where ρ (dimensionless, ρ=1 for a mirror) is the local reflectivity and μ [cm^-1] is the exponential decay constant. The theory was developed to uniquely map the experimentally determined μ and ρ into the optical scattering properties μ_s and g. The values of μ_s and g depend on the composition and microstructure of tissues, and allow characterization of a tissue.

Multi-spectral scatter visualization of tissue ultra-structure in situ can provide a unique tool for guiding surgical resection, but since changes are subtle and the data is multi-parametric, an automated methodology was sought to interpret these data, in order to classify their tissue sub-type. Tissue types observed across AsPC-1 pancreatic tumor samples were pathologically classified under three major groups (epithelium, fibrosis and necrosis) and the variations in scattering parameters, i.e. scattering power, scattering amplitude and average scattered intensity, across these groups were analyzed. The proposed scheme uses statistical pre-processing of the scattering parameter images to create additional data features followed by a k-nearest neighbors (kNN) based algorithm for tissue type classification. The classification accuracy inside some predefined regions of interest was determined and the mean region values of scattering parameters turned out to be stronger data sets for classification, rather than the individual pixel values. This presumably indicates that pixel-to-pixel variations in the remitted spectra need to be minimized for reliable classification approaches. Results show a strong correlation between the automated and expert-based classification within the predefined regions of interest.

Investigators have previously attempted to determine the optimal wavelengths to employ for Near-Infrared Spectroscopy (NIRS) which yield the best separation between absorption and scatter and the least influence of noise. Although these are important criteria it is also important that the volume of tissue sampled at each of the wavelengths is the same. In our study we have generated spatial sensitivity profiles at multiple wavelengths for a suitable model of tissue optical properties, and studied the spatial sensitivity overlap for different combinations of wavelengths. It is found that including this condition significantly influences the range of optimal wavelengths.

The study of biological tissue using white light spectroscopy has the potential to be an effective, fast, and inexpensive method for the detection of size changes in cell nuclei. The relationship between the spherical scatterer size and the number of oscillation peaks in the optical spectrum (intensity of scattered light versus wavelength) has been observed by many researchers. To this point, there was not a detailed theoretical model describing this dependence for elliptical particles, a common shape of cell nuclei at lower tissue layers. In this paper, we report a theoretical model, valid for both spheres and ellipsoids, detailing the scattering intensity as a function of the wavelength and the scatterer's diameter. Supporting this theory, we experimentally test mixtures of scatterers of different sizes and provide density analysis.

The bio-media are made of anisotropic molecules. Using a simple ellipsoid model, the scattering of bio-medium with anisotropic bio-molecule is investigated theoretically. The scattering fields and Mueller matrices are derived from fundamental electromagnetism theory. The bio-medium is modeled as a system of non-correlated anisotropic molecules. Based upon a statistical model of anisotropic distribution, the scattering Mueller matrix is derived. The single and double photon scattering models are investigated. Double scattering is more important for high density scattering medium. For incident light with pure polarization, such as linear and circular polarizations, the results of molecular shape-dependent differential and total scattering cross-sections are reported. This theory can provide a simulation tool for investigating the scattering and polarization/depolarization effect in the highly scattering bio-medium.

Diffuse reflectance spectroscopy is one of the different optical techniques that are used to extract optical properties of tissues such as the scattering and the absorption coefficients. These properties can in turn be linked to physiological parameters. Therefore optical probes are of interest to extract physiological parameters from tissue that derive from diffuse reflectance spectroscopy. Investigating to which extend theses models are valid is of importance in order to assess the reliability of the estimated parameters. We present different models that are used to extract optical parameters from spatially resolved diffuse reflectance measurement.

Diffuse reflectance was applied to the biomedical studies (muscles, cardiac tissues etc.) in a form of either a direct pseudo-optical spectrum or its second derivative. The first derivative adopts advantages of both direct spectrum (high signal-to-noise ratio) and its second derivative (simplifying the consideration of light scattering contribution, S). In contrast to spectrophotometry of solutions, diffuse reflectance application to the analysis of turbid medium chromophores leads to non-trivial problems of contribution of light scattering, the choice of reference, and light pathlength. Under certain conditions, the first approximation of the Taylor series of S results in the known linear dependence of S on wavelength in the 650-1050 nm wavelength range. Then the light scattering contribution to the first derivative becomes a wavelength-independent offset. In contrast to the second derivative, the information on light scattering inside the tissue is not lost. Effect of reference on the measured spectra becomes negligible. Application of the first derivative allowed (i) determination of NIR light pathlength in muscle tissue, and (ii) quantification of hemoglobin + myoglobin absolute concentration (in mM) in cardiac tissue during open-heart surgery. The first derivative approach may in general be applied to any chromophores in turbid (biological) media.

Light scattering methods for assessing structural properties of cells and tissues quantitatively measure morphometric parameters directly without the need for staining. We demonstrate an optical scattering filtering method used in a biological setting that is sensitive to quantifying object orientation and aspect ratio. These parameters are measured in cells both sensitive to and resistant to mitochondrial-mediated apoptosis, the latter having been demonstrated to have shorter mitochondria than apoptosis competent cells. The implementation of the digital micromirror device (DMD) allows for robust filtering of the scatter data, which we implement with Gabor-like filters chosen for their ability to intelligently confine the filter response both in the image and in the scatter regimes. By strategically applying Gabor-like filters to the specific frequencies and orientations in the scatter data, relative changes in object size, orientation and aspect ratio may be derived. Furthermore, using a DMD and filtering the optical scatter data in analog allows us to decouple image resolution from frequency resolution and measure these parameters with high sensitivity for objects within the resolution of the optical system despite an undersampled, lower resolution digital image. As a result, this measurement may be made at lower magnifications with higher throughput and ultimately on a larger population of living and unstained cells imaged simultaneously.