This PDF file contains the front matter associated with SPIE Proceedings volume 7573, including the Title Page, Copyright information, Table of Contents, Introduction, and the Conference Committee listing.

The light scattering effects of turbid media causing opacity may be undone via Optical Phase Conjugation (OPC). Here we rigorously simulate light scattering through a macroscopic random using the pseudospectral time-domain (PSTD) technique. The OPC phenomenon of multiply scattered light can be quantitatively analyzed which is not feasible otherwise. Specifically, factors affecting the electromagnetic energy propagation and refocusing phenomenon is analyzed. The reported simulation study allows accurate characterization of the optical properties of the OPC phenomenon for practical biomedical applications.

In both industry and medicine there is no optical technique to measure distance through light scattering media. Such a technique may be useful for localizing embedded structures, or may be a non-contact method of measuring turbid media. The limits of a frequency domain based technique were explored in three polyurethane optical phantoms. We have demonstrated a simple method to measure the distance between an intensity modulated light source and detector in turbid media based on the proportionality of the phase lag to the distance. The limits of the technique were evident for distances less than 5 mm, particularly when μ¹_s <0.1mm^-1 and distances greater than 55mm for the phantoms studied. This method may prove useful in industry and medicine as a non destructive way measure distance through light scattering media.

A common strategy to study breast cancer is the use of the preclinical model. These models provide a physiologically relevant and controlled environment in which to study both response to novel treatments and the biology of the cancer. Preclinical models, including the spontaneous tumor model and mammary window chamber model, are very amenable to optical imaging and to this end, we have developed a wide-field functional imaging (WiFI) instrument that is perfectly suited to studying tumor metabolism in preclinical models. WiFI combines two optical imaging modalities, spatial frequency domain imaging (SFDI) and laser speckle imaging (LSI). Our current WiFI imaging protocol consists of multispectral imaging in the near infrared (650-980 nm) spectrum, over a wide (7 cm x 5 cm) field of view. Using SFDI, the spatially-resolved reflectance of sinusoidal patterns projected onto the tissue is assessed, and optical properties of the tissue are determined, which are then used to extract tissue chromophore concentrations in the form of oxy-, deoxy-, and total hemoglobin concentrations, and percentage of lipid and water. In the current study, we employ Monte Carlo simulations of SFDI light propagation in order to characterize the penetration depth of light in both the spontaneous tumor model and mammary window chamber model. Preliminary results suggest that different spatial frequency and wavelength combinations have different penetration depths, suggesting the potential depth sectioning capability of the SFDI component of WiFI.

Motility contrast imaging (MCI) detects dynamic speckle from living tissue using digital holography. It detects sub-cellular motion in living tissue as a fully endogenous imaging contrast agent. Three-dimensional imaging assays of anti-mitotic cancer drugs extract label-free functional signatures in tumors.

A comprehensive three-parameter statistical model is presented for the refractive index fluctuations in continuous homogeneous random media, and the light-scattering properties of these media are investigated in the Born (or single-scattering) approximation. Because biological media are usually weakly scattering, the results are applicable to many biomedical light-scattering problems. A rigorous error analysis is presented for the scattering coefficient under the Born approximation in a biologically-relevant, albeit more simplified geometry. The finitedifference- time-domain (FDTD) computational electromagnetic analysis is used to obtain the exact solutions for this error analysis. The ranges for the correlation length and the refractive index fluctuation strength under which Born approximation is valid are clearly identified.

Stochastic Huygens describes a method of propagating a partially coherent source by sampling the Huygens wavelets that evolve from each point of the wavefront. The amplitude and phase of each wavelet is tracked as the light passes through the optical system. We have previously described how a partially coherent wavefront may be simulated by propagating an ensemble of wavefronts with specified first and second-order statistics through simple optical systems. In this work we extend the modeling effort to include an ensemble of phase (or scattering) screens that characterize thin microscope tissue samples in the optical path.

Backscattered light spectra have been used to extract size distribution of cell nuclei in epithelial tissues for noninvasive detection of precancerous lesions. In existing experimental studies, size estimation is achieved by assuming nuclei as homogeneous spheres or spheroids and fitting the measured data with models based on Mie theory. However, the validity of simplifying nuclei as homogeneous spheres has not been thoroughly examined. In this study, we investigate the spectral characteristics of backscattering from models of spheroidal nuclei under plane wave illumination using three-dimensional finite-difference time-domain (FDTD) simulation. A modulated Gaussian pulse is used to obtain wavelength dependent scattering intensity with a single FDTD run. The simulated model of nuclei consists of a nucleolus and randomly distributed chromatin condensation in homogeneous cytoplasm and nucleoplasm. The results show that backscattering spectra from spheroidal nuclei have similar oscillating patterns to those from homogeneous spheres with the diameter equal to the projective length of the spheroidal nucleus along the propagation direction. The strength of backscattering is enhanced in heterogeneous spheroids as compared to homogeneous spheroids. The degree of which backscattering spectra of heterogeneous nuclei deviate from Mie theory is highly dependent on the distribution of chromatin/nucleolus but not sensitive to nucleolar size, refractive index fluctuation or chromatin density.

Three-dimensional localization of protein conformation changes in turbid media using Förster Resonance Energy Transfer (FRET) was investigated by tomographic fluorescence lifetime imaging (FLIM). FRET occurs when a donor fluorophore, initially in its electronic excited state, transfers energy to an acceptor fluorophore in close proximity through non-radiative dipole-dipole coupling. An acceptor effectively behaves as a quencher of the donor's fluorescence. The quenching process is accompanied by a reduction in the quantum yield and lifetime of the donor fluorophore. Therefore, FRET can be localized by imaging changes in the quantum yield and the fluorescence lifetime of the donor fluorophore. Extending FRET to diffuse optical tomography has potentially important applications such as in vivo studies in small animal. We show that FRET can be localized by reconstructing the quantum yield and lifetime distribution from time-resolved non-invasive boundary measurements of fluorescence and transmitted excitation radiation. Image reconstruction was obtained by an inverse scattering algorithm. Thus we report, to the best of our knowledge, the first tomographic FLIM-FRET imaging in turbid media. The approach is demonstrated by imaging a highly scattering cylindrical phantom concealing two thin wells containing cytosol preparations of HEK293 cells expressing TN-L15, a cytosolic genetically-encoded calcium FRET sensor. A 10mM calcium chloride solution was added to one of the wells to induce a protein conformation change upon binding to TN-L15, resulting in FRET and a corresponding decrease in the donor fluorescence lifetime. The resulting fluorescence lifetime distribution, the quantum efficiency, absorption and scattering coefficients were reconstructed.

Early pre-cancerous conditions in tissue can be studied as mixture of cancerous and healthy cells. White light spectroscopy is a promising technique for determining the size of scattering elements, which, in cells are the nuclei. However, in a mixture of different sized scatterers, possibly between healthy and cancerous cells, the white light spectroscopy spatial data is not easily analyzed, making it difficult to determine the individual components that comprise the mixture. We have previously found by obtaining spatial limited data by using an optical filter and converting this spatial data into the Fourier domain, we can determine characteristic signature frequencies for individual scatterers. In this paper, we show analysis of phantom tissues representing esophagus tissue. We examine phantom tissue representing pre-cancerous conditions, when some of the cell nuclei increase in size. We also experimentally show a relationship between the particle concentration and the amplitude of the Fourier signature peak. In addition, we discuss the frequency peak amplitude dependency based on the Tyndall Effect, which describes particles aggregating into clusters.

We propose a fiber-optic system for Fourier-domain angle-resolved low coherence interferometry. The system is based on singlemode fiber couplers and employs a scanning fiber to collect the angular scattering distribution of the sample. The measured distribution is then fitted to Mie theory to estimate the size of the scatterers. Depth resolution is achieved with Fourier-domain low coherence Mach-Zehnder interferometry. The sample arm of the interferometer illuminates the specimen with polarized light and collects back-scattered light from the sample. The probe's optical performance is evaluated and its depth-resolved sizing capability is demonstrated with subwavelength accuracy using a two-layer microsphere phantom.

A fiber-coupled microfluidic cytometer has been recently developed in our group for the obtaining of 2D light scatter patterns from single biological cells. The obtained scatter patterns may be used for label-free characterization of biological cells. Understanding of these 2D scatter patterns can be better achieved by comparing the experimental results with those obtained by theoretical simulations, such as the finite-difference time-domain (FDTD) simulation of light scattering from biological cells. In this paper, we provide detailed study for applying the FDTD method in our fibercoupled microfluidic cytometer. The FDTD simulation results agree reasonably well with the experimentally obtained THP-1 cell 2D scatter patterns. Methods for scatter pattern analysis are under development in our group for new light scattering parameters that may potentially be used in clinics.

The high quantum yield and exclusively photo-stable excitation of gold nanoparticles combined with their bio-inert characteristics make them ideal cellular markers. The aim of the study was to visualise gold nanoparticles size-dependently as colloid and in cells after co-incubation. We show the quantification of colloidal gold particles by standard confocal microscopy down to the single particle level. A calibration is demonstrated for pixel numbers in dilution series of uncoated gold nanoparticles. We give implications for practical use of advanced cellular imaging in cultured cells.

A particular application of interest for tissue reflectance spectroscopy in the UV-Visible is intraoperative detection of residual cancer at the margins of excised breast tumors, which could prevent costly and unnecessary repeat surgeries. Our multi-disciplinary group has developed an optical imaging device, which is capable of surveying the entire specimen surface down to a depth of 1-2mm, all within a short time as required for intraoperative use. In an IRB-approved study, reflectance spectral images were acquired from 54 margins in 48 patients. Conversion of the spectral images to quantitative tissue parameter maps was facilitated by a fast scalable inverse Monte-Carlo model. Data from margin parameter images were reduced to image-descriptive scalar values and compared to gold-standard margin pathology. The utility of the device for classification of margins was determined via the use of a conditional inference tree modeling approach, and was assessed both as a function of type of disease present at the margin, as well as a function of distance of disease from the issue surface. Additionally, the influence of breast density on the diagnostic parameters, as well as the accuracy of the device, was evaluated.

Improved methods for detecting dysplasia, or pre-cancerous growth are a current clinical need, particularly in the esophagus. The currently accepted method of random biopsy and histological analysis provides only a limited examination of tissue in question while being coupled with a long time delay for diagnosis. Light scattering spectroscopy, in contrast, allows for inspection of the cellular structure and organization of tissue in vivo. Fourier-domain angle-resolved low-coherence interferometry (a/LCI) is a novel light scattering spectroscopy technique that provides quantitative depth-resolved morphological measurements of the size and optical density of the examined cell nuclei, which are characteristic biomarkers of dysplasia. Previously, clinical viability of the a/LCI system was demonstrated through analysis of ex vivo human esophageal tissue in Barrett's esophagus patients using a portable a/LCI, as was the development of a clinical a/LCI system. Data indicating the feasibility of the technique in other organ sites (colon, oral cavity) will be presented. We present an adaptation of the a/LCI system that will be used to investigate the presence of dysplasia in vivo in Barrett's esophagus patients.

Epithelial biologists studying human skin diseases such as cancer formation and psoriasis commonly utilize mouse models to characterize the interplay among cells and intracellular signal transduction pathways that result in programmed changes in gene expression and cellular behaviors. The information obtained from animal models is useful only when phenotypic presentations of disease recapitulate those observed in humans. Excision of tissues followed by histochemical analysis is currently the primary means of establishing the morphological presentation. Non invasive imaging of animal models provides an alternate means to characterize tissue morphology associated with the disease of interest in vivo. While useful, the ability to perform in vivo imaging at different time points in the same tissue location has been a challenge. This information is key to understanding site specific changes as the imaged tissue can now be extracted and analyzed for mRNA expression. We present a method employing a micro-tattoo to guide optical coherence tomography (OCT) imaging of ultraviolet induced inflammation over time in the same tissue locations.

Light scattering signal is a potential indicator of tissue viability in brain because cellular and subcellular structural integrity should be associated with cell viability in brain tissue. We previously performed multiwavelength diffuse reflectance measurement for a rat global ischemic brain model and observed a unique triphasic change in light scattering at a certain time after oxygen and glucose deprivation. This triphasic scattering change (TSC) was shown to precede cerebral ATP exhaustion, suggesting that loss of brain tissue viability can be predicted by detecting scattering signal. In the present study, we examined correlation between light scattering signal and tissue reversibility in rat brain in vivo. We performed transcranial diffuse reflectance measurement for rat brain; under spontaneous respiration, hypoxia was induced for the rat by nitrogen gas inhalation and reoxygenation was started at various time points. We observed a TSC, which started at 140 ± 15 s after starting nitrogen gas inhalation (mean ± SD, n=8). When reoxygenation was started before the TSC, all rats survived (n=7), while no rats survived when reoxygenation was started after the TSC (n=8). When reoxygenation was started during the TSC, rats survived probabilistically (n=31). Disability of motor function was not observed for the survived rats. These results indicate that TSC can be used as an indicator of loss of tissue reversibility in brains, providing useful information on the critical time zone for treatment to rescue the brain.

We have investigated the potential application of elastic light single-scattering spectroscopy (ELSSS) as an adjunctive tool for screening of cervical precancerous lesions non-invasively and in real time. Ex-vivo measurements were performed on 95 cervix biopsy tissue of 60 patients. Normal cervix tissue from 10 patients after hysterectomy was used as a control group. Correlation between ELSSS spectra and histopathology results were investigated. It was found that the spectral slope was positive for all the spectra taken on normal cervix tissue samples from the control group. We assumed that if there is only one spectrum with a negative spectral slope among the all spectra taken on a biopsy specimen, the biopsy specimen is pathologically abnormal. This shows that pap smear and ELSSS results are in good agreement. Most biopsy tissue samples had both positive and negative spectral slopes. Therefore, we calculated the percentage of negative spectral slopes and hypothesized that this was correlated to dysplastic percentage of the epithelial tissue of the biopsy material. The ROC curve was calculated using the dysplastic percentage and high squamous intraepithelial lesion (HSIL) and low squamous intraepitherlial lesions (LSIL) biopsy specimens were differentiated from non HSIL and LSIL with a sensitivity and specificity of 70.4% and 66.7% respectively, with p < 0.05.

A unique optical property of biological tissue is high anisotropy so that light is scattered in the same direction with respect to the incident direction. We demonstrate that simple back-directional gating allows us to take advantage of such an intrinsic property of biological tissue to significantly suppress unwanted diffuse light. In back-directional gated imaging, the high anisotropic property of the surrounding medium can serve as a waveguide in a moderate depth. Although this idea is straightforward, it has not been utilized for diffuse light suppression and imaging quality improvement such as contrast and resolution in large-area imaging for biological tissue. We further show that by combining a spectral analysis with back-directional gated imaging, image contrast and depth can be dramatically enhanced for visualizing stromal microvascular blood content in a relatively large area. Because microvasculature can be heterogeneous, our imaging approach can permit detailed visualization of microvascularity in a relatively large area up to ~20 mm x 20 mm.

We report on experimental results and theoretical investigation on probing the structure of turbid medium using ultra low coherence enhanced backscattering spectroscopy where the spatial coherence length of the incident line light is not greater than 25 μm. The periodic structure contained in the low coherence enhanced backscattering spectroscopy is found to decrease with the dominant scatterer size. A theoretical model is proposed to explain the observations and is verified by Monte Carlo simulations.

We study coherent and non-coherent backscattering of circularly polarized light from turbid media. We find that the sign of helicity of circular polarized light does not change for a medium of point-like scatterers and can change significantly for the medium with high anisotropy of scatterers. The helicity flip is observed when the light scattering is described in terms of the Henyey-Greenstein scattering phase function. The angular dependence of the sum of coherent and non-coherent parts of backscattering also exhibits a helicity flip.

Light scattering spectroscopy (LSS) and Fourier domain low coherence interferometry (fLCI) are used in combination with the dual window method (DW) to measure scattering features from a thick turbid sample. By processing with the DW method, the trade off that hinders spectroscopic OCT is avoided, thus yielding depth resolved spectra with simultaneously high spatial and spectral resolution. The capabilities of the method are demonstrated by analyzing a double layer phantom, where the top layer contains polystyrene beads of diameter d = 4.00 μm, and the bottom layer contains beads of d = 6.98 μm. A white light parallel frequency domain OCT system is used to image the sample. The results show that scattering structure can be assessed accurately and precisely throughout the whole OCT image using LSS and fLCI.

Tissues are optically anisotropic and highly photon scattering media. By using a simple ellipsoid model of bio-molecules with anisotropic distribution and using an effective mean-field theory, the principal anisotropic index of refraction n_j(ω) and absorption extinction coefficient K_aj(ω), (j = x,y,z) are derived. The scattering extinction coefficient K_sj is calculated from our scattering depolarization theory of highly scattering bio-medium. The criterion of optical isotropy and anisotropy of a bio-medium is derived. The non-vanishing ▵n, ▵K_a and ▵K_s exist for medium with anisotropic molecules only. ▵K_s/<K_s> is larger for medium with higher density.

Optical properties of biological tissue such as reduced scattering and absorption coefficients can be determined from the temporal or spatial reflectance curve of the diffusion process. Owing to its non-homogenous nature, the assumption of uniform optical parameters may not be valid in practice. We propose a new scheme to resolve the optical effective attenuation profile from the spatial reflectance curve of a non-homogeneous tissue. The algorithm reconstructs the linear attenuation profile along the line of measurement, rather than giving one single value for the coefficient for each reflectance curve. The technique was applied to the reconstruction of a 2-dimensional attenuation image.

Fluorescence optical imaging use one or several (in multiplexing) injected fluorescent markers which specifically bind to targeted compounds. Near infrared light illuminates the region of interest and the emitted fluorescence is analyzed to localize fluorescence sources. A spectroscopic approach and a separation source method (Nonnegative matrix factorization) are explored to separate different fluorescence sources and remove the unwanted biological tissues autofluorescence. We present unmixing results on overlapping spectra of interest, and show that autofluorescence removal improves Fluorescent Diffuse Optical Tomography.

A photon-cell interactive Monte Carlo ("pciMC") model was developed to quantify the intracellular optical propagation in a 3-dimensional biconcave red blood cell (RBC) model having a finite volume and intracellular hemoglobin. The orientation of RBCs with respect to the incident photons was randomized to allow either extra- or intra-cellular propagation depending on the incident point and angles where the photon propagation at the plasma-cell interface was determined by the Snell's law and Fresnell's law. In this study, the photon propagation through single RBC using the pciMC was compared against the Henyey-Greenstein phase function. The absorption dependent intracellular optical path length was evaluated in comparison to Mie theory. Both results showed good agreement. The pciMC can contribute to photo-spectroscopy of blood and tissues by quantifying both extra- and intra-cellular optical propagation.

The objective of this work is to compare quantitatively the imaging capabilities of a laminar optical tomography (LOT) system with those of a spatially modulated imaging (SMI) system. LOT is a three dimensional optical imaging technique that achieves depth sensitivity by measuring multiple-scattered light at different source-detector separations. The SMI method is based on spatially modulated illumination-detection patterns, which encode both optical properties and depth information. In this work, simulation studies are carried out at different noise levels, to obtain the figures of merit of tomographic reconstructions for both systems. Experiments on phantoms are performed to demonstrate the validity of the numerical results.

We present a novel approach to measure the surface effect of a small scattering object in a highly scattering medium by using the amplitude and phase signal of diffuse photon-pairs density wave (DPPDW). The results demonstrate that DPPDW has high sensitivity for resolving the surface effect of a small object. Imaging in highly scattering media with the developed DPPDW method can potentially increase the spatial resolution of small scattering inclusions.

The scattering and absorption properties of human breast are very important for cancer diagnosis based on diffuse optical tomography (DOT). In this study, the dynamics of photon migration in three-dimensional human breast model with various source-detector separations is simulated based on a Monte Carlo algorithm. The three-dimensional human breast structure is obtained from in vivo MRI image. The breast model consists of skin, fatty tissue, glandular tissue, sternum and ribcage. The backscattered diffuse photons from each layer in breast are recorded by marking the deepest layer which every photon can reach. The experimental results indicate that the re-emitted photons contain more information from deep tissue regions with the source-detector separations because of the strong dependence to the resolution and sensitivity in DOT imaging. The geometric position of the source-detector separations were optimized in this study. The different sizes of breast tumor were modeled to analysis of optical image characterizations. Finally, the tumor images from different deep information were obtained with temporal profiles.