Influence of the phase function in generalized diffuse reflectance models: review of current formalisms and novel observations.

Diffuse reflectance spectroscopy, which has been demonstrated as a noninvasive diagnostic technique, relies on quantitative models for extracting optical property values from turbid media, such as biological tissues. We review and compare reflectance models that have been published, and we test similar models over a much wider range of measurement parameters than previously published, with specific focus on the effects of the scattering phase function and the source-detector distance. It has previously been shown that the dependence of a forward reflectance model on the scattering phase function can be described more accurately using a variable, γ, which is a more predictive variable for reflectance than the traditional anisotropy factor, g. We show that variations in the reflectance model due to the phase function are strongly dependent on the source-detector separation, and we identify a dimensionless scattering distance at which reflectance is insensitive to the phase function. Further, we evaluate how variations in the phase function and source-detector separation affect the accuracy of inverse property extraction. By simultaneously fitting two or more reflectance spectra, measured at different source-detector separations, we also demonstrate that an estimate of γ can be extracted, in addition to the reduced scattering and absorption coefficients.

Gender variations in the optical properties of skin in murine animal models.

Gender is identified as a significant source of variation in optical reflectance measurements on mouse skin, with variation in the thickness of the dermal layer being the key explanatory variable. For three different mouse strains, the thickness values of the epidermis, dermis, and hypodermis layers, as measured by histology, are correlated to optical reflectance measurements collected with elastic scattering spectroscopy (ESS). In all three strains, males are found to have up to a 50% increase in dermal thickness, resulting in increases of up to 80% in reflectance values and higher observed scattering coefficients, as compared to females. Collagen in the dermis is identified as the primary source of these differences due to its strong scattering nature; increased dermal thickness leads to a greater photon path length through the collagen, as compared to other layers, resulting in a larger scattering signal. A related increase in the observed absorption coefficient in females is also observed. These results emphasize the importance of considering gender during experimental design in studies that involve photon interaction with mouse skin. The results also elucidate the significant impact that relatively small thickness changes can have on observed optical measurements in layered tissue.

Temporal variations of skin pigmentation in C57BL/6 mice affect optical bioluminescence quantitation.

PURPOSE: Depilation-induced skin pigmentation in C57Bl/6 mice is a known occurrence, and presents a unique problem for quantitative optical imaging of small animals, especially for bioluminescence. The work reported here quantitatively investigated the optical attenuation of bioluminescent light due to melanin pigmentation in the skin of transgenic C57Bl/6 mice, modified such that luciferase expression is under the transcription control of a physiologically and pharmacologically inducible gene. PROCEDURE: Both in vivo and ex vivo experiments were performed to track bioluminescence signal attenuation through different stages of the mouse hair growth cycle. Simultaneous reflectance measurements were collected in vivo to estimate melanin levels. RESULTS: Biological variability of skin pigmentation was found to dramatically affect collected bioluminescent signal emerging through the skin of the mice. When compared to signal through skin with no pigmentation, the signal through highly pigmented skin was attenuated an average of 90%. Positive correlation was found between reflectance measurements and bioluminescence signal loss. A correction scheme is proposed based on this correlation, but signal variation due to non-melanin scattering and absorption sources introduce significant errors. Advanced spectral reflectance analysis will be necessary to develop a more reliable correction method in the future. CONCLUSION: Skin pigmentation is a significant variable in bioluminescent imaging, and should be considered in experimental design and implementation for longitudinal studies, and especially when sensitivity to small signal changes, or differences among animals, is required.

Scanning elastic scattering spectroscopy detects metastatic breast cancer in sentinel lymph nodes.

A novel method for rapidly detecting metastatic breast cancer within excised sentinel lymph node(s) of the axilla is presented. Elastic scattering spectroscopy (ESS) is a point-contact technique that collects broadband optical spectra sensitive to absorption and scattering within the tissue. A statistical discrimination algorithm was generated from a training set of nearly 3000 clinical spectra and used to test clinical spectra collected from an independent set of nodes. Freshly excised nodes were bivalved and mounted under a fiber-optic plate. Stepper motors raster-scanned a fiber-optic probe over the plate to interrogate the node's cut surface, creating a 20x20 grid of spectra. These spectra were analyzed to create a map of cancer risk across the node surface. Rules were developed to convert these maps to a prediction for the presence of cancer in the node. Using these analyses, a leave-one-out cross-validation to optimize discrimination parameters on 128 scanned nodes gave a sensitivity of 69% for detection of clinically relevant metastases (71% for macrometastases) and a specificity of 96%, comparable to literature results for touch imprint cytology, a standard technique for intraoperative diagnosis. ESS has the advantage of not requiring a pathologist to review the tissue sample.

Analysis of changes in reflectance measurements on biological tissues subjected to different probe pressures.

Spectral reflectance measurements of biological tissues have been studied for early diagnoses of several pathologies such as cancer. These measurements are often performed with a fiber optic probe in contact with the tissue surface. We report a study in which reflectance measurements are obtained in vivo from mouse thigh muscle while varying the contact pressure of the fiber optic probe. It is determined that the probe pressure is a variable that affects the local optical properties of the tissue. The reflectance spectra are analyzed with an analytical model that extracts the tissue optical properties and facilitates the understanding of underlying physiological changes induced by the probe pressure.