<p>Mueller microscopy studies of fixed unstained histological cuts of human skin models were combined with an analysis of experimental data within the framework of differential Mueller matrix (MM) formalism. A custom-built Mueller polarimetric microscope was used in transmission configuration for the optical measurements of skin tissue model adjacent cuts of various nominal thicknesses (5 to 30 μm). The maps of both depolarization and polarization parameters were calculated from the corresponding microscopic MM images by applying a logarithmic Mueller matrix decomposition (LMMD) pixelwise. The parameters derived from LMMD of measured tissue cuts and the intensity of transmitted light were used for an automated segmentation of microscopy images to delineate dermal and epidermal layers. The quadratic dependence of depolarization parameters and linear dependence of polarization parameters on thickness, as predicted by the theory, was confirmed in our measurements. These findings pave the way toward digital histology with polarized light by presenting the combination of optimal optical markers, which allows mitigating the impact of tissue cut thickness fluctuations and increases the contrast of polarimetric images for tissue diagnostics.</p>
Mueller transmission microscopy has been used for both theoretical and experimental studies of anisotropic scattering biological tissue. In our prior study, the linear dependence of retardance and quadratic dependence of depolarization on thickness was demonstrated for a dermal layer of skin model. During the primary analysis of polarimetric images of histological cuts both epidermal and dermal layers were delineated manually in order to calculate the spatially averaged values of retardance and depolarization parameters. Consequently, these average values contained the contribution of outliers (noise, not correctly identified pixels, etc.) which produces large standard deviation and biased mean values of the parameters mentioned above. For preventing the errors, the normalized maps of optical properties were calculated pixel-wise taking into account local optical density (e. i. logarithm of M11 element of Mueller matrix at each image pixel) to compensate varying tissue thickness across the cut area. Furthermore, the DBSCAN (Density-based spatial clustering of applications with noise) algorithm was applied for segmentation of microscopic images using the normalized values of retardance, depolarization, and intensity. From the results of image segmentation, we could discriminate the regions of dermal and epidermal layers in Muller microscopic images of skin cuts more accurately and obtain more reliable values of tissue’s optical properties.
The combined approach including experiments (Mueller polarimetry) and theory (differential Mueller matrix formalism) for the studies of anisotropic scattering media was tested on the model system of human skin. Custom-build Mueller polarimetric microscope was used for the studies of histological cuts of full-thickness skin equivalents generated from epidermal keratinocytes forming a multilayered epidermis on top of collagen I hydrogel with dermal fibroblasts. The sets of fixed unstained tissue cuts of different thicknesses (5μm - 30μm) were measured in transmission configuration. The values of polarimetric (dichroism and retardance) and depolarization parameters were calculated by applying pixel-wise the logarithmic decomposition of Mueller matrices. The parabolic dependence of depolarization parameters and linear dependence of polarimetric parameters on thickness, as predicted by theory, was confirmed by measurements. It proves that phenomenological modeling of complex anisotropic scattering medium (e.g., biological tissue) may effectively disentangle the polarimetric and depolarizing properties of the system understudies and may be used for analysis and diagnostics of tissue.
At the present time a definite assessment of cancer diagnosis can only be made after the microscopic analysis of histological cuts of tissue biopsies. Pathologists have to prepare and analyze a considerable amount of histological slides. The accuracy of diagnostics strongly relies on the experience of medical doctor performing histological analysis. Hence, the search for new efficient techniques helping pathologists to sort out the vast majority of histological slides and significantly reduce the time of diagnostic is of paramount importance.
The potential of Mueller polarimetry to become a new optical tool for digitally assisted automated polarized light histology was exploited within the framework of differential Mueller matrix formalism. The measurements of thin scattering anisotropic phantoms and histological slides of fixed unstained healthy and cancerous human tissue (basal cell carcinoma of skin, adenocarcinoma of colon) were performed in transmission configuration with custom-build Mueller polarimetric microscope. In-house Monte Carlo software for the solution of vector radiative transfer equation in scattering anisotropic media was used for the interpretation of experimental Mueller matrices.
The use of phenomenological theory of anisotropic scattering fluctuating medium based on differential Mueller matrix formalism (i. e. logarithmic decomposition of Mueller matrices) combined with an appropriate algorithm of polarimetric image segmentation and statistical analysis of experimental data provide new insight on set of optical markers (e.g. linear/circular retardance, linear/circular depolarization) which increase significantly the contrast between cancerous and healthy zones of tissue histological cuts and assure high sensitivity and specificity of polarimetric optical diagnostics of cancer.