Polarized hyperspectral microscopic imaging system for enhancing the visualization of collagen fibers and head and neck squamous cell carcinoma

Significance: Polarized hyperspectral microscopes with the capability of full Stokes vector imaging have potential for many biological and medical applications.

Aim: The aim of this study is to investigate polarized hyperspectral imaging (PHSI) for improving the visualization of collagen fibers, which is an important biomarker related to tumor development, and improving the differentiation of normal and tumor cells on pathologic slides.

Approach: We customized a polarized hyperspectral microscopic imaging system comprising an upright microscope with a motorized stage, two linear polarizers, two liquid crystal variable retarders (LCVRs), and a compact SnapScan hyperspectral camera. The polarizers and LCVRs worked in tandem with the hyperspectral camera to acquire polarized hyperspectral images, which were further used to calculate four Stokes vectors: $S0$, $S1$, $S2$, and $S3$. Synthetic RGB images of the Stokes vectors were generated for the visualization of cellular components in PHSI images. Regions of interest of collagen, normal cells, and tumor cells in the synthetic RGB images were selected, and spectral signatures of the selected components were extracted for comparison. Specifically, we qualitatively and quantitatively investigated the enhanced visualization and spectral characteristics of dense fibers and sparse fibers in normal stroma tissue, fibers accumulated within tumors, and fibers accumulated around tumors.

Results: By employing our customized polarized hyperspectral microscope, we extract the spectral signatures of Stokes vector parameters of collagen as well as of tumor and normal cells. The measurement of Stokes vector parameters increased the image contrast of collagen fibers and cells in the slides.

Conclusions: With the spatial and spectral information from the Stokes vector data cubes ($S0$, $S1$, $S2$, and $S3$), our PHSI microscope system enhances the visualization of tumor cells and tumor microenvironment components, thus being beneficial for pathology and oncology.