Validation of a polarized hyperspectral imaging (PHSI) probe using phantoms and biological tissues

Polarized hyperspectral imaging (PHSI) combines wavelength-resolved reflectance with polarization sensitivity to probe tissue absorption, scattering, and microstructural organization in a label-free manner. We developed a compact handheld PHSI probe that integrates two liquid crystal variable retarders (LCVRs), a linear polarizer, and a hyperspectral snapshot camera to achieve full Stokes imaging over the range of 460-600 nm. In this work, we establish a comprehensive calibration and validation framework for this probe to improve its quantitative spectral and polarization performance. Individual LCVRs were first calibrated at four wavelengths (480, 530, 580, and 633 nm) using a crossed-polarizer method to obtain voltage-retardance characteristic curves and to determine the driving voltages corresponding to 0, λ/4, and λ/2 retardance at 530 nm. We then adapted a full-Stokes polarimeter calibration method to identify systematic errors at 520 nm, using six calibration samples and a Stokes-Mueller model that account for LCVR axis offsets, residual retardance errors, and polarizer misalignment. Spatial and spectral performance were evaluated using a USAF target, a ruler, and eight color tiles with known reflectance spectra, resulting in a field of view of 7 mm × 11 mm, a resolution of 7.13 lines per millimeter (lp/mm), and spectral angles below 5°. Additional validation on Intralipid-dye phantoms and ex vivo mouse tissues demonstrated that the calibrated probe could capture dye- and tissue-specific reflectance and degree of polarization spectra. These results highlight the potential of the calibrated handheld PHSI probe as a portable tool for quantitative tissue characterization in biomedical research and diagnostics.