Significance Mueller polarimetric imaging shows great promise for differentiating neoplastic from healthy brain tissue during neurosurgery. However, validating algorithmic approaches is limited by the scarcity of substantial tumor border zones in ex vivo samples, limiting comprehensive analysis of tumor margins.Aim We propose a protocol to build a database of histologically annotated polarimetric images from formalin-fixed whole-brain sections. We focus on validating the image alignment pipeline on healthy tissue.Approach To address the size mismatch between samples and the field of view of imaging instruments, we developed an automatic reconstruction pipeline to create large-scale polarimetric images from smaller raster-scanned tiles. Matching points between reference photographs and tile images allowed precise alignment. Similarly, fractionated histological sections were reconstructed and accurately aligned with the polarimetric data to serve as ground truth.Results The integrated reconstruction and alignment approach enabled large-scale, spatially co-registered polarimetric and histological imaging, supporting a more detailed investigation of tissue polarimetric parameters. The database thus created will facilitate the training and evaluation of segmentation models.Conclusions The developed method improved polarimetry-based brain tissue mapping by linking polarimetric parameters with histological features, enhancing the quality and quantity of data available for training and evaluating segmentation models. Although initially applied to brain tissue, the protocol could be extended to other organs to support broader studies of polarimetric tissue characterization.
