Design and Validation of a Custom-Made Hyperspectral Microscope Imaging System for Biomedical Applications

Hyperspectral microscope imaging (HMI) is an emerging modality that integrates spatial information collected by standard laboratory microscopy and the spectral-based contrast obtained by hyperspectral imaging and may be instrumental in establishing novel quantitative diagnostic methodologies, particularly in histopathology. Further expansion of HMI capabilities hinges upon the modularity and versatility of systems and their proper standardization. In this report, we describe the design, calibration, characterization, and validation of the custom-made laboratory HMI system based on a Zeiss Axiotron fully motorized microscope and a custom-developed Czerny-Turner-type monochromator. For these important steps, we rely on a previously designed calibration protocol. Validation of the system demonstrates a performance comparable to classic spectrometry laboratory systems. We further demonstrate validation against a laboratory hyperspectral imaging system for macroscopic samples, enabling future comparison of spectral imaging results across length scales. An example of the utility of our custom-made HMI system on a standard hematoxylin and eosin-stained histology slide is also shown.