Roadmap for the Characterization and Validation of Hyperspectral Microscopic Systems

Hyperspectral (HS) imaging (HSI) is a powerful image technique that allows capturing spatial and spectral information, being able to characterize materials, tissues, and elements in a non-invasive manner. HSI technology is well established at the macroscopic level, but there are still technical challenges to overcome before it can be applied to the microscopic world, such as the lack of standardized characterization methodologies to HS microscopic systems that allow the correct data acquisition as well as ensure the repeatability of the experiments. In this work, we propose a comprehensive roadmap for characterizing and validating such systems, integrating essential parameters highlighted in the current state of the art. Furthermore, we provide a list of the materials needed for their characterization and testing the methodology on two different HS microscopic systems chosen as representative of common configurations in the field, where a HS camera is integrated into a bright-field microscope. Our proposed roadmap assesses the following parameters: dynamic range, noise quantification, pixel size, spatial frequency response, spatial scanning accuracy, spatial repeatability, flat-field correction, tone transfer, and spectral sensitivity. We address the challenge of unifying these parameters into a unified and standardized roadmap. All data used to characterize both systems have been captured by the authors. In summary, this comprehensive analysis provides a guideline for the scientific community to develop and characterize HS microscopic systems to ensure reliability, efficiency, and accuracy.