Evaluation of illumination systems for wide-field hyperspectral imaging in biomedical applications

Hyperspectral imaging (HSI) systems collect both morphological and chemical characteristics from a sample by simultaneously acquiring spatial and spectral information. HSI has potential to advance cancer diagnostics by characterizing reectance and uorescence properties of a tissue, as well as extracting microstructural in- formation, all of which are altered through the development of a tumor. Illumination uniformity is a critical pre-condition for extracting quantitative data from an HSI system. Spatial, angular, or spectral non-uniformity can cause glare, specular reection and unwanted shading, which negatively impact statistical analysis tech- niques used to extract abundance of different chemical species. This is further exacerbated when imaging three-dimensional structures, such as tumors, whose appearance can cast shadows and form other occlusions. Furthermore, as HSI can be used simultaneously for white light and uorescence imaging, a exible system, which multiplexes narrowband and broadband illumination is necessary to fully utilize the capabilities of a biomedical HSI system. To address these challenges, we modeled illumination systems frequently used in wide-field biological imaging with the software LightTools and FRED. Each system is characterized for spectral, spatial, and angular uniformity, as well as total effciency. While all three systems provide high spatial and spectral uniformity, the highest angular uniformity is achieved using a diffiuse scattering dome, yielding a contrast of 0.503 and average deviation of 0.303 with a 3.91% model error. Nonetheless, results suggest that conventional systems may not be suitable for low-light-level applications, where tailoring illumination to match spatial and spectral requirements may be the best approach to maximize the performance.