Multispectral quantitative epi-illumination deep-ultraviolet imaging with pseudo-colorization for fresh tissues

High-resolution, label-free, and slide-free histological imaging methods are highly desired in clinical pathology as efficient alternatives to the laborious histopathology workflow. Deep-ultraviolet (UV) microscopy offers high-resolution, label-free contrast of biomolecules due to its short wavelength and the intrinsic absorption of various biomolecules, but traditional approaches are limited to thin, sectioned samples. Here, we present a light-emitting diode (LED)-based, simple, fast, and low-cost deep UV imaging technique for fresh, thick tissues using epi-illumination at 255 nm and 280 nm (targeting nucleic acid and protein absorption peaks) and autofluorescence to enhance biomolecular contrast. Our method also quantifies pixel-wise nucleic acid mass, protein mass, and tryptophan quantum yield, and introduces more efficient pseudo-colorization, generating pseudo-H&E images familiar to pathologists. This approach could bypass the slow and labor-intensive traditional histopathology workflows and potentially provide a rapid, accurate, and label-free alternative for many clinical applications.