Label- and slide-free multispectral quantitative epi-illumination deep-UV microscopy

Abstract

Label-free and slide-free imaging is highly desired in clinical pathology because it holds the potential to avoid time- and labor-intensive tissue processing and chemical staining while preserving molecular information for downstream analyses. Deep-ultraviolet (UV) microscopy offers high-resolution, label-free molecular contrast via short wavelengths and intrinsic biomolecular absorption, but prior implementations have been limited to the analysis of thin sections and cell monolayers. Here, we present a fast, low-cost, LED-based, epi-illumination deep-UV microscope (epi-DUV) for label- and slide-free imaging of fresh, thick tissues. Using 255 nm and 280 nm absorption images, and tryptophan autofluorescence, the method yields quantitative maps of nucleic acid mass, protein mass, and quantum yield. Moreover, H&E-like contrast can be generated using native 255-nm absorption images. The system achieves 0.5 {micro}m lateral resolution with an effective slice thickness of [~]6 {micro}m across a 707 {micro}m x 707 {micro}m field of view and uses [~]330-ms exposure. To the best of our knowledge, this is the first demonstration of quantitative deep-UV molecular imaging of fresh, unlabeled thick tissues. Epi-DUV has significant potential to streamline the histopathology workflow while adding objective molecular readouts, enabling point-of-care assessment of unprocessed specimens (e.g., rapid intraoperative evaluation).