Label-free Super-resolution Imaging Enabled by Vibrational Imaging of Swelled Tissue and Analysis

The universal utilization of fluorescence microscopy, especially super-resolution microscopy, has greatly advanced knowledge about modern biology. Conversely, the requirement of fluorophore labeling in fluorescent techniques poses significant challenges, such as photobleaching and non-uniform labeling of fluorescent probes and prolonged sample processing. In this protocol, the detailed working procedures of vibrational imaging of swelled tissue and analysis (VISTA) are presented. VISTA circumvents obstacles associated with fluorophores and achieves label-free super-resolution volumetric imaging in biological samples with spatial resolution down to 78 nm. The procedure is established by embedding cells and tissues in hydrogel, isotropically expanding the hydrogel sample hybrid, and visualizing endogenous protein distributions by vibrational imaging with stimulated Raman scattering microscopy. The method is demonstrated on both cells and mouse brain tissues. Highly correlative VISTA and immunofluorescence images were observed, validating the protein origin of imaging specificities. Exploiting such correlation, machine learning-based image-segmentation algorithm was trained to achieve multicomponent prediction of nuclei, blood vessels, neuronal cells, and dendrites from label-free mouse brain images. The procedure was further adapted to investigate pathological poly-glutamine (polyQ) aggregates in cells and amyloid-beta (A beta) plaques in brain tissues with high throughput, justifying its potential for large-scale clinical samples.