Fluorescent Labeling Methods for Super-resolution and Optical-sectioning Imaging

Abstract

Throughout the development of light and fluorescence microscopy, a variety of labeling methods have evolved; from mostly affinity-based labeling used in histological staining to specific labeling using antibodies and genetically encoded fluorescent proteins. Recently, there is a growing interest in the de novo design of proteins as an alternative to repurposing naturally occurring protein scaffolds. Here I present the method used to characterize the photophysical properties of de novo fluorescence-activating β-barrel that are genetically encodable. Also, inspired by well-established histological staining methods, I present a simple labeling method that uses commercially available fluorophores to efficiently, and uniformly, label abundant chemical functional groups on biological specimens for expansion microscopy and cleared tissue microscopy. Amine-reactive fluorophores could effectively label distributions of proteins while aldehyde-reactive fluorophores could show distributions of oxidized carbohydrates. Despite the relatively abundance of such chemical groups, the combination of these covalent chemical stains can powerfully reveal key nanoscale features and general physiological landmarks in a wide variety of thin or thick tissue specimens and fixed cells that were either expanded or cleared.