Imaging intracellular componentsin situusing super-resolution cryo-correlative light and electron microscopy

Abstract

AbstractSuper-resolution cryo-correlative light and electron microscopy (SRcryoCLEM) is emerging as a powerful method to enable targetedin situstructural studies of biological samples. By combining the high specificity and localization accuracy of single-molecule localization microscopy (cryoSMLM) with the high resolution of cryo-electron tomography (cryoET), this method enables accurately targeted data acquisition and the observation and identification of biomolecules within their natural cellular context. Despite its potential, the adaptation of SRcryoCLEM has been hindered by the need for specialized equipment and expertise. In this chapter, we outline a workflow for cryoSMLM and cryoET-based SRcryoCLEM, and we demonstrate that, given the right tools, it is possible to incorporate cryoSMLM into an established cryoET workflow. Using Vimentin as an exemplary target of interest, we exemplify all stages of an SRcryoCLEM experiment: performing cryoSMLM, targeting cryoET acquisition based single-molecule localization maps, and correlation of cryoSMLM and cryoET datasets using scNodes, a software package dedicated to SRcryoCLEM. By showing how SRcryoCLEM enables the imaging of specific intracellular components in situ, we hope to facilitate the further adaptation of the technique within the field of cryoEM.