Imaging intracellular components in situ using super-resolution cryo-correlative light and electron microscopy
bioRxiv (Cold Spring Harbor Laboratory)
Abstract
Abstract Super-resolution cryo-correlative light and electron microscopy (SRcryoCLEM) is emerging as a powerful method to enable targeted in situ structural 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.
Authors 3
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Leiden University · Leiden University Medical Center
Affiliation as printed
Department of Cell and Chemical Biology, Leiden University Medical Centre, 2300 RC Leiden, The Netherlands
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Leiden University · Leiden University Medical Center
Affiliation as printed
Department of Cell and Chemical Biology, Leiden University Medical Centre, 2300 RC Leiden, The Netherlands
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Leiden University · Leiden University Medical Center
Affiliation as printed
Department of Cell and Chemical Biology, Leiden University Medical Centre, 2300 RC Leiden, The Netherlands
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