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Sample preparation and image registration for correlative cryo-FM and cryo-FIB-SEM of plunge-frozen mammalian cells.

ABSTRACT: We recently demonstrated how lipid droplets can serve as in situ fiducials for correlating cryo-fluorescence microscopy (cryo-FM) and cryo-focused ion beam scanning electron microscopy (cryo-FIB-SEM) datasets of mammalian cells grown on grids. Here we describe a step-by-step protocol for correlative cryo-FM and cryo-FIB-SEM, starting from sample preparation of C2C12 cell line, followed by imaging with cryo-FM and cryo-FIB-SEM. Finally, we detail how to perform the 3D-correlation with sub-micron accuracy. For complete details on the use and execution of this profile, please refer to Scher et al. (2021).

SUBMITTER: Scher N

PROVIDER: S-EPMC8844706 | biostudies-literature | 2022 Mar

REPOSITORIES: biostudies-literature

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Sample preparation and image registration for correlative cryo-FM and cryo-FIB-SEM of plunge-frozen mammalian cells.

Scher Nadav N Rechav Katya K Paul-Gilloteaux Perrine P Avinoam Ori O

STAR protocols 20220208 1

We recently demonstrated how lipid droplets can serve as <i>in situ</i> fiducials for correlating cryo-fluorescence microscopy (cryo-FM) and cryo-focused ion beam scanning electron microscopy (cryo-FIB-SEM) datasets of mammalian cells grown on grids. Here we describe a step-by-step protocol for correlative cryo-FM and cryo-FIB-SEM, starting from sample preparation of C2C12 cell line, followed by imaging with cryo-FM and cryo-FIB-SEM. Finally, we detail how to perform the 3D-correlation with sub- ...[more]

PMID: 35199027

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