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Methods Cell Biol. 2017;140:303-320. doi: 10.1016/bs.mcb.2017.03.009. Epub 2017 Apr 18.

triCLEM: Combining high-precision, room temperature CLEM with cryo-fluorescence microscopy to identify very rare events.

Author information

1
MRC Laboratory of Molecular Biology, Cambridge, United Kingdom; National Institutes of Health, Bethesda, MD, United States.
2
MRC Laboratory of Molecular Biology, Cambridge, United Kingdom.

Abstract

Fiducial-based correlation of fluorescence and electron microscopy data from high-pressure frozen and resin-embedded samples allows for high-precision localization of fluorescent signals to subcellular ultrastructure. Here we introduce the triCLEM procedure to facilitate the identification of very rare events for high-precision correlation. We present a detailed protocol to screen high-pressure frozen cell monolayers on sapphire disks for very rare signals by cryo-fluorescence microscopy, relocate the cells of interest after freeze substitution and Lowicryl embedding, and perform fiducial-based correlation of the identified fluorescent signals to high-magnification electron tomograms. We show the applicability of the protocol to localize and image damaged mitochondria marked by the presence of Parkin, a protein involved in initiating mitophagy. We discuss how this extension to previously published fiducial-based correlation procedures has potential to both allow identifying very rare events and assess the quality of preservation in high-pressure frozen samples.

KEYWORDS:

CLEM; Cryo-fluorescence microscopy; Electron tomography; Fiducials; Fluorescent proteins; High-pressure freezing; Sapphire disks; triCLEM

PMID:
28528638
DOI:
10.1016/bs.mcb.2017.03.009
[Indexed for MEDLINE]

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