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

Methods in Cell Biology
Nicholas R Ader, Wanda Kukulski

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.

Citations

Feb 28, 2019·Proceedings of the National Academy of Sciences of the United States of America·Felipe MoserRainer Kaufmann
May 15, 2020·The Journal of Cell Biology·Natalia Gomez-NavarroElizabeth A Miller
Nov 27, 2020·Nature Communications·John J H ShinSean Munro
Aug 13, 2021·The Journal of Cell Biology·Olivia MurielSophie G Martin
Sep 19, 2021·Life Science Alliance·Caroline S SimonJulien Guizetti

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