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Abstract

Biochemistry

Sample Preparation for In Situ Cryotomography of Mammalian Cells

Published: December 15th, 2023

DOI:

10.3791/65697

1College of Liberal Arts, University of Minnesota, 2Department of Biochemistry, Molecular Biology and Biophysics, Medical School, University of Minnesota, 3College of Biological Sciences, University of Minnesota

* These authors contributed equally

Abstract

In situ cellular cryotomography is a powerful technique for studying complex objects in their native frozen-hydrated cellular context, making it highly relevant to cellular biology and virology. The potential of combining cryotomography with other microscopy modalities makes it a perfect technique for integrative and correlative imaging. However, sample preparation for in situ cellular tomography is not straightforward, as cells do not readily attach and stretch over the electron microscopy grid. Additionally, the grids themselves are fragile and can break if handled too forcefully, resulting in the loss of imageable areas. The geometry of tissue culture dishes can also pose a challenge when manipulating the grids with tweezers. Here, we describe the tips and tricks to overcome these (and other) challenges and prepare good-quality samples for in situ cellular cryotomography and correlative imaging of adherent mammalian cells. With continued advances in cryomicroscopy technology, this technique holds enormous promise for advancing our understanding of complex biological systems.

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Keywords Sample Preparation

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