High-quality ultrastructural preservation using cryofixation for 3D electron microscopy of genetically labeled tissues
- University of California, San Diego, United States
Abstract
Electron microscopy (EM) offers unparalleled power to study cell substructures at the nanoscale. Cryofixation by high-pressure freezing offers optimal morphological preservation, as it captures cellular structures instantaneously in their near-native states. However, the applicability of cryofixation is limited by its incompatibilities with diaminobenzidine labeling using genetic EM tags and the high-contrast en bloc staining required for serial block-face scanning electron microscopy (SBEM). In addition, it is challenging to perform correlated light and electron microscopy (CLEM) with cryofixed samples. Consequently, these powerful methods cannot be applied to address questions requiring optimal morphological preservation. Here we developed an approach that overcomes these limitations; it enables genetically labeled, cryofixed samples to be characterized with SBEM and 3D CLEM. Our approach is broadly applicable, as demonstrated in cultured cells, Drosophila olfactory organ and mouse brain. This optimization exploits the potential of cryofixation, allowing quality ultrastructural preservation for diverse EM applications.
Data availability
A source data file has been provided for Figure 4 (Figure 4-source data 1). The SBEM volume of a Drosophila antenna presented in this study has been deposited to the Cell Image Library. The SBEM volume, the tdTomato confocal volume and the DRAQ5 confocal volume used for 3D CLEM in a mouse brain (corresponding to Figure 5) have also been deposited to the Cell Image Library. The video of 3D CLEM in a mouse brain expressing tdTomato that corresponds to Figure 5-video supplement 1 has been deposited to the Cell Image Library.
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SBEM of Drosophila antenna (10x UAS-myc-APEX2-Orco; Or47b-GAL4)Publicly available at the Cell Image Library under accession number CIL:50452.
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SBEM volume used for 3D CLEM in mouse brainPublicly available at the Cell Image Library under accession number CIL:50451.
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tdTomato confocal volume used for 3D CLEM in a mouse brainPublicly available at the Cell Image Library under accession number CIL:50453.
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DRAQ5 confocal volume used for 3D CLEM in a mouse brainPublicly available at the Cell Image Library under accession number CIL:50454.
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Video of 3D CLEM in a mouse brain expressing tdTomatoPublicly available at the Cell Image Library under accession number CIL:50401.
Article and author information
Author details
Funding
National Institute on Deafness and Other Communication Disorders (R01DC015519)
- Chih-Ying Su
National Institute of General Medical Sciences (P41GM103412)
- Mark H Ellisman
Croucher Foundation
- Tin Ki Tsang
Kavli Foundation (2015-004)
- Chih-Ying Su
- Mark H Ellisman
Ray Thomas Edwards Foundation
- Chih-Ying Su
Frontiers of Innovation Scholars Program
- Tin Ki Tsang
National Institute of General Medical Sciences (R01GM086197)
- Daniela Boassa
Kavli Foundation (2016-038)
- Daniela Boassa
- Davide Dulcis
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2018, Tsang et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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High-quality ultrastructural preservation using cryofixation for 3D electron microscopy of genetically labeled tissues
eLife 7:e35524.
https://doi.org/10.7554/eLife.35524
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