Correlative cryo super-resolution light and electron microscopy on mammalian cells using fluorescent proteins
10.1038/s41598-018-37728-8
Crossref journal-article
Springer Science and Business Media LLC
Scientific Reports (297)
Abstract

AbstractSample fixation by vitrification is critical for the optimal structural preservation of biomolecules and subsequent high-resolution imaging by cryo-correlative light and electron microscopy (cryoCLEM). There is a large resolution gap between cryo fluorescence microscopy (cryoFLM), ~400-nm, and the sub-nanometre resolution achievable with cryo-electron microscopy (cryoEM), which hinders interpretation of cryoCLEM data. Here, we present a general approach to increase the resolution of cryoFLM using cryo-super-resolution (cryoSR) microscopy that is compatible with successive cryoEM investigation in the same region. We determined imaging parameters to avoid devitrification of the cryosamples without the necessity for cryoprotectants. Next, we examined the applicability of various fluorescent proteins (FPs) for single-molecule localisation cryoSR microscopy and found that all investigated FPs display reversible photoswitchable behaviour, and demonstrated cryoSR on lipid nanotubes labelled with rsEGFP2 and rsFastLime. Finally, we performed SR-cryoCLEM on mammalian cells expressing microtubule-associated protein-2 fused to rsEGFP2 and performed 3D cryo-electron tomography on the localised areas. The method we describe exclusively uses commercially available equipment to achieve a localisation precision of 30-nm. Furthermore, all investigated FPs displayed behaviour compatible with cryoSR microscopy, making this technique broadly available without requiring specialised equipment and will improve the applicability of this emerging technique for cellular and structural biology.

Bibliography

Tuijtel, M. W., Koster, A. J., Jakobs, S., Faas, F. G. A., & Sharp, T. H. (2019). Correlative cryo super-resolution light and electron microscopy on mammalian cells using fluorescent proteins. Scientific Reports, 9(1).

Authors 5
  1. Maarten W. Tuijtel (first)
  2. Abraham J. Koster (additional)
  3. Stefan Jakobs (additional)
  4. Frank G. A. Faas (additional)
  5. Thomas H. Sharp (additional)
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Dates
Type When
Created 6 years, 6 months ago (Feb. 4, 2019, 6:04 a.m.)
Deposited 2 years, 8 months ago (Dec. 17, 2022, 12:37 a.m.)
Indexed 6 hours, 24 minutes ago (Aug. 21, 2025, 12:56 p.m.)
Issued 6 years, 6 months ago (Feb. 4, 2019)
Published 6 years, 6 months ago (Feb. 4, 2019)
Published Online 6 years, 6 months ago (Feb. 4, 2019)
Funders 0

None

@article{Tuijtel_2019, title={Correlative cryo super-resolution light and electron microscopy on mammalian cells using fluorescent proteins}, volume={9}, ISSN={2045-2322}, url={http://dx.doi.org/10.1038/s41598-018-37728-8}, DOI={10.1038/s41598-018-37728-8}, number={1}, journal={Scientific Reports}, publisher={Springer Science and Business Media LLC}, author={Tuijtel, Maarten W. and Koster, Abraham J. and Jakobs, Stefan and Faas, Frank G. A. and Sharp, Thomas H.}, year={2019}, month=feb }