Precision <i>in situ</i> cryogenic correlative light and electron microscopy of optogenetically positioned organelles
Unambiguous targeting of cellular structures for in situ cryo-electron microscopy in the heterogeneous, dense and compacted environment of the cytoplasm remains challenging. Here, we have developed a cryogenic correlative light and electron microscopy (cryo-CLEM) workflow that utilizes thin cells grown on a mechanically defined substratum for rapid analysis of organelles and macromolecular complexes by cryo-electron tomography (cryo-ET). We coupled these advancements with optogenetics to redistribute perinuclear-localised organelles to the cell periphery, allowing visualisation of organelles that would otherwise be positioned in cellular regions too thick for cryo-ET. This reliable and robust workflow allows for fast in situ analyses without the requirement for cryo-focused ion beam milling. Using this protocol, cells can be frozen, imaged by cryo-fluorescence microscopy and be ready for batch cryo-ET within a day.
Authors
- Renée Whan (ORCID: https://orcid.org/0000-0002-6664-6344)
- Juanfang Ruan (ORCID: https://orcid.org/0000-0001-8994-5235)
- Yin Yao (ORCID: https://orcid.org/0000-0002-0307-8749)
- Robert G. Parton (ORCID: https://orcid.org/0000-0002-7494-5248)
- James Rae (ORCID: https://orcid.org/0000-0003-0525-2154)
- Gregory Redpath (ORCID: https://orcid.org/0000-0002-1990-6457)
- Edna C. Hardeman (ORCID: https://orcid.org/0000-0003-1649-7712)
- Peter W. Gunning (ORCID: https://orcid.org/0000-0003-0833-3128)
- Nicholas Ariotti (ORCID: https://orcid.org/0000-0003-3901-0831)
- María Lastra Cagigas (ORCID: https://orcid.org/0000-0001-5464-2349)
- Vikas A. Tillu (ORCID: https://orcid.org/0000-0002-1034-9543)
- Vaishnavi Ananthanarayanan (ORCID: https://orcid.org/0000-0003-2936-7853)
Institutions
- The University of Queensland (AU)
- UNSW Sydney (AU)
- EMBL Australia (AU)
- Kensington Health (CA)
- Centre for Microscopy and Microanalysis (AU)
Publication Details
- Journal
- Journal of Cell Science
- Published
- 2024-09-23
- DOI
- https://doi.org/10.1242/jcs.262163
- Citations
- 6
- Primary Topic
- Advanced Electron Microscopy Techniques and Applications
- Type
- article
- Field-Weighted Citation Impact
- 3.13
Funders
- Victor Chang Cardiac Research Institute
- EMBL Australia
- Analytical Center for the Government of the Russian Federation
- Australian Cancer Research Foundation
- University of New South Wales
- University of Queensland
- New South Wales Government
- Medical Research Council
- Australian Research Council
- National Health and Medical Research Council