3D correlative microscopy methods have revolutionized biomedical research, allowing the acquisition of multidimensional information to gain an in-depth understanding of biological systems. With the advent of relevant cryo-preservation methods, correlative imaging of cryogenically preserved samples has led to nanometer resolution imaging (2–50 nm) under harsh imaging regimes such as electron and soft X-ray tomography. These methods have now been combined with conventional and super-resolution fluorescence imaging at cryogenic temperatures to augment information content from a given sample, resulting in the immediate requirement for protocols that facilitate hassle-free, unambiguous cross-correlation between microscopes. We present here sample preparation strategies and a direct comparison of different working fiducialization regimes that facilitate 3D correlation of cryo-structured illumination microscopy and cryo-soft X-ray tomography. Our protocol has been tested at two synchrotron beamlines (B24 at Diamond Light Source in the UK and BL09 Mistral at ALBA in Spain) and has led to the development of a decision aid that facilitates experimental design with the strategic use of markers based on project requirements. This protocol takes between 1.5 h and 3.5 d to complete, depending on the cell populations used (adherent cells may require several days to grow on sample carriers).
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Original imaging data referenced in the manuscript are deposited at the BioImage Archive (https://www.ebi.ac.uk/biostudies/BioImages) and EMPIAR (https://www.ebi.ac.uk/pdbe/emdb/empiar/). The accession numbers for the data deposited at EMPIAR are EMPIAR-10617, EMPIAR-10618, EMPIAR-10619, EMPIAR-10620, EMPIAR-10621, EMPIAR-10622 and EMPIAR-10624, and the accession numbers for the data deposited at the BioImage Archive are S-BIAD36, S-BIAD37, S-BIAD38, S-BIAD39, S-BIAD40, S-BIAD41 and S-BSST576.
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We thank P. Paul-Gilloteaux for her invaluable help with eC-CLEM and previous and current members of the B24 and BL09 teams, with special thanks to M. Spink for instrumentation support and A. Taylor and A. Prescott for technical support. We also thank A. Clayton for suggestions in trying new reagents and M. Dumoux for help with laboratory techniques, advice and training. We acknowledge the support of Micron in the development, application and maintenance of the B24 super-resolution facility. We also thank A. Aires Trapote (CIC BiomaGUNE, San Sebastian, Spain), A. V. Villar, A. R. Palanca, D. Maestro Lavín (IBBTEC, Santander, Spain) and J. Conesa (ALBA and CNB-CSIC). This work was carried out with the support of the Diamond Light Source, instrument B24 (proposals MX18737, MX20321, BI22274, BI23046 and BI25162). We acknowledge ALBA for allocated MISTRAL beamtimes 2018093099 and 2019093739. This project has received funding from the European Commission Horizon 2020 iNEXT-Discovery project and the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement 75439 and Wellcome awards 091911/Z/11/Z and 107457/Z/15/Z. S.B. is supported by ERC AdG670930.
The authors declare no competing interests.
Peer review information Nature Protocols thanks Gerd Schneider and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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Key references using this protocol
Kounatidis, I. et al. Cell 182, 1–16 (2020): https://doi.org/10.1016/j.cell.2020.05.051
Phillips, M. et al. Optica 7, 802–812 (2020): https://doi.org/10.1364/OPTICA.393203
Harkiolaki, M. et al. Emerg. Top. Life Sci. 2, 81–92 (2018): https://doi.org/10.1042/ETLS20170086
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Okolo, C.A., Kounatidis, I., Groen, J. et al. Sample preparation strategies for efficient correlation of 3D SIM and soft X-ray tomography data at cryogenic temperatures. Nat Protoc 16, 2851–2885 (2021). https://doi.org/10.1038/s41596-021-00522-4