Abstract
Correlative light and electron microscopy (CLEM) combines light microscopy, for identifying a target via genetic labels, dyes, antibodies and morphological features, with electron microscopy, for analyzing high-resolution subcellular ultrastructures. Here, we describe step-by-step instructions to perform a CLEM experiment, optimized for the investigation of ultrastructural features in human brain tissue. The procedure is carried out at room temperature and can be adapted to other human and animal tissue samples. The procedure requires 8 d to complete and includes the stages of sample fixation for optimal ultrastructural preservation, immunofluorescence staining, image acquisition and multimodal image correlation and is executable within standard electron microscopy laboratories. Serving as a critical tool for characterizing human tissue and disease models, room-temperature CLEM facilitates the identification and quantification of subcellular morphological features across brain regions.
Key points
The protocol for correlative light and electron microscopy (CLEM) is optimized for analyzing chemically fixed human brain tissues. It focuses on maintaining the integrity of ultrastructural features, thereby minimizing artefacts and structural alterations.
Examining brain tissues at the ultrastructural level can provide an unprecedented amount of detail, which may help advance our understanding of the mechanisms underlying neurodegenerative disorders.
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Fig. 1: Schematic of the CLEM workflow.
Fig. 2: Fluorescent mapping of free-floating sections.
Fig. 3: Schematic of the resin-embedding process on free-floating sections.
Fig. 4: Preparation of blocks for CLEM sectioning.
Fig. 5: CLEM sectioning and IHC staining of brain tissue sections on glass slides.
Fig. 6: Staining of glass slides.
Fig. 7: Image correlation using BigWarp.
Fig. 8: IHC and EM correlation.
Fig. 9: The full CLEM pipeline shown for the fluorescence and EM correlation of a single neuron within postmortem human brain tissue.
Data availability
The original microscopy images in this manuscript can be found at BioImage Archive under accession number S-BSST1770.
Code availability
All codes are available on github. CLEM4CINA.py can be accessed through the public repository at https://github.com/LBEM-CH/clem4cina. The FIJI macro can be accessed through the public repository at https://github.com/LBEM-CH/clem-for-human-brain.
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Acknowledgements
We thank the Netherlands brain bank autopsy team and J. Hench at the University Hospital Basel for the collection of postmortem human brain samples used for the development of this protocol; F. Pane from LGB, EPFL for printing the resin autoprocessor components; the Leica Research & Development team for assistance in the optimization of the cutting of resin samples with the laser-capture microdissection instrument; the staff at the Electron Microscopy Facility (UNIL) for training on equipment and transmission electron microscopes; and R. Righetto (University of Basel) for assistance with the CLEM4CINA program. The final metal holders for the autoprocessor components were machined by Laser Automation (La-Chaux-de-Fond, Switzerland). A.J.L. was supported by an EMBO short-term fellowship (Grant no. 8878), the Synapsis Foundation Switzerland (Grant no. 2019-CDA01) and Parkinson Schweiz. This work was in part supported by the Swiss National Science Foundation (SNF Grants CRSII5_177195 and 310030_188548). W.D.J.v.d.B. is supported by the Dutch Parkinson association (Grant no. 2020-G01). Fig. 1, Fig. 6b and Supplementary Fig. 7 were created with BioRender.com with agreement numbers CC275RDBFP, BF26TAAJ8K and GQ26TAAMU1, respectively.
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Author notes
These authors contributed equally: Notash Shafiei, Daniel Stӓhli.
Authors and Affiliations
Laboratory of Biological Electron Microscopy, Institute of Physics, School of Basic Sciences, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland
Notash Shafiei, Daniel Stӓhli, Domenic Burger, Marta Di Fabrizio, Lukas van den Heuvel, Henning Stahlberg & Amanda J. Lewis
Department of Fundamental Microbiology, Faculty of Biology and Medicine, University of Lausanne, Lausanne, Switzerland
Notash Shafiei, Daniel Stӓhli, Domenic Burger, Marta Di Fabrizio, Lukas van den Heuvel, Henning Stahlberg & Amanda J. Lewis
Electron Microscopy Facility, Biophore, University of Lausanne, Lausanne, Switzerland
Jean Daraspe & Christel Genoud
C-CINA, Biozentrum, University of Basel, Basel, Switzerland
Carolin Böing
Brain Institute and Cancer Center, UT Southwestern Medical Center, Dallas, TX, USA
Sarah H. Shahmoradian
Department of Anatomy and Neurosciences, section Clinical Neuroanatomy and Biobanking, Amsterdam Neuroscience, Amsterdam University Medical Centre, Vrije University Amsterdam, Amsterdam, The Netherlands
Wilma D. J. van de Berg
Amsterdam Neuroscience, program Neurodegeneration, Amsterdam University Medical Centre, Vrije University Amsterdam, Amsterdam, The Netherlands
Wilma D. J. van de Berg
School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland
Christel Genoud
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Notash Shafiei
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Contributions
A.J.L., S.H.S., C.G., W.D.J.v.d.B. and H.S. conceived the strategy for carrying out CLEM on postmortem human brain samples. W.D.J.v.d.B. collected, dissected and processed the brain tissue samples for CLEM. A.J.L. and W.D.J.v.d.B. developed the strategy for fluorescent staining, imaging and mapping of free-floating brain sections, with further optimization by D.S., N.S. and D.B. A.J.L. optimized the IHC of resin sections on glass slides, with contributions from M.D.F for the toluidine blue staining. C.G. optimized the manual en bloc staining and alternate serial sectioning protocol for 60-µm human brain sections. J.D. designed, printed and tested the sample holder components and optimized the embedding protocol for the tissue auto-processor. L.v.d.H. wrote the macro to create .xml files. A.J.L. and M.D.F. optimized the cutting of resin samples by laser-capture microdissection. D.B. and L.v.d.H. optimized the strategy for the image correlation of fluorescence and LM with EM. C.B. and H.S. wrote the CLEM4CINA program. A.J.L. tested the protocol on mouse brain sections and cell monolayers. N.S., D.S., M.D.F., D.B., L.v.d.H. and A.J.L. wrote the manuscript and prepared the figures. All authors approved the final version of the manuscript.
Corresponding author
Correspondence to Amanda J. Lewis.
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Nature Protocols thanks Ben Giepmans, Andreas Müller, Marc C. A. Stuart and Anouk Wolters for their contribution to the peer review of this work.
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Key references
Shahmoradian, S. H. et al. Nat. Neurosci. 22, 1099–1109 (2019): https://doi.org/10.1038/s41593-019-0423-2
Böing, C. et al. Brain 147, 3727–3741 (2024): https://doi.org/10.1093/brain/awae137
Lam, I. et al. Neuron 112, 2886–2909.e16 (2024): https://doi.org/10.1016/j.neuron.2024.06.002
Supplementary information
Supplementary Information
Supplementary Figures 1–9, Supplementary Table 1, Supplementary Protocols: Autoprocessor, Laser microdissection, Hematoxylin counterstaining, CLEM4CINA
Supplementary Data 1
Files needed for 3D printing of an autoprocessor basket in resin
Supplementary Data 2
Files needed for machining an autoprocessor basket in surgical metal
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Shafiei, N., Stӓhli, D., Burger, D. et al. Correlative light and electron microscopy for human brain and other biological models. Nat Protoc (2025). https://doi.org/10.1038/s41596-025-01153-9
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Received:16 May 2024
Accepted:17 January 2025
Published:31 March 2025
DOI:https://doi.org/10.1038/s41596-025-01153-9
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