Diverse protocols for correlative super-resolution fluorescence imaging and electron microscopy of chemically fixed samples

Abstract

Our groups have recently developed related approaches for sample preparation for super-resolution imaging within endogenous cellular environments using correlative light and electron microscopy (CLEM). Four distinct techniques for preparing and acquiring super-resolution CLEM data sets for aldehyde-fixed specimens are provided, including Tokuyasu cryosectioning, whole-cell mount, cell unroofing and platinum replication, and resin embedding and sectioning. The choice of the best protocol for a given application depends on a number of criteria that are discussed in detail. Tokuyasu cryosectioning is relatively rapid but is limited to small, delicate specimens. Whole-cell mount has the simplest sample preparation but is restricted to surface structures. Cell unroofing and platinum replication creates high-contrast, 3D images of the cytoplasmic surface of the plasma membrane but is more challenging than whole-cell mount. Resin embedding permits serial sectioning of large samples but is limited to osmium-resistant probes, and is technically difficult. Expected results from these protocols include super-resolution localization (10–50 nm) of fluorescent targets within the context of electron microscopy ultrastructure, which can help address cell biological questions. These protocols can be completed in 2–7 d, are compatible with a number of super-resolution imaging protocols, and are broadly applicable across biology.

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Figure 1: Flowchart of the protocols featured in this paper.
Figure 2: Example of using gold nanoparticles to align fluorescence and electron microscopy data sets.
Figure 3: Tools facilitating location of the regions of interest to be imaged using different microscopy techniques.
Figure 4: Example 3D alignment of iPALM-FIB-SEM of thick cryosections.
Figure 5: Identifying imaged areas when using platinum replicas.
Figure 6: Examples of anticipated results.

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Acknowledgements

We thank R. Fetter and W.-P. Li of the HHMI-Janelia Research Campus for assistance with electron microscopy. We thank M. Daniels and the National Heart, Lung, and Blood Institute (NHLBI) EM core facility for help with platinum replica TEM. U.S. Army Medical Research Institute of Infectious Disease (USAMRIID) disclaimer: Opinions, interpretations, conclusions and recommendations are those of the authors and are not necessarily endorsed by the U.S. Army. M.G.P.-S., G.S., C.S.X., L.L.L. and H.F.H. were supported by the Howard Hughes Medical Institute. J.W.T. was supported by the Intramural Research Program of the NHLBI, US National Institutes of Health. This research was supported in part by funds from the Boettcher Foundation's Webb-Waring Biomedical Research Awards program and The National Institute of Allergy and Infectious Disease, NIH_1R21AI127192-01 to S.B.vE.

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Tokuyasu protocol: B.G.K., G.S., C.S.X. and H.F.H. Whole-mount protocol: S.B.v.E., G.S. and H.F.H. Unroofing protocol: K.A.S., G.S., S.B.v.E., H.F.H. and J.W.T. Resin-embedding protocol: M.G.P.-S., M.G.S., G.S., H.F.H., Y.W. and L.L.L. iPALM/FIB-SEM protocol: C.S.X., G.S. and H.F.H. All authors contributed to writing the paper. Correspondence should be addressed to B.G.K. (kopek@hope.edu) for the Tokuyasu cryosectioning protocol, S.B.v.E. (schuyler.vanengelenburg@du.edu) for the whole-mount protocol, J.W.T. (justin.taraska@nih.gov) for the platinum replica/TEM unroofing protocol, L.L.L. (loogerl@janelia.hhmi.org) for the fluorescent protein engineering/resin embedding protocol and H.F.H. (hessh@janelia.hhmi.org) for iPALM and FIB-SEM.

Corresponding authors

Correspondence to Benjamin G Kopek or Loren L Looger or Harald F Hess.

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Harald Hess is part owner of a patent and license on PALM.

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Kopek, B., Paez-Segala, M., Shtengel, G. et al. Diverse protocols for correlative super-resolution fluorescence imaging and electron microscopy of chemically fixed samples. Nat Protoc 12, 916–946 (2017). https://doi.org/10.1038/nprot.2017.017

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