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Correlative super-resolution fluorescence and metal-replica transmission electron microscopy

Abstract

We combine super-resolution localization fluorescence microscopy with transmission electron microscopy of metal replicas to locate proteins on the landscape of the cellular plasma membrane at the nanoscale. We validate robust correlation on the scale of 20 nm by imaging endogenous clathrin (in two and three dimensions) and apply the method to find the previously unknown three-dimensional position of the endocytic protein epsin on clathrin-coated structures at the plasma membrane.

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Figure 1: iPALM and TEM correlation imaging pipeline.
Figure 2: Correlation of clathrin fluorescence and TEM structures.
Figure 3: Location of epsin 1 in clathrin-coated structures.

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Acknowledgements

We thank M. Daniels and the US National Heart, Lung, and Blood Institute (NHLBI) electron microscopy core for help with EM; H. Shroff, J. Shaw and K. Neuman for critical reading of the manuscript; W. Li and Y. Wang (Janelia Farm) for EM grid preparation; L. Greene (NHLBI) for antibodies; S. Yu (NHLBI) for plasmid preparation; A. Nagy for acquiring atomic force microscopy (AFM) images; and E. Tyler and A. Hoofring of NIH Medical Arts for design work on Figure 1. J.W.T. is supported by the Intramural Research Program of the NHLBI, NIH. G.S. and H.F.H. are supported by the Howard Hughes Medical Institute.

Author information

Authors and Affiliations

Authors

Contributions

K.A.S., G.S. and J.W.T. designed the experiments. K.A.S. and G.S. performed the experiments. G.S. and K.A.S. processed data. K.A.S. analyzed the results. K.A.S. and J.W.T. wrote the manuscript. S.B.v.E. designed plasmids. J.W.T. and H.F.H. oversaw the project. All authors discussed the results and commented on the manuscript.

Corresponding authors

Correspondence to Harald F Hess or Justin W Taraska.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–8 (PDF 1489 kb)

Correlated clathrin-AF647 tomograms viewed in XY

Tomograms of six clathrin structures correlated with clathrin-AF647 (clathrin-AF647 is magenta, myristoylated psCFP2 is cyan). The tomograms are viewed in the XY plane and scan the Z dimension over time. Each voxel is 2.3 nm in all dimensions but a frame is shown every 6.9 nm in the Z-dimension. Each tomogram is 459 nm wide and tall. (MOV 14766 kb)

Correlated clathrin-AF647 tomograms viewed in YZ

The tomograms from Supplementary Video 1 are viewed in the YZ plane and scan the X dimension over time (clathrin-AF647 is magenta, myristoylated psCFP2 is cyan). Each voxel is 2.3 nm in all dimensions but a frame is shown every 6.9 nm in the X-dimension. Each tomogram is 459 nm wide in the Y dimension. (MOV 24262 kb)

Membrane iPALM/EM alignment validation

A tomogram of clathrin data is viewed in the YZ plane and scans the X dimension over time (clathrin-AF647 is magenta, myristoylated psCFP2 is cyan). An example of membrane misalignment between fluorescence and EM is shown in frames 19-106 on the right hand side. Regions with membrane misalignment were not used for analysis. Each voxel is 2.3 nm in all dimensions but a frame is shown every 6.9 nm in the X-dimension. The tomogram is 215.7 nm high in the Z dimension. (MOV 25780 kb)

Correlated epsin-AF647 tomograms viewed in XY

Tomograms of six clathrin structures correlated with epsin-AF647 (epsin-AF647 is magenta, myristoylated psCFP2 is cyan). The tomograms are viewed in the XY plane and scan the Z dimension over time. Each voxel is 2.3 nm in all dimensions but a frame is shown every 6.9 nm in the Z-dimension. Each tomogram is 459 nm wide and tall. (MOV 13360 kb)

Correlated epsin-AF647 tomograms viewed in YZ

The tomograms from Video 4 are viewed in the YZ plane and scan the X dimension over time (epsin-AF647 is magenta, myristoylated psCFP2 is cyan). Each voxel is 2.3 nm in all dimensions but a frame is shown every 6.9 nm in the X-dimension. Each tomogram is 459 nm wide in the Y dimension. (MOV 20493 kb)

Isosurface models of epsin-AF647 tomograms

TEM tomograms (grey) and their corresponding epsin-AF647 fluorescence (magenta) are represented as isosurface models (Fig. 3f–h). Two models are shown, including three CCSs. Each model is 459 nm in the X and Y dimension and is rotated around the X-axis from 0-90°. (MOV 17873 kb)

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Sochacki, K., Shtengel, G., van Engelenburg, S. et al. Correlative super-resolution fluorescence and metal-replica transmission electron microscopy. Nat Methods 11, 305–308 (2014). https://doi.org/10.1038/nmeth.2816

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