Abstract
Biologists have long been fascinated with the organization and function of intricate protein complexes. Therefore, techniques for precisely imaging protein complexes and the location of proteins within these complexes are critically important and often require multidisciplinary collaboration. A challenge in these explorations is the limited resolution of conventional light microscopy. However, a new microscopic technique has circumvented this resolution limit by making the biological sample larger, thus allowing for super-resolution of the enlarged structure. This ‘expansion’ is accomplished by embedding the sample in a hydrogel that, when exposed to water, uniformly expands. Here, we present a protocol that transforms thick expansion microscopy (ExM) hydrogels into sections that are physically expanded four times, creating samples that are compatible with the super-resolution technique structured illumination microscopy (SIM). This super-resolution ExM method (ExM–SIM) allows the analysis of the three-dimensional (3D) organization of multiprotein complexes at ~30-nm lateral (xy) resolution. This protocol details the steps necessary for analysis of protein localization using ExM–SIM, including antibody labeling, hydrogel preparation, protease digestion, post-digestion antibody labeling, hydrogel embedding with tissue-freezing medium (TFM), cryosectioning, expansion, image alignment, and particle averaging. We have used this approach for 3D map** of in situ protein localization in the Drosophila synaptonemal complex (SC), but it can be readily adapted to study thick tissues such as brain and organs in various model systems. This procedure can be completed in 5 d.
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Acknowledgements
We thank the Hawley Lab for invaluable feedback on the manuscript, particularly A. Miller for figure preparation and proofreading, C. Lake for valuable discussion, and S. Hughes. We thank J. Lange for proofreading, and P. Ji and the histology core (Stowers Institute) for valuable discussion on chemistry and histotechniques. This work was supported by the Stowers Institute for Medical Research. R.S.H. is an American Cancer Society Research Professor.
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Y.W., Z.Y., and C.K.C. developed the ExM–SIM protocol; C.K.C. and R.S.H. designed the research project; N.T. and T.P. provided technical expertise and advice; J.R.U. and B.D.S. analyzed data and wrote analysis software; Y.W., Z.Y., C.K.C., J.R.U., B.D.S., and R.S.H. wrote the paper.
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1. Cahoon, C. K. et al. Proc. Natl. Acad. Sci. USA 114, E6857–E68E66 (2017) https://doi.org/10.1073/pnas.1705623114
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Supplementary Figure 1 Quantification of expansion distortion.
The plot shows the average RMS distance error for all pairs of points as a function of distance in an overlaid SIM and expanded confocal image of C(3)G in Box 3 panels a–c (a) and of Corolla in Box 3 panels d–f (b). Both distances are in expanded units. The distance errors in each subregion were determined by cross-correlation analysis as opposed to feature comparison as in Chen et al.3. Note that given the sample manipulations in our protocol (for example: slicing, relabeling), this error represents both expansion distortion and changes in local labeling density.
Supplementary Figure 2 Screenshots of the single-particle averaging analysis.
Screenshots illustrating the details of steps (a) 65—color alignment, (b) 68—making an interpolated three-dimensional line profile, (c) 69—computational straightening of the SC along the Y axis, and (d) 75–76—Gaussian fitting.
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Wang, Y., Yu, Z., Cahoon, C.K. et al. Combined expansion microscopy with structured illumination microscopy for analyzing protein complexes. Nat Protoc 13, 1869–1895 (2018). https://doi.org/10.1038/s41596-018-0023-8
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DOI: https://doi.org/10.1038/s41596-018-0023-8
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