Light microscopy of proteins in their ultrastructural context
Posted on: 8 April 2020
Preprint posted on 14 March 2020
Article now published in Nature Communications at http://dx.doi.org/10.1038/s41467-020-17523-8
Revolutionary imaging - Pan-ExM: imaging the proteome at the nanoscale within the structural context of the cell.
Selected by Mariana De NizCategories: biochemistry, cell biology
Background
With the advent of super-resolution microscopy, the 3D distribution of proteins of interest can be imaged at spatial resolutions down to 10nm, achieving sub-cellular organization at the nanoscale. However, showing proteins in the ultrastructural context of the cell has relied on correlative microscopy (eg. CLEM), which combines electron and fluorescence microscopy. Despite its many advantages, CLEM relies on the use of highly specialized instruments and training, as well as long acquisition and processing times. Relatively recently, the method of expansion microscopy was developed, which is based on embedding and hybridizing a sample to a swellable polyacrylamide or sodium polyacrylate co-polymer (1-2). Following water absorption, the gel physically expands by a factor of 4 in 3D. Iterative microscopy allows further expansion by 20-fold (3). However, the proteases that allow homogeneous expansion, also result in degradation of the cellular content. To overcome the challenges associated with CLEM and with conventional expansion microscopy in their work, M’Saad and Bewersdorf established a method based on expansion microscopy, to resolve the distribution of specific proteins at the nanoscale in the structural context of the cell (4).
Key findings and developments
- The authors developed a method they call pan-ExM, in reference to the idea of labelling the whole proteome. Pan-ExM works by de-crowding the intracellular space through up to 21-fold physical expansion while simultaneously retaining the cellular proteome in the expanded hydrogel.
- The method is based on embedding a dense expanded sample prepared with a cleavable crosslinker in a second dense superabsorbent hydrogel. Entanglements between polymer chains of the first and final hydrogels will physically interlock protein-polymer hybrids in this latter polymer network, thereby preserving the proteome while iteratively expanding it.
- Pan-ExM combined with global fluorescent labelling, which targets all separated proteins, reveals the overall landscape of the cell with a light microscope- resembling the contrast of heavy-metal EM stains.
- Expanding cells twice by an overall factor of 13-21 allows to spatially separate organelles which originally were less than 20nm apart by a standard diffraction-limited (250nm resolution) confocal microscope.
- The protocol is compatible with staining methods such as MitoTracker, and DNA-intercalating dyes. The authors also show that pan-ExM is compatible with immunofluorescence, with no decrease in fluorescent signal upon expansion; while allowing good structural preservation.
- Using pan-ExM, the authors successfully visualized various other organelles, including the nucleus, ER and Golgi. However, they reported that some antibodies did not work, and propose this can be overcome in the future by using antibodies with good outcomes in Western blot, as well as by optimising the pH and temperature for several steps in the preparations.
- The authors suggest that methods to possibly image lipids in the future, would be to use cross-linkable lipid labels.
- They also suggest the use of reversible protein crosslinking reagents as an alternative to conventional fixatives worth investigating.
- The authors further discuss pan-ExM as a technique well-suited for automated segmentation and classification algorithms to identify organelles of interest, and emphasize the potential of using machine learning to identify other organelles. Altogether, this possibility can help answer how different sub-proteomes are distributed in the cell.
- In contrast to optical super-resolution microscopy techniques which are limited in resolution by the size of the fluorescent labels, ExM is not constrained by label-size as long as it is done after expansion and proteins are preserved in the hydrogel.
- Altogether the authors propose pan-ExM as a revolutionary method for light microscopy, capable of revealing nanoscopic structural hallmarks that allow users to identify organelles without the need for specific staining.
What I like about this preprint
I like that the authors explored the use of expansion microscopy- an already revolutionary tool, and developed it in a way that it bridges a significant gap in cell biology. With many sub-fields of cell biology moving to ask questions at the whole-cell level with very high resolution, this development is timely and exciting. I think the preprint is very detailed, and the authors discuss the shortcomings while suggesting methods to tackle such shortcomings.
Acknowledgements
Thank you to Joerg Bewersdorf and Ons M’Saad for their input and engagement.
References
- Chen, F. et al., Expansion Microscopy, Science 347, 543–548 (2015).
- Chozinski T. J., et al., Expansion microscopy with conventional antibodies and fluorescent proteins, Nature Methods 13, 485-488 (2016).
- Chang, J.B. et al., Iterative expansion microscopy, Nature Methods 14, 593-599 (2017).
- M’Saad, O., and Bewersdorf, J., Light microscopy of proteins in their ultrastructural context, bioRxiv, (2020).
doi: https://doi.org/10.1242/prelights.18309
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