Light microscopy of proteins in their ultrastructural context

Ons M’Saad, Joerg Bewersdorf

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 Niz

Categories: 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.

Figure 1. Pan-ExM methodology and achieved imaging on HeLa cell a) prior to expansion; b) with one round of expansion; c) pan-ExM-expanded HeLa cell revealing Golgi cisternae.

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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Author's response

Joerg Bewersdorf and Ons M’Saad shared

Open questions

1.Are there dyes or secondary antibodies that better withstand the process involved in pan-ExM?
In most cases – Mitotracker is the exception – we label post expansion. The expansion process therefore does not affect the dyes used. Any dye that works well with the microscope intended to be used for imaging should be fine. The same applies to secondary antibodies. The bigger problem is the choice of primary antibodies since the proteins they have been raised against have most likely been denatured by heat and SDS in the expansion protocol. Antibodies that have been validated for Western blots are good candidates for pan-ExM.

2.Given the possibility of imaging sub-proteomes, how many simultaneous markers can be used in the protocol, without loss of specificity or resolution? For instance, in your work, you explored one of the Golgi markers – is it envisaged that the entire Golgi network could be simultaneously explored?
We usually consider immunostainings which target specific proteins as different applications than bulk labeling of sub-groups of the proteome – even though the two concepts can of course be combined. What is currently limiting us is the difficulty to distinguish different colors in a microscope. A practical limit here is typically about 5 colors per sample. To go substantially beyond 5 colors, pan-ExM can in the future be combined with multiplexing approaches such as barcoding or multi-round immunostaining. These approaches have demonstrated more than 1000 and roughly 50 “colors”, respectively.
With regards to sub-proteome labelling, we have so far explored palmitoylated, glycosylated and cysteine proteomes in whole cells and we noticed distinct labeling patterns for example at the Golgi apparatus. These patterns were not discernible when the labelling was performed on non-expanded or only 4x expanded cells. We are currently exploring these patterns with the aid of computer vision tools.

3.How well preserved are inter-cellular structures? For instance, can the sub-proteome involved in endothelial cell junctions be maintained, and visualized?
What is preserved with conventional formaldehyde and glutaraldehyde fixation should in theory be retained in the hydrogel. We have not looked at cell-junction markers, but we validated immunofluorescence staining for several membrane proteins on membrane-bound organelles. We are therefore confident that plasma membrane proteins should be well preserved.

4.Besides lipids, are there other components of the cell, whose integrity is not maintained if the pan-ExM protocol is applied?
Cellular components that are not crosslinked with formaldehyde are not retained by design. This includes most lipids, metabolites (ATP, Ca++, NADH etc.), and mRNA transcripts (which might be entangled though). However, there have been several papers in the field describing engineered probes designed to crosslink lipids and RNA nucleobases to acrylamide gels. We plan to explore this approach in the future.

5.Did you face any significant limitations (or envisage any for future work) regarding automatic image segmentation and machine learning approaches? To what extent is it feasible to analyse the cellular sub-proteome automatically and accurately.
Our preliminary results with machine learning approaches are very encouraging so far. Our exploration into machine learning is still ongoing, however, and we do not have a full overview yet of what is feasible and what not. This line of research is certainly of high interest to us because of its high potential.

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Light microscopy of proteins in their ultrastructural context

Background

Key findings and developments

What I like about this preprint

Acknowledgements

References

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