Context

Since the seminal work from the group of Ed Boyden in 2015 [1], expansion microscopy (ExM) has literally turned the idea of resolution limit in microscopy upside down. While the field of super-resolution microscopy is trying to use optical methods to break the diffraction barrier of microscopy, Boyden asked: what if we expanded the sample instead? This is possible by first embedding the sample with polyelectrolytes that polymerise to form a hydrogel throughout the sample. This gel has an extremely fine mesh size of only a few nanometers, which allow the trapping of the molecules of interest in place. Importantly, this gel can also be expanded by osmotic pressure when the sample is subsequently flooded with water.

There are however two main difficulties to do this:

(1) making sure that the expansion is uniform in all direction and in all locations of the sample

and (2) retaining the capacity to specifically visualise the molecules of interest.

The first issue is commonly addressed by using detergent or enzymatic digestion to ensure that the mechanical properties of the sample is uniform throughout prior to the swelling step. So, the developments of ExM in the past few years have focussed on designing anchoring systems that can preserved the molecules of interest itself or a proxy for it (such as an antibody against a protein of interest) throughout the different stages of ExM [2,3]. These approaches have proven successful for a range of molecules of interest (e.g. proteins and RNA), cell types and tissue types.

The method promises the prospect of observing bio-molecular assemblies in situ in cells at the nanoscale, especially when combined with optical super-resolution microscopy methods [4]. However, to achieve this, it requires a robust visualisation of every single molecule of interest within the assembly, and this is currently limited by losses of labels and/or fluorescence during the polymerisation and digestion stages of ExM.

Key findings

Both Shi et al. (from the Huang lab at UCSL) and Wen et al. (from the Hofkens lab at KULeuven) concommitently address one of the main hurdle of ExM: the fact that many labels and much of the fluorescence can be lost in conventional ExM protocols. For this, the teams independently developed a set of trifunctional anchors that are capable of binding to the molecule of interest (1), to the hydrogel (2) and to the fluorescent reporter (3) simultaneously. Importantly, these anchors can also sustain the process of digestion necessary for ExM, therefore retaining a high number of labels and fluorescence molecules available for the post-expansion imaging.

In the method from Shi et al. (called LR-ExM, for label-retention ExM), two orthogonal labelling methods were shown using either biotin/streptavidin or digoxigenin (DIG)/anti-DIG reporter systems in order to add the fluorescent molecules after the expansion step. This avoids the issue of loss of fluorescence signal. The connector to the molecule of interest was demonstrated with either antibodies, SNAP or CLIP tags.

Here, the authors show that their method is compatible with super-resolution microscopy methods such as SIM and dSTORM, the latter showing resolution down to ~12 nm (~5 nm localisation precision). These approaches allow for the direct observation the hollow structure of microtubules, the relative nanoscale organisation of nuclear proteins (LaminA and nuclear pore complex) and, impressively, the lattices from clathrin-coated pits.

On the other hand, Wen et al. developed a large palette of chemistry for this trifunctional approach and showed that it works by conjugating phalloidin for actin labelling, antibody against alpha-tubulin for microtubule visualisation and DSPE for cellular phospholipid membranes labelling. Their approach here focusses on single-step fluorescent labelling prior to expansion where the choice of fluorescent dye is optimised to sustain the process.

Secondly they then extend the approach to DNA oligo labelling, either for fluorescent labelling of the anchor post-expansion or for in situ labelling of mRNA transcripts (similar to FISH), compatible with further signal amplification such as RollFISH and HCR. They therefore show an impressive versatility of chemistry and capabilities.

What I like about these works

These works elegantly and robustly provide a method that allows for direct in-situ observation of bio-cellular structures at the molecular level. This actually bridges with the observation obtained by (cryo-)electron microscopy and fills a gap in molecular observation. Also, this work shows that ExM is gaining maturity and promises to become widely available and robust. This will in turn enable life scientist and labs from all disciplines to study molecular life at unprecedented scale.

Questions to the authors?

Currently, the methods shows trifunctional anchors for 2 individual colours simultaneously. Are there ways to expand the palette to more orthogonal channels? Can both of these methods be combined to expand the capabilities for multi-colour imaging?

With this approach, the direct anchoring to the mesh and molecule of interest allows to get the fluorescent label very close to the molecule of interest? What approaches will give you the smallest effective linker error?

This method will have the highest impact to biomedical research if it is made available on a large scale such as via commercialisation. Is this an avenue that would be explored?

References

[1] F. Chen, P. W. Tillberg, and E. S. Boyden, “Expansion microscopy,” Science (80-. )., vol. 347, no. 6221, pp. 543–548, Jan. 2015.

[2] P. W. Tillberg, F. Chen, K. D. Piatkevich, Y. Zhao, C.-C. J. Yu, B. P. English, L. Gao, A. Martorell, H.-J. Suk, F. Yoshida, E. M. DeGennaro, D. H. Roossien, G. Gong, U. Seneviratne, S. R. Tannenbaum, R. Desimone, D. Cai, and E. S. Boyden, “Protein-retention expansion microscopy of cells and tissues labeled using standard fluorescent proteins and antibodies,” Nat. Biotechnol., vol. 34, no. 9, pp. 987–992, Jul. 2016.

[3] T. J. Chozinski, A. R. Halpern, H. Okawa, H.-J. Kim, G. J. Tremel, R. O. L. Wong, and J. C. Vaughan, “Expansion microscopy with conventional antibodies and fluorescent proteins.,” Nat. Methods, vol. 13, no. April, pp. 1–7, 2016.

[4] D. Gambarotto, F. U. Zwettler, M. Le Guennec, M. Schmidt-Cernohorska, D. Fortun, S. Borgers, J. Heine, J.-G. Schloetel, M. Reuss, M. Unser, E. S. Boyden, M. Sauer, V. Hamel, and P. Guichard, “Imaging cellular ultrastructures using expansion microscopy (U-ExM),” Nat. Methods, vol. 16, no. January, p. 1, 2018.

 

doi: https://doi.org/10.1242/prelights.12459

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