All-optical visualization of specific molecules in the ultrastructural context of brain tissue

Background

Expansion microscopy (ExM) was first introduced by the Boyden Lab in 2015 (Chen et al., 2015). It combines immunofluorescence microscopy with a charged polymer hydrogel that expands when soaked in water. This allows physical magnification of the embedded sample and therefore otherwise diffraction limited structural details can be resolved. This is an alternative take on super-resolution microscopy. Other techniques have tried to outplay the physical diffraction limit of light (Abbe, 1873) by optical modifications and algorithms (Betzig, 1995; Betzig et al., 2006; Heilemann et al., 2008; Hell and Wichmann, 1994; Hess et al., 2006; Rust et al., 2006). In ExM, the microscopy setup remains unchanged, making the method accessible for labs that already use confocal microscopes. Since its first publication, many modifications and iterations have been made to the method (see for example Gambarotto et al., 2019; Sarkar et al., 2020; Tillberg et al., 2016; Truckenbrodt et al., 2018).

In this preprint, Saad et al. show how they adopt ExM to image brain tissue with a pan-protein staining. This leads to contrast similar to electron microscopy (EM) and provides ultrastructural context to specific antibody labelling.

 

Key findings and comments

The authors adopt their previously published pan-ExM method (M’Saad and Bewersdorf, 2020) to mouse brain sections and term the technique pan-ExM-t. The staining is based on an NHS-ester-dye conjugate that reacts with primary amines of proteins. In unexpanded samples, NHS-ester staining leads to a broad overall staining that does not add information. Only when the sample is expanded intensively (16-fold), the uniform protein staining reveals nanoscopic structural details based on protein density.

A comprehensive journey of optimization steps leads to pan-ExM-t protocol

The paper includes a detailed description of the optimization process for pan-ExM-t. I found it very informative and refreshing to follow all the different conditions that the authors tested in order to have an optimal outcome. Their publication shows again the importance of the correct fixation, dependent on the target composition. An example is the addition of glutaraldehyde (GA) in the fixative, which preserves fine structures in EM samples. The authors also show that GA improves the staining of lipids with BODIPY TR Methyl Ester. For pan-ExM-t however, GA reduces the overall expansion factor and masks the antigens for immunostaining. GA was therefore omitted from the fixative.

For those who want to learn more about the principles and chemistry of expansion microscopy, the publication provides information on the effects of different components. In their optimization, the authors describe how the fixation, monomer concentration and denaturation conditions can affect the quality of the staining and preservation of the expanded tissue. To judge the effect of their optimization, the authors needed a reference structure.

I found the choice of the extracellular space and lipid membrane (ECS+) fraction of neuropil very clever. By comparing the fraction of the ECS+ across the different conditions, the authors can tell if the tissue was retained. This indicates a more or less successful protein fixation for pan-ExM-t. In general, this part shows that for a new set of ExM experiments, it is crucial to optimize the conditions specifically for the target. Published EM or super-resolution microscopy data can help in assessing the degree of preservation of the structure of interest.

Post-fixation visualization of lipids made possible by photo-crosslinkable lipid-probe

Another interesting part of the study is the employment of a new photo-crosslinkable molecule to stain lipids. Labelling lipids in ExM, similar to immunofluorescence, has been challenging. The main problem is to retain the lipids in the polymer, as in contrast to proteins they cannot be fixed and crosslinked into the gel by formaldehyde. Even though there are strategies to label lipids, for example by feeding cells with clickable lipid derivatives (see for example Gotz et al., 2020; Sun et al., 2021; Wen et al., 2020), the probe introduced here allows for a more straightforward staining. The sphingosine pacSph is incubated with the fixed tissue and then photo-crosslinked to the polymer by UV-irradiation. After the gel processing, the probe is linked to a fluorescent dye via Click-chemistry.

Pan-ExM-t combines specific immunolabelling with ultrastructural context

The intensive optimization procedure and the introduction of the new lipid probe allow the authors to visualize in great detail different types of synapses and to distinguish the post synaptic density and the dense projections of synapses. The gallery of synapses is very impressive. Even more exciting is the combination of the NHS-ester pan staining with selective antibody labelling. Here, the authors show the location of synaptic marker proteins such as Bassoon, Homer and PSD-95 within the synaptic ultrastructure. As a proof of the high resolution, the authors determine the distance of these markers across the synaptic cleft.

Lastly, the authors show the use of their technique in the tracing of a Thy1-GFP positive neuron in a transgenic mouse brain. The possibility to follow an individual neuron within its tissue context makes pan-ExM-t an exciting tool for brain connectome studies.

Pan-ExM-t is an approachable alternative to CLEM

Overall, the new pan-ExM-t is a great alternative to correlative light and electron microscopy (CLEM). It can achieve a very high spatial resolution and provides the ultrastructural context to a selective visualization of target structures such as proteins or DNA. The chemicals used in pan-ExM-t are cheap and widely available. I imagine the handling of the gelled tissue to be a lot easier than the sample preparation for CLEM. Therefore, the technology should gain importance in labs that want to investigate a specific target in its ultrastructural context without establishing CLEM. A drawback to the pan-ExM-t is the staining, which requires high amounts of antibodies. However, the authors comment in this study that a 50% reduction of antibody concentration actually decreased background without influencing the specific staining.

 

Future directions and questions for the authors

What is the actually achievable resolution of pan-ExM-t?

The authors compare their NHS-ester pan-protein staining to EM. In terms of contrast, I can see how EM and pan-ExM-t data look very similar. However, I wondered about the actually achievable resolution. The authors mentioned they calculated the expansion factor to be 24-fold. This would lead to an effective resolution of ~ 300 nm/24 = 12.5 nm for a red fluorescent dye on a standard fluorescence microscope. This does not reach the resolution of an electron microscope. It would be nice to see a more systematic approach to determine the resolution of pan-ExM-t.

How comes the NHS-ester does not compete with formaldehyde for primary amines?

Since the first introduction of pan-protein staining by NHS-ester, I wondered how the NHS-staining does not compete with formaldehyde used in the fixative. Formaldehyde reacts with primary amines and acrylamide to form acrylate-anchors on the proteins (Ku et al., 2016). NHS-ester also reacts with primary amines. Are there enough primary amines left after fixation? Or is a sufficient amount of primary amines made accessible during the denaturation of the proteins, which is then targeted by the NHS-ester?

Is it possible to visualize synaptic vesicles with this method?

Electron microscopy is commonly used to image synaptic vesicles. Does pan-ExM-t also enable the visualization of these structures? Maybe the protein content is too low to be detected by the NHS-ester staining, but what about the lipid-membrane staining?

 

References

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Tags: expansion microscopy, nanoscopy, tissue context

doi: https://doi.org/10.1242/prelights.31932

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