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Expansion microscopy at one nanometer resolution

Ali H. Shaib, Abed Alrahman Chouaib, Vanessa Imani, Rajdeep Chowdhury, Svilen Veselinov Georgiev, Nikolaos Mougios, Mehar Monga, Sofiia Reshetniak, Daniel Mihaylov, Han Chen, Parisa Fatehbasharzad, Dagmar Crzan, Kim-Ann Saal, Claudia Trenkwalder, Brit Mollenhauer, Tiago F. Outeiro, Julia Preobraschenski, Ute Becherer, Tobias Moser, Edward S. Boyden, A Radu Aricescu, Markus Sauer, Felipe Opazo, Silvio O. Rizzoli

Posted on: 24 November 2022

Preprint posted on 5 August 2022

The shape of single proteins seen with light microscopy – ONE microscopy combines the power of expansion microscopy with SRRF analysis.

Selected by Nadja Hümpfer

Categories: biochemistry, cell biology, neuroscience

Background

Expansion microscopy (ExM) is an upcoming technique that circumvents the diffraction limit of light microscopy by physically expanding the sample [1]. In ExM, the specimen is embedded in a swellable hydrogel that expands when soaked in water. This leads to physical separation of the structures of interest and the introduced fluorophores. The factor of expansion determines the increase in resolution. Researchers have tried to combine ExM with other super-resolution imaging methods to push the resolution even further.

Shaib and colleagues are the first to introduce SRRF to 10-fold ExM. SRRF stands for super-resolution radial fluctuations and this approach benefits from a separation of fluorophores [2], as is the case in 10-fold ExM. Similarly, the background fluorescence is diluted to an almost neglectable level. Measuring the intensity fluctuations of well-separated fluorophores in almost zero-background conditions allows for super-resolution imaging based on an SRRF-algorithm. Combining the 10-fold increase in resolution due to expansion with the increased resolution due to SRRF results in a final resolution of 1 nm. At this resolution, the shape of individual proteins becomes apparent when labelled with a pan-protein staining. The authors show that their method is applicable in a broad diagnostic context.

Key findings

The shape of single proteins or protein complexes is resolved

The most exciting finding of this preprint is the visualization of purified proteins. To this end, proteins, such as antibodies, were embedded in the gel and then homogenized to allow the 10-fold expansion. In the process of gel-homogenization, the samples were digested by protease cleavage. The digestion introduced reactive amine groups that could be labelled by reacting with NHS-ester coupled dyes. An entire IgG antibody could therefore be labelled with NHS-ester coupled fluorophores. Due to the high resolution below 1 nm, the shape of the antibody became apparent. It was possible to show the architecture of antibodies of different classes. Remarkably, the authors were able to show the shape of different proteins, such as GABA-receptors and calmodulin. Moreover, the structure of the protein otoferlin, which has not been determined until now, could be resolved. This structure fitted the prediction of AlphaFold, showing the potential of ONE microscopy in the uncovering of thus far unknown protein structures.

 

Figure 1: Schematic of the steps taken in ONE microscopy: The antibody is anchored into a X10 gel and digested. After labelling with fluorophores, the signal is detected and analysed with SRRF: The shape of the antibody becomes visible with fluorescence microscopy. (C) Rizzoli Lab

A GFP-based ruler shows the resolution is below 1 nm

To prove the resolution to be below one nanometer, the authors generated a ‘triangulate smart ruler’ (TSR). This protein complex consists of a GFP-molecule with an ALFA-tag. Fluorescently labelled nanobodies against GFP as well as the tag were incubated with the protein. After the ONE procedure, it was possible to visualize the individual fluorophores on the nanobodies. Strikingly, the architecture of a GFP molecule bound by three different nanobodies was reflected in the distribution of the fluorescence signal. Distances as low as 2 nm could be visualized, as proven by the appearance of two separate fluorophores per nanobody, 2 nm apart. Full width at half-maximum measurements showed the resolution to be below 1 nm for different fluorophores.

Applications of ONE microscopy for diagnostics

As Parkinson’s disease is characterized by the appearance of clusters or aggregates of the protein alpha-synuclein (ASYN) and ONE microscopy could impressively show the shape of individual proteins, the authors used their method to directly visualize the ASYN-clusters in samples from patients. They sampled the cerebral spinal fluid (CSF) of persons diagnosed with Parkinson’s disease and compared it to a control group. The detection of the aggregates in the CSF was dependent on a nanobody against alpha-synuclein. The increased resolution thanks to ONE microscopy enabled the detection of clusters and aggregates of different shapes and sizes. The authors found significant differences in the appearance and number of ASYN-complexes in the group diagnosed with Parkinson’s disease compared to the control group. This opens up the route of using ONE microscopy as a diagnostic tool for neurodegenerative diseases.

What I like about this work

The work impressively shows the potential of expansion microscopy when combined with other imaging (analysis) techniques. After the introduction of ExM, it was readily combined with light sheet imaging [3], single molecule localization microscopy (SMLM)[4] or stimulated emission depletion (STED) microscopy [5]. However, due to some prerequisites of the methods, it had not been possible to reach the resolution level reported in this preprint. SMLM, for example, requires blinking of fluorophores, which depends on buffer conditions. Buffers that facilitate blinking, however, contain salts that cause the expanded gel to shrink. STED on the other hand requires very strong fluorescent signal – which is difficult to achieve when samples expand and therefore signal is diluted. I found it very innovative that the authors of this preprint made use of this ‘disadvantage’ of diluted fluorophores and combined it with an imaging method that does not rely on high photon-count but instead makes use of signal-fluctuations such as SRRF.

When introducing a groundbreaking technique like this, the evaluation and control is of utmost importance. I really enjoyed the introduction of the TSR as ruler for the achievable resolution. Seeing individual fluorophores on a protein complex only a few nanometers in size was astonishing to me.

In general, the application of ONE to single proteins or protein-complexes with a pan-staining is very impressive. The results are reminiscent of single particle cryo-EM, but based on fluorescence microscopy. It was mind-blowing to see the molecular shape of a protein visualized by a light microscope. I also expect the ONE approach to be more accessible than single-particle cryo-EM, due to the availability of standard fluorescence microscopes and a public analysis pipeline.

Future directions

The introduction of ONE microscopy will be of major importance to the light microscopy field. It is a cheap alternative to other super-resolution techniques. It also provides resolution that could not be achieved previously. Therefore, it bridges the gap between light microscopy and electron microscopy. The plethora of applications, only a few covered in this preLight, speaks for a potential in different areas of both research as well as diagnostics. I envision many laboratories and researchers around the world using this technology to visualize the shape of their proteins of interest without going through the time- and resource-consuming process of crystallography or cryo-EM.

 

Questions for the authors

Q1: You show that a general amine-staining with fluorescein can reveal the shape of proteins. Can you explain why in your initial experiments with the TSR, the shape of the GFP barrel was not visible (Figure 1D)? It rather looks like a single fluorophore signal, which is bound to GFP.

Q2: In Figure 3, you show how the extraction of cholesterol induces spreading of synaptic molecules in the presynapse. However, you mention that synaptic vesicles stay intact and the number of synaptotagmin molecules per individual vesicle remains the same. I was wondering how it is possible to still count synaptotagmin molecules on those dispersed vesicles?

Q3: What does one need to consider when choosing the fluorophores for staining? I was particularly interested in the fluorescein molecule; as this seems to be rather unusual for fluorescent light microscopy.

Q4: How do you practically find the individual proteins in the gel? I can imagine a 10-fold expanded gel is quite difficult to handle and to look for a region of interest must take a very long time.

 

References

  1. Chen, F., P.W. Tillberg, and E.S. Boyden, Optical imaging. Expansion microscopy. Science, 2015. 347(6221): p. 543-8.
  2. Culley, S., et al., SRRF: Universal live-cell super-resolution microscopy. Int J Biochem Cell Biol, 2018. 101: p. 74-79.
  3. Burgers, J., et al., Light-sheet fluorescence expansion microscopy: fast mapping of neural circuits at super resolution. Neurophotonics, 2019. 6(1): p. 015005.
  4. Zwettler, F.U., et al., Molecular resolution imaging by post-labeling expansion single-molecule localization microscopy (Ex-SMLM). Nat Commun, 2020. 11(1): p. 3388. link to preLight
  5. Gao, M., et al., Expansion Stimulated Emission Depletion Microscopy (ExSTED). ACS Nano, 2018. 12(5): p. 4178-4185.

Tags: diffraction barrier, expansion microscopy, nanoscopy, super-resolution microscopy

doi: https://doi.org/10.1242/prelights.33191

Read preprint (1 votes)

Author's response

Ali Shaib and Silvio Rizzoli shared

Q1:

We are not sure as to why this is the case, but one can speculate, for instance, GFP molecule is a rather small and compact beta-barrel. This allows robust anchoring into the gel. The signal is then rather compact, limited to a small area, as expected for the well-folded GFP protein. GFPs may also be somewhat resistant to the homogenization steps, as they are known to survive many types of expansion microscopy and to still provide a fluorescent signal after expansion.

Q2:

In this experimental paradigm, membrane-bound synaptotagmin is blocked with anti-synaptotagmin without a fluorophore, prior to incubating with the anti-synaptotagmin antibody coupled with fluorophores. The procedure we use will result in the exocytosis of one vesicle in a subset of synapses, with most probably not releasing any vesicles. The synaptotagmin signal will have a hard time diffusing out of the synapse, as we showed in other papers before, and as also observed by other groups, such as that of Volker Haucke in Berlin. Therefore, it is very likely that the membrane domains containing vGluT1 and synaptotagmin, after fusion, are indeed single vesicles – as their intensity in the synaptotagmin channel confirmed after our analysis.

Q3:

Pre-expansion labeling limits the choices of fluorophores to those that actually survive the heat and the alkaline homogenization process, as well as the anchoring and gelation reactions. However, post-expansion labeling allows a wider selection of fluorophores. We chose NHS-Ester fluorescein to directly label single molecules because fluorescein is inexpensive. Luckily, it is photostable enough to enable the fluctuation analysis, although it does bleach faster than many modern dyes, as observed by many in the past.

Q4:

Single molecule experiments were literately the easiest of all experiments, in terms of finding the samples and imaging them. One needs to control the protein concentration to have just enough proteins to see them in the gel (ideally 15-20 protein molecule per field of view), but not too many, as otherwise purified proteins may end up aggregating. Normally, 1 to 2 μl of 1 mg/ml protein is needed per gel. To find the focal plane, we used a line accumulation to enhance the signal. Usually, the protein molecules are within the first 3 microns from the glass-gel index mismatch.

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