Periodic Reporting for period 1 - ULTRARESOLUTION (Beyond super-resolution: ultra-resolution imaging provides solutions for synapse physiology and brain pathology)
Berichtszeitraum: 2021-06-01 bis 2022-11-30
Neurons contain hundreds of specialized proteins, whose topology reflects activity, plasticity and disease. Present imaging techniques are unable to present this topology accurately, since even the best super-resolution tools are limited to at least 20-30 nm, many times the size of individual proteins (~3-7 nm). To solve this problem, our ground-breaking objective is to develop reliable ultra-resolution imaging, with true molecular resolution of 1-5 nm, or even below these values. We will combine optics-based super-resolution with a recent innovation, pioneered by our team – physical expansion of the samples. Our efforts will be aided by several imaging tools we have generated, from super-resolution modalities to nano-affinity probes, which, thanks to their power and ease-of-use, are already employed by hundreds of research groups. We will apply ultra-resolution to reveal the functional organization of key components of the synapse, in health and disease. We will also develop protocols for brain pathology samples, for future use in medical diagnostics.
During the first reporting period we followed this goal very closely. We generated new gel chemistries and we optimized multiple procedures linked to the gel imaging procedures, from the fluorophores used to the optics involved. Among other results, we are close to a first solution to the resolution problem, albeit not yet in all 3 dimensions, but only in the 2D plane. This takes the form of a combination of expansion microscopy with fluorescence fluctuation analyses, which appears to achieve resolutions down to 1 nm or better. In our collaborative work, which has been introduced as a pre-print already, we have successfully applied this approach to image cultured cells, tissues, viral particles, molecular complexes and single proteins. At the cellular level, using immunostaining, our technology revealed detailed nanoscale arrangements of synaptic proteins. At the single molecule level, we could visualise the shape of individual membrane and soluble proteins. We could also study Parkinson’s Disease samples, as indicated in our initial application, obtaining interesting first results. Overall, our work follows exactly the initial plans, and is making excellent progress along the lines we indicated in our proposal.
We have already showed that a fluorescence microscopy procedure based on a combination of expansion microscopy and advanced optics can provide a spatial resolution of ≤ 1 nm. In practical terms, most of the data we have obtained up to now were acquired with a standard confocal microscope. Improvements in this direction offer the possibility of far superior resolution, far beyond the state of the art.
The only major limitation we see is that expansion-based approaches cannot be applied to live samples. Nevertheless, future developments in this direction are likely to enable 3D structural analysis of proteins, either purified or in cells and tissue samples, at resolutions approaching electron cryo-microscopy and tomography techniques, at room temperature and at a fraction of the croy-EM cost. Developments envisaged include a refined anchoring chemistry of proteins into the gel structure, development of gels that are homogeneous to sub-nanometer levels, as well as imaging automation, to enable the analysis of tens of thousands of particles in a time-efficient manner.
Overall, our proposed technology provides a simple, robust and easily applied technique for the investigation of the domain beyond super-resolution, which could be termed ultra-resolution, bridging the gap to X-ray crystallography and electron microscopy-based technologies.
The only major limitation we see is that expansion-based approaches cannot be applied to live samples. Nevertheless, future developments in this direction are likely to enable 3D structural analysis of proteins, either purified or in cells and tissue samples, at resolutions approaching electron cryo-microscopy and tomography techniques, at room temperature and at a fraction of the croy-EM cost. Developments envisaged include a refined anchoring chemistry of proteins into the gel structure, development of gels that are homogeneous to sub-nanometer levels, as well as imaging automation, to enable the analysis of tens of thousands of particles in a time-efficient manner.
Overall, our proposed technology provides a simple, robust and easily applied technique for the investigation of the domain beyond super-resolution, which could be termed ultra-resolution, bridging the gap to X-ray crystallography and electron microscopy-based technologies.