Periodic Reporting for period 3 - ULTRARESOLUTION (Beyond super-resolution: ultra-resolution imaging provides solutions for synapse physiology and brain pathology)
Reporting period: 2024-06-01 to 2025-11-30
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.
Optical microscopy has been one of the most valuable tools in biology for more than two centuries, and has been considerably enhanced by the introduction of super-resolution microscopy, two decades ago. Nevertheless, optical imaging remains difficult to perform below 10-20 nm, and is limited by two fundamental problems. First, the achievable structural resolution is determined by the labeling density, which is limited by the size of the fluorescent probes (typically 1 nanometer or larger). Second, fluorophores can interact via energy transfer at distances below 10 nm, which results in accelerated photoswitching (blinking) and photobleaching, and thus in substantially lower localization probabilities.
The solution to these two problems would be to separate the fluorophores spatially by the physical expansion of the specimen, in what is termed expansion microscopy (ExM). To then reach the molecular scale, one would combine ExM with optics-based super-resolution. This has been attempted numerous times, but the resulting performance typically reached only ~10 nm. The ExM gels are dim, because the fluorophores are diluted by the third power of the expansion factor, thus limiting optics techniques that prefer bright samples, as stimulated emission depletion (STED), or saturated structured illumination (SIM). In addition, the ExM gels need to be imaged in distilled water, which reduces the performance of techniques that rely on special buffers, as single molecule localization microscopy, SMLM.
Optical microscopy has been one of the most valuable tools in biology for more than two centuries, and has been considerably enhanced by the introduction of super-resolution microscopy, two decades ago. Nevertheless, optical imaging remains difficult to perform below 10-20 nm, and is limited by two fundamental problems. First, the achievable structural resolution is determined by the labeling density, which is limited by the size of the fluorescent probes (typically 1 nanometer or larger). Second, fluorophores can interact via energy transfer at distances below 10 nm, which results in accelerated photoswitching (blinking) and photobleaching, and thus in substantially lower localization probabilities.
The solution to these two problems would be to separate the fluorophores spatially by the physical expansion of the specimen, in what is termed expansion microscopy (ExM). To then reach the molecular scale, one would combine ExM with optics-based super-resolution. This has been attempted numerous times, but the resulting performance typically reached only ~10 nm. The ExM gels are dim, because the fluorophores are diluted by the third power of the expansion factor, thus limiting optics techniques that prefer bright samples, as stimulated emission depletion (STED), or saturated structured illumination (SIM). In addition, the ExM gels need to be imaged in distilled water, which reduces the performance of techniques that rely on special buffers, as single molecule localization microscopy, SMLM.
A third class of optical super-resolution approaches is based on determining the higher-order statistical analysis of temporal fluctuations measured in a movie, e.g. super-resolution optical fluctuation imaging (SOFI) or super-resolution radial fluctuations (SRRF). The resolution of these approaches is inversely correlated to the distance between the fluorophores, and they do not require especially bright samples or special buffers, implying that they should benefit from ExM. To test this hypothesis, we combined X10 expansion microscopy with SRRF and established a technique we term one-step nanoscale expansion (ONE) microscopy. ONE was implemented using conventional confocal or epifluorescence microscopes and reached an imaging performance that enables imaging individual protein shapes. In October 2024, we published the concept of ONE microscopy (Shaib et al., 2024, doi: 10.1038/s41587-024-02431-9) in a collaborative effort of our three laboratories.
We then proceeded to increase the expansion factor of the gels, to improve their ability to describe molecules (Wang et al., 2024, doi: 10.1038/s41592-024-02454-9) and to enable the application of antibodies and/or other affinity probes after expansion, in conditions in which the size of the antibodies is no longer limiting for the labelling procedure (Kang et al., 2024, doi: 10.1038/s41467-024-53729-w; Eilts et al., 2023, doi: 10.1117/1.NPh.10.4.044412).
To enable a thorough evaluation of our methods, we also introduced expansion microscopy approaches to structures that have very clear sizes (Helmerich et al., 2024, doi: 10.1002/adma.202310104; Chowdhury et al., 2025, doi: 10.1038/s42003-025-07967-3) and new probes that are compatible with expansion procedures (Huang et al., 2025, doi: 10.21769/BioProtoc.5273; Wen et al., 2025, doi: 10.1021/jacs.4c15608).
Finally, we introduced a speed-optimized variant of DNA-based point accumulation for imaging in nanoscale topography (DNA-PAINT), to enhance the speed and precision of our imaging approaches (Ghosh et al., 2025, doi: 10.1126/science.adq4510.).
We then proceeded to increase the expansion factor of the gels, to improve their ability to describe molecules (Wang et al., 2024, doi: 10.1038/s41592-024-02454-9) and to enable the application of antibodies and/or other affinity probes after expansion, in conditions in which the size of the antibodies is no longer limiting for the labelling procedure (Kang et al., 2024, doi: 10.1038/s41467-024-53729-w; Eilts et al., 2023, doi: 10.1117/1.NPh.10.4.044412).
To enable a thorough evaluation of our methods, we also introduced expansion microscopy approaches to structures that have very clear sizes (Helmerich et al., 2024, doi: 10.1002/adma.202310104; Chowdhury et al., 2025, doi: 10.1038/s42003-025-07967-3) and new probes that are compatible with expansion procedures (Huang et al., 2025, doi: 10.21769/BioProtoc.5273; Wen et al., 2025, doi: 10.1021/jacs.4c15608).
Finally, we introduced a speed-optimized variant of DNA-based point accumulation for imaging in nanoscale topography (DNA-PAINT), to enhance the speed and precision of our imaging approaches (Ghosh et al., 2025, doi: 10.1126/science.adq4510.).
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.