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.
