Super-resolution microscope with fluorescence fluctuation and expansion gel imaging capabilities

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Fluorescence microscopes have been limited for decades by the diffraction barrier, to approximately half of the wavelength of the imaging light (200-350 nm in most biological experiments). Several super-resolution approaches have been developed to overcome the diffraction barrier, but fluorescence microscopy still fails to image the morphology of single proteins or small molecular complexes, either purified or in a cellular context. Combining optical super-resolution technologies with expansion microscopy, in which the sample is enlarged after embedding in a swellable gel, should, in principle, reach molecular resolution. This failed, as the gels limited the effectiveness of most super-resolution tools, including both coordinate-targeted approaches (as STED or SIM) and single-molecule based approaches (as STORM). We recently obtained a solution to this problem, employing a third class of optical super-resolution approaches, which is based on determining the higher-order statistical analysis of temporal fluctuations measured in a movie. We combined expansion microscopy with a super-resolution radial fluctuations (SRRF) analysis and obtained 0.8 to 1 nm resolutions across different samples and color channels. We applied this technique, which we termed one-nanometer expansion microscopy (ONE) to many issues, from diagnostics to the analysis of the shape of single molecules. ONE microscopy opens many of new avenues in biological sciences, from an all-optical analysis of protein structure to various combinations of live super-resolution imaging and structural analyses. Higher performance, to resolutions substantially better than 1 nm, is possible, since the current results are only limited by the signal-to-noise ratio. To achieve this, we apply here for a microscope that will replace the setup on which we established ONE microscopy, while offering substantial advantages. In addition, we require the same setup for many other projects, dealing especially with living samples, to replace the general functionality of our previous setup. In short, we require a setup that will perform the following: 1) provide a dynamic analysis of the expanded gels, with excellent speed and signal-to-noise ratio, to obtain molecular-scale resolution; 2) allow the routine testing of the samples at super-resolution (50 nm or better), both in fixed and living cells; 3) enable sample analysis procedures as fluorescence recovery after photobleaching (FRAP); 4) be compatible with approaches that enable strong multiplexing, e.g. by use of fluorescence lifetime imaging. The required setup will be placed in the Biomedical Microscopy Unit of the University Medical Center Göttingen, and will replace a heavily used microscope installed in 2007, which will no longer be fully operational after 2023.