Nanometer Precision Single Molecule Localization with Raster Scanning Fluorescence Microscopes: Benchmarking RASTMIN, RASTMAX, and RASTED

Super-resolution fluorescence microscopy and single-molecule tracking are true discovery tools for biology. MINFLUX has set the standard for localizing single fluorescent molecules with highest precision and photon efficiency. A shared theoretical framework for methods like MINFLUX, that localize molecules using spatially displaced structured beams, has shown that similar precisions may be achieved with commonly available raster scanning microscopes. Namely, three methods—RASTMIN, RASTMAX, and RASTED—emerge as practical alternatives. In this talk, I will present the common framework for these methods and a systematic experimental comparison of RASTMIN, RASTMAX, and RASTED realized on the same microscope, evaluating localization precision, photon efficiency, background robustness, throughput, and practical implementation. RASTMIN offers MINFLUX-like precision, photon efficiency and throughput. RASTMAX is simpler to implement and provides slightly reduced precision and higher throughput. RASTED is more complex as it requires STED type microscopes and compatible fluorophores, but delivers a 20–50× throughput increase. Overall, nanometer precise single molecule localization and super-resolution microscopy can be broadly achieved on standard scanning microscopes with different practical regimes for each raster beam method.