Simple and robust 3D MINFLUX excitation with a variable phase plate.

MINFLUX has achieved extraordinary resolution in superresolution imaging and single fluorophore tracking. It is based on localizing single fluorophores by rapid probing with a patterned beam that features a local intensity minimum. Current implementations, however, are complex and expensive and are limited in speed and robustness. Here, we show that a combination of an electro-optical modulator with a segmented birefringent element such as a spatial light modulator produces a variable phase plate for which the phase can be scanned on the MHz timescale. Bisected or top-hat phase patterns generate high-contrast compact excitation point-spread functions for MINFLUX localization in the x, y, and z-direction, respectively, which can be scanned across a fluorophore within a microsecond, switched within 60 microseconds and alternated among different excitation wavelengths. We discuss how to compensate for non-optimal performance of the components and present a robust 3D and multi-color MINFLUX excitation module, which we envision as an integral component of a high-performance and cost-effective open-source MINFLUX.