3.7.1 MINFLUX, RASTMIN, and MINSTED
Minimising fluorescence fluxes nanoscopy (MINFLUX) utilises a principle somewhat similar to that of STED. However, instead of quenching fluorophores outside the central excitation point, MINFLUX uses an excitation PSF with zero intensity in the centre that makes subnanometer searching steps around the emitting molecule to minimise its fluorescence (Figure 2d) (Gwosch et al., 2020). This allows the use of much lower laser intensities compared to STED and achievement of a spatial resolution of 1-3 nm in 3D and a temporal resolution of 100 µs compatible with very accurate SPT. MINFLUX possesses enormous potential and currently its implementation is primarily limited by the high cost of the required equipment. MINFLUX can be combined with other SRN techniques including DNA-PAINT (Ostersehlt et al., 2022) or single-molecule resonance energy transfer (Cole et al., 2023) further expanding its capabilities. RASTMIN aims to alleviate the equipment cost issue of MINFLUX by using the raster scanning of a sample with spatially modulated light comprising a local minimum of intensity (Masullo et al., 2022). RASTMIN can be implemented on regular scanning microscopes (confocal or two-photon (2P)) and provides a 3D spatial resolution comparable to MINFLUX. In MINSTED, another MINFLUX competitor, the STED beam is used not to separate fluorophores, which are photoswitched independently, but to find the fluorophore position (Weber et al., 2021). MINSTED provides a similar spatial resolution to MINFLUX, but its advantages include lower background noise and the ability to attenuate the desired resolution of the system. The true potential of MINFLUX, RASTMIN, and MINSTED is yet to be fully revealed.