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.