MINFLUX represented a breakthrough showing that single fluorescent molecules could be localized with 1-2 nm precision with moderate photon counts.Such level of localization efficiency enables single-molecule tracking and super-resolution imaging with true molecular resolution. The key concept was to estimate the molecular position from a sequence of exposures to spatially displaced a light minimum. Expanding this concept to multi-photon regimes may be a useful complement to reach even higher localization precision and get deeper into biological specimens.
In a recent News & Views article in Light: Sience and Applications, we review a recent paper with theoretical calculations of a MINFLUX scheme using two-photon excitation, and expand the discussion showing calculations for RASTMIN in two-photon and three-photon excitation. RASTMIN, is a method related to MINFLUX that uses regular raster scans of a minimum of light that we presented theoretically in a recent paper in Biophysical Reports. RASTMIN is equivalently efficient but has the advantage that can be readily implemented in almost any laser scanning microscope.
More to read:
- Luciano A. Masullo, Fernando D. Stefani
“Multiphoton single-molecule localization by sequential excitation with light minima”
- Luciano A. Masullo, Lucía F. Lopez, Fernando D. Stefani
“A common framework for single-molecule localization using sequential structured illumination”
