FPALM (Fluorescence PhotoActivation Localization Microscopy), or fluorescence photoactivation localization microscopy, is one of the methods of super-resolutionlocalization microscopybased on stochastic (random) imaging of individual glowing points. Like PALM, FPALM is used to accurately localize and image cellular structures labeled with photoactivatable fluorescent proteins (PAFPs) in an image, and certain prerequisites must be met for the method to be correct and applicable:
- select the optimal number of PAFPs, whose distance must be greater than the conventional resolution of the objective
- PAFPs have two states (active - fluorescent and inactive - dark), which can be switched (toggled) using light beams with different wavelengths. PAFPs are also capable of switching stochastically
- photons taken from a single PAFPs can be detected by CCD, with the location being influenced by the point spread function (PSF)
- the centre of the PSF is determined mathematically - a more accurate position is then assigned to a given PAFP
Using this method, as well as PALM, STORM, STED, SRRF-Stream and SIM,super-resolution results with resolution in the units of nm canbe achieved. The disadvantage of these methods is usually the longer scanning time of the whole image, since a large number of sub-images have to be scanned.
