Photoactivated localization microscopy

Too spatially resolve closely spaced fluorescing dye molecules (fluorophores), a variety of techniques such as photoactivated localization microscopy (PLM), and work by separating the fluorescence of each emitter in time.  Alter of imaging all the fluorophores simultaneously; these techniques image each individual fluorophore one at a time, making it possible to search the position of each molecule with high precision. Once all of the positions have been create, they are plotted as points in space to construct an image. The spatial resolution is not limited by diffraction, but only by the precision with which each fluorophore can be localized.

To observe each protein individually the  excitation light  illuminates the  entire sample but  at low intensity so that only a few fluorophores are excited at a time,  and  this fluorophore excitation is stochastic; that is, whether a given dye molecule is excited at a given time  is random. This enables different fluorophores to be excited at various times so they can be imaged individually. Computer algorithms are used to construct an image of the sample from the locations of each fluorophore.

The  precision of the  position measurement is dependent on  the  contrast between the brightness of the  fluorophore compared with  the  background; the  greater the  contrast, the higher the precision. An advantage of this stochastic fluorophore imaging is that each dye molecule only undergoes a few photoexcitation cycles and this avoids a problem called photobleaching. The major disadvantage is the time it takes to acquire an image. The greater the fluorophore density in the sample, the longer the imaging time.  Because imaging time  is determined by how  rapidly each fluorophore turns on and  off, acquisition  time  can  be reduced by using higher excitation intensity (the  fluorophore turns off within nanoseconds of it being excited), but this can limit resolution.