Photoactivated Localization Microscopy
Stochastic Optical Reconstruction Microscopy
The basic principle of PALM and STORM dictates that all molecules are prepared in the off state, then, a small subset of individual single molecules are stochastically switched into the on state. Typically, both states are realized via a photoizomerisation between molecular states (e.g. cis/trans isomerization)
After precise readout of the location of each individual molecule, they are switched back to the dark state or bleached with readout light. Sequential readout of stochastically switched molecule subsets results in a high resolution image of the label distribution.
PALM and STORM implement a stochastic readout of the molecule positions. As such, the image is taken iteratively by overlapping all identified positions.
On the implementation side, a PALM or STORM microscope is quite simple: a widefield-setup with a single-photon sensitive area detector.
The PALM and STORM methods provide theoretically unlimited localization accuracy of individual dye (clusters). The localization accuracy depends on the number of photons (N) being emitted by one dye (cluster):
where Δx denotes the localization accuracy of each individual molecule, λ denotes the excitation wavelength, and NA is the numerical aperture of the objective lens.
To deliver a high-resolution image the PALM and STORM concept must leverage numerical methods to calculate and overlay the identified positions of individual molecules.