An Overview of Photo-Activated Localization Microscopy

Photo-Activated Localization Microscopy (PALM) is an ultra resolution technique that significantly enhances the spatial resolution of the optical microscope through an order of magnitude which has 10 to 20 nanometer resolution. This as a result allows the analysis of biological procedures at confined molecular scale. This method banks on the curbed activation and sampling of thin subsets of photo-convertible fluorescent molecules, either genetically-encoded or synthetically-encoded. PALM was developed by Eric Betzig and Harald Hess to surmount the diffraction resolution limit of an optical microscope. According to the Rayleigh criteria determines the limit of diffraction as - R=0.61λ/NA or ~ 200 nm (Hess). PALM has the capacity to resolute on the order of tens of nanometers (Betzig).

PALM

PALM makes use of photoactivatable proteins with the objective to consecutively localize sole molecules which have high accuracy and eventually defeats the limit. Two unique lasers are attained with the help of a discovery of single fluorophores. The activated beam stimulates the photoactivatable fluorophores such as the pamCherry or photo-activatable GFP (paGFP) so as to change it from the dark state to a fluorescent state and the output bean is put to use to develop the fluorescence required for imaging and also to bleach the molecules that have been activated. The main aim is to activate just one molecule in every diffraction limited area.

With the help of the activated photoactivable fluorescent proteins, it is potentially probable to exclusively switch on thousands of thin/sparse subsets of molecules in a consecutive manner. The vital principle behind PALM is to begin with the widespread molecules in the dormant state. A tiny fraction is photoactivated with the help of a brief pulse of violet and/or ultraviolet light to provide that particular subset fluorescent. The activated molecules are later imaged and localized in order to develop precise coordinates at the nanometer level. This is then accompanied by removal from the bigger set of not-activated molecules by photobleaching.