Separate labeled features that are closer than the diffraction barrier
Due to diffraction there is always an area larger than half of the light wavelength illuminated; all molecules in this area emit light and cannot be distinguished.
Keep adjacent fluorophors sequentially dark, so that one can register them separately und thus record adjacent features sequentially. Thus, the basic idea of nanoscale resolution is to introduce a controllable on/off transition of the dye label.
Consequently, the dye is the key for nanoscale resolution. Abberior is specialized in designing dyes with on/off transitions that can be controlled via a second light source.
A: Coordinate-Targeted Readout
STED: STED light forces the molecules into the ground state via stimulated emission. Thus fluorescence cannot occur under STED irradiation which prepares a controllable 'off'-state
RESOLFT:Generalized concept of switching all previously excited molecules off, except the very center of the focus. A particular implementation concept is a light controllable conformational change of the molecule. One isomerized form represents the non-fluorescent 'off'-state
B: Spatially Stochastic Readout
PALM, STORM: Molecules are randomly switched into the 'on'-state, while most molecules stay in the 'off'-state at a certain point of time. Switching different molecule subsets is realized either reversably (e.g. conformational changes) or irreversably (e.g. uncaging)
GSDIM: In contrast to the previous implememtation GSDIM (also referred to as dSTORM) realizes switching of molecule subsets via exploiting purely electronic states of a fluorescent molecule. An electronical dark-state with a sufficiently long lifetime represents the 'off'-state