Single-Molecule Localization Microscopy (SMLM). How to overcome the Abbe diffraction limit? The Challenge The Abbe diffraction barrier, which fundamentally restricted the lateral optical resolution to 200 – 300 nm until the early 1990s, can be overcome by super-resolution optical fluorescence imaging techniques. These techniques have been so groundbreaking that the Nobel Prize 2014 in Chemistry was awarded to Eric Betzig, Stefan Hell and W. E. Moerner “for the development of super-resolved. Background There are two different techniques commonly used so far. One technique is based on point spread function engineering, the other on single-molecule localization. Stefan Hell’s stimulated emission depletion (STED) microscopy belongs to the first class and is a so called deterministic super-resolution technique. In contrast, Eric Betzig’s photoactivated localization microscopy (PALM) is a stochastic technique How does SMLM work? The fundamental principle of all SMLM implementations is the random photoactivation and subsequent localization of individual fluorescence emitters by detecting their photon emission at a certain position and time. This is done in the complete absence of emission from neighboring emitters. The centroid of the diffraction-limited distribution of photons is a measure of the location of the emitter. This is far better defined than the photon distribution itself. Thus, whereas a standard widefield image is an overlay of the photon distributions from a large number of emitters, a super-resolved SMLM image is a map of their individual locations that Read full whitepaper here.