Fluorescence microscopy allows users to dynamically observe phenomena in living cells; however, the wave nature of light and its associated diffraction restrict the resolution of light microscopy: When light of wavelength λ is focused by a lens with a numerical aperture NA, objects that are closer together than d = λ/(2NA) cannot be easily discerned. This is the Abbe diffraction limit, and it has until recently restricted the resolution of fluorescence microscopy to ~250 nm. New methods, collectively grouped under the term super-resolution microscopy, have been developed to overcome this physical limit, gaining over two orders of magnitude in precision, allowing direct observation of processes at spatial scales much more compatible with the regime that biomolecular interactions take place on. Radically, different approaches have so far been proposed, including limiting the illumination of the sample to regions smaller than the diffraction limit (targeted switching and readout) or stochastically separating single fluorophores in time to gain resolution in space (stochastic switching and readout). The latter also described as follows: Single-molecule active control microscopy, or single-molecule localisation microscopy (SMLM) allows imaging of single molecules which cannot only be precisely localised, but also followed through time and quantified.
The essential step leading to SMLM was the ability to control the activation of point emitters, and the techniques now distinguish themselves based on how emitters are spatially isolated. The first SMLM techniques were all demonstrated in 2006, and these were photo-activated localization microscopy (PALM), stochastic optical reconstruction microscopy (STORM), and fluorescence photo-activation localization microscopy (fPALM). The techniques all work by finding the centre position of the point emitters by fitting to a mathematical function. As the point emitters “blink” on and off, they are each “localised” and a super-resolution image is generated by plotting the co-ordinates of the centre positions (see figure below).