Localizing a single molecule means fitting an ideal point spread function (PSF) to its recorded intensity pattern, which is often done with a 2D Gaussian function of the form The main image processing steps consist of localizing each recorded emission of a single molecule and store it in an output file that finally contains the coordinates of thousands to millions of single molecules, followed by rendering this intermediate result into an image. Finally, the data is analysed and converted into a graphical representation that is no longer limited by diffraction. All of these concepts acquire image stacks over time that contain stochastic single-molecule emissions. An alternative version of SMLM without employing stationary photoswitches is points accumulation for imaging in nanoscale topography (PAINT) and DNA-PAINT, which utilize fluorescent probes that are transiently bound to the structure of interest through diffusion. Under experimental conditions the majority of fluorophores is kept non-fluorescent while sparse subsets are stochastically transferred to their fluorescent state. Д¬ommon representatives of SMLM are (fluorescence) photoactivated localization microscopy (PALM/FPALM) and (direct) stochastic optical reconstruction microscopy (STORM/dSTORM), which are all based on separating single photoswitchable fluorophores in a densely labelled sample in time and localizing them with a precision much higher than the optical resolution of the imaging system.

fluorophores that can be reversibly or irreversibly transferred from a non-fluorescent to a fluorescent state, paved the way for the development of SMLM. Among other powerful super-resolution imaging techniques such as stimulated emission depletion (STED) microscopy and structured illumination microscopy (SIM), single-molecule localization microscopy (SMLM) stands out as it provides access to quantitative information. Super-resolution microscopy has the potential to widely enhance our understanding of the structural organisation of cells.

Imagej tracking