SINGLE MOLECULE LOCALIZATION MICROSCOPY (SMLM)
The phenomenon of diffraction has long been thought to set an inevitable physical limit to resolution of light microscopy. According to Abbe’s diffraction limit approximation, structures smaller than half the wavelength of light cannot be resolved. The last decades however, have seen developments in fluorescence microscopy that enable to study cellular structures on the nanometer length scale.
One of these approaches has been termed single molecule localization microscopy (SMLM). In 2006, different realizations of the technique were developed simultaneously: photoactivated localization microscopy (PALM), fluorescence photoactivation localization microscopy (fPALM) and stochastic optical reconstruction microscopy (STORM). More recent approaches include direct stochastic optical reconstruction microscopy (dSTORM) and point accumulation for imaging in nanoscale topography (PAINT), including DNA-PAINT (see further reading for information about SMLM techniques).
The basic idea of all SMLM techniques is to separate the signal of individual emitters in time, which allows to determine their positions with nanometer precision. This is typically achieved by exploiting stochastic blinking or binding phenomena, leaving only a sparse subset of molecules visible at a certain time point. Hence, for image acquisition thousands of individual frames need to be recorded. The raw data is subsequently analyzed, yielding a list of localization coordinates as a final result.
