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The resolution of light microscopy is limited by diffraction to approximately half the wavelength of light (i.e., about 250 nm). Objects smaller than this cannot be resolved from one another using conventional light microscope techniques. The , but the example below shows how adjacent objects smaller than the diffraction limit can be resolved using Single Molecule Localisation Microscopy (SMLM).
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Normally when a fluorescent sample is illuminated using techniques like widefield or confocal microsopy , effectively all its fluorophore molecules will emit light simultaneously. For SMLM, a method is required to ensure only a few molecules emit in a single image, ; that those molecules do not emit in the next image; and that a different set of molecules emits in subsequent images, so that all the molecules can be localised with as little duplication as possible. The on/off switching of fluorophores between frames is often described as 'blinking', as seen in the GIF below.
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