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The problem illustrated above is that the fluorescent signals of adjacent objects overlap if they are closer than the limit set by diffraction, which is why two adjacent fluorophores can seem to form a single spot in panel B. SMLM solves this problem by imaging the fluorophores one at a time and not simultaneously. It does this by ensuring that in each image, light is only emitted from a few sparsely distributed fluorophores, which can then be precisely localised without their emission overlapping with that of a neighbour. The fluorophores only emit for a short time, so in the next image a different set of fluorophores is localised. This process is then repeated, usually for >10000 images and a final super-resolution image is constructed from the full set of localisations.Image Removed

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.Image RemovedImage Removed

SMLM Techniques

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