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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.
SMLM Techniques
The principles above are effectively the same for all SMLM techniques. The main practical difference between experimental approaches is in how the blinking is achieved. The most common SMLM techniques are summarised below.
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