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Panel A shows a light microscope image of fluorescently labelled DNA origami structures immobilized on glass. Each structure appears as a bright dot (scale bar 5 µm). Panel B shows a zoomed image of the square outlined in white in panel A. The underlying structure of the DNA origami cannot be resolved because the diffraction limited spots are too large (scale bar 0.3 µm). Panel C shows a single molecule localization microscopy image of the same region. SMLM allows us to see that each spot is actually a DNA origami structure labelled with two fluorescent molecules, which are 94 nm apart (scale bar 0.3 µm).

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The principle of SMLM

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 final super-resolution image is constructed from all the precise total set of localisations.

In normal circumstances Normally when a fluorescent sample is illuminated using widefield or confocal microsopy effectively all its fluorophore molecules will emit light simultaneously. For SMLM, so some method is required to ensure only a few molecules emit at a timein a single image, and that a different set of molecules . The on/off switching of fluorophores between frames is often described as 'blinking', as seen in the GIF below. SMLM techniques mainly differ in how the blinking is achieved.

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