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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 seem to form a single spot in panel B. SMLM solves this problem by imaging the fluorophores one at a time. It does this by ensuring that in each image, light is only emitted from a few sparsely distributed fluorophores, which are then precisely localised. Another set of fluorophores are turned on and localised in the next image and this process is repeated, usually for >10000 images.
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