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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 emitted from only a few sparsely distributed fluorophores are emitting in a single image. Each emitting fluorophore in an image , which can be precisely localised, usually by fitting to a gaussian function. The fluorophores in that image are then photobleached and another . Another set of fluorophores are turned on and localised in the next image . This and this process is then repeated, usually for >10000 images.
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