The resolution of light microscopy is limited by diffraction to approximately half the wavelength of light (i.e., about 250 nm). Objects smaller than this cannot be resolved from one another using conventional light microscope techniques. The example below shows how adjacent objects smaller than the diffraction limit can be resolved using Single Molecule Localisation Microscopy (SMLM).

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).

How is it done?

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

The above case is relatively trivial because there are very few molecules and they are well spaced apart. In fluorescently labelled cells the problem is usually more challenging because there are more fluorescently labelled molecules and they are crowded together in higher order structures like organelles, membranes, etc.