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).