Introduction to Single Molecule Localisation Microscopy (SMLM)
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 whose size is defined by the resolution of the optics and the wavelength of light and is usually referred to as the Point Spread Function (PSF) of the microscope (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 PSFs 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).
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 a final super-resolution image is constructed from the full set of localisations.
Normally when a fluorescent sample is illuminated using techniques like widefield or confocal microsopy, effectively all its fluorophore molecules will emit light simultaneously. For SMLM, a method is required to ensure only a few molecules emit in a single image, that those molecules do not emit in the next image and that a different set of molecules emits in subsequent images so that all the molecules can be localised with as little duplication as possible. 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.
