SMLM Primer
SMLM is still diffraction-limited but uses a trick to achieve nanometer resolution. Since the main problem of conventional imaging is that all fluorophores emit at the same time, the different techniques of SMLM have the goal to separate individual molecules so that they can be localized at a much higher precision.
How an individual molecule looks like through a microscope can be precisely predicted and/or experimentally determined. A single emitter seen through the optical system of a microscope all the way to the detector will create a spot. More precisely, the spot is a 3D volume that is called a point spread function (PSF). The dimensions and shape of the PSF depend on the microscope but can be measured by imaging a diffraction-limited object such as a single fluorophore or a fluorescent nanosphere.
There are three popular SMLM techniques that use different mechanisms to induce photoswitching.
STochastic Optical Reconstruction Micropscopy (STORM), uses the chemically-induced photoswitching of red cyanine dyes.
PhotoActivation Localization Microscopy (PALM), uses the light-indiced photo-conversion or -activation of fluorescent proteins.
Point Acculumation In Nanoscale Topography (PAINT), uses the controlled transient hybridisation of short DNA oligonucleotides.
