SMLM Primer
SMLM still produces diffraction-limited images but uses a trick to achieve nanometer resolution. Since the main drawback 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 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 2D spot is a projected 3D volume 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. In the presence of thiols in an aqueous buffer and through excitation laser light, cyanine dyes can reversibly photoswitch between a non-emitting (dark) state and an emitting (on) state. The dark state can be excited with UV light, which can be used to control photoswitching. (Rust, 2006, Heilemann, 2008)
PhotoActivation Localization Microscopy (PALM), uses the light-induced photo-conversion or -activation of fluorescent proteins. A number of different derivatives of the green fluorescent protein GFP have been developed initially for intracellular diffusion studies. Remarkably, many of them are suitable for SMLM. (Betzig, 2006, Hess, 2006)
DNA - Point Acculumation In Nanoscale Topography (PAINT), relies on the controlled transient hybridization of short DNA oligonucleotides to achieve single-molecule blinking. The two complementary oligos, the docking and the imager strand, are coupled to the sample (e.g. an antibody) and a fluorescent reporter respectively. (Jungmann, 2010)
This is a short list of three widely-used SMLM techniques. Various variations were developed during the last decade. Please see the References section for further reading.
The workflow when using the different SMLM techniques can be broken down into a few general steps. Following the sample preparation, which is specific to the used method, the blinking sample is imaged using a suitable microscope. Connected to the microscope is a sensitive camera detector with a build-in pixelated sensor chip. A series of images is then acquired, with each frame containing individual blinking events in the form of PSFs. Each PSF on the pixelated sensor can then be localized with sub-pixel precision. The resulting localization list can later be used to generate a super-resolved image or for other quantitative analysis as also presented on this wiki.