Single Molecule Localization Microscopy – SMLM

The super-localization microscopy (SMLM Single Molecule Localization Microscopy achieves a lateral resolution of about 20 nm, with the possibility to acquire at different times the emission of the fluorophores which are in the response function of the microscope (PSF). Different describe these acronyms such super-localization approaches the dSTORM (direct Stochastic Optical Reconstruction Microscopy) when organic fluorophores or PALM when using photoactivatable fluorescent proteins (Photo Activation Localization Microscopy ). The general idea in both cases is to allow in the PSF only one fluorophore to be in a condition that could lead to fluorescence emission. The fluorophore produces a fluorescence spot whose size is given by the diffraction limit. However, the centroid of this spot associated with a single molecule can be located with an accuracy well above the diffraction limit of the order of several nanometers. It bears repeating that process of localization for retrieving the position of all the fluorophore present in the biological sample. In the case of dSTORM to allow the successive localization of these unique molecules, initially a maximum of fluorophores (type Alexa) are carried in a non-fluorescent state, and return to the ground state stochastically, which allows then an observation of the fluorescence of single molecules. The setup developped by Nicolas Bourg during his thesis allows us to achieve multicolor image (excitation @ 640 nm or 488 or 561) A dSTORM image is shown below, it shows that depending of the number of acquired images, the recosntruction of the sample improved. You can see the final image (6500 acquisitions) in the actin network is thus extremely visible on dSTORM image, whereas it is unclear in the diffraction limited epifluorescence image