Fluorescence in Microscopy


Fluorescence microscopy is widely used and offers great specificity. Various techniques make it possible to address different problems and even to circumvent the diffraction limit that was described by Ernst Abbe.

The localization of a molecule species can be determined with a co-staining of organelles, e.g. the cytoskeleton or membranes. Confocal laser scanning microscopy (CLSM) makes it possible to observe areas in the specimen without signals from the outside of the focal plane and allows optical sectioning. Total internal reflection (TIRF) microscopy is a technique that allows the observation of a thin region close to the cell surface. An evanescent field excites the fluorochromes in this area.

A technique for observing the dynamics of a molecule species is fluorescence recovery after photobleaching (FRAP). Fluorochromes in a restricted area are photobleached and the diffusion of unbleached molecules into this area can be measured. Interaction studies can be performed with fluorescence energy transfer (FRET) microscopy. An excited donor chromophore can transfer energy to an acceptor fluorochrome and excite it. This is only possible if both are brought together very closely. If these dyes are coupled to different proteins, they will only be able to transfer energy and fluoresce if the proteins interact with each other.  

Techniques that allow sub-resolution images are stimulated emission depletion (STED) microscopy, ground-state depletion (GSD) microscopy, single molecule and ground state depletion microscopy followed by individual molecule return (



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