Abstract
Although fluorescence microscopy has had a major impact on biomedical research, the resolution barrier inherent in light microscopy restricts the ability to differentiate between objects closer together than ∼250 nm and prevents the true sizing of structures smaller than this limit. Recent innovations have led to the development of three main commercially available options for super-resolution microscopy that effectively break this diffraction limit: structured illumination microscopy (SIM), stochastic optical reconstruction microscopy (STORM)/photoactivation localization microscopy (PALM), and stimulated emission depletion microscopy (STED). The goal of this chapter is to describe the physical basis for these techniques as well as practical information for each, to provide the potential user with a basis for comparison and determination of the optimal choice for specific applications. Finally, innovative variations of these techniques for particular biological studies, as well as descriptions of new alternative techniques are presented.
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