Stimulated emission depletion (STED) microscopy is in its simplest form an extension of confocal fluorescence microscopy that offers much enhanced spatial resolution in both 2D and 3D. This chapter provides a basic overview of the theory behind STED microscopy and the technology developments and modern design of the STED microscope. Like with any advanced imaging technology, it is important to implement simple testing procedures of the overall performance. This chapter provides detailed examples of the testing procedures that have proven useful to ensure optimal performance of a range of STED microscopes. Finally, this chapter includes a few application image examples.
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