Breaking the resolution limit in light microscopy.

The advancement in fluorescence microscopy has dramatically enhanced the obtainable optical resolution enabling the users to inspect the structures of interest at finer and finer level of detail. This chapter describes some of these methods and how they break the classical resolution limit. The labeling of targets, such as individual genetic loci, specific proteins, or organelles, is possible inside living cells, which led to the extensive use of fluorescence microscopy in life sciences. Other microscopic modes usually lack this high specificity but sometimes provide other useful information such as the orientation of molecular species in polarization microscopy. Modes, such as differential interference contrast, phase contrast, or dark field, are useful to discriminate and follow cells or structures within them without the need for specific labeling. However, classically the resolution of all of these light microscopic modes was far below that of the electron microscope, and only some recent approaches have made significant progress in resolution increase. Recently, many microscopy methods have dramatically enhanced the resolution. Gradually, these methods are now applied to solve biological problems. The most promising approaches are all based on fluorescence and use either nonlinear interaction of light with the sample (STED, nonlinear structured illumination, dynamic saturation optical microscopy, or saturation in the time domain) or precise localization of individual particles or molecules with subsequent image generation.