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August 12th, 2007

Nanoscopy - nanoscale resolution in light microscopy

In the early 1870s, the German physicist Ernst Karl Abbé formulated a rigorous criterion for being able to resolve two objects in a light microscope. According to his equation, the best resolution achievable with visible light is about 200 nanometers. This theoretical resolution limit of conventional optical imaging methodology was the primary factor motivating the development of recent higher-resolution scanning probe techniques. The interaction of light with an object results in the generation of what is called 'near-field' and 'far-field' light components. The far-field light propagates through space in an unconfined manner and is the visible light utilized in conventional light microscopy. The near-field (or evanescent) light consists of a nonpropagating field that exists near the surface of an object at distances less than a single wavelength of light. So called near-field microscopy beats light's diffraction limit by moving the source very close to the subject to be imaged. When the first theoretical work on a new technique called "scanning near-field optical microscopy" (SNOM or NSOM) appeared in the 1980's, Abbé's classical diffraction limit was overcome, and resolution even down to single molecule level became feasible. However, light microscopy is still the only way to observe the interior of whole, or even living, cells. The use of fluorescent dyes makes it possible to selectively obtain images of individual cell components, for example, proteins. Today, the wavelength dogma has been overcome with the development of the stimulated emission depletion (STED) microscope. Now, the German team that developed STED is reporting layer-by-layer light microscopic nanoscale images of cells and without having to prepare thin sections with a technique called optical 3D far-field microscopy. They use a chemical marker for fluorescence nanoscopy that relies on single-molecule photoswitching.


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