Bewersdorf Laboratory
Resolution is limited in standard light microscopy.
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The fundamental physical phenomenon of diffraction prevents focusing of any light source, whether it is a laser beam or fluorescence emitted from a single molecule, to spots smaller than about half the wavelength of light (about 250 nm, or ¼ of a micron). This results in blurring of any regular microscopy image and conceals structures below this size scale.
Resolution in standard light microscopy is limited
One approach to overcoming the resolution barrier is based on targeted switching of fluorescence and point-spread function engineering where the effective volume from which light can be emitted is narrowed down. Another approach is based on stochastic switching of fluorescence and single molecule localization utilizing the knowledge that the molecule causing the blurry image is located in the center of that spot which can be determined with a much better accuracy than the spot width.
The diffraction
limit can be overcome by optical switching of fluorophores
While these two strategies approach the problem of overcoming the diffraction limit from different angles, they share common ingredients. Most prominently, both methods rely on the combination of optics with photophysics: by optically switching fluorescent molecules “on” and “off”, super-resolution below ~100 nm becomes possible. Both strategies, the point-spread function engineering and the single molecule localization approach, are currently pursued in our laboratory in form of STED and FPALM (aka PALM or STORM) microscopy, respectively.