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Proc Natl Acad Sci U S A. 2005 Sep 13;102(37):13081-6. Epub 2005 Sep 2.

Nonlinear structured-illumination microscopy: wide-field fluorescence imaging with theoretically unlimited resolution.

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  • 1Department of Physiology and Program in Bioengineering, University of California, San Francisco, CA 94143-2532, USA.


Contrary to the well known diffraction limit, the fluorescence microscope is in principle capable of unlimited resolution. The necessary elements are spatially structured illumination light and a nonlinear dependence of the fluorescence emission rate on the illumination intensity. As an example of this concept, this article experimentally demonstrates saturated structured-illumination microscopy, a recently proposed method in which the nonlinearity arises from saturation of the excited state. This method can be used in a simple, wide-field (nonscanning) microscope, uses only a single, inexpensive laser, and requires no unusual photophysical properties of the fluorophore. The practical resolving power is determined by the signal-to-noise ratio, which in turn is limited by photobleaching. Experimental results show that a 2D point resolution of <50 nm is possible on sufficiently bright and photostable samples.

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