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

      Proceedings of the National Academy of Sciences of the United States of America

      Photochemistry, standards, instrumentation, Microscopy, Fluorescence, Lasers, Fluorescence

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          Abstract

          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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          Most cited references 26

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          Two-photon laser scanning fluorescence microscopy

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            Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy. SHORT COMMUNICATION

             M. Gustafsson (2000)
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              On/off blinking and switching behaviour of single molecules of green fluorescent protein.

              Optical studies of individual molecules at low and room temperature can provide information about the dynamics of local environments in solids, liquids and biological systems unobscured by ensemble averaging. Here we present a study of the photophysical behaviour of single molecules of the green fluorescent protein (GFP) derived from the jellyfish Aequorea victoria. Wild-type GFP and its mutant have attracted interest as fluorescent biological labels because the fluorophore may be formed in vivo. GFP mutants immobilized in aereated aqueous polymer gels and excited by 488-nm light undergo repeated cycles of fluorescent emission ('blinking') on a timescale of several seconds-behaviour that would be unobservable in bulk studies. Eventually the individual GFP molecules reach a long-lasting dark state, from which they can be switched back to the original emissive state by irradiation at 405 nm. This suggests the possibility of using these GFPs as fluorescent markers for time-dependent cell processes, and as molecular photonic switches or optical storage elements, addressable on the single-molecule level.
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                Author and article information

                Journal
                10.1073/pnas.0406877102
                1201569
                16141335

                Chemistry

                Photochemistry, standards, instrumentation, Microscopy, Fluorescence, Lasers, Fluorescence

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