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Research Article

Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission

Thomas A. Klar, Stefan Jakobs, Marcus Dyba, Alexander Egner, and Stefan W. Hell
  1. Max-Planck-Institute for Biophysical Chemistry, High Resolution Optical Microscopy Group, 37070 Göttingen, Germany

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PNAS July 18, 2000 97 (15) 8206-8210; https://doi.org/10.1073/pnas.97.15.8206
Thomas A. Klar
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Stefan Jakobs
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Marcus Dyba
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Alexander Egner
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Stefan W. Hell
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  1. Edited by Daniel S. Chemla, E. O. Lawrence Berkeley National Laboratory, Berkeley, CA, and approved May 12, 2000 (received for review March 10, 2000)

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Abstract

The diffraction barrier responsible for a finite focal spot size and limited resolution in far-field fluorescence microscopy has been fundamentally broken. This is accomplished by quenching excited organic molecules at the rim of the focal spot through stimulated emission. Along the optic axis, the spot size was reduced by up to 6 times beyond the diffraction barrier. The simultaneous 2-fold improvement in the radial direction rendered a nearly spherical fluorescence spot with a diameter of 90–110 nm. The spot volume of down to 0.67 attoliters is 18 times smaller than that of confocal microscopy, thus making our results also relevant to three-dimensional photochemistry and single molecule spectroscopy. Images of live cells reveal greater details.

Footnotes

    • ↵† To whom reprint requests should be addressed. E-mail: shell{at}gwdg.de.

    • This paper was submitted directly (Track II) to the PNAS office.

  • Abbreviations

    PSF,
    point-spread-function;
    STED,
    stimulated emission depletion;
    3D,
    three-dimensional;
    FWHM,
    full-width-at-half-maximum
    • Received March 10, 2000.
    • Copyright © 2000, The National Academy of Sciences
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    Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission
    Thomas A. Klar, Stefan Jakobs, Marcus Dyba, Alexander Egner, Stefan W. Hell
    Proceedings of the National Academy of Sciences Jul 2000, 97 (15) 8206-8210; DOI: 10.1073/pnas.97.15.8206

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    Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission
    Thomas A. Klar, Stefan Jakobs, Marcus Dyba, Alexander Egner, Stefan W. Hell
    Proceedings of the National Academy of Sciences Jul 2000, 97 (15) 8206-8210; DOI: 10.1073/pnas.97.15.8206
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    • Shattering the diffraction limit of light: A revolution in fluorescence microscopy?
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    Proceedings of the National Academy of Sciences: 97 (15)
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