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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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    Figure 1

    Microscope. (a) Excitation pulses are followed by stimulated emission depletion pulses for fluorescence inhibition. After passing dichroic mirrors and emission filters, fluorescence is detected through a confocal pinhole by a counting photodiode. (b) Measured excitation PSF. (c) Measured STED-beam-PSF featuring local minimum at the center and intense maxima above and below the focal plane. Z denotes optic axis. The measurements of b and c are carried out with the pinhole removed.

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    Figure 2

    (a) Fluorescence is a nonlinear function of stimulating intensity; 10% remaining fluorescence is obtained for ISTEDT corresponding to PSTED of 2.2 mW in the focus. (b) Surface plot of XZ-section (Inset) of confocal fluorescence spot for 1.4 oil immersion lens. (d) Same as b but with STED-beam PSF switched on. (c) Corresponding axial intensity profiles demonstrate 5.1-fold reduction of the axial width (FWHM) from 490 nm down to 97 nm.

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    Figure 3

    XZ-images of 100-nm-diameter fluorescent beads (a and b) and of 100-nm-diameter negatively stained glass beads agglomerations (c and d) as observed in the confocal (a and c) and the STED-fluorescence (b and d) microscope. Note the artifacts indicated by arrows induced by the elongated spot in the confocal image and their reduction in the STED counterpart.

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    Figure 4

    Resolution improvement in live cells. XZ-images of a S. cerevisiae yeast cell with labeled vacuolar membranes with standard confocal resolution (a) and with axial resolution improved by STED (b). Whereas the confocal mode fails in resolving the membrane of small vacuoles, the STED microscopy better reveals their spherical structure. XZ-images of membrane-labeled E. coli show a 3-fold improvement of axial resolution by STED in d as compared with their simultaneously recorded confocal counterparts in c.

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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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