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Applied Optics

Applied Optics


  • Vol. 42, Iss. 25 — Sep. 1, 2003
  • pp: 5123–5129

Photostability of a fluorescent marker under pulsed excited-state depletion through stimulated emission

Marcus Dyba and Stefan W. Hell  »View Author Affiliations

Applied Optics, Vol. 42, Issue 25, pp. 5123-5129 (2003)

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Saturated stimulated-emission depletion (STED) of a fluorescent marker has been shown to break the diffraction barrier in far-field fluorescence microscopy and to facilitate spatial resolution down to a few tens of nanometers. Here we investigate the photostability of a fluorophore that, in this concept, is repeatedly excited and depleted by synchronized laser pulses. Our study of bacteria labeled with RH-414, a membrane marker, reveals that increasing the duration of the STED pulse from ∼10 to 160 ps fundamentally improves the photostability of the dye. At the same time the STED efficiency is maintained. The observed photobleaching of RH-414 is due primarily to multiphoton absorption from its ground state. One can counteract photobleaching by employing STED pulses that range from 150 ps to approximately half of the lifetime of the excited state. The results also have implications for multiphoton excitation microscopy.

© 2003 Optical Society of America

OCIS Codes
(180.2520) Microscopy : Fluorescence microscopy
(260.5130) Physical optics : Photochemistry
(300.2530) Spectroscopy : Fluorescence, laser-induced
(350.1820) Other areas of optics : Damage

Original Manuscript: March 21, 2003
Revised Manuscript: May 23, 2003
Published: September 1, 2003

Marcus Dyba and Stefan W. Hell, "Photostability of a fluorescent marker under pulsed excited-state depletion through stimulated emission," Appl. Opt. 42, 5123-5129 (2003)

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  1. S. W. Hell, “Increasing the resolution of far-field fluorescence light microscopy by point-spread-function engineering,” in Topics in Fluorescence Spectroscopy, J. R. Lakowicz, eds. (Plenum, New York, 1997), pp. 361–426.
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  9. H. J. Koester, D. Baur, R. Uhl, S. W. Hell, “Ca2+ fluorescence imaging with pico- and femtosecond two-photon excitation: signal and photodamage,” Biophys. J. 77, 2226–2236 (1999). [CrossRef] [PubMed]
  10. A. Hopt, E. Neher, “Highly nonlinear photodamage in two-photon fluorescence microscopy,” Biophys. J. 80, 2029–2036 (2001). [CrossRef] [PubMed]
  11. K. König, T. W. Becker, P. Fischer, I. Riemann, K.-J. Halbhuber, “Pulse-length dependence of cellular response to intense near-infrared laser pulses in multiphoton microscopes,” Opt. Lett. 24, 113–115 (1999). [CrossRef]

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