OSA's Digital Library

Applied Optics

Applied Optics

APPLICATIONS-CENTERED RESEARCH IN 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)
http://dx.doi.org/10.1364/AO.42.005123


View Full Text Article

Acrobat PDF (150 KB)





Browse Journals / Lookup Meetings

Browse by Journal and Year


   


Lookup Conference Papers

Close Browse Journals / Lookup Meetings

Article Tools

Share
Citations

Abstract

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

Citation
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)
http://www.opticsinfobase.org/ao/abstract.cfm?URI=ao-42-25-5123


Sort:  Author  |  Year  |  Journal  |  Reset

References

  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.
  2. T. A. Klar, S. Jakobs, M. Dyba, A. Egner, and S. W. Hell, “Fluorescence microscopy with diffraction resolution limit broken by stimulated emission,” Proc. Natl. Acad. Sci. USA 97, 8206–8210 (2000).
  3. T. A. Klar, E. Engel, and S. W. Hell, “Breaking Abbe’s diffraction resolution limit in fluorescence microscopy with stimulated emission depletion beams of various shapes,” Phys. Rev. E 64, 066613 (2001).
  4. M. Dyba and S. W. Hell, “Focal spots of size 1/23 open up far-field fluorescence microscopy at 33-nm axial resolution,” Phys. Rev. Lett. 88, 163901 (2002).
  5. F. P. Schäfer, Dye Lasers (Springer-Verlag, Berlin, 1973).
  6. A. Grinvald, B. M. Salzberg, V. Lev-Ram, and R. Hildesheim, “Optical recording of synaptic potentials from processes of single neurons using intracellular potentiometric dyes,” Biophys. J. 51, 643–651 (1987).
  7. V. Y. Artyukhov and E. I. Sinchenko, “Photoisomerization effect on fluorescence quenching in molecules containing a styryl group,” Russ. Phys. J. 44, 718–722 (2001).
  8. P. E. Hänninen, M. Schrader, E. Soini, and S. W. Hell, “Two-photon excitation fluorescence microscopy using a semiconductor laser,” Bioimaging 3, 70–75 (1995).
  9. H. J. Koester, D. Baur, R. Uhl, and S. W. Hell, “Ca2+ fluorescence imaging with pico- and femtosecond two-photon excitation: signal and photodamage,” Biophys. J. 77, 2226–2236 (1999).
  10. A. Hopt and E. Neher, “Highly nonlinear photodamage in two-photon fluorescence microscopy,” Biophys. J. 80, 2029–2036 (2001).
  11. K. König, T. W. Becker, P. Fischer, I. Riemann, and K.-J. Halbhuber, “Pulse-length dependence of cellular response to intense near-infrared laser pulses in multiphoton microscopes,” Opt. Lett. 24, 113–115 (1999).

Cited By

Alert me when this paper is cited

OSA is able to provide readers links to articles that cite this paper by participating in CrossRef's Cited-By Linking service. CrossRef includes content from more than 3000 publishers and societies. In addition to listing OSA journal articles that cite this paper, citing articles from other participating publishers will also be listed.


« Previous Article  |  Next Article »

OSA is a member of CrossRef.

CrossCheck Deposited