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Combining optical trapping and single-molecule fluorescence spectroscopy: enhanced photobleaching of fluorophores.

Publication ,  Journal Article
Dijk, MAV; Kapitein, LC; Mameren, JV; Schmidt, CF; Peterman, EJG
Published in: The journal of physical chemistry. B
May 2004

To obtain high-resolution information on position or conformation of a molecule and at the same time apply forces to it, one can combine optical trapping with single-molecule fluorescence microscopy. The technical challenge in such an experiment is to discriminate a minute fluorescence signal from the much larger background signals caused by the trap and the fluorescence excitation laser light. We show here that this is feasible even when the fluorophore is directly attached to the trapped particle, by using optimized optical filters. We found, however, that the photostability of the fluorophores we tested suffered from the presence of the additional laser light used for trapping. We found that bleaching rates increased linearly with both the intensity of the trapping laser and the intensity of the fluorescence excitation light. Photobleaching rates were unaffected by the presence or absence of oxygen, but were significantly diminished in the presence of antioxidants. Our results indicate that the enhanced photobleaching is caused by the absorption of a visible photon followed by the excited-state absorption of a near-infrared photon. The higher excited singlet states generated in this way readily form nonfluorescent dye cations. We found that different dyes suffer to a different extent from the excited-state absorption, with Cy3 being worst and tetramethylrhodamine least affected.

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Published In

The journal of physical chemistry. B

DOI

EISSN

1520-5207

ISSN

1520-6106

Publication Date

May 2004

Volume

108

Issue

20

Start / End Page

6479 / 6484

Related Subject Headings

  • 51 Physical sciences
  • 40 Engineering
  • 34 Chemical sciences
  • 09 Engineering
  • 03 Chemical Sciences
  • 02 Physical Sciences
 

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Dijk, M. A. V., Kapitein, L. C., Mameren, J. V., Schmidt, C. F., & Peterman, E. J. G. (2004). Combining optical trapping and single-molecule fluorescence spectroscopy: enhanced photobleaching of fluorophores. The Journal of Physical Chemistry. B, 108(20), 6479–6484. https://doi.org/10.1021/jp049805+
Dijk, Meindert A van, Lukas C. Kapitein, Joost van Mameren, Christoph F. Schmidt, and Erwin J. G. Peterman. “Combining optical trapping and single-molecule fluorescence spectroscopy: enhanced photobleaching of fluorophores.The Journal of Physical Chemistry. B 108, no. 20 (May 2004): 6479–84. https://doi.org/10.1021/jp049805+.
Dijk MAV, Kapitein LC, Mameren JV, Schmidt CF, Peterman EJG. Combining optical trapping and single-molecule fluorescence spectroscopy: enhanced photobleaching of fluorophores. The journal of physical chemistry B. 2004 May;108(20):6479–84.
Dijk, Meindert A. van, et al. “Combining optical trapping and single-molecule fluorescence spectroscopy: enhanced photobleaching of fluorophores.The Journal of Physical Chemistry. B, vol. 108, no. 20, May 2004, pp. 6479–84. Epmc, doi:10.1021/jp049805+.
Dijk MAV, Kapitein LC, Mameren JV, Schmidt CF, Peterman EJG. Combining optical trapping and single-molecule fluorescence spectroscopy: enhanced photobleaching of fluorophores. The journal of physical chemistry B. 2004 May;108(20):6479–6484.
Journal cover image

Published In

The journal of physical chemistry. B

DOI

EISSN

1520-5207

ISSN

1520-6106

Publication Date

May 2004

Volume

108

Issue

20

Start / End Page

6479 / 6484

Related Subject Headings

  • 51 Physical sciences
  • 40 Engineering
  • 34 Chemical sciences
  • 09 Engineering
  • 03 Chemical Sciences
  • 02 Physical Sciences