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Applied Optics

Applied Optics

APPLICATIONS-CENTERED RESEARCH IN OPTICS

  • Editor: James C. Wyant
  • Vol. 46, Iss. 30 — Oct. 20, 2007
  • pp: 7401–7411

Role of three-dimensional bleach distribution in confocal and two-photon fluorescence recovery after photobleaching experiments

Davide Mazza, Francesca Cella, Giuseppe Vicidomini, Silke Krol, and Alberto Diaspro  »View Author Affiliations


Applied Optics, Vol. 46, Issue 30, pp. 7401-7411 (2007)
http://dx.doi.org/10.1364/AO.46.007401


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Abstract

The quantitative analysis of fluorescence perturbation experiments such as fluorescence recovery after photobleaching (FRAP) requires suitable analytical models to be developed. When diffusion in 3D environments is considered, the description of the initial condition produced by the perturbation (i.e., the photobleaching of a selected region in FRAP) represents a crucial aspect. Though it is widely known that bleaching profiles approximations can lead to errors in quantitative FRAP measurements, a detailed analysis of the sources and the effects of these approximations has never been conducted until now. In this study, we measured the experimental 3D bleaching distributions obtained in conventional and two-photon excitation schemes and analyzed the deviations from the ideal cases usually adopted in FRAP experiments. In addition, we considered the non-first-order effects generated by the high energy pulses usually delivered in FRAP experiments. These data have been used for finite-element simulations mimicking FRAP experiments on free diffusing molecules and compared with FRAP model curves based on the ideal bleach distributions. The results show that two-photon excitation more closely fits ideal bleaching patterns even in the event of fluorescence saturation, achieving estimations of diffusion coefficients within 20 % accuracy of the correct value.

© 2007 Optical Society of America

OCIS Codes
(180.1790) Microscopy : Confocal microscopy
(180.2520) Microscopy : Fluorescence microscopy
(190.4180) Nonlinear optics : Multiphoton processes
(260.2510) Physical optics : Fluorescence
(300.6460) Spectroscopy : Spectroscopy, saturation

ToC Category:
Microscopy

History
Original Manuscript: May 8, 2007
Revised Manuscript: August 21, 2007
Manuscript Accepted: August 22, 2007
Published: October 10, 2007

Virtual Issues
Vol. 2, Iss. 11 Virtual Journal for Biomedical Optics

Citation
Davide Mazza, Francesca Cella, Giuseppe Vicidomini, Silke Krol, and Alberto Diaspro, "Role of three-dimensional bleach distribution in confocal and two-photon fluorescence recovery after photobleaching experiments," Appl. Opt. 46, 7401-7411 (2007)
http://www.opticsinfobase.org/ao/abstract.cfm?URI=ao-46-30-7401

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