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Proceedings Paper

Saturation in two-photon microscopy
Author(s): Gianguido C. Cianci; Keith M. Berland
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Paper Abstract

Excitation saturation and other photophysical dynamics can have a dramatic influence on the effective imaging point spread function (psf) in fluorescence microscopy. Specifically, saturation leads to increased fluorescence observation volumes and altered spatial profiles for the psf. These changes have important implications for both fluorescence correlation spectroscopy (FCS) and imaging applications. A detailed characterization of these changes is required for accurate interpretation of FCS measurements. We here introduce a method to calculate molecular excitation profiles that represent the true fluorescence observation volume under the influence of excitation saturation in two-photon microscopy. An analytical model that accounts for pulsed excitation is developed to calculate the influence of saturation at any location within the excitation laser profile, and the overall saturation influenced molecular excitation profiles are evaluated numerically. Fluorescence signals measured with a solution of Rhodamine 6G are presented, showing good agreement with these calculations.

Paper Details

Date Published: 21 June 2004
PDF: 8 pages
Proc. SPIE 5323, Multiphoton Microscopy in the Biomedical Sciences IV, (21 June 2004); doi: 10.1117/12.537954
Show Author Affiliations
Gianguido C. Cianci, Emory Univ. (United States)
Keith M. Berland, Emory Univ. (United States)

Published in SPIE Proceedings Vol. 5323:
Multiphoton Microscopy in the Biomedical Sciences IV
Ammasi Periasamy; Peter T. C. So, Editor(s)

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