Fluorescence in two-photon-excited diffusible samples exposed to photobleaching: a simulation-based study

Imre B. Juhász; Árpád I. I. Csurgay

doi:10.1117/1.JBO.20.1.015001

9 January 2015 Fluorescence in two-photon-excited diffusible samples exposed to photobleaching: a simulation-based study

Imre B. Juhász, Árpád I. I. Csurgay

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Journal of Biomedical Optics, Vol. 20, Issue 1, 015001 (January 2015). https://doi.org/10.1117/1.JBO.20.1.015001

Abstract

We created a simulation model to investigate the characteristics of fluorescence in two-photon-excited samples. In the model, the sample is a diffusible solution of fluorophore molecules, which is divided into cubic cells and illuminated by a train of focused laser pulses described as a Gaussian beam. Simulating the state transitions according to a multilevel photodynamic model (also including photobleaching and intersystem crossing), the simulator provides the expected number and the spatial distribution of emitted photons over time. Our simulations demonstrated how the illumination laser power, diffusion, and the photodynamic parameters of the fluorophore affect fluorescence. We revealed the unusual fluorescent profile that evolves as photobleaching progresses: the most photons are not emitted from the focus (where a “dark hole” appears) but from an ellipsoid around the focus. The model could be adapted to several fluorescent techniques (such as two-photon microscopy and fluorescence recovery after photobleaching). Furthermore, it might help to optimize the operating parameters of the measurement devices (e.g., in order to reach higher image quality and lower photobleaching).

CC BY: © The Authors. Published by SPIE under a Creative Commons Attribution 4.0 Unported License. Distribution or reproduction of this work in whole or in part requires full attribution of the original publication, including its DOI.

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Imre B. Juhász and Árpád I. I. Csurgay "Fluorescence in two-photon-excited diffusible samples exposed to photobleaching: a simulation-based study," Journal of Biomedical Optics 20(1), 015001 (9 January 2015). https://doi.org/10.1117/1.JBO.20.1.015001

Published: 9 January 2015

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