Simulation-based investigation of the three-dimensional distribution of fluorescence and photobleaching in multi-photon excited samples

Imre B. Juhász; Árpád I. Csurgay

doi:10.1117/12.2052243

1 May 2014 Simulation-based investigation of the three-dimensional distribution of fluorescence and photobleaching in multi-photon excited samples

Imre B. Juhász, Árpád I. Csurgay

Author Affiliations +

Proceedings Volume 9131, Optical Modelling and Design III; 91310S (2014) https://doi.org/10.1117/12.2052243
Event: SPIE Photonics Europe, 2014, Brussels, Belgium

Abstract

We present a numerical study on the spatial distribution of fluorescence and photobleaching occurring in samples subject to multi-photon excitation. We developed a simulation model and implemented a simulator program. Its quantitative predictions can help to find the optimal operating parameters (such as laser power, pulse length, pulse repetition rate) of the two-photon microscope to reach higher image quality, to reduce undesired photobleaching, and to pave the way for optimized photoswitching-based super-resolution imaging. Conversely, the simulator might also be useful when photodynamic parameters are searched for. Furthermore, such simulations can promote the evaluation of the results of other fluorescence-based techniques [e.g. fluorescence recovery after photobleaching (FRAP) measurements]. The photodynamic model of the fluorophore contains a ground state, an excited state, a triplet state, and several photobleached states; the state transitions are characterized by absorption cross sections and lifetimes. The sample is modeled as a fluorophore solution divided into cubic cells among which diffusion takes place. The illumination is simulated as a focused laser pulse train described by a pulsed Gaussian beam. As a demonstration of the capabilities of the simulator, an example is presented that reveals the spatial distribution of photon emission in the sample investigated by a two-photon microscope in the case of different laser and photobleaching parameters, assuming one-photon absorption induced photobleaching. The simulation demonstrates quantitatively how photobleaching affects the spatial distribution of fluorescence and the resolution of the microscope.

Citation Download Citation

Imre B. Juhász and Árpád I. Csurgay "Simulation-based investigation of the three-dimensional distribution of fluorescence and photobleaching in multi-photon excited samples", Proc. SPIE 9131, Optical Modelling and Design III, 91310S (1 May 2014); https://doi.org/10.1117/12.2052243

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