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

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.