Photobleaching and photoenhancement of endogenous fluorescence observed in two-photon microscopy with broadband laser sources

We examine the effects of pulse duration tuning on the photodamage inflicted by laser light illumination on the imaged sample and, thereby, explore the optimization of optical pulse parameters for multiphoton microscopy imaging under variable conditions. We discuss the dependence of the nonlinear excitation efficiency and associated photodamage rates on pulse energy and duration, and use the controlled amount of second-order dispersion (linear chirp), introduced by a pulse shaper, to adjust the pulse duration at the imaging plane of the microscope. The pulse energy is varied to maintain a constant two-photon excitation efficiency when switching between short (∼14 fs) and long (∼280 fs) pulses, and the damage is assessed by monitoring the photobleaching rates and sample morphology. We have found that in addition to the well-known photobleaching effects, significant enhancement of the two-photon excited autofluorescence intensity can be observed. Photobleaching rates at the onset of the laser light exposure are shown to be independent of the pulse shape under our experimental conditions, which indicates that the primary damage (bleaching) mechanism stems from the two-photon excitation process. The photoenhancement, however, is found to occur more readily with longer pulses, having higher energies per pulse. Experiments are carried out on human melanoma tissue and on rabbit red blood cells.