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