Pulse shaping for reducing photodamage in multiphoton microscopy

We formulate a simple strategy for mitigation of laser-induced damage through pulse shaping and demonstrate experimentally the effect of laser pulse duration on the degree of optically induced damage for two-photon microscopy imaging. We use a broadband Ti:Sapphire laser source, aided with a shaper, and adjust both the laser pulse duration and energy to maintain constant two-photon excitation efficiency. The damage is assessed by the dynamics of two-photon excited autofluorescence intensity and sample morphology during prolonged laser exposure. We observe that for a 5-μm layer of skin tissue the damage rate is independent of the pulse shape, which suggests that the primary damage (bleaching) mechanism stems from the two-photon excitation itself. For optically thick dried blood samples, taken as another example, the data suggests that the damage is driven by one-photon absorption. In the later case, it is favorable to use shorter laser pulses to mitigate photodamage while maintaining adequate intensity of two-photon excited autofluorescence.