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We demonstrate efficient generation of wide-field fluorescence signals in two-photon microscopy exploiting diffractive optical elements and short pulses by using a dispersion-compensated beam delivery optics module. Computer-generated holograms are codified onto a phase-only spatial light modulator, which allows for arbitrary single-shot patterning of the sample. Spatiotemporal shaping of the pulse is mandatory to overcome spatial chirp and pulse-front tilt effects that spread both in space and time the irradiance patterns, thus limiting not only the spatial resolution but also the signal-to-noise ratio in two-photon microscopy. By using a multipass amplifier delivering 30 fs, 0.8 mJ pulses at 1 kHz repetition rate, we experimentally demonstrated arbitrary single-shot fluorescence irradiance patterns in Rhodamine B.

Dispersion management in two-photon microscopy by using diffractive optical elements