Optical sectioning in wide-field two-photon microscopy using temporal focusing and random illumination.

Temporal focusing (TF) is a key tool in wide-field two-photon excitation fluorescence microscopy for confining fluorescence excitation to a thin layer around the focal plane. TF is typically implemented with 100 femtosecond laser pulses and large microscope magnifications (>40). In this work, we demonstrate that performing TF with a random speckled illumination rather than a collimated beam significantly improves the optical sectioning for long pulses and small magnifications. We derive simple formulas of the optical sectioning as a function of speckle angular divergence, pulse bandwidth, and microscope parameters. Our approach paves the way for optically sectioned wide-field nonlinear imaging using >200 fs pulses and low magnifications enabling large fields of view.