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We demonstrate a significant resolution enhancement beyond the conventionallimit in multiphoton microscopy (MPM) using saturated excitation offluorescence. Our technique achieves super-resolved imaging by temporallymodulating the excitation laser-intensity and demodulating the higher harmonicsfrom the saturated fluorescence signal. The improvement of the lateral andaxial resolutions is measured on a sample of fluorescent microspheres. Whilethe third harmonic already provides an enhanced resolution, we show that afurther improvement can be obtained with an appropriate linear combination ofthe demodulated harmonics. Finally, we present in vitro imaging of fluorescentmicrospheres incorporated in HeLa cells to show that this technique performswell in biological samples.

3D super-resolved in vitro multiphoton microscopy by saturation of excitation