Improvement of the spatial resolution in multiphoton microscopy by saturated excitation of fluorescence

We demonstrate, for the first time to the best of our knowledge, a significant resolution improvement in multiphoton microscopy (MPM) using saturated excitation of fluorescence. At high illumination power, saturation of the two-photon excitation probability of the fluorophore occurs and induces a strong nonlinear response. By temporally modulating the excitation laser-intensity and demodulating high-order harmonics from the saturated fluorescence signal, images with higher spatial resolution than with standard MPM can be obtained. We show, as expected from a two-photon excitation process, that resolution improvement arises when demodulating at least at the third harmonic and that linear combinations of harmonics provide further improvement of the resolution. Images of 200 nm fluorescent microspheres confirm the improvement of the spatial resolution.