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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. |
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Luminescence
Spatial resolution
Multiphoton microscopy
Modulation
Point spread functions
Signal detection
Confocal microscopy
