Non-degenerate 2-photon excitation (ND-2PE) of a fluorophore with two laserbeams of different photon energies offers an independent degree of freedom intuning of the photon flux for each beam. This feature takes advantage of theinfrared wavelengths used in 3-photon microscopy to achieve an increasedpenetration depth, while preserving a relatively high degenerate 2-photonexcitation (D-2PE) cross section, exceeding that achievable with 3-photonexcitation. Here, using spatially and temporally aligned Ti:Sapphire laser andoptical parametric oscillator beams operating at near infrared (NIR) andshort-wavelength infrared (SWIR) optical frequencies, respectively, we providea practical demonstration that the emission intensity of a fluorophore excitedin the non-degenerate regime in a scattering medium is more efficient than thecommonly used D-2PE.
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