Non-degenerate 2-photon excitation in scattering medium for fluorescence microscopy

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