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 degenerate 3-photon excitation (D-3PE) microscopy to achieve increased penetration depths, while preserving a relatively high 2-photon excitation cross section in comparison to that of D-3PE. 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, we employ ND-2PE and provide a practical demonstration that a constant fluorophore emission intensity is achievable deeper into a scattering medium using ND-2PE as compared to the commonly used degenerate 2-photon excitation (D-2PE).