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

TL;DR

This paper demonstrates that non-degenerate 2-photon excitation (ND-2PE) in scattering media enhances fluorescence microscopy by allowing deeper tissue imaging with higher efficiency than traditional degenerate 2-photon excitation.

Contribution

The study provides a practical demonstration of ND-2PE using aligned laser beams, showing improved emission efficiency over degenerate 2-photon excitation in scattering media.

Findings

ND-2PE achieves higher fluorescence emission than D-2PE in scattering media.

Using NIR and SWIR laser beams enables deeper tissue imaging.

Practical setup with Ti:Sapphire and OPO lasers demonstrates improved excitation efficiency.

Abstract

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.