Two-color (two-photon) excitation fluorescence with two confocal beams and a Raman shifter

Abstract

Two-color (two-photon) excitation fluorescence (2CF) with two confocal excitation beams is demonstrated with a Raman shifter as excitation light source. Coumarin 6H dye sample (peak absorption≈394 nm, peak fluorescence≈490 nm) is excited using the first two Stokes outputs (683, 954 nm, two-color excitation=398 nm) of a Raman shifter pumped by a 6.5 ns-pulsed 532 nm-Nd:YAG laser (repetition rate=10 Hz). The two Stokes pulses overlap for about 6 ns and two-color fluorescence is generated even with focusing objectives of low numerical apertures (NA<0.4). We observed the linear dependence of the two-color fluorescence signal with the product of the average intensities of the two Stokes excitation beams. The two-color fluorescence distribution is strongly localized around the common focus of the confocal excitation beams.