2ND HARMONIC DETECTION OF SPATIALLY FILTERED 2-PHOTON EXCITED FLUORESCENCE

Spatial filtering of two-photon excited fluorescence combined with second harmonic detection has demonstrated a reduction in the signal blank resulting in improved detection limits. The second harmonic detection fluorometer is based on the recovery of the second harmonic produced as a result of nonlinear absorption of two photons from a sinusoidally modulated laser. This instrument is capable of acquiring corrected two-photon excitation spectra rapidly while simultaneously providing detection limits as low as 200 pM (S/N = 3) for strong absorbers such as p-bis(o-methyistyryl)benzene with an 82 MHz train of 12-ps pulses having an average power of 150 mW. Various instrumental contributions to the noise spectrum are examined. A titanium:sapphire laser was used to demonstrate the compatibility of very short laser pulses with second harmonic detection. A detection limit of 7.55 nM was obtained for the laser dye coumarin-480 (S/N = 3) with an 82 MHz train of 1-ps pulses having an average power of 650 mW.