MULTIPHOTON-EXCITED FLUORESCENCE OF FLUOROGEN-LABELED NEUROTRANSMITTERS

Fluorescence detection of fluorogen-labeled neurotransmitters is demon strated using 100 fs pulses from a titanium-sapphire mode-locked laser to achieve molecular excitation by simultaneous absorption of two and three photons of near-IR radiation. Two-photon excitation spectra are determined for the naphthalene-2,3-dicarboxaldehyde derivative of gly cine and the fluorescamine derivative of leucine enkephalin, with the peak excitation cross section (sigma(2)) approximately equal to 1 x 10 (-50) cm(4) s/photon for both species. Three-photon-excited fluorescen ce is demonstrated for o-phthaldialdehyde-labeled glutamate using exci tation wavelengths between 965 and 1012 nm. The three-photon excitatio n cross section (sigma(3)) remains nearly constant in this wavelength range, with an absolute value of similar to 10(-84)-10(-85) cm(6) s(2) /photon(2). Rapid cycling of analytes through the fluorescent excited state and detection that is free from background caused by Rayleigh an d Raman scatter combine to make multiphoton-excited fluorescence a hig hly sensitive approach for detecting trace amounts of neurotransmitter s. Measurements of two-photon-excited fluorescence of fluorescamine-la beled bradykinin and analysis of multiphoton-excited background reveal the potential of this method to detect fewer than 1000 neurotransmitt er molecules.