Multiphoton-Excited Fluorescence of Fluorogen-Labeled Neurotransmitters

Abstract

Fluorescence detection of fluorogen-labeled neurotransmitters is demonstrated 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 glycine and the fluorescamine derivative of leucine enkephalin, with the peak excitation cross section (σ₂) approximately equal to 1 × 10^-50 cm⁴ s/photon for both species. Three-photon-excited fluorescence is demonstrated for o-phthaldialdehyde-labeled glutamate using excitation wavelengths between 965 and 1012 nm. The three-photon excitation cross section (σ₃) remains nearly constant in this wavelength range, with an absolute value of ∼10^-84−10^-85 cm⁶ s²/photon². Rapid cycling of analytes through the fluorescent excited state and detection that is free from background caused by Rayleigh and Raman scatter combine to make multiphoton-excited fluorescence a highly sensitive approach for detecting trace amounts of neurotransmitters. Measurements of two-photon-excited fluorescence of fluorescamine-labeled bradykinin and analysis of multiphoton-excited background reveal the potential of this method to detect fewer than 1000 neurotransmitter molecules.