Characterizing Spectrally Diverse Biological Chromophores Using Capillary Electrophoresis with Multiphoton-Excited Fluorescence

Minute quantities of native cellular fluorophores can be quantitatively assayed using ultraviolet fluorescence detection with microcolumn separations, but spectral diversity of biological chromophores imposes serious limitations on the use of this strategy to investigate biological components. We present an approach for rapid characterization of picoliter samples containing dissimilar cellular fluorophoresincluding amino acids, monoamine neurotransmitters, flavins, and pyridine nucleotidesusing multiphoton excited fluorescence detection coupled to capillary electrophoresis separations. In this highly versatile approach, biological fluorophores are excited through the nearly simultaneous absorption of different numbers of low-energy photons. Because spectrally distinct species all can be excited with a single, long-wavelength laser source, fluorescence throughout the ultraviolet and visible regions can be detected efficiently with extremely low background. Samples containing serotonin, melatonin, FAD, and ...