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 fluorophoresincluding amino acids, monoamine neurotransmitters, flavins, and pyridine nucleotidesusing 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 NADH can be reproducibly analyzed in 5-μm and 2-μm i.d. channels. Detection limits in 5-μm capillaries range from 350 zmols (38 nM) for FAD to 27 amols (1.0 μM) for serotonin. Use of 2-μm channels is shown to improve the mass detection limit for serotonin approximately as the decrease in capillary cross-sectional area (LOD ≈ 4 amol), and further reductions in mass detection limits are projected for analysis with even smaller diameter channels that better match the submicron size of the diffraction-limited multiphoton focal spot.