CHARACTERIZING SPECTRALLY DIVERSE BIOLOGICAL CHROMOPHORES USING CAPILLARY ELECTROPHORESIS WITH MULTIPHOTON-EXCITED FLUORESCENCE

Minute quantities of native cellular fluorophores can be quantitativel y assayed using ultraviolet fluorescence detection with microcolumn se parations, but spectral diversity of biological chromophores imposes s erious limitations on the use of this strategy to investigate biologic al components. We present an approach for rapid characterization of pi coliter samples containing dissimilar cellular fluorophores-including amino acids, monoamine neurotransmitters, flavins, and pyridine nucleo tides-using multiphoton excited fluorescence detection coupled to capi llary electrophoresis separations. In this highly versatile approach, biological fluorophores are excited through the nearly simultaneous ab sorption of different numbers of low-energy photons. Because spectrall y distinct species all can be excited with a single, long-wavelength l aser source, fluorescence throughout the ultraviolet and visible regio ns can be detected efficiently with extremely low background. Samples containing serotonin, melatonin, FAD, and NADH can be reproducibly ana lyzed in 5-mu m and 2-mu m i.d. channels. Detection Limits in 5-mu m c apillaries range from 350 zmols (38 nM) for FAD to 27 amols (1.0 mu M) for serotonin. Use of 2-mu m channels is shown to improve the mass de tection limit for serotonin approximately as the decrease in capillary cross-sectional area (LOD approximate to 4 amol), and further reducti ons in mass detection Limits are projected for analysis with even smal ler diameter channels that better match the submicron size of the diff raction-limited multiphoton focal spot.