Development and characterization of a capillary electrophoresis fluorimeter based on two-photon excitation

A capillary electrophoresis fluorimeter has been built based on two-photon excitation. Signal-to-noise enhancements have been investigated in three areas: electronic, optical, and chemical. Major findings include the nonideal behavior in the two-photon detection volume size, the presence of a two-photon absorbing impurity in the walls of quartz capillaries, and the discovery of enzymatic tags suitable for two-photon excitation. The two-photon detection volume was measured by photography and by fitting the fluorescence to a cumulative distribution function and was found to be a factor of seven larger than the size predicted by Gaussian optics. When two-photon excitation was applied to microbore capillaries, a significant chemical blank was observed which originated from the walls of the capillary. The background fluorescence was eventually removed with a spatial filter. Using a $90\sp\circ$ excitation/collection arrangement with 0.25 numerical aperture microscope objectives, it was determined that the two-photon detection volume was larger than the inner diameter of a 75-$\rm\mu m$ i.d. capillary. The truncation allowed the observation of less than 15% of the total observable fluorescence. Six amine-containing fluorophores were evaluated for their use in enzymatic assays using two-photon excitation. After investigating the spectral and chemical properties of 2-naphthylamine, 4-methoxy-2-naphthylamine, 7-amino-4-methylcoumarin, 2-aminoacridone, 6-aminoquinoline, and 5-dimethylamino-1-naphthalenesulfonyl chloride, it was found that the coumarin tag provided the highest absolute sensitivity while providing the spectral characteristics to allow the rejection of much of the interference caused by the a tagged substrate