Study on multi-photon excited fluorescence combined with capillary electrophoresis

This paper describes a new method, multiphoton excitation fluorescence detection combined with capillary electrophoresis separation. The excitation source was a self mode-locked femtosecond titanium-sapphire laser (Spectra-physics Inc.), producing a stream of pulses with a pulse duration of about 100 fs at 82 MHz repetition rate. Its average power was about 200 mW at 750 nm. The laser beam was focused into a thin wall flow cell of capillary electrophoresis. A high numerical aperture objective (100 X NA 1.25) was chosen to focus the laser beam and to collect the fluorescence emitted by detecting molecules. Then the fluorescence was detected by a fast response PMT. All data were acquired and processed by a microcomputer. For three biological molecules, 5HT, FAD and NADH, it was demonstrated that they can be separated and detected efficiently by this method with three-photon and two-photon excitation respectively using only one 750 nm laser beam. The detection limits were 1.0 x 10(-6) mol (.) L-1 for 5HT, 7.4 x 10(-7) mol (.) L-1 for FAD and 9.8 x 10(-7) mol (.) L-1 for NADH using the criterion of three standard deviations above background. The results are much better than those with UV absorption detection by one or two magnitudes.