Live cell ultraviolet microscopy: A comparison between two- and three-photon excitation

We compare conventional infrared laser based three-photon excitation with a visible laser based two-photon excitation scheme for imaging the ultraviolet fluorophore serotonin in solution and in live cells. To obtain a signal level of 1,000 photons per second per mM serotonin solution, we need a back aperture power of 5 mW at 550 nm (for two-photon excitation) and 33 mW at 740 nm (for three-photon excitation). The detectivity of serotonin (defined as the concentration of serotonin that yields a signal equivalent to three times the standard deviation of the signal obtained from the buffer alone) is 12 muM for two-photon, and 220 muM for three-photon excitation. Surprisingly, for live cell imaging of vesicular serotonin in serotonergic cells, three-photon excitation appears to provide better image contrast than two-photon excitation. The origin of this is traced to the concentration-dependent shift of the serotonin emission spectrum.