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

Abstract

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 μM for two‐photon, and 220 μM 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.