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 1000 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 microM for two-photon, and 220 microM 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.
Balaji
J
J
Department of Chemical Sciences, Tata Institute of Fundamental Research, Homi Bhabha Road, Colaba, Mumbai 400005, India.
Desai
R
R
Maiti
S
S
D000818:Animals; D004867:Equipment Design; D031541:Fluorescence Resonance Energy Transfer / Q000295:instrumentation / Q000379:methods; D036641:Microscopy, Fluorescence, Multiphoton / Q000379:methods*; D008860:Microscopy, Ultraviolet / Q000295:instrumentation / Q000379:methods*; D009474:Neurons / Q000166:cytology; D017785:Photons; D011506:Proteins / Q000032:analysis*; D012701:Serotonin / Q000032:analysis
D011506:Proteins; D012701:Serotonin