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Microsc Res Tech. 2004 Jan 1;63(1):67-71.

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

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Department of Chemical Sciences, Tata Institute of Fundamental Research, Homi Bhabha Road, Colaba, Mumbai 400005, India.


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.

[Indexed for MEDLINE]

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