On the possibility of calcium imaging using Indo-1 with three-photon excitation.


We show that the calcium fluorophore Indo-1 can be excited by simultaneous absorption of three-photons at 885 nm, a wavelength readily available from Ti:sapphire lasers. Three-photon excitation was demonstrated by the emission intensity of Indo-1 which depended on the cube of the laser power, and by a higher anisotropy than was observed for two-photon excitation. Excitation of Indo-1 becomes a two-photon process when the wavelength is decreased to 820 nm. Three-photon excitation was accomplished at a low 17 microM concentration of Indo-1. Examination of the spatial profile of the excited Indo-1 showed a smaller volume for three- versus two-photon excitation. These results suggest that three-photon excitation may be useful in fluorescence microscopy using the long wavelength output of Ti:sapphire lasers, and may provide higher spatial resolution than available using two-photon excitation.

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@article{Gryczyski1995OnTP, title={On the possibility of calcium imaging using Indo-1 with three-photon excitation.}, author={Ignacy Gryczyński and Henryk Szmacinski and Joseph. R. Lakowicz}, journal={Photochemistry and photobiology}, year={1995}, volume={62 4}, pages={804-8} }