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.
D002118:Calcium / Q000032:analysis*; D002614:Chelating Agents; D005454:Fluorescence Polarization / Q000295:instrumentation / Q000379:methods; D005456:Fluorescent Dyes; D007211:Indoles; D008856:Microscopy, Fluorescence / Q000295:instrumentation / Q000379:methods; D017785:Photons; D013050:Spectrometry, Fluorescence / Q000295:instrumentation / Q000379:methods
D002614:Chelating Agents; D005456:Fluorescent Dyes; D007211:Indoles; C048960:indo-1; D002118:Calcium