RAPID COMMUNICATION: ON THE POSSIBILITY OF CALCIUM IMAGING USING INDO‐1 WITH THREE‐PHOTON EXCITATION

Abstract— We show that the calcium fiuorophore Indo‐1 can be excited by simultaneous absorption of three‐photons at 885 nra, 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 17μ 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 Tksapphire lasers, and may provide higher spatial resolution than available using two‐photon excitation.