Near infrared two-photon excitation cross-sections of voltage-sensitive dyes

Microscopy based on voltage-sensitive dyes has proven effective for revealing spatio-temporal patterns of neuronal activity in vivo and in vitro. Two-photon microscopy using voltage-sensitive dyes offers the possibility of wide-field visualization of membrane potential on sub-cellular length scales, hundreds of microns below the tissue surface. Very little information is available, however, about the utility of voltage-sensitive dyes for two-photon imaging purposes. Here we report on measurements of two-photon fluorescence excitation cross-sections for nine voltage-sensitive dyes in a solvent, octanol, intended to simulate the membrane environment. Ultrashort light pulses from a Ti:sapphire laser were used for excitation from 790 to 960 nm, and fluorescein dye was used as a calibration standard. Overall, dyes RH795, RH421, RH414, di-8-ANEPPS, and di-8-ANEPPDHQ had the largest two-photon excitation cross-sections (∼15 × 10−50 cm4 s photon−1) in this wavelength region and are therefore potentially useful for two-photon microscopy. Interestingly, di-8-ANEPPDHQ, a chimera constructed from the potentiometric dyes RH795 and di-8-ANEPPS, exhibited larger cross-sections than either of its constituents.