Fluorinated Voltage Sensitive Dyes for SHG and Multiphoton Microscopy

Abstract

Dyes based on hemicyanine chromophores have high membrane affinities and the ability to report on local membrane environment and act as sensors of membrane voltage. Laser-scanning second harmonic generation (SHG) microscopy utilizing such voltage sensitive dyes has shown considerable promise as an imaging modality, possessing several advantages over fluorescence for the optical mapping of membrane potentials. The addition of electronegative fluorine atoms to the chromophore is intended to lower both the ground and excited state energies, so as to make dyes less susceptible to photobleaching. Improved photostability will allow extension of the duration of optical recording measurements, permit the use of more intense laser excitation, and minimize photodamage to the biological sample. The effect of fluorination on photostability and dye performance has been systematically investigated for a series of newly synthesized dyes and found to depend critically on the location of the substituent within the chromophore. Voltage-clamped neuroblastoma cells stained with these dyes were imaged with 1064nm excitation, allowing sensitivities and response kinetics of SHG and two-photon fluorescence to be determined simultaneously for several fluorinated dyes. Our results suggest that voltage sensitive dyes can be developed which have large SHG signal changes, sufficient photostability, and the requisite speed for use as a practical tool for measuring electrical activity in biological systems.(Supported by NIH grant EB001963)