Improving the Photostability of Membrane Probes through Fluorination

Abstract

High photostability is a desired property for fluorescent probes, especially when long recording time is necessary. It becomes more crucial for long wavelength fluorescent dyes because they generally have lower fluorescence quantum yields and are more vulnerable to chemical and photochemical attacks. We have developed a series of optical probes with high membrane affinities that are able to report on their local membrane environment and act as sensors of membrane voltage. These dyes have hemicyanine chromophores with donor-bridge-acceptor structures. Following our recent success in developing near-infrared fluorescent membrane probes, we further tried to improve the performance of these dyes through fluorination. Synthetic pathways to hemicyanines with fluorine substitution at various positions have been developed and the performances of these fluorinated dyes have been evaluated. It was found that the effect of fluorination depends critically on the site of fluorination: donor, acceptor, or bridge. Fluorination on the vinylene bridge appears to give optimal outcomes: improvement in photostability, red-shifts in absorption and emission spectra, and increase in absorption cross section. On the other hand, fluorination on the pyridinium acceptor results in an undesirable outcome: the dye is much less stable than the nonfluorinated counterpart in PBS buffer. Fluorinated membrane dyes generally show response kinetics and voltage sensitivities similar to their nonfluorinated counterparts when tested in a voltage-clamped hemispherical lipid bilayer (HLB) apparatus and in nonlinear optical imaging. (Supported by NIH grants EB001963 and U54RR022232)