Near infrared voltage sensitive dyes based on chromene electron donors.

Abstract

Excellent hemicyanine voltage sensitive dyes (VSDs) are available for optical electrophysiology over a wide spectral range out to the far red. Near infrared fluorescent VSDs would be advantageous because they allow deeper light penetration into tissue because of decreased light scattering and also because they can be used in combination with other fluorescent indicators or optogenetic actuators. The usual approach to constructing long wavelength chromophores is to lengthen the π-system; however, this is generally accompanied by a significant decrease in fluorescence quantum efficiency and can also decrease aqueous solubility, making it difficult to deliver the probes to the target tissue. The VSDs developed in our lab are generally composed of an amino-naphthyl electron donor moiety linked to a pyridinium acceptor. Here we show that replacing the naphthalene with chromene induces a 60-80 nm red-shift in the absorption and emission spectra while maintaining the structure at the same size and fluorescence quantum yield at the same level. The voltage sensitivity of these chromene-based VSDs is also comparable to our older VSDs and they display opposite polarity sensitivity when excited at the short and long wavelength edges of the absorbance spectra; thus they can be used in dual wavelength ratio measurements to eliminate artifacts from motion and bleaching. We have tested one of these VSDs in voltage imaging of a whole murine heart at 730 nm, by far the longest wavelength reported in voltage imaging. It resolves action potentials in single sweep scans with 10.1% ΔF/F per action potential. Chromene-based electron donors are likely to find wide applications in generating long wavelength hemicyanines or cyanines. (Supported by NIH grant MH116830. Conflict of Interest statement: LML, CDA, and PY are founders and owners of Potentiometric Probes LLC.)