UC Berkeley

Light can be used to provide an optical readout of relative changes in membrane potential in a minimally invasive manner with high spatiotemporal resolution via the use of fluorescent voltage reporters. These reporters localize to cell membranes and are composed of three primary elements: a fluorophore, a molecular wire, and an electron rich aniline donor. The donor modulates the fluorescence of the reporter in response to changes in the membrane potential of a cell through a photo induced electron transfer (PeT) process. The existing color palette of these reporters is limited to the visible range of light (< 700 nm). Near-infrared (NIR) fluorescent dyes are favorable since the use of NIR light avoids cellular autofluorescence, is less damaging than visible light, and can be used simultaneously with other tools in the visible spectrum such as calcium indicators and optogenetic actuators. To take advantage of these properties, we synthesized and characterized four NIR voltage reporters using a phosphine oxide rhodamine fluorophore with a maximum absorbance at 704 nm and peak emission at 723 nm. These NIR phosphine oxide rhodamine voltage reporters, poRhoVRs, exhibit fractional changes in fluorescence ranging from 13-43% per 100 mV with good signal to noise ratios. We have successfully used poRhoVR to monitor spontaneous neuronal activity in cultured neurons as well as activity evoked through field stimulation or via optogenetic activation. We have also used poRhoVR in mouse retina in conjunction with GCaMP6f, to monitor pharmacologically induced changes in activity, while offering greater spatial resolution than possible with multi-electrode arrays. These poRhoVR dyes can easily be modified to be genetically targetable by appending chemical handles for proteins such as HaloTag.