Fluorogenic Targeting of Voltage-Sensitive Dyes to Neurons.

We present a method to target voltage-sensitive fluorescent dyes to specified cells using an enzyme-catalyzed fluorogenic reaction on cell surfaces. The dye/enzyme hybrids are composed of a photoinduced electron transfer (PeT)-based fluorescent voltage indicator and a complementary enzyme expressed on the cell surface. Action of the exogenous enzyme on the dye results in fluorogenic activation of the dye, enabling fast voltage imaging in defined neurons with sensitivity surpassing those of purely genetically encoded approaches. We employ a bulky methylcyclopropylacetoxymethyl ether to diminish the fluorescence of a PeT-based voltage-sensitive dye, or VoltageFluor. The hydrolytically stable ether can be removed by the action of porcine liver esterase (PLE) to reveal the bright unmodified VoltageFluor. We established that the chemically modified VoltageFluor is a substrate for PLE in vitro and in live cells. When PLE is targeted to the external face of cell membranes, it controls the apparent staining of cells. The use of neuron-specific promoters can direct staining to mammalian neurons to provide clear detection of neuronal action potentials in single trials. All of the new VoltageFluors targeted by esterase expression (VF-EXs) report single spikes in cultured mammalian neurons. The best, VF-EX2, does so with a signal-to-noise ratio nearly double that of comparable genetically encoded voltage reporters. By targeting PLE to neurons, VF-EX2 can interrogate the neuromodulatory effects of serotonin in cultured hippocampal neurons. Taken together, our results show that a combination of synthetic chemistry and biochemistry enables bright and fast voltage imaging from genetically defined neurons in culture.