| Summary: | Abstract An improved genetically encoded voltage indicator (GEVI) was achieved by altering the charge composition of the region linking the voltage-sensing domain of the GEVI to a pH-sensitive fluorescent protein. Negatively charged linker segments reduced the voltage-dependent optical signal while positively charged linkers increased the signal size. Arginine scanning mutagenesis of the linker region improved the signal size of the GEVI, Bongwoori, yielding fluorescent signals as high as 20% ΔF/F during the firing of action potentials. The speed of this new sensor was also capable of optically resolving action potentials firing at 65 Hz. This large signal size enabled individual pixels to become surrogate electrodes. Plotting the highest correlated pixels based only on fluorescence changes reproduced the image of the neuron exhibiting activity. Furthermore, the use of a pH-sensitive fluorescent protein facilitated the detection of the acidification of the neuron during the firing of action potentials.
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