Positive-going hybrid indicators for voltage imaging in excitable cells and tissues

Membrane potential dynamics are crucial to the physiology of electrically excitable cells, such as neurons, cardiomyocytes, and pancreatic islet cells. Fluorescence voltage indicators have enabled the visualization of electrical activities at the single-cell level across large cell populations. However, most existing indicators have a negative fluorescence-voltage response, resulting in a high background signal when cells are at their resting state. To reduce background interference during voltage imaging, we developed a positive-going hybrid voltage indicator (HVI⁺), which has lower intensity at the resting state and becomes brighter upon membrane depolarization. HVI⁺-Cy3b exhibits a remarkable voltage sensitivity of 55% ΔF/F₀ per 100 mV, enabling accurate reporting of action potential waveforms in beating cardiomyocytes and neurons. In addition, by combining HVI⁺-Cy3b with HVI-Cy3b, we demonstrate the capability to simultaneously assess the effects of glucose on the spike activities of two cell types in pancreatic islets.