Bright and Photostable Voltage Sensors Derived from mBaoJin

Genetically encoded voltage indicators (GEVIs) are powerful tools for monitoring neuronal activity, but their application, particularly for long-term recordingsin vivo, is often limited by photobleaching under the required high illumination intensities. This constraint restricts the total duration of continuous or trial-based experiments, crucial for studying processes like synaptic plasticity or circuit dynamics during behavior. Here, we introduce ElectraON and ElectraOFF, a pair of green fluorescent eFRET-based GEVIs engineered by incorporating a photostability-enhanced derivative of the bright monomeric fluorescent protein mBaoJin with Ace opsin variants. Critically, Electras demonstrate over 6-fold improved photostability compared to state-of-the-art eFRET GEVIs, pAce, and Ace-mNeon2, under one-photon illumination, while characterized by bright green fluorescence, millisecond kinetics, and good membrane localization. This enhanced stability translates to a 3-to >10-fold extension in functional recording duration, maintaining reliable spike detection in both cultured neuronsin vitroand sparsely labeled neurons in the awake mouse cortexin vivo. We demonstrated sustainedin vivorecordings exceeding 30 minutes, with instances surpassing one hour. Furthermore, Electras show functionality under scanless two-photon excitation in cultured cells. These highly photostable indicators significantly extend the temporal window for voltage imaging, broadening the scope of accessible biological questions.