Stimulation and continuous monitoring of neural activities at cellular resolution are required for the understanding of the sensory processing of stimuli and development of effective neuromodulation therapies. We present bioluminescence multi-characteristic opsin (bMCOII), a hybrid optogenetic actuator, and a bioluminescence Ca2+ sensor for excitation-free, continuous monitoring of neural activities in the visual cortex, with high spatiotemporal resolution. An exceptionally low intensity (10 μW/mm2) of light could elicit neural activation that could be detected by Ca2+ bioluminescence imaging. An uninterrupted (>14 h) recording of visually evoked neural activities in the cortex of mice enabled the determination of strength of sensory activation. Furthermore, an artificial intelligence-based neural activation parameter transformed Ca2+ bioluminescence signals to network activity patterns. During continuous Ca2+-bioluminescence recordings, visual cortical activity peaked at the seventh to eighth hour of anesthesia, coinciding with circadian rhythm. For both direct optogenetic stimulation in cortical slices and visually evoked activities in the visual cortex, we observed secondary delayed Ca2+-bioluminescence responses, suggesting the involvement of neuron-astrocyte-neuron pathway. Our approach will enable the development of a modular and scalable interface system capable of serving a multiplicity of applications to modulate and monitor large-scale activities in the brain.
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