The development of in vivo imaging and optogenetic tools makes it possible to control neural circuit activities in an all-optical, closed-loop manner , but such applications are limited by the lack of software for online analysis of neuronal imaging data. We developed ORCA (Online Real-time activity and offline Cross-session Analysis), which performs image registration, neuron segmentation, and activity extraction at over 100 frames per second, fast enough for real-time readout of neural activity. Using ORCA, we demonstrate closed-loop control of neurons in the mouse auditory cortex by two-photon targeted optogenetic inhibition of sound-evoked activity in selected neurons. ORCA also includes an offline module that processes hour-long imaging sessions within minutes and efficiently tracks neurons across multiple sessions. With a modular design, ORCA can be easily integrated into existing analysis toolboxes. In summary, ORCA is a powerful toolbox for closed-loop neuronal manipulation and fast data analysis.
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