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Recent advances combining two-photon calcium imaging and two-photon optogenetics with digital holography now allow us to read and write neural activity in vivo at cellular resolution with millisecond temporal precision. Such “all-optical” techniques enable experimenters to probe the impact of functionally defined neurons on neural circuit function and behavioural output with new levels of precision. This protocol describes the experimental strategy and workflow for successful completion of typical all-optical interrogation experiments in awake, behaving head-fixed mice. We describe modular procedures for the setup and calibration of an all-optical system, the preparation of an indicator and opsin-expressing and task-performing animal, the characterization of functional and photostimulation responses and the design and implementation of an all-optical experiment. We discuss optimizations for efficiently selecting and targeting neuronal ensembles for photostimulation sequences, as well as generating photostimulation response maps from the imaging data that can be used to examine the impact of photostimulation on the local circuit. We demonstrate the utility of this strategy using all-optical experiments in three different brain areas – barrel cortex, visual cortex and hippocampus – using different experimental setups. This approach can in principle be adapted to any brain area for all-optical interrogation experiments to probe functional connectivity in neural circuits and for investigating the relationship between neural circuit activity and behaviour.

Original publication




Journal article


Cold Spring Harbor Laboratory

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