Multi-site optical recording of neuronal activity with complex light patterns

We use complex light patterns to simultaneously record the neuronal activity along the dendrites of a single neuron. We use holographic projection to produce multiple foci directed onto different dendritic regions of the neuron. Each focus excites neuronal activity reporters via either two-photon (2P) or single-photon (1P) excitation. The fluorescence emanating from all foci are simultaneously recorded using an electron-multiplying charge-coupled device (EMCCD) camera thereby enabling simultaneous multi-channel recording of the neuronal activity from multiple sites at high frame rates (up to 400Hz). We report recording of neuronal activity from two types of reporters: (1) Ca2+ indicator, Cal-520; and (2) voltage indicator, JPW-1114. We optically recorded the activity evoked by the neuron following injection of current onto the soma. Holographic multi-site Ca2+ imaging resulted in high signal-to-noise ratio but with poor temporal resolution. On the other hand, multi-site voltage imaging produced noisy and low SNR signals but with high temporal resolution that is able to resolve action potentials.