Large-scale optical voltage imaging in behaving animals

Recent improvements in genetically encoded voltage indicators have enabled optical imaging of action potentials and subthreshold membrane voltage from single neurons in the mammalian brain. However, most current voltage imaging techniques can only simultaneously sample a few cell, limited either by strong background or small field-of-view. We show that, both theoretically and experimentally, targeted illumination with a widefield microscopy can significantly improve voltage imaging performance by improving signal contrast and reducing background cross-contamination. With such improvements, we demonstrated large-scale voltage imaging with fully genetically encoded voltage indicator SomArchon from tens of neurons over a large anatomical area, while maintaining signal contrast over a prolonged recording duration of several continuous minutes.