Acquisition of voltage and calcium responses to visual motion. (a) Setup for simultaneous electrophysiology and calcium imaging by multifocal two-photon and conventional wide-field microscopy. Insets show the recording site (top left) and the multifocal beamsplitter for two-photon microscopy (bottom left). The motion stimulus consists of a drifting square wave grating generated by a light emitting diode board. (b) Response of a vertical system (VS)2/3 neuron to a grating moving in preferred (bottom left) and antipreferred (top left) direction and directional tuning (right). Arrow directions indicate the direction of motion; arrow lengths represent response amplitudes averaged over an interval of 4 s starting at motion onset minus the mean response during 2 s before motion onset. Black arrows signal increases (depolarization); red arrows signal decreases (hyperpolarization). (c) Calcium response to downward motion of the same VS cell stained with Oregon Green BAPTA-1. Time course of the calcium signal integrated over the whole dendrite (left) and series of colour-coded ΔF/F0 images showing local differences in fluorescence intensity for various time points (right). Resting fluorescence F0 was determined by averaging the last three frames before start of pattern motion. Images were taken at 10 Hz and 512 × 512 pixel resolution.