Using simultaneous voltage and calcium imaging to study fast Ca(2+) channels

The combination of fluorescence measurements of membrane potential and intracellular [Formula: see text] concentration allows correlating the electrical and calcium activity of a cell with spatial precision. The technical advances allowing this type of measurement were achieved only recently and represent an important step in the progress of the voltage imaging approach pioneered over 40 years ago by Lawrence B. Cohen. Here, we show how this approach can be used to investigate the function of [Formula: see text] channels using the foreseen possibility to extract [Formula: see text] currents from imaging experiments. The kinetics of the [Formula: see text] current, mediated by voltage-gated [Formula: see text] channels, can be accurately derived from the [Formula: see text] fluorescence measurement using [Formula: see text] indicators with [Formula: see text] that equilibrate in [Formula: see text]. In this respect, the imaging apparatus dedicated to this application is described in detail. Next, we illustrate the mathematical procedure to extract the current from the [Formula: see text] fluorescence change, including a method to calibrate the signal to charge flux density. Finally, we show an example of simultaneous membrane potential and [Formula: see text] optical measurement associated with an action potential at a CA1 hippocampal pyramidal neuron from a mouse brain slice. The advantages and limitations of this approach are discussed.