Optogenetic activation during detector "dead time" enables compatible real-time fluorescence imaging.

Optogenetic tools, such as channelrhodopsin2 (ChR2), have enabled the behavior of whole organisms by light-mediated manipulation of neuronal activities. Fluorescent indicators have been used to aid in the understanding of what is happening in living cells. To date, optogenetic stimulation and imaging acquisition were sequentially performed during detector "live time." However, there is a problem with interrupting acquisition time sequences because such stimulation invades the time territory of fluorescent imaging. Here, our purpose was to show that optogenetic stimulation can be performed within the "dead time" of the charge-coupled device camera, the short interval of data transfer between frames. We show the kinetic measurement of Ca(2+) dynamics in neuron-like cells upon ChR2 stimulation, by which we reveal the biphasic property of the Ca(2+) increase in response to optical stimulation.