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 Ca2+ dynamics in neuron-like cells upon ChR2 stimulation, by which we reveal the biphasic property of the Ca2+ increase in response to optical stimulation.

► Use of “dead-time” of CCD camera for optogenetic activation maximizes time resolution of fluorescence image acquisition.
► Exclusion of leak signal from optogenetic stimulation minimizes background signal.
► We successfully captured in-depth kinetic information of Ca2+ increase during optogenetic stimulations.