Protocol for using the multi-cellular analysis toolbox in ImageJ for single-cell calcium imaging analysis

Functional imaging using genetically encoded indicators (GEIs) allows researchers to understand neuronal-, circuit-, and organism-level mechanisms that govern sensory processing and brain function. Here, we present a protocol to extract calcium signals and significant changes in neuronal activity from zebrafish calcium imaging data using the multi-cellular analysis (MCA) toolbox in ImageJ. We describe steps for installing MCA, using all functions within MCA, and plotting MCA outputs. This pipeline can be adapted for use in diverse model systems. For complete details on the use and execution of this protocol for multiple model systems, please refer to Hageter et al.¹.