TACI: an ImageJ plugin for 3D calcium imaging analysis

Abstract

Research in the field of neuroscience has evolved to use complex imaging and computational tools to extract comprehensive information from data sets. Calcium imaging is a widely used technique that requires sophisticated software to obtain reproducible results, but many laboratories struggle to adopt computational methods when updating protocols to meet modern standards. Difficulties arise due to the lack of computational knowledge and paywalls for software. In addition, most calcium imaging analysis approaches ignore motion on the z-axis. Here, we described a workflow to use ImageJ to analyze 3D calcium imaging. We applied TrackMate, an open-source ImageJ plugin, to track neurons in the lateral (x/y) direction, detect regions of interest (ROIs), and extract fluorescence intensities. To track motion on the z-axis, we developed a new ImageJ plugin, TrackMate Analysis of Calcium Imaging (TACI). For neurons appearing on multiple z-positions, maximum fluorescence values were identified to represent neurons’ intensities of corresponding z-stacks. This workflow does not require coding ability, avoids human bias, and increases reproducibility. We validated this workflow using fly larval thermosensitive neurons that displayed movements in all directions during temperature fluctuation and a 3D calcium imaging dataset acquired from the fly brain.