An open source toolbox for analysis of calcium imaging data


Fluorescence calcium imaging is emerging as one of the preferred methods to record neuronal activity. Because changes in the fluorescence intensity of genetically-encoded or chemical calcium indicators correlate with action potential firing in neurons, data analysis is based on extracting pixel intensity values across time for different regions of interest. Unfortunately, the algorithms necessary to extract biologically relevant information from these fluorescent signals are complex and require significant expertise in programming to develop robust analysis pipelines.

Currently, the publicly available methods for performing these analyses are custom-written, often poorly annotated and lack intuitive graphical user interfaces (GUI). As a result, there is a barrier for many laboratories to adopt these tools, especially for potential users without sufficient programming knowledge, despite the increasing access to affordable miniscopes and 2-photon microscopes. Indeed, the bottleneck is no longer the hardware, but the software to analyze the calcium data.

We have created an open-source, easy to use, GUI-based, intuitive and automated data analysis software (EZcalcium) for analyzing dynamic fluorescence calcium signals from defined regions of interest.


EZcalcium automates all of the core aspects of calcium imaging analysis:

- image registration (motion correction)
- image segmentation (and refinement) into distinct regions of interest (ROI)
- signal extraction and deconvolution (dimension reduction)
- data exporting and plotting


3 distinct modules:

1. Motion Correction
2. ROI Detection
3. ROI Refinement


EZcalcium is fully automated
Allows for batch processing
Compatible with standard desktop or laptop computers
The code is well-documented The code is wrapped in a set of user-friendly and intuitive GUIs The toolbox is optimized for speed


To download the latest version of EZcalcium please go to Github:





A screenshot of the program.