Introduction: Calcium (Ca) sparks are elementary units of subcellular Ca release in cardiomyocytes and other cells. Accordingly, Ca spark imaging is an essential tool for understanding physiology and pathophysiology of Ca handling and is used to identify new drugs targeting Ca-related cellular dysfunction (e.g., cardiac arrhythmias). The large volumes of imaging data produced during such experiments require accurate and high-throughput analysis.
Objective: To produce a new improved software tool for the analysis of Ca sparks imaged by confocal line-scan microscopy, combining high accuracy, flexibility, and user-friendliness.
Methods and Results: We created a new open-source Python-based software tool SparkMaster 2 (SM2) for the analysis of line-scan spark imaging data, with the following key strengths: 1) high accuracy at identifying Ca release events, clearly outperforming previous highly successful software SparkMaster; 2) multiple types of Ca release events can be identified using SM2: Ca sparks, waves, mini-waves, and long sparks; 3) SM2 can accurately split and analyze individual sparks within spark clusters, a capability not handled adequately by prior tools. We demonstrate the practical utility of SM2 on two case studies, investigating how Ca levels affect spontaneous Ca release, and how large-scale release events may promote release refractoriness. Broad usefulness across cell types and imaging conditions is also shown. SM2 is distributed as a stand-alone application requiring no installation. It can be controlled using a simple-to-use graphical user interface, or using Python scripting.
Conclusions: SparkMaster 2 is a new and much improved user-friendly software for accurate high-throughput analysis of line-scan Ca spark imaging data. It is free, easy to use and provides valuable built-in features to facilitate visualization, analysis and interpretation of Ca spark data. It should enhance the quality and throughput of Ca spark and wave analysis across cell types, particularly in the study of arrhythmogenic Ca release events in cardiac myocytes.