CalQuo ² : Automated Fourier-space, population-level quantification of global intracellular calcium responses

Intracellular calcium acts as a secondary messenger in a wide variety of crucial biological signaling processes. Advances in fluorescence microscopy and calcium sensitive dyes has led to the routine quantification of calcium responses in non-excitable cells. However, the automatization of global intracellular calcium analysis at the single-cell level within a large population simultaneously remains challenging. One software, CalQuo (Calcium Quantification), offers some automatic features in calcium analysis. Here, we present an advanced version of the software package: CalQuo ² . CalQuo ² analyzes the calcium response in the Fourier-domain, allowing the number of user-defined filtering parameters to be reduced to one and a greater diversity of calcium responses to be recognized, compared to CalQuo that directly interprets the calcium intensity signal. CalQuo ² differentiates cells that release a single calcium response and those that release oscillatory calcium fluxes. We have demonstrated the use of CalQuo ² by measuring the calcium response in genetically modified Jurkat T-cells under varying ligand conditions, in which we show that peptide:MHCs and anti-CD3 antibodies trigger a fraction of T cells to release oscillatory calcium fluxes that increase with increasing k_off rates. These results show that CalQuo ² is a robust and user-friendly tool for characterizing global, single cell calcium responses.