Imaging Initial Ca²⁺ Microdomains in Primary T Cells

Local, sub-second Ca²⁺ signals, termed Ca²⁺ microdomains, are highly dynamic and short-lived Ca²⁺ signals, which result in a global [Ca²⁺]_i elevation and might already determine the fate of a T cell. Upon T cell receptor activation, NAADP is formed rapidly, binding to NAADP binding proteins (HN1L/JPT2, LSM12) and their respective receptors (RyR1, TPC2) sitting on intracellular Ca²⁺ stores, like the ER and lysosomes, and leading to subsequent release and elevation of [Ca²⁺]_i. To capture these fast and dynamically occurring Ca²⁺ signals, we developed a high-resolution imaging technique using a combination of two Ca²⁺ indicators, Fluo-4 AM and FuraRed AM. For postprocessing, an open-source, semi-automated Ca²⁺ microdomain detection approach was developed based on the programming language Python. Using this workflow, we are able to reliably detect Ca²⁺ microdomains on a subcellular level in primary murine and human T cells in high temporal and spatial resolution fluorescence videos. This method can also be applied to other cell types, like NK cells and murine neuronal cell lines.