Calcium is a universal second messenger present in all eukaryotic cells. The mobilization and storage of Ca²⁺ ions drives a number of signaling-related processes, stress-responses or metabolic changes, all of which are relevant for the development of immune cells and their adaption to pathogens. Here, we introduce the FRET-reporter mouse YellowCaB expressing the genetically encoded calcium indicator TN-XXL in B lymphocytes. Calcium-induced conformation change of TN-XXL results in FRET-donor quenching measurable by two-photon fluorescence lifetime imaging. For the first time, using our novel numerical analysis, we extract absolute cytoplasmic calcium concentrations in activated B cells during affinity maturation in vivo. We show that calcium in activated B cells is highly dynamic and that activation introduces a persistent calcium heterogeneity to the lineage. A characterization of absolute calcium concentrations present at any time within the cytosol is therefore of great value for the understanding of long-lived beneficial and detrimental (auto)immunity.

Absolute calcium values calculated per cell out of microscopy data. The data were collected as fluorescent lifetime values with a time correlated single photon counting device in each pixel of an image. The images have been processed with Imaris software (bitplane) for segmentation of single cells/objects. Values were exported over time per object in excel spreadsheets. Out of each fluorescent lifetime value, absolute calcium concentration was calculated according to the formula given in the original publication.

A README file is available, explaining variables.