RESUMEN
The assessment of leukocyte activation in vivo is mainly based on surrogate parameters, such as cell shape changes and migration patterns. Consequently, additional parameters are required to dissect the complex spatiotemporal activation of leukocytes during inflammation. Here, we showed that intravital microscopy of myeloid leukocyte Ca2+ signals with Ca2+ reporter mouse strains combined with bioinformatic signal analysis provided a tool to assess their activation in vivo. We demonstrated by two-photon microscopy that tissue-resident macrophages reacted to sterile inflammation in the cremaster muscle with Ca2+ transients in a distinct spatiotemporal pattern. Moreover, through high-resolution, intravital spinning disk confocal microscopy, we identified the intracellular Ca2+ signaling patterns of neutrophils during the migration cascade in vivo. These patterns were modulated by the Ca2+ channel Orai1 and Gαi-coupled GPCRs, whose effects were evident through analysis of the range of frequencies of the Ca2+ signal (frequency spectra), which provided insights into the complex patterns of leukocyte Ca2+ oscillations. Together, these findings establish Ca2+ frequency spectra as an additional dimension to assess leukocyte activation and migration during inflammation in vivo.