Abstract
Calcium (Ca2+) imaging aims at investigating the dynamic changes in live cells of its concentration ([Ca2+]) in different pathophysiological conditions. Ca2+ is an ubiquitous and versatile intracellular signal that modulates a large variety of cellular functions thanks to a cell type-specific toolkit and a complex subcellular compartmentalization. Many Ca2+ sensors are presently available (chemical and genetically encoded) that can be specifically targeted to different cellular compartments. Using these probes, it is now possible to monitor Ca2+ dynamics of living cells not only in the cytosol but also within specific organelles. The choice of a specific sensor depends on the experimental design and the spatial and temporal resolution required. Here we describe the use of novel Förster resonance energy transfer (FRET)-based fluorescent Ca2+ probes to dynamically and quantitatively monitor the changes in cytosolic and mitochondrial [Ca2+] in a variety of cell types and experimental conditions. FRET-based sensors have the enormous advantage of being ratiometric, a feature that makes them particularly suitable for quantitative and in vivo applications.