Intracellular Ca2+ signaling controls numerous cellular functions. Mitochondria respond to cytosolic Ca2+ changes by adapting mitochondrial functions and, in some cell types, shaping the spatiotemporal properties of the cytosolic Ca2+ signal. Numerous methods have been developed to specifically and quantitatively measure the mitochondrial-free Ca2+ concentrations ([Ca2+ ]m ), but there are still significant discrepancies in the calculated absolute values of [Ca2+ ]m in stimulated live cells. These discrepancies may be due to the distinct properties of the methods used to measure [Ca2+ ]m , the calcium-free/bound ratio, and the cell-type and stimulus-dependent Ca2+ dynamics. Critical processes happening in the mitochondria, such as ATP generation, ROS homeostasis, and mitochondrial permeability transition opening, depend directly on the [Ca2+ ]m values. Thus, precise determination of absolute [Ca2+ ]m values is imperative for understanding Ca2+ signaling. This review summarizes the reported calibrated [Ca2+ ]m values in many cell types and discusses the discrepancies among these values. Areas for future research are also proposed.
