Imaging mitochondrial calcium signalling with fluorescent probes and single or two photon confocal microscopy

The concentration of calcium ions in the mitochondria has a profound impact on mitochondrial function, modulating respiratory activity at physiological concentrations, while causing lethal damage during calcium overload. The "rhod" series of calcium sensitive fluorescent dyes tend to accumulate preferentially in mitochondria, although the reliability of mitochondrial calcium measurements depends critically on the partitioning of dye within the mitochondria which can vary between preparations. Methods are described to aid verification and quantification of the mitochondrial calcium concentration using single or two photon confocal microscopy and combining the imaging with another cytosolic calcium sensing dye. The method of linear unmixing to separate fluorescent signals based on either differing excitation or emission spectra is outlined and for the purposes of illustration is applied to the separation of rhod-2 signals originating from dye within the mitochondrial and nucleoli.