Mitochondrial Calcium Imaging with GCaMP6m

To test resting mitochondrial Ca²⁺ concentrations with high sensitivity, we used a new Ca²⁺ probe based on the last-generation GCaMP probe (Chen et al., 2013 (link)) targeted to the mitochondrial matrix. We chose the GCaMP6m version because it had the highest Ca²⁺ affinity. To measure the signal independent of variations in basal fluorescence intensity due to the variable expression levels of the probe, we took advantage of the isosbestic point in the GCaMP6m excitation spectrum; exciting GCaMP6m at 406 nm led to fluorescence emission that was not Ca²⁺ dependent. As a consequence, the ratio between the excitation wavelengths of 494 and 406 nm was proportional to the Ca²⁺ concentration and independent of probe expression levels. Cells were imaged with an IX-81 automated epifluorescence microscope (Olympus) equipped with a 40× oil immersion objective (numerical aperture 1.35; Olympus) and an ORCA-R2 charge-coupled device camera (Hamamatsu Photonics).