Confocal microscopic determination of geometric and affinity parameters required for the calculation of ΔψM in rat primary β-cells.

(A) The mitochondria:cell volume fraction (VF) was calculated from confocal micrographs of live rat primary β-cells loaded with MitoTracker Red CMX and Calcein-AM (a-b). β-Cells were identified by insulin immunocytochemistry (ICC; c). MitoTracker images were high pass filtered (d) and both Calcein and MitoTracker images were binarized (e) to determine cellular and mitochondrial cross sections, respectively. Each β-cell was imaged in one of 10 serial z-planes to provide a balanced representation of all planes, indicated here by the three rows of images of separate cells. Cross sections of non-β cells were manually removed as indicated in the midplane images (the boundaries of the β-cell is marked by the dashed line). VF was calculated by the indicated formula after summing the number of all mitochondrial and all cellular (including mitochondrial) pixels in all cells in a recording of 50–100 cells (represented by column e). (B) aR’ was calculated from TMRM fluorescence after depolarization of mitochondria by FCCP (1 μM), oligomycin (1 μg/ml) and antimycin A (1 μM). aR’ is the slope of the linear relationship between nuclear and mitochondrial fluorescence (F; as marked by the dashed line in the raw and gated fluorescence images, respectively) as TMRM fluorescence intensity decays as it leaks out of the cell, and b is background fluorescence. The identity of β-cells was determined by post-hoc immunofluorescence imaging, revisiting stored coordinates (right). Scale bars, 5 μm.