(A) Wide-field fluorescence micrographs of TMRM in isolated rat skeletal muscle mitochondria immobilised on glass in the presence of the indicated substrates. The two cropped frames show the same mitochondria before and after glycerol 3-phosphate addition with individual mitochondria marked by automated morphological segmentation. (B) Fluorescence intensity time courses of TMRM as in (A), recorded over individual mitochondria during the sequential addition of the indicated substrates. Data were normalised between the frame background and the maximal fluorescence for the entire time course for each mitochondrion. (C) Scatter plot analysis of ΔψM responses of individual mitochondria to glycerol 3-phosphate (when added after glutamate plus malate (G + M)) as a function of polarisation by glutamate plus malate alone. The polarisation by glutamate plus malate was referenced to the ΔψM provided by succinate at the end of the experiment (see (B)), therefore, smaller values (difference) indicate a larger initial polarisation by glutamate plus malate, assuming that succinate was maximally polarising. Coloured regions indicate arbitrarily chosen classes of mitochondria with differing extent of response to the two substrates. Mean ± SE values describe the size of these classes for experimental replicates (n = 4). (D) Wide-field fluorescence micrographs of NAD(P)H autofluorescence recorded in the same mitochondria as in (A) following the TMRM frames, before and after the addition of rotenone. (E) Fluorescence intensity time courses of TMRM, followed by recording of NAD(P)H autofluorescence over the same mitochondria while substrates or rotenone were added as indicated. (F) Scatter plot analysis of ΔψM responses of individual mitochondria to glycerol 3-phosphate (added after 50 µM glutamate plus malate) as a function of response to rotenone in the presence of glutamate plus malate + glycerol 3-phosphate. Mean ± SE values describe experimental replicates (n = 4). Note that only 4.2% of mitochondria responded to rotenone by significant oxidation of NAD(P)H (orange line defined at log₂ of 1–2 times the average SE of the repeated autofluorescence determinations after rotenone addition), and the 1% of mitochondria that did not polarise well with glycerol 3-phosphate responded to rotenone with a stronger increase in autofluorescence (pink 1.0 ± 0.1 vs grey 0.53 ± 0.1 log₂-fold values, P < 0.01, n = 4). G + M, 50 µM glutamate plus 50 µM malate; G3P, 12.5 mM glycerol 3-phosphate; succ, 5 mM succinate.