FIGURE 3.

The TR technique estimates the diameter of mitochondria. (A) Typical imaged mitochondria (upper row) and model mitochondrion capsule bodies (lower row) are shown at the same printed pixel size. The modeling parameters were dependent on the optical configuration as indicated. Model mitochondria have diameter d = 0.34 μm and are scaled to match the resolution of the acquisition and are blurred by the theoretical MTF of the given optical configuration. (B) Model mitochondrion images were generated with increasing diameter and correspond to the confocal microscopic configuration. (C and D) Effect of mitochondrial diameter on the TR in the three indicated optical configurations using spheres (C) or capsules of l = 50 pixels (D) as models. The model images were blurred with the MTFs given in Fig. 2, B–D. Although the slopes are not intended to be used for calibration of real biological data (because the real MTF of the microscope could differ from the theoretical), the reciprocal slopes are given for the estimation of sensitivity in pixels/TR for C (▵, 2.8; □, 1.6; ○, 17.8) and for D (▵, 4.3; □, 3.1; ○, 22.2). (E) TR as a function of subpixel changes of the diameter. Model mitochondria were 50 pixels long and were placed in the same positions and orientations in each image (black trace) or in a different manner (gray trace). The latter simulation was repeated five times and the mean ± SE is shown. Comparison of all data points by ANOVA with Tukey post hoc test at p < 0.05 indicates that data points farther away from each other than an average 0.15 pixels or 15 nm had significantly different TRs. This corresponds to the optical configuration given in Fig. 2 D. (F) TR (as mean ± SE of n = 3) calculated from fluorescent bead images acquired by mean-intensity projected z-stacking wide-field microscopy in conditions similar to the images in Figs. 2 D and 10.