Figure 3. Lens cells rich in actively respiring mitochondria have high endogenous hydrogen peroxide (H2O2). A: E10 chick embryonic lens in organ culture exposed simultaneously to both MitoTracker Red CMXRos, a mitochondrial probe that
detects mitochondria with active membrane potential (red), and PF6-AM (green). Image shows close overlap of actively respiring
mitochondria and production of endogenous H2O2 in the EQ zone of the lens. (i) Low magnification image in which the whole lens was viewed in an anterior–posterior direction.
Co-localization also occurred in the EC zone, but the label was most intense in cells of the EQ zone. The right panel in (i)
is an overlay of MitoTracker Red (red), peroxyfluor-6 acetoxymethyl ester (PF6-AM, green) and differential interference contrast
(DIC) imaging (greyscale). The boxed region in (i) is shown in (ii) at higher magnification. (ii) EQ zone of the E10 lens
showing overlap of H2O2 production (PF6-AM, green) with cells rich in active mitochondria (MitoTracker Red CMXRos, red), (arrows). Overlay of PF6-AM
and Mitotracker is shown in the right panel. Note fiber cell regions are negative for both probes. B: Primary lens epithelial cells in culture exposed simultaneously to MitoTracker Red CMXRos (red) and PF6-AM (green) often
showed a pattern where cells rich in active mitochondria have the highest levels of endogenous H2O2 (i). Right panel shows overlay of MitoTracker Red CMXRos (red) and PF6-AM (green). Boxed area in (i) is shown below at a
higher magnification in (ii), to highlight the mitochondria (red) in lens epithelial cells with production of endogenous H2O2 (green). Right panels show the mitotracker/PF6-AM overlay. Arrows indicate individual active mitochondria. Results suggested
that the majority of the lens cells rich in actively respiring mitochondria are generating endogenous H2O2. Images were acquired live using confocal microscopy. Each image is a single optical slice of 1 μm thickness; scale bar =
20 μm. Results are representative of three independent experiments.