Figure 1. Detection of endogenous hydrogen peroxide (H₂O₂) in lens cells with peroxyfluor-6 acetoxymethyl ester (PF6-AM). A: Diagram of the embryonic day 10 (E10) chick lens (top panel) depicting four distinct regions of differentiation including: EC, undifferentiated lens epithelial cells; EQ, equatorial epithelium which is the zone of differentiation initiation; FP, the zone of lens fiber cell morphogenesis; and FC, the zone of fiber cell maturation. Lens model highlighting the EC and EQ zones (blue, bottom panel). B: E10 chick lens placed in organ culture and loaded with PF6-AM (green) to detect endogenous H₂O₂ production, shown as an overlay of PF6-AM (green) to detect endogenous H₂O₂ production, and the differential interference contrast (DIC) image. Whole lens was viewed live from the equatorial region providing an anterior–posterior view. The detection of H₂O₂ production was limited to the lens epithelium and was highest in the EQ zone. C: E10 chick lens exposed to PF6-AM (green) and viewed en-face as an overlay with DIC, demonstrating H₂O₂ production was greatest in the EQ zone. D: Line scan intensity analysis across the image profile from (B) (created using LSM5 Image Examiner along [i] an EQ to EQ line) to quantify the level of H₂O₂ production showed that highest intensity labeling for endogenous H₂O₂ occurred in the EQ zone of the lens. (ii) Surface plot histogram (plotted against EC and EQ zones of the lens in B) showing the relative intensities of the H₂O₂ signal across the lens confirmed that the greatest endogenous H₂O₂ production occurred in the EQ zone. E: Line scan intensity analysis across the image profile from (C) demonstrated that the greatest production of endogenous H₂O₂ occurred in the EQ zone. F: E10 lens with a puncture in the equatorial region (boxed area) incubated with PF6-AM and imaged en-face as an overlay with the DIC image. Results confirmed the specificity of H₂O₂ production to the lens epithelium, as detected by PF6-AM. G: (i) Primary lens epithelial cells in culture loaded with PF6-AM showed production of endogenous H₂O₂ (PF6-AM, green); (ii): DIC of image shown in (i). Panel just below (i) shows the line scan intensity analysis for H₂O₂ in lens epithelial cells in (i). Images were acquired live by confocal microscopy. Each optical slice is 3 μM in thickness in (B) and (C); scale bar = 200 μm. Optical slice thickness for images (F) and (G) is 1 μm; scale bar in (F) is 500 μm and in (G) is 20 μm. Results are representative of three independent experiments. Studies provided direct evidence for the presence of endogenous H₂O₂ in lens epithelial cells.