A method for quantifying pigment position in retinal pigment epithelium

In wildtype mice, the pigment granules in the retinal pigment epithelium aggregate in the dark towards Bruch's membrane and disperse towards the photoreceptors in the light. We have developed a repeatable method amenable for quantifying pigment position in the RPE from wild type mice by estimating the population density of pigment granules, or pigment density, within 4 μm² areas in the basal part of cells examined by transmission electron microscopy. To measure pigment position, 2 μm × 2 μm squares were aligned along the apical ends of the basal microvilli. The pigment granules within each 4 μm² area were counted, and the average pigment density was calculated for each mouse. The average pigment density for light-adapted mice (n = 3 mice) was 1.3 pigment granules/μm² (± 0.2 pigment granules/μm²). For dark-adapted wildtype mice (n = 3 mice), pigment density was 1.9 pigment granules/μm² (± 0.3 pigment granules/μm²). Pigment density was statistically significantly different (p < 0.02) between light-adapted and dark-adapted mice, with pigment density higher in the dark-adapted mice. This method was implemented by four observers and their results were compared. No statistically significant differences were found in the measurements acquired by the different observers, illustrating the repeatability of the method.