Quantification of cells expressing M-opsin or L-opsin mRNA in adult retinas and validation.

(A) Schematic of retina with regions isolated using a 5 mm biopsy punch (as in Fig 2G with additional detail). White circle = optic nerve. Red lines = blood vessels. Yellow circle = macular pigment. (B–E) Validation of quantification of % of cells expressing M-opsin or L-opsin with HALO semi-automated image analysis software. (B) Images from 20 μm sections were probed for M-opsin (blue) and L-opsin (pink) mRNA. Images as in Fig 2H–2J. (C) HALO software deconvoluted the colorimetric M-opsin or L-opsin mRNA signals to generate pseudo fluorescent images (from Fig S3B). (D) Manually counted average ratios of M and L cones as percent of M/L total cones across 3 individuals. One-way ANOVA with Tukey’s multiple comparisons test: Center L versus Middle L = no significance; Center L versus Periphery L p < 0.01; Middle L versus Periphery L p < 0.01; ** indicates p < 0.01. Data as in Fig 2K for comparison to Fig S3E. (E) HALO semi-automated software analysis of single deconvoluted representative images from the center, middle, and periphery regions (Fig S3C), showed similar ratios of M and L cones as manually scored retinas (Fig 2K). (F–H) Ratios of M and L cones as percent of M/L total cones for each individual; n > 850 cones for each region for each individual. Averages ratios are shown in Fig 2K.

(PDF)