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Purpose  To investigate the influence of various technical factors on the variation of cone packing density estimates in adaptive optics flood illuminated retinal images.    Methods  Adaptive optics images of the photoreceptor mosaic were obtained in fifteen healthy subjects. The cone density and Voronoi diagrams were assessed in sampling windows of 320×320 µm, 160×160 µm and 64×64 µm at 1.5 degree temporal and superior eccentricity from the preferred locus of fixation (PRL). The technical factors that have been analyzed included the sampling window size, the corrected retinal magnification factor (RMF corr ), the conversion from radial to linear distance from the PRL, the displacement between the PRL and foveal center and the manual checking of cone identification algorithm. Bland-Altman analysis was used to assess the agreement between cone density estimated within the different sampling window conditions.    Results  The cone density declined with decreasing sampling area and data between areas of different size showed low agreement. A high agreement was found between sampling areas of the same size when comparing density calculated with or without using individual RMF corr . The agreement between cone density measured at radial and linear distances from the PRL and between data referred to the PRL or the foveal center was moderate. The percentage of Voronoi tiles with hexagonal packing arrangement was comparable between sampling areas of different size. The  boundary  effect, presence of any retinal vessels, and the manual selection of cones missed by the automated identification algorithm were identified as the factors influencing variation of cone packing arrangements in Voronoi diagrams.    Conclusions  The sampling window size is the main technical factor that influences variation of cone density. Clear identification of each cone in the image and the use of a large buffer zone are necessary to minimize factors influencing variation of Voronoi diagrams of the cone mosaic.

Technical Factors Influencing Cone Packing Density Estimates in Adaptive Optics Flood Illuminated Retinal Images