Images of all layers were exported and analyzed using the FIJI software (an expanded version of ImageJ, version 1.51a, available at fiji.sc, provided in the public domain by the National Institute of Health, Bethesda, MD, USA).
20,21 The superficial and the deep plexus were binarized using the maximum intensity of the foveolar avascular zone as a threshold determined by two masked readers (ST and CM). After binarization, images were skeletonized. Vessel density (VD), skeleton density (SD), and vessel diameter index (VDI) were calculated from the resulting images using the FIJI software as previously described.
22 Fractal dimension (FD) was calculated using the box counting method with FracLac, a plug-in for ImageJ (Karperien A, FracLac for ImageJ, available at
http://rsb.info.nih.gov/ij/plugins/fraclac/FLHelp/Introduction.htm).
23 In MacTel, pathologic alterations usually arise and are most pronounced in the temporal parafovea, predisposing this area to study both early and progressed disease-related changes. In order to determine the position of the temporal parafovea, a customized template following the subfields of the ETDRS-grid (as used in the Early Treatment Diabetic Retinopathy Study)
24,25 was centered on the foveolar avascular zone. Quantitative analyses of the superficial and deep retinal plexus were primarily conducted in the temporal parafoveal subfield (
Fig. 1, indicated in red). The size of this standardized template was customized (
Fig. 1: size of the inner, yellow circle: 0.9 mm diameter, size of the outer, red circle: 2.4 mm diameter) in order to fit into the borders of the image and include the area of interest. In cases, where the FAZ was irregularly distorted (e.g., in eyes with progressed disease stages), the center of the FAZ was determined following neighboring vessels of the nasal, superior and inferior parafovea. In a secondary analysis, quantitative measures were also conducted within the nasal, inferior and superior parafoveal subfields (
Fig. 1, indicated in blue). For binarization of the avascular layer, the borders of the FAZ were projected within the avascular layer and thresholding was conducted using the maximum intensity that was determined within this area (see
Supplementary Fig. S1, the projection of the FAZ is indicated with a yellow-dotted outline). The avascular layer usually does not show signals of blood flow in healthy subjects. Vessel density (VD) was subsequently calculated from the resulting complete image (3 × 3 mm) using the FIJI software as previously described.
20,22