2.2.2. Image Processing and Analysis

Logarithmic-transformed display of OCT was exported as tagged image file format (TIFF) using the integrated proprietary software. Semi-automated quantification of all three OCT biomarkers in the open-source software Fiji (US National Institutes of Health, Bethesda, Rockville, MD, USA. https://imagej.net/software/fiji/ (accessed on 25 January 2022) has been described in the literature [15,23,24,25]. All images underwent a preprocessing of image registration for alignment and histogram-matched normalization before quantification. The region of interest (ROI) was defined as the central 3 mm area. For EZR, we applied the same methods we described in our previous work, where, briefly, a longitudinal reflectance profile was obtained at every 200 µm [11]. Ellipsoid zone reflectivity ratio (EZR) was calculated as the ratio of EZ reflectivity to the retinal pigmented epithelium (RPE) reflectivity, which is considered the most hyperreflective band in OCT. Quantification of hyperreflective foci and the measurement of choroidal vascularity index required a manual selection of the ROI. This mask was outlined in the EDI image and then transferred to the conventional OCT image to ensure that the ROI is the same in both images (Figure 1). For hyperreflective foci, we followed the protocol from Midena et al. [23]. That is, foveal OC B-scan was denoised followed by enhancement of local contrasts and the integrated tool Spot Counter was used for automated quantification of hyperreflective foci in the area between the retinal nerve fiber layer and the external limiting membrane. For choroidal vascularity index (CVI), we followed the protocol from Agrawal et al. [15]. The ROI, which represented the entire choroid thickness in the central 3 mm area, was first defined in the EDI scan. The image was binarized to distinguish luminal and stromal areas within the ROI. By thresholding and composition of selected ROIs, the luminal area within the total choroidal area was measured. The mask of ROI was transferred to the conventional OCT B-scan to conduct the same analysis.

Spectral domain-OCT without (A) and with (B) enhanced depth imaging mode and (C) region of interest according to OCT biomarkers of interest. ROI for CVI and HF was derived from the EDI image. C Left: Choroidal vascularity index was calculated according to the protocol of Agrawal et al. [15]. Middle: For the quantification of hyperreflective foci (red circles), we applied the protocol described by Midena et al. [23] Right: EZR was quantified automatically in 200 µm distances between each ROI (in total 16 measurements). Horizontal line in the upper part of the image represents the central 3 mm area. White scale bar at the bottom left corner in A and C represent 200 µm. White arrow indicates the ellipsoid zone.