ROI analysis

We postulated whether analysis of nerve density in the ROI could be a substitute for wide-area image analysis of the SBNP. An ROI was selected as a region in the central cornea that would be easiest to access and image reproducibly in a clinical IVCM examination. The ROI was superior to the whorl region, with its location referenced to the whorl center. As the whorl region is the most significant landmark in the SBNP, referencing the ROI to the whorl center would enable consistency and standardization in localizing the same corneal region relative to the whorl center in each examined eye. This could be relevant for comparisons of nerve parameters across individuals, but also for repeated within-subject examinations, for example in subsequent clinical follow-up visits. In this manner, we aimed to investigate the utility of the ROI for possible clinical use.

The ROI was selected by first creating a rectangular ROI box with the dimensions 1,000 µm (width) and 650 µm (height) using ImageJ. This ROI represents an area of four times the standard IVCM image frame size and was deemed suitably large to yield a broader view than a single frame while being small enough to be feasible to obtain in most eyes within a clinical setting. The coordinates of the whorl center were then identified from the mosaic image, and the ROI box was placed superior to the whorl center such that the centre of the lower horizontal side of the rectangle was positioned directly 650 µm superior to the whorl center (Fig. 1). With the ROI rectangular box positioned above the whorl centre, the ROI area could be cropped from the wide-area mosaic image. Two experienced observers conducted the ROI analysis; the first observer analysed 100% of ROI images and the second observer analysed 25% of images in each of the four subgroups. The cropped ROI region was then subjected to quantitative analyses of the following parameters.

Number of main nerves: main nerves were defined as nerves that traversed the height of the ROI area from top to bottom.

Main nerve fibre length density: main nerves in the ROI were traced using the NeuronJ²⁹ plugin for ImageJ. Subsequently, the total length of the main nerves in the ROI was determined and divided by the ROI area.

Total nerve fibre length density: all nerve fibres in the ROI, both the main nerves and secondary branches were identified and traced. The total length of all nerves in the ROI was divided by the ROI area, and considered the CNFL in the ROI.

Secondary nerve fibre length density: secondary nerve fibres were defined as all the nerve fibres other than the main nerves. The combined length of all secondary nerve fibres in the ROI was divided by the ROI area.

Nerve branch density: this refers to the sum of the number of points within the ROI where nerves bifurcate into branches, divided by the ROI area.

Inter-nerve distance: this is the average distance between the main nerves in the ROI area. First, a horizontal line bisecting the short sides of the ROI box area was drawn. Then, the distance between each main nerve and the adjacent main nerve was measured in ImageJ using the line tool. The mean value of all such distances in the ROI was then determined.

Illustration of ROI box. The ROI box (1,000 × 650 µm) is illustrated (yellow rectangle) in a mosaic as it is placed in the predefined position in relation to the whorl center (650 µm superior to the whorl center) where the center of the lower horizontal side of the ROI is positioned exactly 650 µm above the whorl center. The vertical distance between the two is marked by the yellow square bracket. The ROI was subsequently cropped from the mosaic image.

Figure 2 illustrates these definitions graphically. The above parameter values were determined for the right and left eyes of each subject, with the observer masked to the subject’s identity. Subsequently, the parameters across both eyes of the same subject were averaged. In an earlier study²⁸, we measured the values for total nerve fiber length density in the entire (non-cropped) mosaic (mCNFL) and similarly averaged them across both eyes of a given subject. These values served as a comparison to the corresponding nerve density measures in the ROI.

ROI image illustrating different parameters. A region of interest (ROI) cropped from an original wide-area mosaic image is shown here. The main nerves that transverse from the top to the bottom of the image are traced (marked in purple). Red arrows indicate the secondary nerves. Short yellow arrows indicate nerve bifurcations (branches). White double-headed arrows indicate the inter-nerve distance (IND). The mid-horizontal line (in yellow) divides the image at the height of 325 µm. One nerve to the right of the image (in white) was not included among main nerves because it did not satisfy the condition of traversing from the top to the bottom of the ROI image. The ROI image size is 1,000 µm (width) × 650 µm (height).