A novel method for standardised imaging of corneal subbasal nerves by in vivo confocal microscopy – a pilot validation study

Abstract In vivo confocal microscopy is a valuable tool for evaluating corneal subbasal nerve plexus (SBNP). However, lack of standardised, repeatable, and reliable methods is a challenge, particularly in clinical, multi-centre, and longitudinal settings. To address this, we developed a standardised imaging method using the inferior whorl (IW) region of the SBNP as a reference for imaging a predefined 1.5 mm 2 central region of interest (ROI). No systematic differences were observed either between the two operators or between test–retest assessments performed by the same operator. A full 400 μm diameter circle around the IW was imaged in 85% of the mosaics. The inter-operator repeatability for the x- and the y-coordinates when defining the centre of the IW was 0.996 (95% CI [0.991, 0.999]) and 0.997 (95% CI [0.994, 0.999]), respectively. For 61% of the mosaics, the ROI was accurately positioned and fully imaged. By anchoring the ROI to the IW, the approach enables more repeatable and anatomically consistent imaging, supporting improved comparability across examinations. While the method shows promise, further refinement and technical development are required to enhance its reliability and robustness.