Automated Image Registration Method for In Vivo Confocal Microscopy of the Corneal Sub-basal Nerve Plexus

In vivo confocal microscopy (IVCM) assesses corneal innervation in the sub-basal nerve plexus but is typically quantified manually from a single z-scan, limiting biomarker extrapolation for diagnosing limbal stem cell deficiency (LSCD). We developed an automated 3D reconstruction method of IVCM image volumes to improve sub-basal nerve density quantification. Our dataset comprised 99 IVCM stacks from 63 LSCD eyes (51 patients) and 23 stacks from 15 normal eyes. We designed an image registration algorithm combining phase correlation and homography transformation, which achieved a pairwise image correlation of 0.69 and mutual information of 0.60, significantly outperforming manual registration (0.60 and 0.43, respectively; p<0.001). Validation on an independent dataset of 325 volume scans from 24 eyes of 12 unilateral, severe LSCD patients yielded a correlation of 0.75 and MI of 0.76. This method enhances sequential IVCM scan alignment and supports more accurate, reproducible 3D evaluation of LSC biomarkers.