Panoramic Two-Photon Imaging and Quantitative Analysis of the Corneal Sub-Basal Nerve Plexus

Comprehensive and distortion-free mapping of the entire corneal sub-basal nerve plexus (SBNP) remains a major technical challenge, as standard methods are compromised by limited fields of view (e.g., in vivo confocal microscopy) or architectural flattening (e.g., ex vivo flat-mounts). We aimed to develop and validate an integrated imaging-computational platform that overcomes these limitations by enabling panoramic, 3-dimensional (3D) visualization and objective quantification of the intact SBNP.

Experimental study.

BALB/c mice.

We developed a novel platform integrating large-field 2-photon microscopy of the intact, fluid-supported murine cornea with a semiautomated quantitative algorithm (LF-CNP Metrics). This system preserves native corneal curvature, enables pan-neuronal labeling (β-III-tubulin), and allows projection-based morphometric analysis across entire sub-basal nerve plexuses. For proof-of-concept validation, we applied it to mice subjected to in vivo desiccating stress. Animals were assessed at 1, 2, and 4 weeks postintervention; at each time point, clinical parameters (blink rate, tear secretion, corneal fluorescein staining) were measured, followed by the quantification of ten nerve metrics. Associations between nerve parameters and clinical scores were evaluated using Spearman correlation.

Corneal nerve fiber length (CNFL), density (CNFD), width, branching densities (CNBD, CTBD), arc-to-chord ratio, total curvature, total squared curvature, directional change count, and inflection count metric.

The platform generated panoramic 3D reconstructions of the sub-basal nerve plexus in ex vivo corneal samples, enabling unbiased quantification. Compared to controls, samples from the 1- and 2-week time points exhibited a trend toward reduction in key parameters including CNFL, CNFD, CNBD, CTBD, total curvature, total squared curvature, and absolute direction angle (all η² > 0.14). In the 4-week group, these metrics increased relative to the earlier time points. The corneal fluorescein staining score was negatively correlated with the corneal nerve parameters CNBD, CNFD, CTBD, and the arc length over chord length ratio, with correlation coefficients of -0.74, -0.55, -0.75, and -0.66, respectively.

We present a panoramic imaging and analysis platform that overcomes the critical limitations of field-of-view and tissue distortion, enabling comprehensive and unbiased assessment of the corneal sub-basal nerve plexus for research into neuropathic changes.

The authors have no proprietary or commercial interest in any materials discussed in this article.