Three-dimensional topography of Descemet’s membrane in Fuchs endothelial corneal dystrophy using laser scanning confocal microscopy and white-light interferometry

Aim To evaluate the potential of a three-dimensional microscope combining Laser scanning confocal imaging and white-light interferometry for quantitative topographic characterisation of Descemet’s membrane (DM) in Fuchs endothelial corneal dystrophy (FECD). Methods Descemet’s membranes were collected from 38 FECD patients undergoing endothelial keratoplasty and 4 healthy donors. After flat-mounting on glass slide and drying, specimens were analysed using the VK-X3000 system (KEYENCE). Entire samples were reconstructed by image stitching at low magnification (×10) in white-light interferometry mode (0.01nm axial resolution). Higher magnifications (×20-×150) in confocal mode (12 nm axial resolution) enabled detailed structural analysis. Three-dimensional height maps were generated to calculate standardised surface roughness parameters. Guttae and other DM features were classified according to spatial organisation and elevation profiles. Results White-light interferometry enabled full-field mapping of whole 8mm diameter DMs with nanometric vertical resolution (∼2 hours/sample). Surface roughness (Sa) was higher in FECD than in controls (median±IQR: 0.571±0.259 µm vs 0.239±0.161 µm; p = 0.0018). In FECD, three zones were identified: central (guttae buried in the posterior fibrillar layer; Sa 0.442 ± 0.112 µm), paracentral (large uncovered guttae; Sa 0.562±0.170 µm; p = 0.0423), and outer zone (no confluent guttae; Sa 0.261±0.143 µm; p < 0.0001). Confocal 3D imaging revealed radial striae, embossments and furrows in the DM, confluent central guttae, and fused or buried structures. Conclusions Combining white-light interferometry and confocal microscopy enables label-free, high-resolution surface characterisation of DM in FECD, providing quantitative metrics to compare histological subtypes and supporting the predominance of radial structural organisation.