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.

The authors have declared no competing interest.

This study did not receive any funding

I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.

Yes

The details of the IRB/oversight body that provided approval or exemption for the research described are given below:

The Ethics Committee of the Saint-Etienne University Hospital gave ethical approval for this work (IRB_IORG0007394, Ref_IRBN1142021/CHUSTE)

I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.

Yes

I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).

Yes

I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable.

Yes

All data produced in the present study are available upon reasonable request to the authors