Validation of Confocal Microscopy Through Focusing for Corneal Sublayer Pachymetry

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Abstract
To evaluate the accuracy and precision of confocal microscopy through focusing (CMTF) for corneal sublayer pachymetry. A tandem scanning confocal microscope equipped with a nonapplanating contact objective was used to perform CMTF. The accuracy of CMTF measurements was evaluated using nine custom-made calibration contact lenses (PMMA) with varying thickness (300–600 μm) and radius of curvature (7.0–9.0 mm). The influence of immersion fluid stabilization and the consequence of prolonged corneal examination were assessed by performing CMTF in rabbits. Additionally, factors related to the instrumental setup and to sedation of experimental animals were examined. For all calibration contact lenses, the thickness measured by CMTF was within ±1.0 μm of the certified value. Varying the target thickness or radius of curvature had no consistent impact on the high accuracy of CMTF. When performing CMTF in vivo, z-axis motion was readily identified by sampling and comparing both in- and out-scans. Apart from involuntary eye movements, z-axis motion was due to initial thinning of the immersion fluid with stabilization obtained after approximately 1.5 minutes. Continued confocal examination led to slight but significant swelling of both the stroma (0.5 μm/min) and epithelium (0.1 μm/min). CMTF is an accurate and precise technique for corneal sublayer pachymetry with concurrent display of cellular morphology. The precision of CMTF can be improved by allowing time for methylcellulose stabilization and by performing repeated two-way (in and out) scans to account for z-axis motion.