Epithelial and corneal thickness measurements by in vivo confocal microscopy through focusing (CMTF).

PURPOSE: To study the feasibility of measuring total corneal thickness, as well as the thickness of the epithelium and Bowman's layer, using a novel in vivo confocal microscopy through-focusing (CMTF) methodology. METHODS: The central cornea was scanned from the epithelium to endothelium at an average focal plane speed of 32 microns/sec for rabbits, and 64 microns/sec for humans. Scans were initially video-recorded and later digitized. From digital images, CMTF intensity curves were generated by calculating the average pixel intensity in the central 180 x 180 pixel region (285 microns x 285 microns) of each image in the scan, and plotting as a function of z-depth. Peaks in this intensity profile were then empirically correlated to unique corneal layers using a program which interactively displayed images corresponding to the mouse cursor position along the intensity profile curve. Sublayer thickness values were then calculated from the z-axis positions of the relevant peaks in the intensity curve. Ten normal rabbits and seven human volunteers were evaluated in the study. Both CMTF and ultrasonic pachymetry (UP) measurements were performed on rabbit eyes to determine the agreement between CMTF and UP. RESULTS: Distinct epithelial, basal lamina, and endothelial peaks were identified for all 10 rabbit eyes. The mean central corneal thickness in the rabbit was 381.6 +/- 27.3 microns by CMTF and 384.4 +/- 28.7 microns by UP. The mean difference in central corneal thickness between CMTF and UP was -2.8 +/- 7.1 microns which was not statistically significant (p > 0.2 by paired t-test). Central epithelial thickness in the rabbit measured by CMTF was 47.7 +/- 2.2 microns. The average coefficients of variation for repeated scans were 2.5% and 0.7% for epithelial and corneal thickness, respectively. The standard errors for both epithelial and corneal thickness were less than 1.5 microns for all rabbits. The reproducibilities for epithelial and corneal thickness measurements were 2.2 microns and 2.6 microns, respectively, calculated as the square root of the within group variances of One-Way ANOVA. Intensity profiles for human corneas showed strong epithelial and endothelial peaks, as well as smaller peaks corresponding to the basal-epithelial nerve plexus and the denser anterior layer of stromal keratocyte nuclei. The mean central corneal thickness in the human was 532.1 +/- 18.8 microns; central epithelial thickness was 50.6 +/- 3.9 microns; central Bowman's layer thickness was 16.6 +/- 1.1 microns. The average coefficients of variation for repeated scans were 5.9%, 13.2%, and 1.6% for epithelial, Bowman's layer, and corneal thickness, respectively. The standard errors for all measurements were less than 2.4 microns. The reproducibilities for epithelial, Bowman's layer, and corneal thickness measurements were 3.2 microns, 2.3 microns, and 10.0 microns, respectively. CONCLUSIONS: CMTF is a novel, reproducible technique for obtaining epithelial and corneal thickness measurements during clinical in vivo confocal microscopy of the cornea. More importantly, this methodology provides the first objective, quantitative approach for measurement and analysis of depth and thickness of corneal sub-layers which may prove uniquely valuable in temporally assessing corneal function.