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W. Fledelius, J. Fledelius, T. Moller-Pedersen; A Validated Method for Accurate Morphometric Analysis of the Corneal Endothelium Using In Vivo Confocal Microscopy . Invest. Ophthalmol. Vis. Sci. 2003;44(13):3659.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: The human corneal endothelium has a limited regenerative capacity. Reliable morphometric analysis of these cells is therefore important for evaluating their response to environmental stress, trauma, surgery, and disease. Clinically, the corneal endothelium can be imaged using specular or confocal microscopy, but these methods have not been thoroughly validated. The purpose of the present study is to characterize the imaging properties of the tandem scanning confocal microscope (TSCM) and to calibrate and correct for optical factors that may alter the accuracy of morphometric analysis of the corneal endothelium. Methods: The imaging properties of the TSCM were described using: 1) a contact lens with a uniform spiral pattern to identify lateral optical distortion; 2) polymer microspheres (diameter 25 µm) to give the absolute scale of the captured images; 3) nine PMMA contact lenses with varying thickness (range 300-600 µm) and radius of curvature (range 7.0-9.0 mm) to simulate the impact of variations in corneal anatomy; 4) varying thickness of the immersion fluid (2.5% methylcellulose) to change the distance from the objective surface to the focal plane. Results: To compensate for optical distortion, the image height had to be reduced by 11% and corrected by 2. order polynomiums. The magnification depended on the absolute distance from the objective surface to the focal plane with a linear relationship of 0.0104 % magnification per µm depth. However, the curvature and thickness of the contact lens (i.e. the cornea) did not, per se, influence the absolute size measurements. All calibration and correction factors were integrated in a semi-automated software program, "Endomorph", for calculation and registration of standard morphometric parameters. Conclusions: The imaging properties of the TSCM have been characterized. A validated software program has been developed that allows for accurate morphometric analysis of the corneal endothelium.
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