Optical principles for estimation of endothelial cell density with the non-contact specular microscope

In non-contact specular microscopy of corneal endothelium, endothelial cell density is overestimated due to the angle of observation and the curvature of cornea. On the basis of theoretical considerations, it is concluded that if the angle of observation is kept at a small value the curvature of cornea contributes only insignificantly to this overestimation, the major determinant being the angle of observation. When an angle of 46 degrees is chosen between slit illumination and optical axis of the microscope it is calculated that estimates of endothelial cells should be multiplied with a factor of 0.959 to correct for the angle effect. Six eye were photographed with both a contact and non-contact specular microscope and endothelial cell density estimated. Mean observed difference in cell counts was +5.5% without correction and +1.2% with correction for angle effect. 95% confidence limits for the difference with correction were -3.7 and +6.0% respectively showing that estimates of endothelial cell density obtained with the non-contact specular microscope agree closely with those obtained by contact specular microscopy when corrected for angle of observation.