Corneal cell density measurement in vivo by scanning slit confocal microscopy: method and validation

Method and validation of a technique to quantify cell density in vivo in 6 corneal layers with a scanning slit confocal microscope (SSCM).

A confocal image of a small volume in a corneal layer is registered on videotape. Cells or nuclei according to a layer classification are counted manually using an unbiased frame. Surface cell density is calculated from an image on the screen, and volumetric density is obtained using stereological methods.

Image distortion on the screen is less than 3%. The classification of a cell layer is verified by determining the position of the measurement volume in the cornea. Validation of density measurements is performed by comparing confocal results with those obtained by histology. The difference between the two methods varies from -24.1% (posterior stroma) to +16.4% (basal layer). Intersession and intrasession repeatability are 8.3 and 5.8%, respectively. The cell density (mean +/- SD) in 20 healthy controls in the superficial, basal and endothelial layers was 759 +/- 162, 5,817 +/- 632 and 2,743 +/- 285 cells.mm(-2) (surface), and in the anterior, mid and posterior stroma 28,616 +/- 3,081, 19,578 +/- 4,421 and 26,073 +/- 5,962 cells.mm(-3) (volumetric). These results are in accordance with those of other investigators.

The SSCM can produce repeatable quantitative measurements of corneal cell density in conscious humans.