A method for determining cell number in the undisturbed epithelium of the mouse lens

The anterior face of the mouse lens is covered by a layer of epithelial cells. The epithelial cells serve a barrier function at the lens surface and as a progenitor population from which lens fiber cells, the predominant cell type of the lens, are derived. Decreased epithelial cell density is commonly observed during aging and cataract formation in humans and animal models and may contribute directly to tissue opacification. However, the loss of cells from the epithelium is often not easy to quantify, in part because the cells are arrayed across a near-spherical surface and, as a consequence, are difficult to image and count. Here, we describe a technique for determining epithelial cell number in the undisturbed lens of the mouse, a popular cataract model. The method utilizes orthographic projections of confocal images collected from the anterior and equatorial regions of the lens. The overlapping projections are brought into register using the unique distribution of proliferating cells as fiduciary points. Cell counts are performed using a computer-assisted method. This approach offers several advantages over flat-mount methods employed previously.