Corneal cell size and density are among the primary parameters that determine the structural integrity and normal physiological function of the cornea.Recently, BALB/c mice have become the most widely used laboratory animal in corneal researches and studies.An understanding of normal corneal cell size, morphology and cell density is desirable for future comparisons when mouse models are used in corneal research.

The aim of this study was to measure the cell size and cell density in three layers of the central cornea in the widely used inbred BALB/c mice strain based on in vivo three-dimensional (3D) two-photon (2PH) imaging.

Corneal endothelium layer, corneal stromal layer and corneal epithelium layer were sequentially scanned in six BALB/c mice by using a 2PH laser scanning microscope after staining with plasma membrane stain and Hoechst 33342 by intrastromal injection with the scanning thickness 2 μm for each layer, pixels 512×512 and 12 bit.Good quality 3D images were selected for the cell density and cell size analyses.Cell density was determined by counting the cell nuclei in a predefined cube of 3D images.Cell size measurements including cell surface area, cell volume, nuclear surface area, and nuclear volume were automatically quantified using the Imaris software.The cell and nuclear surface-area-to-volume ratio and the cell nuclear-to-cytoplasmic ratio were calculated.

Corneal basal epithelium layer and endothelium layer appeared the monolayer cells, while middle stromal layer showed the double-layer cells under the 2PH laser scanning microscope.The cell density was (108.00±18.97)×10⁴/mm³ in the basal epithelium layer, which was significantly higher than (9.27±0.48)×10⁴/mm³ in the middle stromal layer and (22.30± 2.28) ×10⁴/mm³ in the endothelium layer (F=141.592, P=0.000). The cell surface area and volume were highest in the middle stromal layer and lowest in the basal epithelium layer, with significant differences among the three layers (F=2 222.000, 598.504, both at P=0.000). The area/volume ratios of cells were (0.80±0.13), (0.51±0.07) and (0.40±0.04)/μm in the basal epithelium layer, middle stromal layer and endothelium layer, with the highest ratio in the basal epithelium layer and lowest ratio in the endothelium layer (F=127.075, P=0.000). The cell nuclear surface area and volume were highest in the endothelium and lowest in the basal epithelium.The cell nuclear surface area/volume ratio was highest in the basal epithelial cell and lowest in the endothelial cell.In addition, the nucleus/cytoplasm ratio was highest in the endothelial cells and lowest in the middle keratocytes.

2PH laser scanning microscope can quantitatively measure the parameters of corneal cells in vivo in BALB/c mice, including cell surface area and volume, cell nuclear surface area and volume.These data provide important cell morphology features for the study of corneal physiology, pathology and disease in mice, particularly in BALB/c mice.

Cornea/anatomy & histology; Cell count; Cell size; Mice, inbred BALB/c; Microscopy, fluorescence, two-photon