Measurement of In Vivo Three-Dimensional Corneal Cell Density and Size Using Two-Photon Imaging in C57BL/6 Mice.

PURPOSE: To measure the cell size and cell density in five layers of the central cornea in the widely used inbred C57BL/6 mouse strain using in vivo three-dimensional (3D) two-photon (2PH) imaging. METHODS: Corneas were scanned using a 2PH laser scanning fluorescence microscope after staining with plasma membrane stain and Hoechst 33342. Good quality 3D images were selected for the cell density and cell size analysis. 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 (S:V ratio) and the cell nuclear-cytoplasmic ratio (N:C ratio) were calculated. RESULTS: The highest cell density was observed in the basal epithelium and the lowest in the posterior stroma. The highest cell surface area was found in the anterior stroma, and the highest cell volume was observed in the superficial epithelium. The lowest cell surface area and cell volume were both found in the basal epithelium. The highest S:V ratio was observed in the basal epithelium and the lowest in the superficial epithelium. The highest cell nuclear surface area and volume were both observed in the superficial epithelium and the lowest in the basal epithelium. The highest cell nuclear S:V ratio was observed in the basal epithelium and the lowest in the superficial epithelium. The highest N:C ratio was found in the basal epithelial cells and the lowest in the posterior keratocytes. CONCLUSIONS: We are the first to quantify the cell density and size parameters, including cell surface area and volume, cell nuclear surface area and volume, and the S:V ratio, in the five layers of the central cornea. These data provide important cell morphology features for the study of corneal physiology, pathology and disease in mice, particularly in C57BL/6 mice.

Corneal sublayer thickness measurements with the Nidek ConfoScan 4 (z Ring).

PURPOSE: To determine the repeatability of corneal sublayer thickness measurements using the Nidek ConfoScan 4 (CS4) with the z ring on a group of young adult subjects. METHODS: Thirty subjects aged 18 to 30 years were invited to have thickness measurements with the CS4 (z ring) on two different days, at similar time of the day to avoid diurnal variation. RESULTS: Only 22 subjects had valid measurements for analysis. The mean ± SD of central corneal thickness (CCT) was 534 ± 26 µm, epithelial thickness was 42 ± 8 µm, Bowman's layer thickness was 19 ± 7 µm, and stromal thickness (ST) was 472 ± 25 µm. There was no significant difference in the between-visit thickness measurements of each layer with the CS4 (z ring) (paired t-tests, p > 0.05). The limits of agreement of between-visit measurements were -41 (8%) to 37 µm (7%) for CCT, -21 (50%) to 19 µm (45%) for epithelial thickness, -13 (68%) to 17 µm (89%) for Bowman layer thickness, and -46 (10%) to 37 µm (8%) for ST. CONCLUSIONS: Measurements of CCT and ST with the CS4 (z ring) showed reasonably good repeatability (7 to 10%). However, the repeatability of measurements of the thinner corneal layers, such as epithelium and Bowman's layer, was poor.

Three-dimensional analysis of collagen lamellae in the anterior stroma of the human cornea visualized by second harmonic generation imaging microscopy.

PURPOSE: The structure of collagen lamellae in the anterior stroma of the human cornea is thought to be an important determinant of corneal rigidity. The three-dimensional structure of such collagen lamellae in normal human corneas was examined. METHODS: The anterior portion of 27 normal human corneas was obtained from donor tissue for Descemet's stripping automated endothelial keratoplasty (DSAEK) surgery, and blocks (∼3-mm square) of the central cornea were examined by second harmonic generation (SHG) imaging microscopy. Each cornea was scanned from the surface of Bowman's layer to a depth of 150 µm, and SHG forward signals were collected. The angles of collagen lamellae immediately below to a depth of 30 µm below Bowman's layer (sutural lamellae) as well as of those at a depth of 50 or 100 µm were measured. The density and width of sutural lamellae were also evaluated. RESULTS: Collagen lamellae in the anterior stroma were evenly distributed and randomly oriented. The angle of sutural lamellae relative to Bowman's layer was 19.19 ± 4.34° (mean ± SD). The angles of collagen lamellae at depths of 50 or 100 µm were 8.91 ± 2.91 and 6.91 ± 2.11°, respectively. The density of sutural lamellae was 910.0 ± 480.4/mm(2), and their width was 13.14 ± 5.03 and 7.11 ± 3.00 µm in the region immediately beneath and 30 µm below Bowman's layer, respectively. CONCLUSIONS: Collagen lamellae in the anterior stroma of the normal human cornea are interwoven in three dimensions and adhere densely to Bowman's layer. This structure may contribute to the rigidity and curvature of the anterior portion of the cornea.

[In vivo laser confocal microscopic analysis of the interface between Bowman's layer and the stroma of the cornea].

Recently, cornea-specific in vivo laser confocal microscopy (Heidelberg Retina Tomograph 2 Rostock Cornea Module (HRT2-RCM), Heidelberg Engineering GmbH, Dossenheim, Germany) has become available, are now possible detailed in vivo observation of corneal and conjunctival microstructure. Using HRT2-RCM, we have demonstrated the presence of "polymorphic structures composed of fibrillar material" beneath Bowman's layer in normal volunteer eyes. We surmise that these microstructures may correspond to the modified and condensed anterior stromal collagen fibers/lamellae that merge into Bowman's layer. We also observed numerous ridges protruding into the epithelial basal and wing cell layers after application of pressure with a Tomo-cap. These ridges corresponded exactly to the orientation of the K-structures beneath the epithelial cells, suggesting that these ridge formations correspond to the pattern of the anterior mosaic formation. The potential association of these microstructures with anterior corneal mosaic formation is also discussed in this review.

In vivo confocal microscopy of human cornea covered with human amniotic membrane.

PURPOSE: Amniotic membrane transplantation has been widely performed to reconstruct the surface of the eye and treat chemical burns or epithelial defects. However, we have difficulty observing the cornea through the opaque transplanted amniotic membrane by slit-lamp biomicroscopy. We investigated the use of confocal microscopy for observation of human corneas covered with amniotic membrane. METHODS: Human amniotic membrane was placed onto the normal corneas of five volunteers aged 22-24 years. Then, all layers of the covered corneas were observed by in vivo confocal microscopy. RESULTS: Confocal microscopy displayed the epithelium, basement membrane, and stroma of the amniotic membrane. It also displayed the corneal epithelium. Furthermore, corneal stromal keratocytes and the corneal endothelium were clearly observed through the amniotic membrane by confocal microscopy. CONCLUSIONS: We demonstrated that in vivo confocal microscopy enabled us to observe all layers of corneas covered with amniotic membrane in normal human eyes. Our findings suggest that confocal microscopy may have advantages for clinical examination of the ocular surface, including all layers of the cornea.

Mapping of normal corneal K-structures by in vivo laser confocal microscopy.

PURPOSE: To produce 2-dimensional reconstruction maps of normal human corneal fibrous structures beneath the Bowman layer (K-structures) by in vivo laser confocal microscopy and to show association of structures with the anterior corneal mosaic (ACM). METHODS: Central corneal regions of 3 healthy volunteers were scanned. Acquired images of K-structures for each eye were arranged and mapped into a subconfluent montage. For each subject, electrical tracings of K-structures were superimposed on a slit-lamp photograph of the ACM produced by rubbing the eyelid. RESULTS: A mean of 677 +/- 211 images of K-structures were obtained for each eye. Mean dimensions of the mapped areas were 5.88 +/- 0.50 (horizontal) and 3.51 +/- 1.37 mm (vertical). In all subjects, K-structures formed a netlike pattern (mean area, 0.082 +/- 0.051 mm), and electrical tracings had good concordance with the ACM. CONCLUSIONS: This is the first study, to our knowledge, to elucidate the overall distribution of K-structures in normal human corneas. The netlike pattern of K-structures corresponded well with ACM pattern. These results support the hypothesis that the K-structures are the anterior collagen fiber bundles running at the posterior surface of the Bowman layer and thus are the structural basis for ACM formation.

In vivo laser confocal microscopy of Bowman's layer of the cornea.

PURPOSE: To investigate in vivo microstructures of Bowman's layer in normal human subjects using a cornea-specific in vivo laser scanning confocal microscope (Heidelberg Retina Tomograph 2 Rostock Cornea Module, HRT2-RCM). DESIGN: Single-center, prospective, observational case series. PARTICIPANTS: Nineteen normal volunteers (10 male, 9 female; mean age, 46.2+/-21.7 years [range, 18-77]). METHODS: The central and peripheral cornea, specifically the epithelium, Bowman's layer, and its subjacent stroma, were examined using the HRT2-RCM. MAIN OUTCOME MEASURES: Selected images of the corneal layers were evaluated qualitatively for the shape and degree of light reflection of the microstructures. RESULTS: In all subjects, normal epithelial (superficial, wing, basal) cells, subbasal nerve plexus, Bowman's layer, and its subjacent stoma were observed clearly. However, in all subjects, polymorphic structures composed of fibrillar materials with less reflectivity than corneal nerves were observed beneath Bowman's layer. After application of pressure by a Tomo-cap, we observed numerous ridges that protruded into the epithelial basal and wing cell layers. Superficial stromal striae were also observed. These ridges and striae corresponded exactly to the orientation of the fibrous structures located beneath the epithelial cells. CONCLUSION: We report for the first time, the presence of polymorphic structures composed of fibrillar materials (K-structures) beneath Bowman's layer in normal human subjects, detected by HRT2-RCM. We surmise that these microstructures may correspond to the modified and condensed anterior stromal collagen fibers/lamellae that merge into Bowman's layer and that these fibrillar materials may be responsible for the formation of the anterior corneal mosaic. Further investigation of these microstructures in diseased eyes may provide insights into their pathophysiologic role in Bowman's layer.

Comparative anatomy of laboratory animal corneas with a new-generation high-resolution in vivo confocal microscope.

PURPOSE: The aim of the current study was to compare the corneas of three commonly used laboratory animals with a new in vivo confocal microscope. METHODS: Six eyes of three adult male New Zealand albino rabbits, six eyes of three adult male Lewis rats, and six eyes of three adult male Swiss mice were used in this study. Corneas were analyzed in vivo using the Rostock Cornea Module of the Heidelberg Retina Tomograph (HRT)-II. For all eyes, 20 confocal microscopic images of each layer, that is, the superficial and basal corneal epithelia, the Bowman layer, the anterior and posterior stroma, and the endothelium, were recorded. The images were then analyzed qualitatively and compared among animals. Cellular densities of anterior and posterior stroma keratocytes of rabbits and endothelium density of the three different animals were also measured and compared. RESULTS: The Rostock Cornea Module of the HRT II was successfully used to analyze all corneal layers of these three commonly used laboratory animals. Although the cellular patterns of the corneal layers of these three animals, as observed with in vivo confocal microscopy, were quite similar, some differences were seen in terms of endothelial cell density and stroma appearance. Superficial cells were seen as hyper- and hyporeflective polygonal cells. Basal cells had dark cytoplasm without visible nuclei and were closely organized. A Bowman layer was observed in all three animals as an amorphous tissue containing fine subepithelial nerve plexus. In rabbits, the stroma consisted of an amorphous ground substance with hyper-reflective structures corresponding with keratocyte nuclei. In rats and mice, numerous reflective stellate structures with no clearly visible nuclei were observed within the stroma. Besides endothelial cell density, the endothelium was similar among the three animals and was seen as hyper-reflective cells with dark limits organized in a honeycomb pattern. CONCLUSIONS: The Rostock Cornea Module of the HRT II can provide high-resolution images of all corneal layers of rabbits, rats, and mice without sacrificing animals or preparing tissue. This new device may be useful for evaluating the cornea during experimental animal studies.