In our study, we showed that the generation of second-harmonic signals from corneal collagen using femtosecond pulsed infrared lasers can be used to assess the lamellar organization of the cornea. Because SHG images identify fibers with an approximate diameter of 1 μm, the SHG signal most likely represents groups of individual collagen fibrils that interact with pulsed laser light to generate second harmonic signals that can then be detected to determine the overall 3-D geometry of the lamellar organization. Using SHIM, three patterns of lamellar organization were detected in the human cornea: transverse, interwoven, and orthogonal. The detection of transverse lamellae supports observations by Komai and Ushiki,2
who report the presence of collagen lamellae that branch and insert into Bowman’s layer. However, because their study used transmission electron microscopy, the extension of these transverse lamellae deeper within the corneal stroma was not reported. The anterior cornea is known to have extensive anteroposterior, interweaving lamellae that obliquely pass from one lamellar layer to another, sometimes passing across several lamellae and occasionally insert to Bowman’s layer.21
These transverse lamellae may contribute to the anterior corneal mosaic that is visible in normal corneas as a polygonal pattern within the anterior stroma after instillation of fluorescein.21
Our findings indicate these transverse lamellae extend much deeper into the anterior cornea and are much more frequent than formerly appreciated.
Recent studies by Müller et al.6
of swollen human corneas also show that the anterior 100 to 120 μm of stroma is resistant to swelling and maintains the corneal curvature. Although it has been proposed that the interwoven nature of the anterior cornea is responsible for this mechanical property, this region also corresponds to the region containing transverse lamellae that extend to a depth of approximately 130 μm. Therefore, it is likely that the presence of transverse lamellae within the anterior cornea provides additional rigidity to the corneal stroma, much greater than that provided by lamellar interweaving, particularly if the lamellae insert into the corneal limbus, as Bron21
Although transverse lamellae were only clearly identified in the anterior quarter of the cornea, it is likely these lamellae extend much deeper into the stroma. If so, the lamellae may represent a structural feature similar to that of “sutural fibers” identified in the dogfish.19,20
Sutural fibers are thought to provide rigidity to the dogfish cornea and play a role in the resistance to swelling following removal of the corneal endothelium, a similar function identified by Müller et al.6
for the anterior region of the human cornea. The importance of this lamellar organizational pattern to the mechanical strength of the cornea is also emphasized by Maurice and Monroe22
and Smolek and McCarey,23
who measured the interlamellar adhesive strength of rabbit and human corneas, respectively. Their studies showed the rabbit cornea is much less resistant than the human cornea to tearing in the lamellar plane. In our studies, lamellar interweaving in rabbit was limited to the anterior one third of the corneal stroma, while in the human, interweaving extended to the anterior two thirds. In addition, transverse, sutural lamellae were detected only in the human. This finding suggests that the transverse, sutural pattern and the more extensive interweaving of lamellae may be important in defining corneal rigidity and resistance to lateral sheer forces.
The distinct organizational pattern in the human cornea may also help explain differences in the risk for corneal ectasia after refractive surgical procedures. It is known that the posterior corneal stroma is mechanically weaker and that this weakness is generally thought to be caused by differences in the proteoglycan composition, collagen crosslinking, and keratocyte density.2,5,24
However, the regional differences in the 3-D collagen geometry, as noted by others and our report, support the hypothesis that the mechanical properties are defined by the microstructural organization and the anterior region of the stroma that contains transverse lamellae is much stiffer than the posterior stroma containing classically orthogonally arranged collagen. Surgical procedures that alter the relative contribution of the two organizational patterns might significantly alter the mechanical properties and susceptibility to ectasia. More important, variation in patient populations in the relative depth or thickness of these structures might also define these patients’ relative risk for ectasia. Although patient variation in corneal structural organization is not known, additional studies are needed to define differences in normal patients and in those with an increased risk for ectasia, such as patients with forme fruste keratoconus.25-27
Because SHG imaging microscopy is noninvasive, this new technology may provide an important approach to evaluating corneal structural organization in patients before refractive surgery. Transverse sutural lamellae were best detected with forward-scattered SHIM; however, outlines of the transverse lamellae were detected using back-scatter SHG signals that could potentially be used to estimate transverse lamellar density and depth in patients. Furthermore, varying the polarization or the angle of the excitation beam may provide better detection of the lamellae using the back-scattered signal detection.
Knowledge of the transverse, sutural lamellar density and how density changes in different patient populations may provide more accurate prediction of the final refractive surgical results as well as prediction of patients at risk for ectasia. Second-harmonic imaging microscopy is a promising in vivo imaging paradigm; however, additional study is needed before evaluation of patients is done.