Structural analysis of normal corneas and diseased corneas by applying second harmonic generation

We have established a second harmonic generation (SHG) microscopy system for imaging of the human cornea with a mode-locked femtosecond laser and a laser confocal microscope. This SHG microscopy system has allowed us to scan corneal tissue noninvasively ex vivo and to obtain three-dimensional images of corneal collagen lamellae. Such three-dimensional imaging of the normal anterior cornea revealed that collagen lamellae at the anterior stroma are inter-woven and adhere to Bowman membrane with these adherent lamellae being designated "sutural lamellae." Sutural lamellae adhere to Bowman membrane at an angle of approximately 19 degrees, whereas the angle of lamellae in the mid-stroma relative to Bowman membrane is smaller. We hypothesize that the structural unit consisting of both Bowman membrane and the sutural lamellae contributes to the rigidity and anterior curvature of the cornea. SHG imaging of keratoconic corneas revealed an either abnormal or a total lack of structure of the sutural lamellae, suggesting that this abnormality might be related to that of the corneal anterior curvature in such corneas. Furthermore, SHG imaging of corneas affected by stromal edema showed that the structure of the sutural lamellae was maintained, although abnormal collagen signals both above and below Bowman membrane were detected in corneas affected by clinical stromal edema for more than 12 months. SHG imaging of the structure of collagen lamellae in normal and diseased corneas thus has the potential to provide insight both into the mechanism for maintenance of corneal curvature as well as into the pathophysiology of corneal diseases.