Detection of Subepithelial Fibrosis Associated with Corneal Stromal Edema by Second Harmonic Generation Imaging Microscopy

Human corneas with or without stromal edema were examined by second harmonic generation (SHG) imaging microscopy to characterize stromal collagen organization. Tissue buttons from 31 corneas with stromal edema and 8 normal corneas were fixed, and 3-mm(2) blocks were cut and stained with phalloidin, to visualize the cytoskeleton. The blocks were examined by SHG imaging with a laser confocal microscope and a mode-locked titanium:sapphire femtosecond laser. Samples were scanned to a depth of 150 microm from the surface of Bowman's layer, and SHG forward- and backscatter signals were collected. Phalloidin staining was detected by conventional laser confocal microscopy. The three-dimensional structure of the anterior segment of the cornea was reconstructed from stacked SHG images. Three-dimensional reconstruction of SHG signals showed adherence of interwoven collagen lamellae in the anterior stroma to Bowman's layer in both normal and edematous corneas. Abnormal SHG signals at the level of Bowman's layer were observed in edematous corneas; three-dimensional images revealed that these signals were actually localized above Bowman's layer and were indicative of subepithelial fibrosis. Phalloidin staining showed transdifferentiation of stromal cells into fibroblastic cells in edematous corneas. The incidence of subepithelial fibrosis or of fibroblastic cells increased beginning 12 months after the onset of clinical stromal edema. SHG imaging of the anterior segment of edematous corneas revealed a normal appearance of interwoven collagen lamellae in the anterior stroma. The development of subepithelial fibrosis beginning 12 months after the onset of edema suggests that stromal edema may be a progressive disease.