Two-photon optical microscopy imaging of endothelial keratoplasty grafts

Purpose: To investigate the microstructure of endothelial keratoplasty grafts using two-photon optical microscopy. Methods: Six endothelial keratoplasty grafts obtained from human donor corneoscleral tissues and prepared by submerged hydrodissection technique were imaged by two-photon optical microscopy. In each graft, two liquid bubbles were created in order to investigate the presence of a conserved cleavage plane regardless of the volume of posterior stroma that remained attached to Descemet's membrane (DM); the first bubble (bubble A) was generated under DM and the second bubble (bubble B) injection was done in order to obtain a layer of deep stroma that kept the two bubbles separated. Six human donor corneoscleral tissues were used as controls. Second harmonic generation and two-photon emitted fluorescence signals were collected from each specimen. Results: Dissection of stroma occurred along the posterior collagen lamellae at variable distance from DM, which ranged between 3 and 16 ?m in bubble A and between 23 and 41 ?m in bubble B. The residual stroma included, anteriorly, bands of collagen lamellae, and thin bundles of stromal collagen fibrils, posteriorly, which were tightly intertwining with the underlying DM. There was no anatomically distinct plane of separation between these pre-Descemetic stromal collagen bundles and the overlying collagen lamellae with this hydrodissection technique. Conclusions: Two-photon optical microscopy provided label-free high-resolution imaging of endothelial keratoplasty grafts, showing that the most posterior stroma changes organization at approximately 10 ?m above the DM. The pre-Descemetic stromal collagen fibrils form an intertwined complex with DM, which cannot be separated using hydrodissection.