Assessment of Human Corneas Prior to Transplantation Using High-Resolution Two-Photon Imaging

Abstract

PURPOSE. The purpose of this study was to evaluate the feasibility of using two-photon imaging (TPI) to assess the condition of human corneas for transplantation.METHODS. Human corneas were imaged after different storage times: short-term (STS), mediumterm (MTS), and long-term (LTS) storage. A high-resolution, custom-built 5-dimensional multiphoton microscope with 12-fs pulsed laser excitation was used for image acquisition. RESULTS.Optical discrimination between different corneal layers and sublayers based on their morphologic characteristics revealed by two-photon autofluorescence (AF) is possible. Furthermore, all layers were characterized based on AF lifetimes to gain information on metabolic activities of cells. The NAD(P)H free to protein-bound ratio (a 1 /a 2 ) of epithelial cells increased significantly in both MTS and LTS corneas compared with STS corneas. In endothelial cells, NAD(P)H a 1 /a 2 was significantly increased in MTS samples. For keratocytes, the NAD(P)H a 1 /a 2 decreased significantly with storage time. This could indicate that the metabolic activity of the epithelial and endothelial cells reduces, whereas the activity of keratocytes increases with storage time. The analysis of the stroma SHG images indicated that the organization of collagen fibers decreases with storage time. The feasibility of measuring the endothelial cell density (ECD) using TPI was demonstrated. An ECD of 1461 6 190 cells/ mm 2 was obtained for MTS samples based on TPI.CONCLUSIONS. TPI can provide information not accessible by current clinical methods, such as the cells' metabolic state and structural organization of the stroma, with subcellular resolution. Thus, it may improve the screening process of corneas prior to transplantation and might help to optimize the storage conditions.

Riboflavin Concentrations at the Endothelium During Corneal Cross-Linking in Humans

“…In the near future, clinical in vivo two-photon imaging of the cornea will be available as an instrument to optimize the treatment parameters. 33,34 In conclusion, the results presented here reveal a riboflavin concentration at the endothelium substantially lower than initially anticipated. Based on our findings, the 400-lm rule is too high.…”

Assessment of the metabolism and morphology of the porcine cornea, lens and retina by 2‐photon imaging

“…The feasibility of TPI to evaluate the cornea cells morphology, metabolic activity and stroma structural organization may provide clinicians with additional information that can aid in patient diagnosis and follow up. The advantages of TPI of the cornea to assess alterations due to pathologies [52,53], storage [17] or medical procedures [54] have already been demonstrated by our and other groups.…”

“…Moreover, our group has described the morphology and metabolism of different layers and sublayers of the porcine cornea based on the AF intensity and lifetime of the metabolic cofactors . Recently, we have also reported on the evaluation of metabolic alterations induced by storage time in ex vivo human corneas as well as the feasibility of improving the screening process of corneal samples prior to transplants using TPI .…”

An Update on Corneal Imaging Techniques: from Macroscale to Nanostructure

“…Both 2PA and SHG signals are complementary in analysing simultaneously all components of the different matrices (cellular and extracellular) in biological tissues as in the case of the cornea. The combination of both twophoton imaging modalities has been employed to assess human corneas prior to transplantation as 2PA provides metabolic information of cells and SHG structural organization of collagen fibres [72].…”