Assessment of Human Corneas Prior to Transplantation Using High-Resolution Two-Photon Imaging
| Autor: | Aisada König, Hans Georg Breunig, Tobias Hager, Karsten König, António Miguel Morgado, Ana Batista, Andreas Schindele, Berthold Seitz |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2018 |
| Předmět: |
Endothelium
Confocal Cell Count 01 natural sciences 010309 optics Corneal Transplantation 03 medical and health sciences 0302 clinical medicine Imaging Three-Dimensional Organ Culture Techniques Two-photon excitation microscopy Cornea 0103 physical sciences Microscopy medicine Humans femtosecond lasers Microscopy Confocal Chemistry optical metabolic imaging Endothelium Corneal fluorescence lifetime imaging Transplantation Autofluorescence medicine.anatomical_structure Preoperative Period 030221 ophthalmology & optometry NAD+ kinase two-photon imaging Biomedical engineering second-harmonic generation |
| Popis: | 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), medium-term (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 (a1/a2) of epithelial cells increased significantly in both MTS and LTS corneas compared with STS corneas. In endothelial cells, NAD(P)H a1/a2 was significantly increased in MTS samples. For keratocytes, the NAD(P)H a1/a2 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 ± 190 cells/mm2 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. |
| Databáze: | OpenAIRE |
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