2 March 2018 High-resolution, label-free two-photon imaging of diseased human corneas
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The diagnosis of corneal diseases may be improved by monitoring the metabolism of cells and the structural organization of the stroma using two-photon imaging (TPI). We used TPI to assess the differences between nonpathological (NP) human corneas and corneas diagnosed with either keratoconus, Acanthamoeba keratitis, or stromal corneal scars. Images were acquired using a custom-built five-dimensional laser-scanning microscope with a broadband sub-15 femtosecond near-infrared pulsed excitation laser and a 16-channel photomultiplier tube detector in combination with a time-correlated single photon counting module. Morphological alterations of epithelial cells were observed for all pathologies. Moreover, diseased corneas showed alterations to the cells’ metabolism that were revealed using the NAD(P)H free to protein-bound ratios. The mean autofluorescence lifetime of the stroma and the organization of the collagen fibers were also significantly altered due to the pathologies. We demonstrate that TPI can be used to distinguish between NP and diseased human corneas, based not only on alterations of the cells’ morphology, which can also be evaluated using current clinical devices, but on additional morphological and functional features such as the organization of the stroma and the cells’ metabolism. Therefore, TPI could become an efficient tool for diagnosing corneal diseases and better understanding the biological processes of the diseases.
© 2018 Society of Photo-Optical Instrumentation Engineers (SPIE)
Ana Batista, Ana Batista, Hans Georg Breunig, Hans Georg Breunig, Aisada König, Aisada König, Andreas Schindele, Andreas Schindele, Tobias Hager, Tobias Hager, Berthold Seitz, Berthold Seitz, Karsten König, Karsten König, "High-resolution, label-free two-photon imaging of diseased human corneas," Journal of Biomedical Optics 23(3), 036002 (2 March 2018). https://doi.org/10.1117/1.JBO.23.3.036002 . Submission: Received: 20 November 2017; Accepted: 8 February 2018
Received: 20 November 2017; Accepted: 8 February 2018; Published: 2 March 2018


Two-photon cryomicroscope
Proceedings of SPIE (February 09 2012)

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