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

Ana Batista1,2, Hans Georg Breunig2, Aisada König1,2

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Two-photon imaging (TPI) assesses human cornea viability for transplantation by analyzing cellular metabolism and stromal structure. This advanced technique offers insights beyond current methods, potentially improving donor cornea selection and storage protocols.

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Area of Science:

  • Ophthalmology
  • Biomedical Imaging
  • Cell Biology

Background:

  • Corneal transplantation relies on donor tissue viability.
  • Current screening methods for donor corneas have limitations in assessing cellular health and stromal integrity.
  • Advanced imaging techniques are needed to optimize the pre-transplantation evaluation of corneas.

Purpose of the Study:

  • To evaluate the feasibility of two-photon imaging (TPI) for assessing the condition of human corneas intended for transplantation.
  • To determine if TPI can provide information on corneal layers, cellular metabolism, and stromal organization.
  • To assess the potential of TPI to improve the screening of donor corneas.

Main Methods:

  • Human corneas were subjected to short-term (STS), medium-term (MTS), and long-term (LTS) storage.
  • A custom-built 5-dimensional multiphoton microscope with pulsed laser excitation was used for imaging.
  • Two-photon autofluorescence (AF) and second-harmonic generation (SHG) imaging were employed to analyze corneal layers and collagen structure.

Main Results:

  • TPI successfully discriminated between corneal layers and sublayers using autofluorescence.
  • Metabolic activity, assessed by NAD(P)H ratios, showed significant changes in epithelial, endothelial, and keratocyte cells with storage time.
  • Stromal collagen organization decreased with storage time, and endothelial cell density (ECD) was measurable via TPI.

Conclusions:

  • TPI provides subcellular resolution insights into corneal metabolic state and stromal structure, surpassing current clinical methods.
  • The metabolic and structural data from TPI can aid in optimizing corneal storage conditions.
  • TPI holds promise for enhancing the pre-transplantation screening process for donor corneas.