Two-photon microscopy of the cornea using intrinsic contrast

Klinische Monatsblätter für Augenheilkunde
A KrügerA Heisterkamp

Abstract

Three-dimensional imaging of the cornea under physiological conditions is best performed with intrinsic contrast mechanisms for the visualisation of cells and extracellular matrix. However, the unique transparency of the cornea goes along with a lack of contrast for the extracellular matrix (ECM) in reflective mode microscopy and optical coherence tomography. Femtosecond laser-based non-linear microscopy provides novel contrast mechanisms for the visualisation of ECM. The confinement of the non-linear contrast to the focus volume provides an intrinsic sectioning property for 3D imaging. Further advantages of the infrared light are lower phototoxicity and higher penetration depth into the tissue. For the visualisation of the cornea and its layered substructures two non-linear contrast mechanisms are of main interest: Two-photon excited autofluorescence of NAD(P)H in the cytoplasma and second harmonic generation (SHG) in the collagen-I fibres of the stroma. Ex-vivo corneas of the rabbit were imaged to demonstrate the abilities of non-linear microscopy. Using the autofluorescence of NAD(P)H the corneal epithelium with squamous cells, wing cells and basal cells is visualised in three dimensions without additional exogenoeus stainin...Continue Reading

Citations

Jun 28, 2012·Der Ophthalmologe : Zeitschrift der Deutschen Ophthalmologischen Gesellschaft·C HussK König
Nov 7, 2017·Advanced Functional Materials·Adam E JakusRamille N Shah

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