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Updated: Jun 16, 2026
06:53
A Custom Multiphoton Microscopy Platform for Live Imaging of Mouse Cornea and Conjunctiva
Published on: May 17, 2020
[Two-photon microscopy of the cornea using intrinsic contrast]
A Krüger1, M Hovakimyan, D F Ramírez
1Biomedizinische Optik, Laser Zentrum Hannover e. V, Hannover. a.krueger@lzh.de
Klinische Monatsblatter Fur Augenheilkunde
|January 29, 2010
Summary
Femtosecond laser-based non-linear microscopy enables label-free 3D imaging of corneal extracellular matrix (ECM) and cells. This technique visualizes corneal structures like collagen and NAD(P)H autofluorescence, offering a new tool for ophthalmic research.
Area of Science:
- Ophthalmic Imaging
- Biophotonics
- Microscopy
Context:
- Corneal transparency limits contrast in traditional microscopy and optical coherence tomography.
- Visualizing the extracellular matrix (ECM) and cellular structures is crucial for understanding corneal physiology and pathology.
- Existing methods often require exogenous staining, which can be invasive or alter tissue properties.
Purpose:
- To demonstrate the capability of femtosecond laser-based non-linear microscopy for intrinsic contrast imaging of the cornea.
- To visualize corneal cellular components and the ECM using two-photon excited autofluorescence of NAD(P)H and second harmonic generation (SHG).
- To assess the potential of this technique for evaluating corneal treatments, such as riboflavin/UV cross-linking.
Summary:
- Femtosecond laser non-linear microscopy utilizes NAD(P)H autofluorescence to visualize corneal epithelium and keratocytes in 3D without staining.
- Second harmonic generation (SHG) imaging reveals the layered, oriented structure of collagen-I fibers within the corneal stroma.
- The technique successfully identified post-treatment alterations in rabbit corneas after riboflavin/UV cross-linking, showing changes in autofluorescence and keratocyte distribution.
Impact:
- Establishes non-linear microscopy as a powerful, dye-free imaging modality for the cornea.
- Complements existing techniques like optical coherence tomography and confocal microscopy by providing intrinsic contrast for ECM visualization.
- Offers a new avenue for research into corneal diseases, wound healing, and treatment efficacy assessment.
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