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Updated: Aug 10, 2026
A Custom Multiphoton Microscopy Platform for Live Imaging of Mouse Cornea and Conjunctiva
Published on: May 17, 2020
Imaging corneal pathology in a transgenic mouse model using nonlinear microscopy
Julia G Lyubovitsky1, Joel A Spencer, Tatiana B Krasieva
1University of California, Beckman Laser Institute, Laser Microbeam and Medical Program, Irvine, California 92612, USA. jlyubovi@uci.edu
Abstract:
A transgenic mouse model with a Clim [co-factor of LIM (a combination of first letters of Lin-11 (C. elegans), ISL1 (rat), and Mec-3 (C. elegans) gene names) domain proteins] gene partially blocked in the epithelial compartment of its tissues is used to establish the sensitivity of intrinsic reflectance nonlinear optical microscopy (NLOM) to stromal and cellular perturbations in the cornea. Our results indicate dysplasia in the squamous epithelium, irregular collagen arrays in the stroma, and a compromised posterior endothelium in the corneas of these mice. As suggested by biochemical data, the collagen alterations are likely due to collagen III synthesis and deposition during healing and remodeling of transgenic mice corneal stromas. All of the topographic features seen in NLOM images of normal and aberrant corneas are confirmed by coregistration with histological sections. In this work, we also use ratiometric redox fluorometry based on two-photon excited cellular fluorescence from reduced nicotinamide adenine dinucleotide (NAD)(P)H and oxidized flavin adenine dinucleotide (FAD) to study mitochondrial energy metabolism. Employing this method, we detect higher metabolic activity in the endothelial layer of cornea compared to an epithelial layer located further away from the metabolites. The combination of two-photon excited fluorescence (TPF) with second harmonic generation (SHG) signals allows imaging to aid in understanding the relationship between alternation of specific genes and structural changes in cells and extracellular matrix.
Insights
Nonlinear optical microscopy (NLOM) reveals corneal abnormalities in transgenic mice with blocked Clim gene function. These changes include epithelial dysplasia, stromal collagen irregularities, and endothelial compromise, impacting corneal structure and metabolism.
Area of Science:
- Ophthalmology
- Biomedical Engineering
- Molecular Biology
Background:
- Gene alterations can lead to significant structural and metabolic changes in tissues.
- Nonlinear optical microscopy (NLOM) offers advanced imaging capabilities for biological tissues.
Purpose of the Study:
- To investigate the sensitivity of NLOM to detect corneal perturbations caused by genetic modification.
- To correlate gene expression changes with structural and metabolic alterations in the cornea.
Main Methods:
- Utilized a transgenic mouse model with partially blocked Clim gene function in the corneal epithelium.
- Employed intrinsic reflectance NLOM to image corneal structure, including epithelium, stroma, and endothelium.
- Applied ratiometric redox fluorometry (two-photon excited cellular fluorescence of NAD(P)H and FAD) to assess mitochondrial energy metabolism.
- Confirmed NLOM findings with histological sections and biochemical data.
Main Results:
- NLOM detected epithelial dysplasia, irregular collagen arrays in the stroma, and a compromised posterior endothelium in transgenic corneas.
- Collagen alterations were linked to increased synthesis and deposition of collagen III during corneal remodeling.
- Ratiometric redox fluorometry revealed higher metabolic activity in the corneal endothelium compared to the epithelium.
- Combined TPF and SHG imaging elucidated the relationship between gene alternation and structural changes.
Conclusions:
- NLOM is sensitive to genetic perturbations affecting corneal structure and cellular metabolism.
- Clim gene modification induces significant corneal abnormalities, including stromal and endothelial damage.
- The combination of NLOM and metabolic imaging provides a powerful tool for studying gene-environment interactions in corneal diseases.
