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Yueh-Feng Wu, Hsin-Yuan Tan, Sung-Jan Lin; Intravital imaging of corneolimbal cell dynamics by two-photon microscope. Invest. Ophthalmol. Vis. Sci. 2018;59(9):5837.
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© ARVO (1962-2015); The Authors (2016-present)
Corneal epithelial stem cells are thought to reside at junctional limbal area, and are responsible for the homeostasis of corneal epithelium. Any pathology that leads to limbal deficiency may result into potential blindness. Currently, there is no imaging system available clinically for monitoring corneolimbal cell dynamics in vivo. In this work, we propose two-photon microscopic technique as an effective intravital imaging system for visualizing and tracking corneolimbal epithelial cell dynamics.
The two-photon microscope system used in this work is a home-built system, with the laser output wavelength set at 880 nm for GFP and 960nm for tdTomato, respectively. The laser power was set to be ~80mW. We used three transgenic mice for tracking different cellular structures: K5-H2B-EGFP, Gt(ROSA26)ACTB-tdTomato-EGFP(mT/mG), and R26R-GR transgenic mice. In order to study cell dynamics, we also designed a mouse holder and stereotaxic platform for continuous imaging ocular surface for a relatively long duration(12h). All images are analyzed by imaging J and 3D reconstruction was performed by AVIZO software.
We found that K5 expressed in basal cells and wing cells of cornea, and also basal cells of limbus. Through mT/mG transgenic mice, all cell membrane, the neuron within stroma, and the capillaries could be outlined. With the stereotaxic platform we designed, the mitotic activity could be monitored in R26R-GR transgenic mice. In addition to the cellular components, the collagenous stroma could be observed using second harmonic generation signals with no need of additional staining.
In this study, we established an intravital imaging platform for corneolimbal region by two-photon microscope. We used various transgenic mice models for visualizing and tracking different cellular components within corneolimbal region. The system could potentially be applied as a useful platform for study cell dynamics of ocular surface in vivo.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
Figure 1. The 3D imaging of corneal and limbal epithelial cells.
Figure 2. The live imaging of various cells in cornea and limbus.
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