1. Intravital multiphoton microscopic imaging platform for ocular surface imaging
- Author
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Yueh-Feng Wu, Chia-Yi Wang, Tsung-Lin Yang, Hsin-Yuan Tan, Sung-Jan Lin, and Po-Nien Tsao
- Subjects
0301 basic medicine ,Genetically modified mouse ,Cell type ,Conjunctiva ,Corneal Stroma ,Diagnostic Techniques, Ophthalmological ,Limbus Corneae ,Green fluorescent protein ,Cornea ,Mice ,03 medical and health sciences ,Cellular and Molecular Neuroscience ,Imaging, Three-Dimensional ,0302 clinical medicine ,In vivo ,medicine ,Animals ,Corneal epithelium ,Chemistry ,Epithelium, Corneal ,eye diseases ,Sensory Systems ,Epithelium ,Cell biology ,Ophthalmology ,Microscopy, Fluorescence, Multiphoton ,030104 developmental biology ,medicine.anatomical_structure ,030221 ophthalmology & optometry ,sense organs - Abstract
The purpose of this study is to provide an intravital noninvasive multiphoton microscopic platform for long-term ocular imaging in transgenic fluorescent mice with subcellular resolution. A multiphoton microscopic system with tunable laser output was employed. We designed a mouse holder incorporated with stereotaxic motorized stage for in vivo three-dimensional imaging of ocular surface in 3 transgenic mouse line with fluorescent protein (FP) expression to visualize distinct structures. With our imaging platform and the expression of FPs, we obtained the three-dimensional images across the whole cornea from epithelium to endothelium and in conjunctiva with subcellular resolution in vivo. Specified EGFP expression in corneal epithelium of K5-H2B-EGFP mice helped to identify both corneal and limbal epithelial cells while ubiquitous nuclear FP expression in R26R-GR mice allowed us to visualized nuclei of all cell types. Universal membrane-localized FP in mT/mG mice outlined all cell boundaries, nerve fibers, and capillaries. The simultaneously collected second harmonic generation signals from collagenous stroma provided architectural contrast. Time-lapsed recording enabled monitoring the mitotic activity of corneal epithelial cells and limbal epithelial cells. We developed an intravital multiphoton microscopic stereotaxic imaging platform and showed that, by incorporating FP-expressing transgenic mice, this platform enables in vivo 4-dimensional ophthalmic study at subcellular resolution.
- Published
- 2019