Intravital multiphoton microscopic imaging platform for ocular surface imaging.
Exp Eye Res
; 182: 194-201, 2019 05.
Article
em En
| MEDLINE
| ID: mdl-30822399
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.
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Base de dados:
MEDLINE
Assunto principal:
Córnea
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Imageamento Tridimensional
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Microscopia de Fluorescência por Excitação Multifotônica
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Técnicas de Diagnóstico Oftalmológico
Limite:
Animals
Idioma:
En
Revista:
Exp Eye Res
Ano de publicação:
2019
Tipo de documento:
Article
País de afiliação:
Taiwan