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Hongmin Zhang, Liya Wang, Susu Liu, Yanting Xie, Xipi Wu, Huiwei Zhao; High-Resolution, Noninvasive, Two-Photon Imaging of the Intact Cornea in Living Mouse. Invest. Ophthalmol. Vis. Sci. 2012;53(14):5008.
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To the best of our knowledge, there are no reports of high-resolution, noninvasive, two-photon maging of the intact cornea in living mouse. Therfore, to image the morphological features and the detailed structure of epithelium, keratocytes, endothelial cell; corneal collagen fibril bundles and nerves within the intact in vivo mouse cornea using two-photon laser microscopy.
Two-photon microscopy (2PM) techniques, including two-photon fluorescence (2PF) and second harmonic generation (SHG), were used to obtain images of the entire cornea in living mouse cornea. Under the anesthesia, fluorescent viability probes (Hoechest 33342 and CellMask™ Orange plasma membrane stain) have been used to visualize the morphology and organisation of the corneal native cells and nerves with intrastromal injection. The intact cornea was scanned at the wavelength of 720 nm and 780nm emitted from femtosecond laser with Zeiss two-photon laser scanning microscope. The broadband two-photon fluorescence (SP 485 nm and 565-610) and second harmonic generation (SHG, 380-430 nm) signals of the corneal collagen fibrils were collected in the reflected light channel by non-descanned detection (NDD). A water immersion objective (Plan-Apochromat, 20X, NA 1.0; Zeiss) was used to facilitate imaging the curve ocular surface.
The 2PM images over entire cornea provided morphological information of epithelium, keratocytes, endothelial cell, corneal collagen fibril bundles and nerves. Specifically, our planar, large area SHG image reveals a concentric pattern of the stroma collagen, indicative of the laminar collagen organization throughout the stroma. In addition, the vital dyes labeling contributed to fluorescence contrast of nucleus and cell outline and facilitated visualizing of native cells and corneal nerves.
Our results show that 2PM imaging of the living dyes labeled mouse cornea manifests both morphological significance and structural details within the intact living cornea without slicing. These results support that 2PM is an appropriate technology for further in vivo investigation and diagnosis of cornea.
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