High resolution three-dimensional imaging of the ocular surface and intact eyeball using tissue clearing and light sheet microscopy.

purposeHigh resolution visualization of the ocular surface and intact eyeball is critical and essential for our understanding and treatment of eye diseases. This study is to achieve this goal using advanced tissue clearing and three-dimensional (3D) imaging technologies.

methodsWild type and fluorescently labeled transgenic mice of Prox-1-GFP (green fluorescent protein) or Thy1-YFP (yellow fluorescent protein) were used in the study. Eyeballs were harvested from normal or a disease model of corneal inflammation. Samples were infused with hydrogel monomers and heated for polymerization. Lipids were removed by electrophoresis. The transparent tissue-hydrogel hybrids of the anterior segments or intact eyeballs with immunolabeling or endogenous fluorescence were imaged by an advanced light sheet fluorescent microscope. High resolution 3D images and videos were captured for a wide array of structures and cell types.

resultsOptical transparency was achieved from intact eyeballs of both normal and diseased conditions. A variety of important structures and cell types, such as blood and lymphatic vessels, Schlemm's canal, nerves and endothelial cells, were detected with their natural morphology, location and organizational network.

conclusionsThis study provides the first comprehensive and 3D high resolution imaging of the intact eyeball using tissue clearing and advanced light sheet microscopy. Given that the eye is the window of the body, we anticipate this advanced technology will facilitate diverse applications in biomedical research inside and outside the eye.