Abstract：
:Immunofluorescent imaging is an indispensable technique to study morphology and molecular aspects in tissues. Classical approaches make it necessary to cut physical sections of tissue samples to overcome the limited penetration depth of light, restricting the available information to two dimensions. Recent advances in tissue-clearing techniques enable imaging of fluorescently labeled organs and entire organisms on a cellular level in three dimensions without the need of sectioning. Volume imaging of immunolabeled and cleared tissues started a new era of systems biology, because these techniques provide information on connectivity and circuits, especially in structures with projections in three dimensions such as vascular or nervous systems. The variety of published clearing protocols allows the imaging of every organ with a single exception: the eye. Whole-eye clearing approaches were unsuccessful so far due to the strong pigmentation of the retinal pigment epithelium. Here, we present a new protocol that combines a highly effective melanin bleaching step with solvent-based clearing, termed EyeCi. The protocol is compatible with immunolabeling as demonstrated by the visualization of ocular and retinal vasculature in the intact mouse eye by means of light-sheet fluorescence microscopy. This novel protocol is rapid (1 week) and inexpensive, hence allowing high-throughput, high resolution analysis of vascular architecture of healthy and diseased eyes, in its native, three-dimensional organization within intact eyeballs. Volume imaging of whole cleared eyeballs further enables three-dimensional surface reconstruction and automated quantification of choroidal and retinal vasculature extending ocular imaging to a global level. Thus, EyeCi represents an extension to state-of-the-art light microscopy techniques and is potentially suitable for the investigation of vascular leakage or neovascularization processes.
journal_name
Exp Eye Resjournal_title
Experimental eye researchauthors
Henning Y,Osadnik C,Malkemper EPdoi
10.1016/j.exer.2018.12.001subject
Has Abstractpub_date
2019-03-01 00:00:00pages
137-145eissn
0014-4835issn
1096-0007pii
S0014-4835(18)30569-4journal_volume
180pub_type
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journal_title：Experimental eye research
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journal_title：Experimental eye research
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journal_title：Experimental eye research
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