EyeCi: Optical clearing and imaging of immunolabeled mouse eyes using light-sheet fluorescence microscopy
EyeCi: Optical clearing and imaging of immunolabeled mouse eyes using light-sheet fluorescence microscopy
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DOI：
10.1016/j.exer.2018.12.001
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发表时间：
2019-03-01
影响因子：
3.4
通讯作者：
Malkemper, Erich Pascal
中科院分区：
文献类型：
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作者：
Henning, Yoshiyuki;Osadnik, Christin;Malkemper, Erich Pascal
免疫荧光成像是研究组织形态和分子方面不可或缺的技术。经典方法需要切割组织样本的物理切片，以克服光的有限穿透深度，将可用信息限制在二维。组织清除技术的最新进展使荧光标记的器官和整个生物体在细胞水平上的三维成像成为可能，而不需要切片。免疫标记和清除组织的体积成像开启了系统生物学的新时代，因为这些技术提供了关于连接和电路的信息，特别是在三维投影结构中，如血管或神经系统。各种已发布的清除协议允许对每个器官进行成像，只有一个例外：眼睛。由于视网膜色素上皮细胞的强烈色素沉着，到目前为止，全眼清洁方法是不成功的。在这里，我们提出了一种新的协议，结合了高效的黑色素漂白步骤与溶剂型清除，称为EyeCi。该方案与免疫标记兼容，如通过光片荧光显微镜观察完整小鼠眼中的眼和视网膜血管所证明的。这种新的协议是快速的（1周）和廉价的，因此允许健康和患病的眼睛的血管结构的高通量，高分辨率的分析，在其原生的，三维组织在完整的眼球。整个透明眼球的体积成像进一步实现了脉络膜和视网膜脉管系统的三维表面重建和自动量化，将眼部成像扩展到全局水平。因此，EyeCi代表了最先进的光学显微镜技术的延伸，并可能适用于血管渗漏或新血管形成过程的调查。
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.