Microsurgical Dissection and Tissue Clearing for High Resolution Intact Whole Retina and Vitreous Imaging
Microsurgical Dissection and Tissue Clearing for High Resolution Intact Whole Retina and Vitreous Imaging
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DOI：
10.3791/61595
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发表时间：
2021-03-01
影响因子：
1.2
通讯作者：
Motamedi, Massoud
中科院分区：
文献类型：
--
作者：
Nazari, Hossein;Ivannikov, Maxim;Motamedi, Massoud
与传统的视网膜平片制备和切片相比，使用完整的全视网膜成像技术可以更好地观察和表征视网膜在生理和病理状态下的神经元和血管结构。然而，完整全视网膜的免疫荧光成像受到眼球不透明涂层（即巩膜、脉络膜和视网膜色素上皮（RPE））和视网膜层的光散射特性的阻碍，这妨碍了全层高分辨率光学成像。色素层的化学漂白和组织清理方案已被描述为解决这些障碍；然而，目前所描述的方法并不适合在完整的全视网膜中成像内源性荧光分子，如绿色荧光蛋白（GFP）。其他方法绕过了这一限制，通过手术切除色素层和眼球前段，尽管周围视网膜和透明体结构被破坏，但可以获得完整的眼部成像。本文介绍了一种完整的全视网膜和玻璃体免疫荧光成像方案，该方案将巩膜/脉络膜/视网膜色素上皮（RPE）层的手术解剖与改进的组织清除方法和光片荧光显微镜（LSFM）相结合。新方法提供了一个前所未有的观点，不受干扰的血管和神经元元素的视网膜，以及玻璃体和透明体血管系统在病理条件下。
Neuronal and vascular structures of the retina in physiologic and pathologic conditions can be better visualized and characterized by using intact whole retina imaging techniques compared to conventional retinal flat mount preparations and sections. However, immunofluorescent imaging of intact whole retina is hindered by the opaque coatings of the eyeball, i.e., sclera, choroid, and retinal pigment epithelium (RPE) and the light scattering properties of retinal layers that prevent full thickness high resolution optical imaging. Chemical bleaching of the pigmented layers and tissue clearing protocols have been described to address these obstacles; however, currently described methods are not suitable for imaging endogenous fluorescent molecules such as green fluorescent protein (GFP) in intact whole retina. Other approaches bypassed this limitation by surgical removal of pigmented layers and the anterior segment of the eyeball allowing intact eye imaging, though the peripheral retina and hyaloid structures were disrupted. Presented here is an intact whole retina and vitreous immunofluorescent imaging protocol that combines surgical dissection of the sclera/choroid/retina pigment epithelium (RPE) layers with a modified tissue clearing method and light sheet fluorescent microscopy (LSFM). The new approach offers an unprecedented view of unperturbed vascular and neuronal elements of the retina as well as the vitreous and hyaloid vascular system in pathologic conditions.