Super-resolution stimulated emission depletion imaging of slit diaphragm proteins in optically cleared kidney tissue
Super-resolution stimulated emission depletion imaging of slit diaphragm proteins in optically cleared kidney tissue
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DOI：
10.1038/ki.2015.308
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发表时间：
2016-01-01
影响因子：
19.6
通讯作者：
Brismar, Hjalmar
中科院分区：
文献类型：
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作者：
Unnersjo-Jess, David;Scott, Lena;Brismar, Hjalmar
肾小球滤过屏障由足细胞足突与桥接狭缝隔膜、肾小球基底膜和内皮组成，是肾功能的关键组成部分。以前，过滤屏障的最细微的元素只能用电子显微镜观察到。然而，电子显微镜主要局限于超薄的二维样品，同时可视化多种不同蛋白质的可能性有限。因此，我们试图实施一种超分辨率免疫荧光显微镜方案来研究肾脏中的滤过屏障。最近，已经开发了几种光学清除方法，使得使用光学显微镜对大量组织甚至整个器官进行成像成为可能。在这里，我们发现基于水凝胶的光学清除是在纳米尺度上研究完整肾组织的有益工具。当使用超分辨率STED显微镜对样品进行成像时，为了评估正确的纳米级信息，染色质量至关重要。光清除组织的信噪比和免疫信号均匀性均得到改善。因此，在荧光标记、光学清除的完整肾脏样本中，裂隙横膈膜的STED是研究健康和疾病肾小球滤过屏障的新工具。
The glomerular filtration barrier, consisting of podocyte foot processes with bridging slit diaphragm, glomerular basement membrane, and endothelium, is a key component for renal function. Previously, the subtlest elements of the filtration barrier have only been visualized using electron microscopy. However, electron microscopy is mostly restricted to ultrathin two-dimensional samples, and the possibility to simultaneously visualize multiple different proteins is limited. Therefore, we sought to implement a super-resolution immunofluorescence microscopy protocol for the study of the filtration barrier in the kidney. Recently, several optical clearing methods have been developed making it possible to image through large volumes of tissue and even whole organs using light microscopy. Here we found that hydrogel-based optical clearing is a beneficial tool to study intact renal tissue at the nanometer scale. When imaging samples using super-resolution STED microscopy, the staining quality was critical in order to assess correct nanoscale information. The signal-to-noise ratio and immunosignal homogeneity were both improved in optically cleared tissue. Thus, STED of slit diaphragms in fluorescently labeled, optically cleared, intact kidney samples is a new tool for studying the glomerular filtration barrier in health and disease.