Finding the best clearing approach- Towards 3D wide-scale multimodal imaging of aged human brain tissue
Finding the best clearing approach- Towards 3D wide-scale multimodal imaging of aged human brain tissue
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DOI：
10.1016/j.neuroimage.2021.118832
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发表时间：
2021-12-21
期刊：
影响因子：
5.7
通讯作者：
Morawski, Markus
中科院分区：
文献类型：
--
作者：
Rusch, Henriette;Brammerloh, Malte;Morawski, Markus
新的大规模多模态成像技术的可访问性导致了过去十年中出现的许多清除技术。然而，清除显微镜大小的老年人脑组织块仍然是一项极具挑战性的任务。使折射率均匀化并减少光吸收和散射是组织透明化的基础。由于其致密和高度髓鞘化的性质，特别是在白色物质中，人脑对清除技术提出了特殊的挑战。在这里，我们提出了七种组织清除方法及其对老年人脑组织块（> 5 mm）的影响的比较研究。目的是确定在宏观透明度、简短的清除时间、与免疫组织化学处理的兼容性和大规模多模态显微成像方面最实用和有效的方法。我们成功地清除了26 × 26 × 5毫米（3）大小的人脑样品与两个亲水性和两个疏水性的清除技术。这些方法之间的光学特性以及光和抗体渗透深度差异很大。除了找到最佳的清除方法，我们还比较了三种显微成像装置，（Zeiss激光扫描显微镜（LSM）880，Miltenyi Biotec Ultramicroscopy II（UM II）和3i Marianas LightSheet显微镜）关于大规模组织样本的最佳成像。我们证明，结合显微镜技术，（透明脂质交换丙烯酰胺杂交刚性成像兼容组织水凝胶）与Zeiss LSM 880并结合iDISCO技术使用Miltenyi Biotec UM II进行的免疫标记（溶剂清除的器官的免疫标记使能的三维成像）是充分清除老化人脑组织并生成3D显微图像的最实用和有效的方法。我们的研究结果指出了应用于非标准化组织样本（如老年人脑组织）的七种清除和三种成像技术所带来的挑战。
The accessibility of new wide-scale multimodal imaging techniques led to numerous clearing techniques emerging over the last decade. However, clearing mesoscopic-sized blocks of aged human brain tissue remains an extremely challenging task. Homogenizing refractive indices and reducing light absorption and scattering are the foundation of tissue clearing. Due to its dense and highly myelinated nature, especially in white matter, the human brain poses particular challenges to clearing techniques.Here, we present a comparative study of seven tissue clearing approaches and their impact on aged human brain tissue blocks (> 5 mm). The goal was to identify the most practical and efficient method in regards to macroscopic transparency, brief clearing time, compatibility with immunohistochemical processing and wide scale multimodal microscopic imaging. We successfully cleared 26 x 26 x 5 mm(3)-sized human brain samples with two hydrophilic and two hydrophobic clearing techniques. Optical properties as well as light and antibody penetration depths highly vary between these methods. In addition to finding the best clearing approach, we compared three microscopic imaging setups (the Zeiss Laser Scanning Microscope (LSM) 880 , the Miltenyi Biotec Ultramicroscope ll (UM ll) and the 3i Marianas LightSheet microscope) regarding optimal imaging of large-scale tissue samples.We demonstrate that combining the CLARITY technique (Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging compatible Tissue hYdrogel) with the Zeiss LSM 880 and combining the iDISCO technique (immunolabeling-enabled three-dimensional imaging of solvent-cleared organs) with the Miltenyi Biotec UM ll are the most practical and efficient approaches to sufficiently clear aged human brain tissue and generate 3D microscopic images. Our results point out challenges that arise from seven clearing and three imaging techniques applied to non-standardized tissue samples such as aged human brain tissue.