Chinese Academy of Science Key Laboratory of Brain Function and Diseases, School of Life Sciences, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, 230026, China.
Department of Neurosurgery, Huashan Hospital, Fudan University, Middle Urumqi Road 12, Shanghai, 200040, China.
BMC Biol. 2022 Mar 29;20(1):77. doi: 10.1186/s12915-022-01275-6.
Tissue-clearing techniques have recently been developed to make tissues transparent for three-dimensional (3D) imaging at different scales, including single-cell resolution. However, current tissue-clearing workflows have several disadvantages, including complex protocols, time-consuming application, and fluorescence quenching. Additionally, they can be used mainly for clearing larger-volume samples, preventing wide and easy applicability in conventional experimental approaches. In this study, we aimed to develop a versatile, fast, and convenient method for clearing thin and semi-thick samples, which can be used for three-dimensional imaging of experimental or even clinical samples.
We developed an alkaline solution (AKS) containing a combination of 2,2'-thiodiethanol (TDE), DMSO, D-sorbitol, and Tris for tissue clearing, as the alkaline environment is suitable for maintaining the fluorescence of most commonly used fluorescence protein GFP and its variants, and tested its clearing effect on samples from mice and human brains. We assessed the clearing speed, the preservation of fluorescence protein and dyes, and the imaging depth and quality. The results showed that AKS treatment rapidly cleared 300-μm-thick brain slices and 1-mm-thick slices from different organs within 5 min and 1 h, respectively. Moreover, AKS was compatible with a variety of fluorescence proteins and dyes. Most importantly, AKS enhanced the fluorescence of YFP, in contrast to the majority of existing tissue-clearing methods which reduce the fluorescence intensity of fluorescent proteins. Using AKS, we performed long-time high-resolution imaging of weak fluorescent protein-labelled tissues, long-distance fibre tracking, larger-scale 3D imaging and cell counting of the entire brain area, neural circuit tracing, 3D neuromorphic reconstruction, and 3D histopathology imaging.
AKS can be used for simple and rapid clearing of samples from mice and human brains and is widely compatible with a variety of fluorescent dyes. Therefore, AKS has great potential to be used as a broad tissue-clearing reagent for biological optical imaging, especially for time-sensitive experiments.
最近开发了组织透明化技术，可使组织在不同尺度下（包括单细胞分辨率）进行三维（3D）成像透明化。然而，目前的组织透明化工作流程存在几个缺点，包括复杂的方案、耗时的应用和荧光猝灭。此外，它们主要用于清除较大体积的样本，在常规实验方法中无法广泛且易于应用。在这项研究中，我们旨在开发一种通用、快速且方便的方法，用于透明化薄和半厚样本，可用于实验甚至临床样本的三维成像。
我们开发了一种包含 2,2'-硫代二乙醇（TDE）、DMSO、D-山梨糖醇和 Tris 的碱性溶液（AKS）用于组织透明化，因为碱性环境适合维持最常用的荧光蛋白 GFP 及其变体的荧光，并用其测试了对来自小鼠和人脑的样本的透明化效果。我们评估了透明化速度、荧光蛋白和染料的保留情况以及成像深度和质量。结果表明，AKS 处理可在 5 分钟和 1 小时内分别快速透明化 300μm 厚的脑切片和来自不同器官的 1mm 厚切片。此外，AKS 与多种荧光蛋白和染料兼容。最重要的是，AKS 增强了 YFP 的荧光，与大多数现有的组织透明化方法相反，后者会降低荧光蛋白的荧光强度。使用 AKS，我们对弱荧光蛋白标记的组织进行了长时间高分辨率成像、远距离纤维追踪、更大规模的 3D 成像和整个脑区的细胞计数、神经回路追踪、3D 神经形态重建和 3D 组织病理学成像。
AKS 可用于简单快速地透明化来自小鼠和人脑的样本，并且与多种荧光染料广泛兼容。因此，AKS 具有成为生物光学成像广泛组织透明化试剂的巨大潜力，特别是对于时间敏感的实验。
