Bekkouche Bo M B, Fritz Helena K M, Rigosi Elisa, O'Carroll David C
Department of Biology, Lund University, Lund, Sweden.
Front Neuroanat. 2020 Nov 20;14:599282. doi: 10.3389/fnana.2020.599282. eCollection 2020.
Improvement of imaging quality has the potential to visualize previously unseen building blocks of the brain and is therefore one of the great challenges in neuroscience. Rapid development of new tissue clearing techniques in recent years have attempted to solve imaging compromises in thick brain samples, particularly for high resolution optical microscopy, where the clearing medium needs to match the high refractive index of the objective immersion medium. These problems are exacerbated in insect tissue, where numerous (initially air-filled) tracheal tubes branching throughout the brain increase the scattering of light. To date, surprisingly few studies have systematically quantified the benefits of such clearing methods using objective transparency and tissue shrinkage measurements. In this study we compare a traditional and widely used insect clearing medium, methyl salicylate combined with permanent mounting in Permount ("MS/P") with several more recently applied clearing media that offer tunable refractive index (): 2,2'-thiodiethanol (TDE), "SeeDB2" (in variants SeeDB2S and SeeDB2G matched to oil and glycerol immersion, = 1.52 and 1.47, respectively) and Rapiclear (also with = 1.52 and 1.47). We measured transparency and tissue shrinkage by comparing freshly dissected brains with cleared brains from dipteran flies, with or without addition of vacuum or ethanol pre-treatments (dehydration and rehydration) to evacuate air from the tracheal system. The results show that ethanol pre-treatment is very effective for improving transparency, regardless of the subsequent clearing medium, while vacuum treatment offers little measurable benefit. Ethanol pre-treated SeeDB2G and Rapiclear brains show much less shrinkage than using the traditional MS/P method. Furthermore, at lower refractive index, closer to that of glycerol immersion, these recently developed media offer outstanding transparency compared to TDE and MS/P. Rapiclear protocols were less laborious compared to SeeDB2, but both offer sufficient transparency and refractive index tunability to permit super-resolution imaging of local volumes in whole mount brains from large insects, and even light-sheet microscopy. Although long-term permanency of Rapiclear stored samples remains to be established, our samples still showed good preservation of fluorescence after storage for more than a year at room temperature.
成像质量的提高有可能使大脑中以前看不见的组成部分可视化，因此是神经科学中的重大挑战之一。近年来，新的组织透明化技术迅速发展，试图解决厚脑样本中的成像难题，特别是对于高分辨率光学显微镜而言，透明化介质需要与物镜浸没介质的高折射率相匹配。在昆虫组织中，这些问题更加严重，因为众多（最初充满空气）的气管在整个大脑中分支，增加了光的散射。迄今为止，令人惊讶的是，很少有研究使用客观透明度和组织收缩测量来系统地量化此类透明化方法的益处。在本研究中，我们将一种传统且广泛使用的昆虫透明化介质——水杨酸甲酯与Permount中的永久封固剂组合（“MS/P”），与几种最近应用的、提供可调折射率（）的透明化介质进行比较：2,2'-硫代二乙醇（TDE）、“SeeDB2”（有与油和甘油浸没相匹配的变体SeeDB2S和SeeDB2G，分别为 = 1.52和1.47）以及Rapiclear（也为 = 1.52和1.47）。我们通过比较刚解剖的大脑与双翅目果蝇经透明化处理后的大脑来测量透明度和组织收缩，无论是否添加真空或乙醇预处理（脱水和再水化）以从气管系统中排出空气。结果表明，无论后续使用何种透明化介质，乙醇预处理对于提高透明度都非常有效，而真空处理几乎没有可测量的益处。经乙醇预处理的SeeDB2G和Rapiclear处理的大脑收缩比使用传统MS/P方法小得多。此外，在较低折射率下，更接近甘油浸没的折射率，与TDE和MS/P相比，这些最近开发的介质具有出色的透明度。与SeeDB2相比，Rapiclear方案的操作更简便，但两者都提供了足够的透明度和折射率可调性，以允许对大型昆虫整装大脑中的局部体积进行超分辨率成像，甚至是光片显微镜成像。尽管Rapiclear储存样本的长期稳定性仍有待确定，但我们的样本在室温下储存一年多后仍显示出良好的荧光保存效果。
