PRIAMOS:一种混合包埋介质的技术,可自由调节pH值和折射率(RI),用于整个组织样品的光学清除(OC)。

IF 1.9 4区 工程技术 Q3 MICROSCOPY
Ulrich Leischner, Martin Reifarth, Monika S Brill, Florian Schmitt, Stephanie Hoeppener, David Unnersjö Jess, Hjalmar Brismar, Ulrich S Schubert, Rainer Heintzmann
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引用次数: 0

摘要

生物样品的研究通常需要样品的透明度,这是通过将样品包埋在高折射率(RI)介质中以获得样品中均匀的RI分布来实现的,称为光学清除(OC)。在这里,我们介绍了一种通过增加分子轨道来设计具有更高RI的嵌入介质的方法,该方法是通过用具有更明显极化性的元素取代通常用于OC的分子中的元素来实现的。简单地说,我们将建立的包埋介质甘油用巯基交换羟基,得到性质非常相似的包埋介质,但折射率更高。我们描述了一个程序-缩写为priamos -使生物样品透明使用ri匹配液体,我们称之为ph值和折射率调整通过混合高度极化的分子轨道物质。我们专注于三维组织样品的光学清除,同时保留荧光标记的荧光。清除过程需要2-3天,产生高度透明的样品,保留黄色荧光蛋白(YFP)等荧光蛋白的荧光。这在小鼠脑样本上得到了证明,用标准共聚焦显微镜成像至1.6 mm深度,以及肾脏样本。单硫甘油酯、二硫甘油酯和三丁基胺的混合物的RI值在1.52至1.57之间,酸度相当于pH值在5至8之间。我们的PRIAMOS方法可以作为优化光清除协议的指导方针。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
PRIAMOS: A technique for mixing embedding media for freely adjusting pH value and refractive index (RI) for optical clearing (OC) of whole tissue samples.

Investigations of biological samples often require sample transparency, which is achieved by embedding the sample in a high-refractive index (RI) medium to obtain a homogenous RI distribution in the sample, referred to as optical clearing (OC). Here, we introduce a method for designing embedding media with an increased RI by increasing molecular orbitals, which is achieved by replacing elements in molecules commonly used for OC with elements possessing a more pronounced polarisability. Briefly, we took the established embedding medium Glycerol and exchanged the OH-groups by Thiol-groups, resulting in an embedding medium with very similar properties, but with a higher refractive index. We describe a procedure-abbreviated PRIAMOS-to render biological samples transparent using an RI-matching liquid, which we refer to as pH-value and Refractive Index Adjustment by Mixing highly polarisable molecular Orbital Substances. We focus on optical clearing in three-dimensional tissue samples whilst preserving fluorescence of fluorescent labels. The clearing procedure requires 2-3 days, yielding highly transparent samples, preserving the fluorescence of fluorescent proteins like the yellow fluorescent protein (YFP). This is demonstrated on mouse brain samples, imaged with standard confocal microscopy down to 1.6 mm depth, as well as on kidney samples. Mixtures of monothioglycerol, dithioglycerol and tributylamine achieve RI values between 1.52 and 1.57, and an acidity equivalent to pH values between 5 and 8. Our PRIAMOS approach can serve as a guideline for optimising optical clearing protocols.

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来源期刊
Journal of microscopy
Journal of microscopy 工程技术-显微镜技术
CiteScore
4.30
自引率
5.00%
发文量
83
审稿时长
1 months
期刊介绍: The Journal of Microscopy is the oldest journal dedicated to the science of microscopy and the only peer-reviewed publication of the Royal Microscopical Society. It publishes papers that report on the very latest developments in microscopy such as advances in microscopy techniques or novel areas of application. The Journal does not seek to publish routine applications of microscopy or specimen preparation even though the submission may otherwise have a high scientific merit. The scope covers research in the physical and biological sciences and covers imaging methods using light, electrons, X-rays and other radiations as well as atomic force and near field techniques. Interdisciplinary research is welcome. Papers pertaining to microscopy are also welcomed on optical theory, spectroscopy, novel specimen preparation and manipulation methods and image recording, processing and analysis including dynamic analysis of living specimens. Publication types include full papers, hot topic fast tracked communications and review articles. Authors considering submitting a review article should contact the editorial office first.
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