In situ X-ray-assisted electron microscopy staining for large biological samples
In situ X-ray-assisted electron microscopy staining for large biological samples
复制标题
大型生物样品的原位 X 射线辅助电子显微镜染色
DOI：
10.1101/2021.06.19.448808
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发表时间：
2021
期刊：
影响因子：
7.7
通讯作者：
A. Wanner
中科院分区：
文献类型：
--
作者：
Sebastian Ströh;Eric W. Hammerschmith;D. Tank;H. Seung;A. Wanner
最近，生物组织的电子显微镜在成像吞吐量方面出现了前所未有的增长，从而将大型组织块（如整个大脑）的超微结构分析带入了可行的领域。然而，大型生物样品的均匀、高质量的电子显微镜染色仍然是一个重大挑战。迄今为止，评估电子显微镜中的染色质量需要将样本端到端地运行整个染色方案，对于大样本来说可能需要数周甚至数月的时间，从而导致此类样本的方案优化效率低下。在这里，我们提出了一种原位延时X射线辅助染色程序，它打开了电子显微镜染色的“黑匣子”，并允许真实的实时观察各个染色步骤。使用这种新的方法，我们测量了重金属的积累，在大的组织样品浸泡在不同的染色液。我们发现，所测得的积累锇在固定的组织服从经验的孵育时间和样本大小之间的二次依赖。我们发现，亚铁氰化钾，一种经典的还原剂四氧化锇，清除组织后，锇染色和组织膨胀，在四氧化锇溶液中，但在还原锇溶液收缩。X-射线辅助染色提供了原位染色动力学的途径，并使我们能够开发一种扩散-反应-平流模型，该模型准确地模拟了组织中锇的实测积累。这些是计算机模拟染色实验和模拟指导的大样本染色方案优化的第一步。因此，X射线辅助染色将是一个有用的工具，为大样本，如小鼠，猴子或人类的整个大脑可靠的染色程序的发展。
Electron microscopy of biological tissue has recently seen an unprecedented increase in imaging throughput moving the ultrastructural analysis of large tissue blocks such as whole brains into the realm of the feasible. However, homogeneous, high quality electron microscopy staining of large biological samples is still a major challenge. To date, assessing the staining quality in electron microscopy requires running a sample through the entire staining protocol end-to-end, which can take weeks or even months for large samples, rendering protocol optimization for such samples to be inefficient. Here we present an in situ time-lapsed X-ray assisted staining procedure that opens the “black box” of electron microscopy staining and allows observation of individual staining steps in real time. Using this novel method we measured the accumulation of heavy metals in large tissue samples immersed in different staining solutions. We show that the measured accumulation of osmium in fixed tissue obeys empirically a quadratic dependence between the incubation time and sample size. We found that potassium ferrocyanide, a classic reducing agent for osmium tetroxide, clears the tissue after osmium staining and that the tissue expands in osmium tetroxide solution, but shrinks in reduced osmium solution. X-ray assisted staining gave access to the in situ staining kinetics and allowed us to develop a diffusion-reaction-advection model that accurately simulates the measured accumulation of osmium in tissue. These are first steps towards in silico staining experiments and simulation-guided optimization of staining protocols for large samples. Hence, X-ray assisted staining will be a useful tool for the development of reliable staining procedures for large samples such as entire brains of mice, monkeys or humans.
影响因子：
25
作者：
Wanner, Adrian A.;Friedrich, Rainer W.
通讯作者：
Friedrich, Rainer W.