Genoud Christel, Titze Benjamin, Graff-Meyer Alexandra, Friedrich Rainer W
Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland.
Faculty of Natural Sciences, University of Basel, Basel, Switzerland.
Front Neuroanat. 2018 Sep 28;12:76. doi: 10.3389/fnana.2018.00076. eCollection 2018.
Fixation and staining of large tissue samples are critical for the acquisition of volumetric electron microscopic image datasets and the subsequent reconstruction of neuronal circuits. Efficient protocols exist for the staining of small samples, but uniform contrast is often difficult to achieve when the sample diameter exceeds a few hundred micrometers. Recently, a protocol (BROPA, brain-wide reduced-osmium staining with pyrogallol-mediated amplification) was developed that achieves homogeneous staining of the entire mouse brain but requires very long sample preparation times. By exploring modifications of this protocol we developed a substantially faster procedure, fBROPA, that allows for reliable high-quality staining of tissue blocks on the millimeter scale. Modifications of the original BROPA protocol include drastically reduced incubation times and a lead aspartate incubation to increase sample conductivity. Using this procedure, whole brains from adult zebrafish were stained within 4 days. Homogenous high-contrast staining was achieved throughout the brain. High-quality image stacks with voxel sizes of 10 × 10 × 25 nm were obtained by serial block-face imaging using an electron dose of ~15 e/nm. No obvious reduction in staining quality was observed in comparison to smaller samples stained by other state-of-the-art procedures. Furthermore, high-quality images with minimal charging artifacts were obtained from non-neural tissues with low membrane density. fBROPA is therefore likely to be a versatile and efficient sample preparation protocol for a wide range of applications in volume electron microscopy.
大组织样本的固定和染色对于获取体积电子显微镜图像数据集以及随后的神经回路重建至关重要。对于小样本的染色，已有高效的方案，但当样本直径超过几百微米时，往往难以实现均匀的对比度。最近，开发了一种方案（BROPA，焦性没食子酸介导扩增的全脑低锇染色），可实现整个小鼠脑的均匀染色，但样本制备时间非常长。通过探索该方案的改进，我们开发了一种更快的程序，即fBROPA，它能够对毫米尺度的组织块进行可靠的高质量染色。原始BROPA方案的改进包括大幅缩短孵育时间以及进行天冬氨酸铅孵育以提高样本导电性。使用该程序，成年斑马鱼的全脑在4天内完成染色。整个大脑实现了均匀的高对比度染色。通过使用约15 e/nm的电子剂量进行连续块面成像，获得了体素大小为10×10×25 nm的高质量图像堆栈。与通过其他先进程序染色的较小样本相比，未观察到染色质量有明显下降。此外，从膜密度低的非神经组织中获得了具有最小充电伪影的高质量图像。因此，fBROPA可能是一种适用于体积电子显微镜广泛应用的通用且高效的样本制备方案。
