Sample Preparation and Warping Accuracy for Correlative Multimodal Imaging in the Mouse Olfactory Bulb Using 2-Photon, Synchrotron X-Ray and Volume Electron Microscopy.
Sample Preparation and Warping Accuracy for Correlative Multimodal Imaging in the Mouse Olfactory Bulb Using 2-Photon, Synchrotron X-Ray and Volume Electron Microscopy.
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DOI：
10.3389/fcell.2022.880696
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发表时间：
2022
影响因子：
5.5
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将生理学与同一神经元回路的结构见解相结合，为理解哺乳动物大脑如何计算信息提供了一种独特的方法。然而，将提供两种数据流的技术结合起来是一项实验挑战。在研究小鼠嗅球中的肾小球柱回路时，这种方法涉及例如使用体内 2 光子 (2P) 钙成像记录神经元活动，使用基于传播的相差 (SXRT) 和/或串行块面扫描电子显微镜 (SBEM) 的同步加速器 X 射线计算机断层扫描检索回路结构，并将这些数据集关联起来。样本准备和数据集关联是该关联工作流程中的两个关键瓶颈。在这里，我们首先量化用重金属对组织切片进行染色以产生 X 射线或电子对比时不同伪影的发生。我们报告了染色程序的改进，最终在之前使用 2P 进行体内成像的 0.6 毫米厚的嗅球切片中约 67% 实现了完美染色。其次，我们表征功能数据集和结构数据集之间空间相关性的准确性。我们证明体内 2P 和 SXRT 组织体积之间的直接、单细胞精确关联是可能的，并且与 2P 和 SBEM 之间的关联一样可靠。总而言之，这些结果为需要检索目标区域的生理学、电路结构和突触特征的实验铺平了道路。这些相关的功能结构研究将带来对跨空间尺度和时间的哺乳动物嗅觉处理的更全面的理解。
Integrating physiology with structural insights of the same neuronal circuit provides a unique approach to understanding how the mammalian brain computes information. However, combining the techniques that provide both streams of data represents an experimental challenge. When studying glomerular column circuits in the mouse olfactory bulb, this approach involves e.g., recording the neuronal activity with in vivo 2-photon (2P) calcium imaging, retrieving the circuit structure with synchrotron X-ray computed tomography with propagation-based phase contrast (SXRT) and/or serial block-face scanning electron microscopy (SBEM) and correlating these datasets. Sample preparation and dataset correlation are two key bottlenecks in this correlative workflow. Here, we first quantify the occurrence of different artefacts when staining tissue slices with heavy metals to generate X-ray or electron contrast. We report improvements in the staining procedure, ultimately achieving perfect staining in ∼67% of the 0.6 mm thick olfactory bulb slices that were previously imaged in vivo with 2P. Secondly, we characterise the accuracy of the spatial correlation between functional and structural datasets. We demonstrate that direct, single-cell precise correlation between in vivo 2P and SXRT tissue volumes is possible and as reliable as correlating between 2P and SBEM. Altogether, these results pave the way for experiments that require retrieving physiology, circuit structure and synaptic signatures in targeted regions. These correlative function-structure studies will bring a more complete understanding of mammalian olfactory processing across spatial scales and time.