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：
10.1101/2022.02.18.481045
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发表时间：
2022-02
影响因子：
5.5
通讯作者：
Yuxin Zhang;Tobias Ackels;A. Pacureanu;M. Zdora;A. Bonnin;Andreas T. Schaefer;C. Bosch
中科院分区：
文献类型：
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作者：
Yuxin Zhang;Tobias Ackels;A. Pacureanu;M. Zdora;A. Bonnin;Andreas T. Schaefer;C. Bosch
将生理学与相同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个实验性的挑战。当研究小鼠嗅球中的肾小球柱电路时，该方法涉及例如用体内2-光子（2 P）钙成像记录神经元活动，用具有基于传播的相位衬度（SXRT）和/或连续块面电子显微镜（SBEM）的同步加速器X射线计算机断层扫描检索电路结构，并将这些数据集相关联。样品制备和数据集关联是这一相关工作流程中的两个关键瓶颈。在这里，我们首先量化不同的文物时，用重金属染色组织切片产生X射线或电子对比度的发生。我们报告的染色程序的改进，最终达到完美的染色在0.67%的0.6毫米厚的嗅球切片，以前在体内成像与2 P。其次，我们验证了功能数据集和结构数据集之间的空间相关性的准确性。我们证明，在体内2 P和SXRT组织体积之间的直接，单细胞精确的相关性是可能的，并且与2 P和SBEM之间的相关性一样可靠。总而言之，这些结果为需要检索目标区域中的生理学、电路结构和突触特征的实验铺平了道路。这些相关的功能-结构研究将带来更完整的理解哺乳动物的嗅觉加工跨越长度尺度和时间。
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 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 length scales and time.