Table3_Sample Preparation and Warping Accuracy for Correlative Multimodal Imaging in the Mouse Olfactory Bulb Using 2-Photon, Synchrotron X-Ray and Volume Electron Microscopy.XLSX

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.

将生理学与同一路径神经元结构的洞察相结合，为理解哺乳动物大脑如何处理信息提供了一种独特的方法。然而，整合提供这两类数据的技术的实验挑战是显而易见的。在研究小鼠嗅球中的肾小球柱状电路时，此方法涉及例如，通过体内双光子（2P）钙成像记录神经元活动，利用基于传播相衬的同步辐射X射线计算机断层扫描（SXRT）和/或串行断面对比扫描电子显微镜（SBEM）检索电路结构，并关联这些数据集。样本制备和数据集关联是该相关工作流程中的两个关键瓶颈。在此，我们首先量化了在用重金属染色组织切片以生成X射线或电子对比时不同伪影的发生率。我们报告了染色程序的改进，最终在约67%的0.6毫米厚的嗅球切片中实现了完美的染色，这些切片之前已通过2P在体内成像。其次，我们表征了功能数据集与结构数据集之间空间关联的准确性。我们证明了直接、单细胞精确关联体内2P与SXRT组织体积是可能的，并且与2P与SBEM关联一样可靠。总之，这些结果为需要获取特定区域生理学、电路结构和突触特征的实验铺平了道路。这些相关功能-结构研究将有助于全面理解哺乳动物在空间和时间尺度上的嗅觉处理。