Biomaterials Science Center, Department of Biomedical Engineering, University of Basel, 4123 Allschwil, Switzerland; Biomaterials Science Center, Department of Clinical Research, University Hospital Basel, 4031 Basel, Switzerland.
The Interface Group, Institute of Physiology, University of Zurich, 8057 Zurich, Switzerland; National Centre of Competence in Research, Kidney.CH, 8057 Zurich, Switzerland.
J Neurosci Methods. 2021 Dec 1;364:109354. doi: 10.1016/j.jneumeth.2021.109354. Epub 2021 Sep 13.
Micrometer-resolution neuroimaging with gold-standard conventional histology requires tissue fixation and embedding. The exchange of solvents for the creation of sectionable paraffin blocks modifies tissue density and generates non-uniform brain shrinkage.
We employed synchrotron radiation-based X-ray microtomography for slicing- and label-free virtual histology of the mouse brain at different stages of the standard preparation protocol from formalin fixation via ascending ethanol solutions and xylene to paraffin embedding. Segmentation of anatomical regions allowed us to quantify non-uniform tissue shrinkage. Global and local changes in X-ray absorption gave insight into contrast enhancement for virtual histology.
The volume of the entire mouse brain was 60%, 56%, and 40% of that in formalin for, respectively, 100% ethanol, xylene, and paraffin. The volume changes of anatomical regions such as the hippocampus, anterior commissure, and ventricles differ from the global volume change. X-ray absorption of the full brain decreased, while local absorption differences increased, resulting in enhanced contrast for virtual histology. These trends were also observed with laboratory microtomography measurements.
Microtomography provided sub-10 μm spatial resolution with sufficient density resolution to resolve anatomical structures at each step of the embedding protocol. The spatial resolution of conventional computed tomography and magnetic resonance microscopy is an order of magnitude lower and both do not match the contrast of microtomography over the entire embedding protocol. Unlike feature-to-feature or total volume measurements, our approach allows for calculation of volume change based on segmentation.
We present isotropic micrometer-resolution imaging to quantify morphology and composition changes in a mouse brain during the standard histological preparation. The proposed method can be employed to identify the most appropriate embedding medium for anatomical feature visualization, to reveal the basis for the dramatic X-ray contrast enhancement observed in numerous embedded tissues, and to quantify morphological changes during tissue fixation and embedding.
使用金标准常规组织学进行微米分辨率神经影像学需要组织固定和包埋。为了制作可切片的石蜡块而交换溶剂会改变组织密度并产生不均匀的脑收缩。
我们使用基于同步辐射的 X 射线微断层扫描技术，对从小鼠大脑在福尔马林固定、通过升序乙醇溶液和二甲苯到石蜡包埋的标准制备方案的不同阶段进行切片和无标记虚拟组织学。解剖区域的分割使我们能够量化不均匀的组织收缩。X 射线吸收的全局和局部变化为虚拟组织学提供了对比度增强的见解。
整个小鼠大脑的体积分别为福尔马林的 60%、56%和 40%，用于 100%乙醇、二甲苯和石蜡。海马体、前连合和脑室等解剖区域的体积变化与整体体积变化不同。全脑的 X 射线吸收减少，而局部吸收差异增加，导致虚拟组织学对比度增强。这些趋势也在实验室微断层扫描测量中观察到。
微断层扫描提供了亚 10μm 的空间分辨率，具有足够的密度分辨率，可以解析嵌入协议的每个步骤中的解剖结构。传统计算机断层扫描和磁共振显微镜的空间分辨率低一个数量级，并且都不符合整个嵌入协议中微断层扫描的对比度。与特征到特征或总体积测量不同，我们的方法允许根据分割计算体积变化。
我们提出了各向同性的微米分辨率成像方法，以量化小鼠大脑在标准组织学制备过程中的形态和成分变化。该方法可用于确定最适合解剖特征可视化的嵌入介质，揭示在许多嵌入组织中观察到的戏剧性 X 射线对比度增强的基础，并量化组织固定和嵌入过程中的形态变化。
