Barbone Giacomo E, Bravin Alberto, Mittone Alberto, Kraiger Markus J, Hrabě de Angelis Martin, Bossi Mario, Ballarini Elisa, Rodriguez-Menendez Virginia, Ceresa Cecilia, Cavaletti Guido, Coan Paola
Department of Medical Physics, Faculty of Physics, Ludwig-Maximilians-Universität München, Garching, Germany.
European Synchrotron Radiation Facility, Grenoble, France.
J Neurosci Methods. 2020 Jun 1;339:108744. doi: 10.1016/j.jneumeth.2020.108744. Epub 2020 Apr 27.
Dense and unbiased cellular-resolution representations of extended volumetric central nervous system soft-tissue anatomy are difficult to obtain, even in experimental post-mortem settings. Interestingly, X-ray phase-contrast computed tomography (X-PCI-CT), an emerging soft-tissue-sensitive volumetric imaging technique, can provide multiscale organ- to cellular-level morphological visualizations of neuroanatomical structure.
Here, we tested different nervous-tissue fixation procedures, conventionally used for transmission electron microscopy, to better establish X-PCI-CT-specific sample-preparation protocols. Extracted rat spinal medullas were alternatively fixed with a standard paraformaldehyde-only aldehyde-based protocol, or in combination with glutaraldehyde. Some specimens were additionally post-fixed with osmium tetroxide. Multiscale X-PCI-CT datasets were collected at several synchrotron radiation facilities, using state-of-the-art setups with effective image voxel sizes of 3.0 to 0.3 μm, and compared to high-field magnetic resonance imaging, histology and vascular fluorescence microscopy data.
Multiscale X-PCI-CT of aldehyde-fixed spinal cord specimens resulted in dense histology-like volumetric representations and quantifications of extended deep spinal micro-vascular networks and of intra-medullary cell populations. Osmium post-fixation increased intra-medullary contrast between white and gray-matter tissues, and enhanced delineation of intra-medullary cellular structure, e.g. axon fibers and motor neuron perikarya.
Volumetric X-PCI-CT provides complementary contrast and higher spatial resolution compared to 9.4 T MRI. X-PCI-CT's advantage over planar histology is the volumetric nature of the cellular-level data obtained, using samples much larger than those fit for volumetric vascular fluorescence microscopy.
Deliberately choosing (post-)fixation protocols tailored for optimal nervous-tissue structural preservation is of paramount importance in achieving effective and targeted neuroimaging via the X-PCI-CT technique.
即使在实验性尸检环境中，也难以获得扩展的中枢神经系统软组织解剖结构的密集且无偏差的细胞分辨率表示。有趣的是，X射线相衬计算机断层扫描（X-PCI-CT）是一种新兴的对软组织敏感的容积成像技术，它可以提供神经解剖结构的多尺度器官到细胞水平的形态可视化。
在这里，我们测试了传统上用于透射电子显微镜的不同神经组织固定程序，以更好地建立X-PCI-CT特定的样品制备方案。提取的大鼠脊髓髓质分别用仅含多聚甲醛的标准醛基方案固定，或与戊二醛联合固定。一些标本还用四氧化锇进行了后固定。在几个同步辐射设施中，使用有效图像体素大小为3.0至0.3μm的最先进设置收集了多尺度X-PCI-CT数据集，并与高场磁共振成像、组织学和血管荧光显微镜数据进行了比较。
醛固定脊髓标本的多尺度X-PCI-CT产生了密集的组织学样容积表示，并对扩展的深部脊髓微血管网络和髓内细胞群进行了定量分析。锇后固定增加了白质和灰质组织之间的髓内对比度，并增强了髓内细胞结构（如轴突纤维和运动神经元胞体）的轮廓。
与9.4T MRI相比，容积X-PCI-CT提供了互补的对比度和更高的空间分辨率。X-PCI-CT相对于平面组织学的优势在于，使用比适合容积血管荧光显微镜的样本大得多的样本，可以获得细胞水平数据的容积性质。
在通过X-PCI-CT技术实现有效和有针对性的神经成像方面，精心选择为最佳神经组织结构保存量身定制的（后）固定方案至关重要。
