Stephan Handschuh, Ursula Reichart, Stefan Kummer, Martin Glösmann
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We show that the spectral contrast of µAngiofil and Micropaque barium sulphate in perfused specimens allows for the effective separation of vasculature from mineralised skeletal tissues. Furthermore, we demonstrate the counterstaining of perfused specimens using established x-ray dense contrast agents to depict blood vessels together with the morphology of soft tissue. Phosphotungstic acid (PTA) is used as a counterstain that shows excellent spectral contrast in both µAngiofil and Micropaque barium sulphate-perfused specimens. A novel Sorensen-buffered PTA protocol is introduced as a counterstain for µAngiofil specimens, as the polyurethane polymer is susceptible to artefacts when using conventional staining solutions. Finally, we demonstrate that counterstained samples can be automatically processed into three separate image channels (skeletal tissue, vasculature and stained soft tissue), which offers multiple new options for data analysis. 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引用次数: 0
摘要
体外 X 射线血管造影术可提供低至毛细血管水平的高分辨率三维血管表型信息。体外血管造影的样品制备首先要清除血管系统中的血液,然后用一种与明胶或聚合物等载体混合的 X 射线致密造影剂进行灌注。随后,在完整固定的器官中对采集器官的血管微观结构进行成像。在本研究中,我们提出了新颖的显微双能 CT(microDECT)成像方案,可以参照硬组织和软组织的形态,在原位观察和分析微血管。我们的研究表明,µAngiofil 和 Micropaque 硫酸钡在灌注标本中的光谱对比可以有效地将血管从矿化骨骼组织中分离出来。此外,我们还演示了使用成熟的 X 射线致密造影剂对灌注标本进行反染色,以描绘血管和软组织形态。磷钨酸(PTA)作为一种反染色剂,在 µAngiofil 和 Micropaque 硫酸钡灌注标本中都能显示出极佳的光谱对比度。由于聚氨酯聚合物在使用传统染色溶液时容易产生伪影,因此我们引入了一种新颖的索伦森缓冲 PTA 方案作为 µAngiofil 标本的染色剂。最后,我们展示了反染色样本可自动处理成三个独立的图像通道(骨骼组织、血管和染色软组织),这为数据分析提供了多种新选择。所介绍的 microDECT 工作流程适合作为筛选和量化微血管的工具,并可在各种相关成像管道中实施,以瞄准感兴趣的区域进行下游的光学显微镜研究。
In situ isotropic 3D imaging of vasculature perfusion specimens using x-ray microscopic dual-energy CT.
Ex vivo x-ray angiography provides high-resolution, three-dimensional information on vascular phenotypes down to the level of capillaries. Sample preparation for ex vivo angiography starts with the removal of blood from the vascular system, followed by perfusion with an x-ray dense contrast agent mixed with a carrier such as gelatine or a polymer. Subsequently, the vascular micro-architecture of harvested organs is imaged in the intact fixed organ. In the present study, we present novel microscopic dual-energy CT (microDECT) imaging protocols that allow to visualise and analyse microvasculature in situ with reference to the morphology of hard and soft tissue. We show that the spectral contrast of µAngiofil and Micropaque barium sulphate in perfused specimens allows for the effective separation of vasculature from mineralised skeletal tissues. Furthermore, we demonstrate the counterstaining of perfused specimens using established x-ray dense contrast agents to depict blood vessels together with the morphology of soft tissue. Phosphotungstic acid (PTA) is used as a counterstain that shows excellent spectral contrast in both µAngiofil and Micropaque barium sulphate-perfused specimens. A novel Sorensen-buffered PTA protocol is introduced as a counterstain for µAngiofil specimens, as the polyurethane polymer is susceptible to artefacts when using conventional staining solutions. Finally, we demonstrate that counterstained samples can be automatically processed into three separate image channels (skeletal tissue, vasculature and stained soft tissue), which offers multiple new options for data analysis. The presented microDECT workflows are suited as tools to screen and quantify microvasculature and can be implemented in various correlative imaging pipelines to target regions of interest for downstream light microscopic investigation.
期刊介绍:
The Journal of Microscopy is the oldest journal dedicated to the science of microscopy and the only peer-reviewed publication of the Royal Microscopical Society. It publishes papers that report on the very latest developments in microscopy such as advances in microscopy techniques or novel areas of application. The Journal does not seek to publish routine applications of microscopy or specimen preparation even though the submission may otherwise have a high scientific merit.
The scope covers research in the physical and biological sciences and covers imaging methods using light, electrons, X-rays and other radiations as well as atomic force and near field techniques. Interdisciplinary research is welcome. Papers pertaining to microscopy are also welcomed on optical theory, spectroscopy, novel specimen preparation and manipulation methods and image recording, processing and analysis including dynamic analysis of living specimens.
Publication types include full papers, hot topic fast tracked communications and review articles. Authors considering submitting a review article should contact the editorial office first.