X-ray micro tomography of pathology tissue samples

在这个项目的前一个版本中，商业和同步辐射x射线微型CT被用来指导组织样本的电子显微镜研究，这些组织样本被四氧化三铯的电子显微镜造影剂严重染色。这种染色还提供了强烈的X射线对比度。最近，我们重新启动了该项目，以满足临床和研究病理学程序的更广泛需求，即在有限的时间窗口内对未染色、完整的组织样本进行3D显微镜检查，以提供标准病理学方案无法获取的相关信息，并将病理程序的切片和光学显微镜引导到样本中的特定位置。 组织标本的病理学测试用于临床诊断和生物医学研究。组织样本通常被包埋在石蜡块中，并切成许多薄片，然后用适当的试剂进行光学显微镜染色。由于每个组织块可以产生数百个薄片，分析所有切片需要大量的时间和人力。完整区块的非侵入性侦察成像有助于指导病理程序。理想的侦察步骤是在几分钟的时间窗口内完成，而不需要特殊的样品准备，这可能会干扰病理程序。挑战是在足够的分辨率下获得一些未染色的组织结构的可见性。 我们开发了x射线微断层合成技术，这是一种用于机场3D行李安检的显微镜版本。X射线成像是应对这一挑战的一种很有前途的工具，因为X射线可以穿透对可见光不透明的厚样品。对于x射线成像，组织可见性的一个决定因素是照射样本的光子的通量密度。我们探索了一种新的X射线断层合成方法，作为最大化这一因素的一种方式。它在5到15分钟的扫描时间内以7.3米的分辨率提供了数千张横断面图像。与微型CT扫描(一种广泛使用的实验室技术)相比，这种方法不需要旋转样品，从而使石蜡块等扁平样品尽可能靠近X射线源。因此，在硬件、扫描时间和分辨率相同的情况下，这种方法最大化了通过样品的光子通量密度，这有助于提高未染色组织在X射线下的可见度。该方法的折衷是不完整的3D信息。 在与其他研究人员的合作下，我们使用显微断层合成技术扫描了100多个未染色的人和动物组织样本，作为各自病理方案的一部分。在所有情况下，层叠的横断面图像显示了指导病理分析或提供相关结构信息的组织结构。这项技术还在一名HIV患者捐赠者的冠状动脉壁内弹力层中发现了孤立的局灶性钙化，这是动脉内侧钙化硬化的开始。 该技术具有普遍性，适用于新冠肺炎患者的组织样本。我们计划探索这一应用，作为我们参与Anthony Suffredini博士的新冠肺炎自然历史方案的成像组件的一部分。 此外，在NHLBI Small Animal MRI Core的Stasia Anderson的帮助下，我们还通过对3D打印的人工膝关节半月板的内部结构和材料分布进行成像，帮助Walter Reed NMMC的文森特·何博士进行了组织工程工作。 除了该项目参考书目中列出的放射学病例报告论文外，研究结果将在北美放射学会2020年会议上公布，一篇论文正在显微镜杂志上进行审查。

In the previous version of this project, commercial and synchrotron x-ray micro CT have been adapted to guide electron microscopy studies of tissue samples, which are heavily stained with the electron microscopy contrast agent of osmium tetroxide. This stain also provided strong x-ray contrast. More recently, we revived the project to meet a broader need in clinical and research pathology procedures, which is 3D microscopy of unstained, intact tissue samples in a limited time window, to provide relevant information that is inaccessible by standard pathology protocols, and to guide the sectioning and light microscopy of the pathology procedures to specific locations within the sample. Pathology tests of tissue samples are used for clinical diagnosis and for biomedical research. The tissue samples are often embedded in paraffin blocks and sectioned into many thin slices, which are then stained with the appropriate agents for light microscopy. Since each tissue block can produce several hundred thin sections, much time and labor is required to analyze all sections. Non-invasive scout imaging of intact blocks can help in guiding the pathology procedure. The scouting step is ideally done in a time window of minutes without special sample preparation that may interfere with the pathology procedures. The challenge is to obtain some visibility of unstained tissue structures at sufficient resolution. We developed x-ray micro tomosynthesis, a microscopy version of the technology for 3D luggage screening at airports. X-ray imaging is a promising tool to meet the challenge since x-rays can penetrate thick samples that are opaque to visible light. With x-ray imaging, a determinant of tissue visibility is the flux density of photons that illuminate the sample. We explored a novel x-ray tomosynthesis method as a way to maximize this factor. It provided a stack of thousands of cross-sectional images at 7.3 m resolution in scans of 5 to 15 minutes. When compared with micro CT scans (a widely-used laboratory technology), this method did not need to rotate the sample, which allowed flat samples such as paraffin blocks to be kept as close as possible to the x-ray source. Thus, given the same hardware, scan time and resolution, this method maximized the photon flux density through the sample, which helped in improving the visibility of unstained tissue under x-ray. The tradeoff of the method is incomplete 3D information. In collaboration with other investigators, we scanned over 100 unstained human and animal tissue samples with micro tomosynthesis, as part of their respective pathology protocols. In all cases, the stack of cross-sectional images showed tissue structures that guided pathology analysis or provided correlative structural information. The technology also made a new discovery of isolated focal calcification in the internal elastic lamina of the coronary artery wall of an HIV patient donor, which was the onset of medial calcific sclerosis in the arteries. The technology is universal and applicable to tissue samples from COVID-19 patients. We plan to explore this application as part of our participation in the imaging components of Dr. Anthony Suffredini's COVID-19 Natural History protocol. Additionally, with the help of Stasia Anderson of NHLBI Small Animal MRI Core, we also helped Dr. Vincent Ho of Walter Reed NMMC in his tissue engineering effort, by imaging the internal structure and material distribution in a 3D printed artificial meniscus of the knee. Besides the Radiology Case Reports paper listed in Bibliography of this project, the results will be presented at the Radiology Society of North America 2020 meeting, and a paper is under review at Journal of Microscopy.