X-ray micro tomography of pathology tissue samples

在该项目的前一个版本中，商业和同步加速器X射线显微CT已被改编为指导组织样本的电子显微镜研究，这是严重染色的电子显微镜造影剂四氧化锇。这种染色也提供了强烈的X射线对比。最近，我们恢复了该项目，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的完整组织样本进行3D显微镜检查，以提供标准病理学协议无法获得的相关信息，并将病理学程序的切片和光学显微镜引导到样本内的特定位置。 组织样本的病理学测试用于临床诊断和生物医学研究。组织样本通常包埋在石蜡块中并切成许多薄片，然后用适当的试剂染色以进行光学显微镜检查。由于每个组织块可以产生数百个薄切片，因此需要大量的时间和劳动来分析所有切片。完整组织块的非侵入性定位成像可以帮助指导病理过程。理想情况下，侦察步骤在几分钟的时间窗口内完成，而无需可能干扰病理学程序的特殊样品制备。挑战是在足够的分辨率下获得未染色组织结构的一些可见性。 我们开发了X射线显微断层合成，这是机场3D行李筛查技术的显微镜版本。X射线成像是一种很有前途的工具，以满足这一挑战，因为X射线可以穿透厚样品是不透明的可见光。对于X射线成像，组织可见性的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，以最大限度地提高这一因素。它提供了数千张7.3米分辨率的横截面图像，扫描时间为5至15分钟。与显微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，这使得平面样品（如石蜡块）尽可能靠近X射线源。因此，给定相同的硬件、扫描时间和分辨率，该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射线下的可见性。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中，我们使用显微断层合成技术扫描了100多个未染色的人类和动物组织样本，作为其各自病理学方案的一部分。在所有情况下，堆叠的横截面图像显示组织结构，指导病理分析或提供相关的结构信息。该技术还在一名艾滋病患者供体的冠状动脉壁内弹性膜中发现了孤立的局灶性钙化，这是动脉中膜钙化硬化的开始。 该技术具有通用性，适用于COVID-19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COVID-19自然史方案成像部分的一部分。 此外，在NHLBI Small Animal MRI Core的Stasia安德森的帮助下，我们还帮助Walter Reed NMMC的Vincent Ho博士进行组织工程研究，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。 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年会议上发表，一篇论文正在《显微镜杂志》上进行评审。

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.