X-ray micro tomography of pathology tissue samples

在这个项目的上一个版本中，商业和同步加速器x射线微型CT已被用于指导组织样本的电子显微镜研究，这些组织样本被四氧化锇的电子显微镜造影剂严重染色。该染色也提供了强烈的x线对比。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，并指导病理程序的切片和光学显微镜到样本内的特定位置。

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.