X-ray micro tomography of pathology tissue samples

National Heart, Lung, And Blood Institute

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Abstract

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 trade-off of the method is incomplete 3D information. Current development includes the x-ray enclosure of the microscope to eliminate emission of radiation into the surrounding, and an inverted configuration for better sample handling. 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.