X-ray microtomosynthesis of unstained pathology tissue samples

In pathology protocols, a tissue block, such as one containing a mouse brain or a biopsy sample from a patient, can produce several hundred thin sections. Substantial time may be required to analyse all sections. In cases of uncertainty regarding which sections to focus on, noninvasive 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 sub-10 µm resolution. We explored a novel x-ray tomosynthesis method as a way to maximise contrast-to-noise ratio, a determinant of tissue visibility. It provided a z-stack of thousands of images at 7.3 μm resolution (10% contrast, half-period of 68.5 line pairs/mm), in scans of 5-15 minutes. When compared with micro-CT scans, the straight-line tomosynthesis scan 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 mode maximised the photon flux density through the sample, which helped in maximising the contrast-to-noise ratio. The tradeoff of tomosynthesis is incomplete 3D information. The microtomosynthesis scanner has scanned 110 unstained human and animal tissue samples as part of their respective pathology protocols. In all cases, the z-stack of images showed tissue structures that guided sectioning or provided correlative structural information. We describe six examples that presented different levels of visibility of soft tissue structures. Additionally, in a set of coronary artery samples from an HIV patient donor, microtomosynthesis made a new discovery of isolated focal calcification in the internal elastic lamina of coronary wall, which was the onset of medial calcific sclerosis in the arteries.