In situ isotropic 3D imaging of vasculature perfusion specimens using x‐ray microscopic dual‐energy CT.
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| Title: | In situ isotropic 3D imaging of vasculature perfusion specimens using x‐ray microscopic dual‐energy CT. |
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| Authors: | Handschuh, Stephan1 (AUTHOR) stephan.handschuh@vetmeduni.ac.at, Reichart, Ursula1 (AUTHOR), Kummer, Stefan1 (AUTHOR), Glösmann, Martin1 (AUTHOR) |
| Source: | Journal of Microscopy. Feb2025, Vol. 297 Issue 2, p179-202. 24p. |
| Subjects: | Blood vessels, X-ray microscopy, Dual energy CT (Tomography), Chemical sample preparation, Three-dimensional imaging, Contrast media, Microcirculation |
| Abstract: | Ex vivo x‐ray angiography provides high‐resolution, three‐dimensional information on vascular phenotypes down to the level of capillaries. Sample preparation for ex vivo angiography starts with the removal of blood from the vascular system, followed by perfusion with an x‐ray dense contrast agent mixed with a carrier such as gelatine or a polymer. Subsequently, the vascular micro‐architecture of harvested organs is imaged in the intact fixed organ. In the present study, we present novel microscopic dual‐energy CT (microDECT) imaging protocols that allow to visualise and analyse microvasculature in situ with reference to the morphology of hard and soft tissue. We show that the spectral contrast of µAngiofil and Micropaque barium sulphate in perfused specimens allows for the effective separation of vasculature from mineralised skeletal tissues. Furthermore, we demonstrate the counterstaining of perfused specimens using established x‐ray dense contrast agents to depict blood vessels together with the morphology of soft tissue. Phosphotungstic acid (PTA) is used as a counterstain that shows excellent spectral contrast in both µAngiofil and Micropaque barium sulphate–perfused specimens. A novel Sorensen‐buffered PTA protocol is introduced as a counterstain for µAngiofil specimens, as the polyurethane polymer is susceptible to artefacts when using conventional staining solutions. Finally, we demonstrate that counterstained samples can be automatically processed into three separate image channels (skeletal tissue, vasculature and stained soft tissue), which offers multiple new options for data analysis. The presented microDECT workflows are suited as tools to screen and quantify microvasculature and can be implemented in various correlative imaging pipelines to target regions of interest for downstream light microscopic investigation. LAY DESCRIPTION: Microscopic 3D imaging of the vasculature of laboratory animal models such as the mouse can help to identify and understand vascular disorders. Microscopic x‐ray computed tomography is one of the most widely used imaging techniques for this purpose. High‐resolution insights are typically gained from the tissues of dead animals, where an x‐ray dense contrast agent was post‐mortem supplied to the bloodstream. This work presents sample preparation and imaging protocols that allow to depict and analyse microvasculature in the structural context of mineralised (bone and teeth) and non‐mineralised (e.g. brain) tissues, thus expanding the toolkit for the quantitative analyses of vascular morphology. [ABSTRACT FROM AUTHOR] |
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| Database: | Engineering Source |
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| Abstract: | Ex vivo x‐ray angiography provides high‐resolution, three‐dimensional information on vascular phenotypes down to the level of capillaries. Sample preparation for ex vivo angiography starts with the removal of blood from the vascular system, followed by perfusion with an x‐ray dense contrast agent mixed with a carrier such as gelatine or a polymer. Subsequently, the vascular micro‐architecture of harvested organs is imaged in the intact fixed organ. In the present study, we present novel microscopic dual‐energy CT (microDECT) imaging protocols that allow to visualise and analyse microvasculature in situ with reference to the morphology of hard and soft tissue. We show that the spectral contrast of µAngiofil and Micropaque barium sulphate in perfused specimens allows for the effective separation of vasculature from mineralised skeletal tissues. Furthermore, we demonstrate the counterstaining of perfused specimens using established x‐ray dense contrast agents to depict blood vessels together with the morphology of soft tissue. Phosphotungstic acid (PTA) is used as a counterstain that shows excellent spectral contrast in both µAngiofil and Micropaque barium sulphate–perfused specimens. A novel Sorensen‐buffered PTA protocol is introduced as a counterstain for µAngiofil specimens, as the polyurethane polymer is susceptible to artefacts when using conventional staining solutions. Finally, we demonstrate that counterstained samples can be automatically processed into three separate image channels (skeletal tissue, vasculature and stained soft tissue), which offers multiple new options for data analysis. The presented microDECT workflows are suited as tools to screen and quantify microvasculature and can be implemented in various correlative imaging pipelines to target regions of interest for downstream light microscopic investigation. LAY DESCRIPTION: Microscopic 3D imaging of the vasculature of laboratory animal models such as the mouse can help to identify and understand vascular disorders. Microscopic x‐ray computed tomography is one of the most widely used imaging techniques for this purpose. High‐resolution insights are typically gained from the tissues of dead animals, where an x‐ray dense contrast agent was post‐mortem supplied to the bloodstream. This work presents sample preparation and imaging protocols that allow to depict and analyse microvasculature in the structural context of mineralised (bone and teeth) and non‐mineralised (e.g. brain) tissues, thus expanding the toolkit for the quantitative analyses of vascular morphology. [ABSTRACT FROM AUTHOR] |
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| ISSN: | 00222720 |
| DOI: | 10.1111/jmi.13369 |
