phaidra.vetmeduni.ac.at o:3870 DOI 10.1111/jmi.13369 In situ isotropic 3D imaging of vasculature perfusion specimens using x-ray microscopic dual-energy CT 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. eng Imaging, Three-Dimensional Methods Animals Contrast Media Perfusion Methods Barium Sulfate Tomography, X-Ray Computed Methods Microvessels Diagnostic Imaging Mice X-Ray Microtomography Methods 2025-02-18T09:33:06.595Z LOMv1.0 Author BEGIN:VCARD VERSION:3.0 N:Handschuh;Stephan; FN:Stephan Handschuh X-ORCID:https://orcid.org/0000-0002-2140-7892 END:VCARD BEGIN:VCARD VERSION:3.0 N:Reichart;Ursula; FN:Ursula Reichart X-ORCID:https://orcid.org/0000-0001-9709-0919 END:VCARD BEGIN:VCARD VERSION:3.0 N:Kummer;Stefan; FN:Stefan Kummer X-ORCID:https://orcid.org/0000-0002-0746-1403 END:VCARD BEGIN:VCARD VERSION:3.0 N:Glösmann;Martin; FN:Martin Glösmann X-ORCID:https://orcid.org/0000-0002-2094-3247 END:VCARD https://w3id.org/kim/hcrt/scheme https://w3id.org/kim/hcrt/text Textdokument Text LOMv1.0 yes https://creativecommons.org/licenses/by-nc/4.0 application/pdf 32555161 https://phaidra.vetmeduni.ac.at/o:3870 https://phaidra.vetmeduni.ac.at/api/object/o:3870/thumbnail LOMv1.0 discipline