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CONtrast through metal-enriched polymer SALTs: novel contrast agents for dual-energy micro-computed tomography

Periodic Reporting for period 1 - CONSALT (CONtrast through metal-enriched polymer SALTs: novel contrast agents for dual-energy micro-computed tomography)

Reporting period: 2016-04-01 to 2018-03-31

3D biomedical imaging has proven to provide additional benefits, which can support pathological findings. Micro and nanoCT technologies offer the possibility to identify tissue morphologies with comparable resolution and quality compared to conventional 2D histology, which is seen as gold standard in diagnosis. However, the availability of tools that enable non-destructive high-resolution 3D histology with laboratory-based microCT and nanoCT devices is currently very limited. Especially staining agents and procedures targeting specific biological (sub)-cellular organelles are missing.

Withinn the MSCA project CONSALT novel specific staining methods tailored for X-ray CT, which allow for an efficient 3D volume screening and analysis at high resolutions were developed. The results obtained in CONSALT allowed for the first time the visualization of soft tissue architecture at high-resolution using laboratory-based microCT and nanoCT devices. Furthermore, full compatibility with 2D conventional histology highlights the potential for modern histopathological applications. Currently, the developed staining methods are tested on human biopsy samples to evaluate the embedding of the methodology into clinical routine.

The design, development (synthesis and characterization) and testing of such new stains to overcome the challenges of contrast enhancement and selectivity for certain tissues and cellular structures (e.g. nucleus, mitochondria or cytoplasm) was proposed within the CONSALT project.

In particular, CONSALT aimed at three ambitious scientific objectives:
1. Design and Development (consisting of synthesis and characterization) of novel contrast agents for X ray (dual-energy) microCT devices.
2. Development of staining protocols
3. Testing the new overview and/or specific stains using X ray microCT devices and result evaluation of those stains through conventional optical microscopy used in histology and/or pathology.
Conference Participation

• 7th EuChemMS European Chemistry Congress; Liverpool, UK; August 2018; Oral talk entitled “Cytoplasm-specific X-ray stain for micro- and nano computed tomography enables three-dimensional virtual histology“ has been accepted.
• XRM – International Conference X ray Microscopy; Saskatoon, Saskatchewan, Canada; August 2018; Poster presentation entitled “Bismuth-Oxo-Clusters for Soft Tissue Staining“ has been accepted.
• Inorganic Chemistry meets Medicine Workshop; Garching, Germany; November 2017; Oral talk entitled “Bismuth-Oxo-Clusters for Soft Tissue Staining“ was presented.
• EMBEC17 & NBC17 – European Medical and Biological Engineering Conference 2017 & Nordic Baltic Conference on Biological Engineering and Medical Physics; Tampere, Finland; June 2017; Poster entitled “Bismuth-Oxo-Clusters for Soft Tissue Staining” was presented.
• SPIE – Medical Imaging Conference; Orlando, Florida, U.S.A.; February 2017; Oral talk entitled “Novel staining methods for X-ray Imaging of soft tissue samples“ was presented.
• XEN ZEISS Meeting; Vienna, Austria; December 2016; Oral talk entitled “3D histology of soft tissue samples using novel staining methods for X-ray imaging“ was presented.

Patent Applications

• Madleen Busse and Franz Pfeiffer, Technical University of Munich, Novel heavy metal ion ligand complexes useful as ex vivo contrast agent for a computed tomography scanning of a biological sample, ex vivo method for investigating a biological sample, and use of the complexes, European Patent Office, Application No. 17196492.7 – 1109.
• Madleen Busse and Franz Pfeiffer, Technical University of Munich, Novel heavy metal ion hematein complexes useful as ex vivo contrast agent for computed tomography scanning of a biological sample, ex vivo method for investigating a biological sample, and use of the complexes, European Patent Office, Application No. 17196491.9 – 1109.
• Madleen Busse and Franz Pfeiffer, Technical University of Munich, Novel heavy metal ion-ligand-complexes useful as ex vivo contrast agent for a computed tomography scanning of a biological sample, ex vivo method for investigating a biological sample, and use of the complexes, European Patent Office, Application No. 18 158 168.7.

Scientific Publications

• Madleen Busse, Mark Müller, Melanie A. Kimm, Simone Ferstl, Sebastian Allner, Klaus Achterhold, Julia Herzen and Franz Pfeiffer, Three dimensional virtual histology enabled through cytoplasm specific stain for microscopic and nanoscopic computed tomography, Proceedings of the National Academy of Sciences of the U.S.A. 115, 2293-2298 (2018).
• Mark Müller, Melanie A. Kimm, Simone Ferstl, Sebastian Allner, Klaus Achterhold, Julia Herzen, Franz Pfeiffer and Madleen Busse, X ray stain puts cell nuclei in the spotlight, manuscript submitted.
Overall, the Milestone M5 has been reached as part of the MSCA project CONSALT.
The results obtained in CONSALT allowed for the first time the visualization of soft tissue architecture at high resolution using laboratory based microCT and nanoCT devices. Furthermore, full compatibility with 2D conventional histology highlights the potential for modern histopathological applications. Currently, the developed staining methods are tested on human biopsy samples to evaluate the embedding of the methodology into clinical routine.

Research results obtained in CONSALT were made accessible to the research community through publications in international peer reviewed high impacted scientific journals and presentations at important conferences within the research field of biomedical imaging (a detailed listing of all publications and presentations can be found in chapter 1.2.6). In particular, preliminary results were presented at conferences, in order to foster rigorous discussions and enhance “knowledge exchange”, while the final results were published and will be published at top international journals in the fields of chemistry, biology, radiology and biomedical imaging (e.g. Angewandte Chemie International Edition, PNAS, PLoS Biology, Nature Biomedical Engineering and/or Radiology). The fellow ensured Open Access publication.

The TUM Patent and Licensing Office provided personal consulting and information on Intellectual Property issues. CONSALT resulted in three patent applications, which were made to the European Patent Office. The applications are still pending.

The dedicated Corporate Communication Center (CCC) of TUM university was supporting the fellow with a press release, which was prepared after successful high impacted publication of the research results in the Proceedings of the National Academy of Sciences of the U.S.A. Furthermore, the fellow ignited the interest in younger students and the general public by acting as a “Marie Skłodowska-Curie Ambassador.” Participation with a project at the “Herbstuniversität” (autumn university) at TUM designed for girls from grade 10 promoted a career in science and gave the students an understanding of learning and research within a university environment. The involvement at the “Lange Nacht der Wissenschaft” and “Tag der offenen Tür” (Long Night of Sciences – Open Day Event at TUM) by staging an experimental lecture for children embedded in a theatre play, whereby the students can interact and get involved, rewarded not only the children and their parents, but also the fellow as a scientist. All activities within CONSALT were performed and explained in an understandable fashion.
Dr. Madleen Busse and Prof. Franz Pfeiffer at the Nano-CT device enabling 3D histology.
These images were created using the new staining method: left: Micro-CT image; right: NanoCT image.
Chemist Dr. Madleen Busse has developved a new X-ray staining procedure to visualize soft tissue.
Franz Pfeiffer mounting a sample on the Nano-CT device.