Exploring contrast-enhancing staining agents for contrast-enhanced computed tomography imaging

The need for high-resolution 3D imaging of biological tissues is a driving force behind the development of innovative technologies. Among these technologies, microfocus computed tomography (MicroCT) stands out for its exceptional spatial resolution, user-friendliness, and cost-effectiveness. While MicroCT excels at providing non-destructive 3D views of tissue samples, it is limited to visualizing dense tissues, such as bone, that strongly interact with X-rays. To address this limitation, researchers have developed methods to enhance the contrast of soft tissues. The approach studied and discussed in this dissertation focuses on the use of heavy atom-containing molecules, known as contrast-enhancing staining agents (CESAs). These molecules passively diffuse into an ex vivo biological tissue sample and disperse throughout the tissues based on their affinity, contributing to improved tissue contrast. This specific methodology is also called contrast-enhanced computed tomography (CECT). Within this thesis, a series of case studies were conducted to shed light on the staining mechanisms and diffusion kinetics of both established and novel CESAs for 3D histopathology of biological tissues. The primary focus was on investigating the chemistry and interactions of these staining systems, with each case study centered on a specific tissue or organ. In the first project, we explored the potential of CECT in revealing the microstructure of healthy murine brain hemispheres, as well as in murine disease models of Alzheimer’s disease and multiple sclerosis. The second project involved screening various CESAs for the study of adipose tissue in the bone marrow and muscle. Additionally, the staining mechanism of Lugol’s iodine components with respect to biological tissues was investigated using both simplified and more complex model systems. The third project centered on studying the diffusion kinetics of different types of polyoxometalates (POMs) through the porcine aorta wall. In summary, this thesis has provided invaluable insights into the staining kinetics and mechanisms of CESAs for biological tissues. Furthermore, it has introduced novel CESAs into various fields of CECT, paving the way for further advancements in CESAs for 3D histopathology of biological tissues using CECT.(FSA - Sciences de l'ingénieur) -- UCL, 202

Distinguishing biofilms from water using X-ray micro-computed tomography

1. Introduction Bacteria colonize porous media and form biofilms affecting the material properties. It is important to visualize biofilms to understand their effect. Within porous media, X-ray micro-computed tomography (µCT) could image them. However, distinguishing biofilms from water is challenging due to similar Xray attenuation. Contrast-enhancing staining agents (CESAs) binding to the biofilms can enhance their attenuation. BaSO4 [1], silver-coated microspheres [2] and 1-chloronaphtalene [3] were successful, but there are drawbacks related to the hydrophobic nature, toxicity, sedimentation, etc. Hence, we assessed other CESAs for their capabilities to visualize biofilms using µCT that avoid these issues. 2. Materials and Methods CESAs (KBr, FeSO4, BaCl2, PTA, Hexabrix, isotonic lugol, Ca4+, Mono-WD POM, Hf-WD POM [4]) were tested to stain submerged biofilms on stones or in between sand. All biofilms were imaged by HECTOR at the Centre for X-ray Tomography (UGCT) [5]. 3. Results and Conclusion From all CESAs, isotonic lugol and Hf-WD POM were most promising. It was possible to visualize cyanobacterial biofilms on rocks (Figure 1), which could lead to the 3D examination of microbial mats. Moreover, biofilms could also be distinguished inside a sand column, allowing to determine their spatial distribution. As a next step, dynamic experiments can be performed to visualize biological weathering or their effect on water transport in situ. 4. Acknowledgements This work was funded by the FWO (11D4518N, 11D4520N and G088218N). 5. References [1] Davit et al, 10.1111/j.1365-2818.2010.03432.x [2] Iltis et al, 10.1029/2010WR009410 [3] Rolland du Roscoat et al, 10.1002/bit.25168 [4] De Bournonville et al, 10.1155/2019/8617406 [5] Masschaele 6596/463/1/012012 et al, 10.1088/174

Distinguishing biofilms from water using X-ray micro-computed tomography

1. Introduction Bacteria colonize porous media and form biofilms affecting the material properties. It is important to visualize biofilms to understand their effect. Within porous media, X-ray micro-computed tomography (µCT) could image them. However, distinguishing biofilms from water is challenging due to similar Xray attenuation. Contrast-enhancing staining agents (CESAs) binding to the biofilms can enhance their attenuation. BaSO4 [1], silver-coated microspheres [2] and 1-chloronaphtalene [3] were successful, but there are drawbacks related to the hydrophobic nature, toxicity, sedimentation, etc. Hence, we assessed other CESAs for their capabilities to visualize biofilms using µCT that avoid these issues. 2. Materials and Methods CESAs (KBr, FeSO4, BaCl2, PTA, Hexabrix, isotonic lugol, Ca4+, Mono-WD POM, Hf-WD POM [4]) were tested to stain submerged biofilms on stones or in between sand. All biofilms were imaged by HECTOR at the Centre for X-ray Tomography (UGCT) [5]. 3. Results and Conclusion From all CESAs, isotonic lugol and Hf-WD POM were most promising. It was possible to visualize cyanobacterial biofilms on rocks (Figure 1), which could lead to the 3D examination of microbial mats. Moreover, biofilms could also be distinguished inside a sand column, allowing to determine their spatial distribution. As a next step, dynamic experiments can be performed to visualize biological weathering or their effect on water transport in situ. 4. Acknowledgements This work was funded by the FWO (11D4518N, 11D4520N and G088218N). 5. References [1] Davit et al, 10.1111/j.1365-2818.2010.03432.x [2] Iltis et al, 10.1029/2010WR009410 [3] Rolland du Roscoat et al, 10.1002/bit.25168 [4] De Bournonville et al, 10.1155/2019/8617406 [5] Masschaele 6596/463/1/012012 et al, 10.1088/174

Revealing the murine brain anatomy by contrast-enhanced computed tomography – a screening study

The brain is a complex organ, which allows species to think, remember, feel and move. Notwithstanding the interest of the scientific community in this organ, many unknowns are still to be solved, such as the structure-function relationship that allows the brain to perform its daily tasks. Today, the main hypothesis is that the anatomical architecture influences, but not fully determines, the dynamics of the neural network. Advanced high-resolution 3D imaging techniques could allow researchers to better visualize the structure of the brain and hence better understand how the structure influences the functioning of the brain. Conventional 2D histology is currently still the gold standard for ex vivo structural assessment of the brain, since it provides high discriminative power, subcellular resolution and the methodologies are well-established. Nevertheless, it lacks in 3D information, is destructive and laborious. Therefore, a novel complementary 3D histological technique, contrast-enhanced microfocus computed tomography (CECT), has been introduced that combines the discriminative power of conventional 2D histology, with the 3D, non-destructive properties of CT. Multiple contrast-enhancing staining agents (CESAs) have been explored for brain CECT, including organic (e.g. Iodixanol) and inorganic (e.g. Lugol’s iodine solution). However, a lack of information exists on the nature of their interactions with different constituents of the brain. In order to provide insights in the choice of CESA for certain applications, we investigated four different CESAs. In this screening study, Hexabrix, CA4+, Preyssler anion and the 1:2 hafnium substituted Wells-Dawson polyoxometalate (Hf-WD 1:2 POM) have been evaluated as CESAs for the imaging of healthy murine hemispheres with CECT. These molecules differ in terms of chemical properties (e.g. charge, size, molecular weight) and heavy atom (Iodine vs Tungsten), which will impact staining specificity, diffusion and attenuation properties. First, the staining specificity of the CESAs was evaluated by determining where they accumulate in the tissue. For this purpose, we compared the CECT images with conventional 2D histological sections and textbook images of the murine brain. Differences in staining specificity were observed for all four CESAs, which highlight that any change in chemical properties alters specificity. Then, a quantitative analysis was performed to determine how the CESAs diffuse through the brain and whether the CESA accumulation in the brain induces tissue shrinkage or swelling (Fig. 1). Results show that next to the size of the molecules, the interactions that occur also play an important role in the diffusion speed of the molecule through the tissue. Finally, we explored the added value of obtaining 3D images by segmentation of certain structures followed by quantitative analysis To conclude, we introduced four CESAs, new to the field of brain CECT imaging, that all exhibit distinct profiles in terms of specificity and speed of diffusion. In future experiments, we envision the use of one or multiple of these CESAs in the study of a pathology with CECT

Virtual 3D histological analysis of soft tissues by contrast-enhanced microfocus computed tomography: screening contrast-enhancing staining agents

The gold standard for studying biological tissues at the microscale (i.e. histology) is tissue sectioning with subsequent colorimetric or fluorescent staining and microscopic evaluation. However, it loses the 3D information of the tissue. Also, the tissue treatment is highly time-consuming, especially for bone because it needs to be decalcified. In the field of tissue engineering and regenerative medicine, it is essential to analyze the 3D distribution of different tissue types and constituents, and therefore to maintain the integrity of the biological tissues. As non-invasive imaging technique, contrast-enhanced microfocus X-ray computed tomography (CECT) is a promising solution to image soft tissues in addition to mineralized tissues in full 3D. However, there is still a lack of systematic screening and comparison of X-ray contrast-enhancing staining agents (CESAs) for their potential to highlight tissues and their constituents in whole organs. Therefore, in this study, fixed murine auricles, incubated with Hafnium-substituted Wells-Dawson polyoxometalate (Hf-WD POM), cationic iodinated contrast agent CA4+ and/or Lugol’s iodine, were scanned with high-resolution microCT. The tissues were then embedded and processed for colorimetric staining. By comparing CECT images and corresponding tissue sections, we identified and segmented tissue types such as the epidermis, dermis, hair follicles, sebaceous glands, adipose tissue, muscle, cartilage and vessels. The three CESAs conferred different contrasts to these tissue types. We are also assessing the effect of CESA staining on subsequent histological assessment by sectioning and colorimetric staining. We found that the CESAs can only be partly washed out (even after 21 days in PBS) and could modify the biochemical properties of the tissues, rendering them more fragile for cutting. Additionally, they could change the binding properties of subsequent histological stains. Taken together, CECT is a promising technique for 3D histological analysis of biological tissues and will give us a better insight in the spatial distribution and interrelationship of tissues. However, it should be considered that the CESAs may influence subsequent colorimetric histological assessment

Design of experiments to evaluate the staining properties of polyoxometalates for contrast-enhanced microCT of aorta tissue

Vascular diseases are one of the leading causes of death in developed countries. To better treat these diseases, tissue engineered (TE) vascular grafts could provide a solution. However, for an improved design of these grafts, a better understanding of the relationship between the microstructure of vascular tissues and their mechanical behavior is required, as this relationship is key to obtain matching properties between the TE construct and the native tissue. The detailed microstructural characterization can be achieved by microfocus X-ray computed tomography (microCT), and in particular contrast-enhanced microCT or CECT. This novel virtual 3D histological technique combines the benefits of microCT imaging with the high discriminative power of conventional 2D histology, by using contrast-enhancing staining agents (CESAs). These CESAs enrich a specific region of the tissue under evaluation with X-ray attenuating atoms, which subsequently allows soft tissue visualization. Recently, two new CESAs have shown promise in this field: Hf-WD 1:2 POM (i.e. two Mono-WD POMs linked together by a Hafnium atom) and Mono-WD POM. However, there is still a great lack of fundamental knowledge about their staining properties i.e. the nature of chemical interactions, specificity and diffusional properties. In this study, we characterized these two CESAs in terms of their contrast enhancement and diffusion properties within the media of porcine aorta tissue. However, it is known that the Hf-WD 1:2 POM can dissociate into the Mono-WD POM and the Hf-WD 1:1 POM, which complicates observations. As a result, we added the Hf-WD 2:2 POM in a preliminary experiment, which is known to dissociate mainly into two Hf-WD 1:1 POMs. Based on this preliminary experiment, we could correlate both diffusion fronts generated by the Hf-WD 1:2 POM with the presence of Mono-WD POM and Hf-WD 1:1 POM. We used Design of Experiments to carry out these experiments efficiently (DoE, JMP software). The DoE method reduces the number of samples required and is also capable of studying complex interactions between parameters. The input parameters used in this study were: 1) the mass percentage of CESA in the staining solution, 2) the volume of the staining solution and 3) the staining time. The results revealed that both CESAs behave differently in terms of the final contrast they offer and their diffusional speed into the tissue. After analysis of the DoE, we found out that the main parameters influencing both observations are the staining time and the mass percentage of CESA. A higher initial mass percentage of CESA resulted in a higher grey value of the tissue and reduced the staining time necessary to completely stain the tissue. In addition, it was observed that for both CESAs, final grey values obtained at the intima side of the tissue, were lower than for the adventitia side. Histological analysis and comparison with high-resolution CT images revealed that the observed grey value could be correlated with the packing of the elastin fibers in the media. Near the adventitia side these fibers are more straight and hence better stacked, whereas near the intima side these fibers have a wavy appearance which inhibits efficient stacking. Moreover, the Hf-containing POMs seemed to have a higher affinity for these fibers, resulting in higher grey values. In conclusion, the Hf-containing POMs result in higher grey values and a slower diffusion, which could be linked to the interaction with the elastin fibers in the media. In future research, interactions with elastin fibers will be confirmed by performing (bio)chemical experiments. Enhancing knowledge on the staining properties of these CESAs will be beneficial for the study of the microstructure of aorta tissue and TE vascular grafts