Human fetal whole-body postmortem microfocus computed tomographic imaging

Perinatal autopsy is the standard method for investigating fetal death; however, it requires dissection of the fetus. Human fetal microfocus computed tomography (micro-CT) provides a generally more acceptable and less invasive imaging alternative for bereaved parents to determine the cause of early pregnancy loss compared with conventional autopsy techniques. In this protocol, we describe the four main stages required to image fetuses using micro-CT. Preparation of the fetus includes staining with the contrast agent potassium triiodide and takes 3–19 d, depending on the size of the fetus and the time taken to obtain consent for the procedure. Setup for imaging requires appropriate positioning of the fetus and takes 1 h. The actual imaging takes, on average, 2 h 40 min and involves initial test scans followed by high-definition diagnostic scans. Postimaging, 3 d are required to postprocess the fetus, including removal of the stain, and also to undertake artifact recognition and data transfer. This procedure produces high-resolution isotropic datasets, allowing for radio-pathological interpretations to be made and long-term digital archiving for re-review and data sharing, where required. The protocol can be undertaken following appropriate training, which includes both the use of micro-CT techniques and handling of postmortem tissue

A multi-technique hierarchical X-ray phase-based approach for the characterization and quantification of the effects of novel radiotherapies

Cancer is the first or second leading cause of premature deaths worldwide with an overall rapidly growing burden. Standard cancer therapies include surgery, chemotherapy and radiotherapy (RT) and often a combination of the three is applied to improve the probability of tumour control. Standard therapy protocols have been established for many types of cancers and new approaches are under study especially for treating radio-resistant tumours associated to an overall poor prognosis, as for brain and lung cancers. Follow up techniques able to monitor and investigate the effects of therapies are important for surveying the efficacy of conventionally applied treatments and are key for accessing the curing capabilities and the onset of acute and late adverse effects of new therapies. In this framework, this doctoral Thesis proposes the X-ray Phase Contrast Im-aging - Computed Tomography (XPCI-CT) technique as an imaging-based tool to study and quantify the effects of novel RTs, namely Microbeam and Minibeam Radiation therapy (MRT and MB), and to compare them to the standard Broad Beam (BB) induced effects on brain and lungs. MRT and MB are novel radiotherapies that deliver an array of spatially fractionated X-ray beamlets issued from a synchrotron radiation source, with widths of tens or hundreds of micrometres, respectively. MRT and MB exploit the so-called dose-volume effect: hundreds of Grays are well tolerated by healthy tissues and show a preferential effect on tumour cells and vasculature when delivered in a micrometric sized micro-plane, while induce lethal effects if applied over larger uniform irradiation fields. Such highly collimated X-ray beams need a high-resolution and a full-organ approach that can visualize, with high sensitivity, the effects of the treatment along and outside the beamlets path. XPCI-CT is here suggested and proven as a powerful imaging technique able to determine and quantify the effects of the radiation on normal and tumour-bearing tissues. Moreover, it is shown as an effective technique to complement, with 3D information, the histology findings in the follow-up of the RT treatments. Using a multi-scale and multi-technique X-ray-based approach, I have visualized and analysed the effects of RT delivery on healthy and glioblastoma multiforme (GBM)-bearing rat brains as well as on healthy rat lungs. Ex-vivo XPCI-CT datasets acquired with isotropic voxel sizes in the range 3.253 – 0.653 μm3 could distinguish, with high sensitivity, the idiopathic effects of MRT, MB and BB therapies. Histology, immunohistochemistry, Small- and Wide-Angle X-ray Scattering and X-ray Fluorescence experiments were also carried out to accurately interpret and complement the XPCI-CT findings as well as to obtain a detailed structural and chemical characterization of the detected pathological features. Overall, this multi-technique approach could detect: i) a different radio-sensitivity for the MRT-treated brain areas; ii) Ca and Fe deposits, hydroxyapatite crystals formation; iii) extended and isolated fibrotic contents. Full-organ XPCI-CT datasets allowed for the quantification of tumour and mi-crocalcifications’ volumes in treated brains and the amount of scarring tissue in irradiated lungs. Herein, the role of XPCI-CT as a 3D virtual histology technique for the follow-up of ex-vivo RT effects has been assessed as a complementary method for an accurate volumetric investigation of normal and pathological states in brains and lungs, in a small animal model. Moreover, the technique is proposed as a guidance and auxiliary tool for conventional histology, which is the gold standard for pathological evaluations, owing to its 3D capabilities and the possibility of virtually navigating within samples. This puts a landmark for XPCI-CT inclusion in the pre-clinical studies pipeline and for advancing towards in-vivo XPCI-CT imaging of treated organs.Weltweit gilt Krebs als häufigste bzw. zweithäufigste Ursache eines zu früh erfolgenden Todes, wobei die Zahlen rasch ansteigen. Standardmäßige Krebstherapien umfassen chirurgische Eingriffe, Chemotherapie und Strahlentherapie (radiotherapy, RT); oft kommt eine Kombination daraus zur Anwendung, um die Wahrscheinlichkeit der Tumorkontrolle zu erhöhen. Es wurden Standardtherapieprotokolle für zahlreiche Krebsarten eingerichtet und es wird vor allem in der Behandlung von strahlenresistenten Tumoren mit allgemein schlechter Prognose wie bei Hirn- und Lungentumoren an neuen Ansätzen geforscht. Nachverfolgungstechniken, welche die Auswirkungen von Therapien überwachen und ermitteln, sind zur Überwachung der Wirksamkeit herkömmlich angewandter Behandlungen wichtig und auch maßgeblich am Zugang zu den Fähigkeiten zur Heilung sowie zum Auftreten akuter und verzögerter Nebenwirkungen neuer Therapien beteiligt. In diesem Rahmenwerk unterbreitet diese Doktorarbeit die Technik der Röntgen-Phasenkontrast-Bildgebung über Computertomographie (X-ray Phase Contrast Imaging - Computed Tomography, XPCI‑CT) als bildverarbeitungs-basiertes Tool zur Untersuchung und Quantifizierung der Auswirkungen neuartiger Strahlentherapien, nämlich der Mikrobeam- und Minibeam-Strahlentherapie (MRT und MB), sowie zum Vergleich derselben mit den herkömmlichen durch Breitstrahlen (Broad Beam, BB) erzielten Auswirkungen auf Gehirn und Lunge. MRT und MB sind neuartige Strahlentherapien, die ein Array räumlich aufgeteilter Röntgenstrahlenbeamlets aus einer synchrotronen Strahlenquelle mit einer Breite von Zehnteln bzw. Hundersteln Mikrometern abgeben. MRT und MB nutzen den sogenannten Dosis-Volumen-Effekt: Hunderte Gray werden von gesundem Gewebe gut vertragen und wirken bei der Abgabe in einer Mikroebene im Mikrometerbereich vorrangig auf Tumorzellen und Blutgefäße, während sie bei einer Anwendung über größere gleichförmige Strahlungsfelder letale Auswirkungen aufweisen. Solche hoch kollimierten Röntgenstrahlen erfordern eine hohe Auflösung und einen Zugang zum gesamten Organ, bei dem die Auswirkungen der Behandlung entlang und außerhalb der Beamletpfade mit hoher Empfindlichkeit visualisiert werden können. Hier empfiehlt und bewährt sich die XPCI‑CT als leistungsstarke Bildverarbeitungstechnik, welche die Auswirkungen der Strahlung auf normale und tumortragende Gewebe feststellen und quantifizieren kann. Außerdem hat sich gezeigt, dass sie durch 3‑D-Informationen eine effektive Technik zur Ergänzung der histologischen Erkenntnisse in der Nachverfolgung der Strahlenbehandlung ist. Anhand eines mehrstufigen und multitechnischen röntgenbasierten Ansatzes habe ich die Auswirkungen der Strahlentherapie auf gesunde und von Glioblastomen (GBM) befallene Rattenhirne sowie auf gesunde Rattenlungen visualisiert und analysiert. Mit isotropen Voxelgrößen im Bereich von 3,53 bis 0,653 μm3 erfasste Ex-vivo-XPCI-CT-Datensätze konnten die idiopathischen Auswirkungen der MRT-, MB- und BB‑Behandlung mit hoher Empfindlichkeit unterscheiden. Es wurden auch Experimente zu Histologie, Immunhistochemie, Röntgenklein- und ‑weitwinkelstreuung und Röntgenfluoreszenz durchgeführt, um die XPCI‑CT-Erkenntnisse präzise zu interpretieren und zu ergänzen sowie eine detaillierte strukturelle und chemische Charakterisierung der nachgewiesenen pathologischen Merkmale zu erhalten. Im Allgemeinen wurde durch diesen multitechnischen Ansatz Folgendes ermittelt: i) eine un-terschiedliche Strahlenempfindlichkeit der mit MRT behandelten Gehirnbereiche; ii) Ca- und Fe-Ablagerungen und die Bildung von Hydroxylapatitkristallen; iii) ein ausgedehnter und isolierter Fibrosegehalt. XPCI‑CT-Datensätze des gesamten Organs ermöglichten die Quantifizierung der Volume von Tumoren und Mikroverkalkungen in den behandelten Gehirnen und der Menge des Narbengewebes in bestrahlten Lungen. Dabei wurde die Rolle der XPCI‑CT als virtuelle 3‑D-Histologietechnik für die Nachverfolgung von Ex-vivo-RT‑Auswirkungen als ergänzende Methode für eine präzise volumetrische Untersuchung des normalen und pathologischen Zustands von Gehirnen und Lungen im Kleintiermodell untersucht. Darüber hinaus wird die Technik aufgrund ihrer 3‑D-Fähigkeiten und der Möglichkeit zur virtuellen Navigation in den Proben als Leitfaden und Hilfstool für die herkömmliche Histologie vorgeschlagen, die der Goldstandard für die pathologische Evaluierung ist. Dies markiert einen Meilenstein für die Übernahme der XPCI‑CT in die Pipeline präklinischer Studien und für den Übergang zur In-vivo-XPCI‑CT von behandelten Organen

Imaging fascicular organisation in mammalian vagus nerve for selective VNS

Nerves contain a large number of nerve fibres, or axons, organised into bundles known as fascicles. Despite the somatic nervous system being well understood, the organisation of the fascicles within the nerves of the autonomic nervous system remains almost completely unknown. The new field of bioelectronics medicine, Electroceuticals, involves the electrical stimulation of nerves to treat diseases instead of administering drugs or performing complex surgical procedures. Of particular interest is the vagus nerve, a prime target for intervention due to its afferent and efferent innervation to the heart, lungs and majority of the visceral organs. Vagus nerve stimulation (VNS) is a promising therapy for treatment of various conditions resistant to standard therapeutics. However, due to the unknown anatomy, the whole nerve is stimulated which leads to unwanted off-target effects. Electrical Impedance Tomography (EIT) is a non-invasive medical imaging technique in which the impedance of a part of the body is inferred from electrode measurements and used to form a tomographic image of that part. Micro-computed tomography (microCT) is an ex vivo method that has the potential to allow for imaging and tracing of fascicles within experimental models and facilitate the development of a fascicular map. Additionally, it could validate the in vivo technique of EIT. The aim of this thesis was to develop and optimise the microCT imaging method for imaging the fascicles within the nerve and to determine the fascicular organisation of the vagus nerve, ultimately allowing for selective VNS. Understanding and imaging the fascicular anatomy of nerves will not only allow for selective VNS and the improvement of its therapeutic efficacy but could also be integrated into the study on all peripheral nerves for peripheral nerve repair, microsurgery and improving the implementation of nerve guidance conduits. Chapter 1 provides an introduction to vagus nerve anatomy and the principles of microCT, neuronal tracing and EIT. Chapter 2 describes the optimisation of microCT for imaging the fascicular anatomy of peripheral nerves in the experimental rat sciatic and pig vagus nerve models, including the development of pre-processing methods and scanning parameters. Cross-validation of this optimised microCT method, neuronal tracing and EIT in the rat sciatic nerve was detailed in Chapter 3. Chapter 4 describes the study with microCT with tracing, EIT and selective stimulation in pigs, a model for human nerves. The microCT tracing approach was then extended into the subdiaphragmatic branches of the vagus nerves, detailed in Chapter 5. The ultimate goal of human vagus nerve tracing was preliminarily performed and described in Chapter 6. Chapter 7 concludes the work and describes future work. Lastly, Appendix 1 (Chapter 8) is a mini review on the application of selective vagus nerve stimulation to treat acute respiratory distress syndrome and Appendix 2 is morphological data corresponding to Chapter 4

Novel Applications and Refinements of Ultrasound Techniques in Perinatal and Infant Death Investigation

The decline in parental consent for perinatal autopsies has led to the development of less invasive autopsy techniques, primarily using imaging and in some cases acquiring tissue samples through laparoscopic techniques via small incisions. Whilst post-mortem MRI (PMMR) has been extensively tested, and shown to be a suitable modality for determining the cause of death/demise in the perinatal cohort, it is not widely available. This thesis explores the utility of a poorly explored, yet more accessible, cheaper, dynamic imaging modality widely used in ‘live’ paediatric imaging for the purposes of a less invasive autopsy – the perinatal post-mortem ultrasound (PMUS). I present a systematic review of the limited literature of PMUS diagnostic accuracy for perinatal death investigation encompassing 4 publications with 455 cases in total. This reveals an overall pooled whole body sensitivity rate of 73.3% [95% CI 59.9, 83.5] and specificity rate of 96.6% [95% CI 92.6, 98.4] . A comprehensive imaging protocol for whole body PMUS is also presented based on my own experience in scanning 272 perinatal deaths. An analysis in 130 of these cases with autopsy as a reference standard, showed that brain and abdominal diagnoses yielded the highest sensitivity rates (90.9% and 92.3% respectively), with spinal, cardiac and thoracic diagnoses yielding the lowest sensitivity rates (50%, 50% and 57.1% respectively). Imaging of the brain and heart however were the least likely to be of diagnostic quality (76.8% and 78.3% diagnostic cases respectively), particularly in macerated fetuses. In a subset of cases where PMUS and 1.5T PMMR were performed, there was no significant difference in whole body diagnostic accuracy rates (concordance rates for PMUS versus PMMR of 86.4% [95%CI 77.7, 92.0] versus 88.6% [95% CI 80.3, 93.7]), although PMMR yielded fewer non-diagnostic brain and cardiac examinations (2.9% and 2.9% non-diagnostic brain and cardiac PMMR cases versus 22.8% and 14.7% non-diagnostic PMUS cases). In the second part of my thesis, I describe the development of an ‘incisionless’ ultrasound guided biopsy method using a single entry site for the biopsy needle – the umbilical vein. This ‘INTACT’ biopsy method allowed for a ‘non-invasive’ autopsy with tissue sampling, with a biopsy success rate of 76.1% overall for all organs, with highest individual organ success rates >90% for heart and lungs. I conclude by discussing how best to incorporate PMUS into clinical practice and suggest areas for future researc

Applications of micro-CT in the Criminal Justice System of England and Wales: an impact assessment

The Criminal Justice System of England and Wales is currently facing major challenges. One is the financial pressure of government funding cuts, the other the increasing need for professionalisation and rigour within the system. This thesis presents the use of micro Computed Tomography, Additive Manufacturing, and 3D visualisation to address both challenges. By drawing on data from live murder investigations the project examines how these digital technologies can be used to improve the investigation of strangulation deaths, sharp force injuries, and fractures. Each of these categories was treated as a separate case in the overall multiple-case study research design. The increased detail enabled by micro-CT assisted pathologists in the diagnosis of strangulation as previously undetected injuries of the larynx could be identified. A validation study comparing injured to uninjured samples was conducted to increase the strength of the interpretations. For sharp force injuries analysis, micro-CT proved useful for providing the necessary injury characteristics and highly accurate measurements to allow weapon identification. The high resolution of micro-CT scanning also enabled the visualisation of trauma on the smallest of skeletal elements, often encountered in non-accidental injuries in children. The cross-case synthesis revealed the main themes of clarity, objectivity, and visualisation which were improved by using micro-CT irrespective of type of homicide. The significance of these themes further crystallised in semi-structured interviews conducted with various stakeholders of the Criminal Justice System. Management concepts proved suitable to assess the project’s success as the themes used in operations management such as quality, delivery, and cost apply to the delivery of justice as well. A good working relationship with West Midlands Police’s homicide investigators and researchers at WMG was crucial to providing the technology and expertise to address real-life problems whilst ultimately saving taxpayers’ money

Optimization and new applications of edge-illumination based X-ray phase contrast CT

The Edge Illumination (EI) method is a phase contrast imaging technique suitable for use with conventional X-ray sources, which has demonstrated its great potential for translation into real-world environments. The increase in image contrast provided by it, in particular for low-absorbing materials, along with its ability to be operated as a computed tomography (CT) modality, make it an especially advantageous technique for biomedical applications. The work presented in this thesis aimed to develop new image acquisition and processing strategies to further advance the current capabilities of EI CT, such that it could be used in a robust way for a wide range of biomedical applications. This was achieved by the development of two retrieval algorithms, which are required for the extraction of quantitative information of various sample properties. The first algorithm targets applications which involve high-resolution, multi-modal lab-based CT scans. It has been shown that, during these long scans, the variation in system parameters across the field-of-view and over time can lead to significant image artefacts in reconstructed CT slices. The proposed algorithm is capable of correcting for both types of variations, leading to the quantitative retrieval of sample properties from long CT scans in non-ideal environments. The second algorithm was aimed at applications which require fast CT scans, possibly at the expense of spatial resolution and quantitative information. A previously developed algorithm enabled a substantial reduction in scan times for homogeneous samples, by reducing the number of required images per angular view. The new algorithm presented here has extended the previous one to non-homogeneous samples, therefore expanding the range of objects which could benefit from a reduction in scan times through use of this approach. In addition, perinatal post-mortem imaging has been identified as a new biomedical application which could benefit from EI CT in the future. Promising results are reported from a proof-of-principle scan carried out using a gold standard X-ray phase contrast imaging method at a synchrotron

3D histopathology-a lung tissue segmentation workflow for microfocus X-ray-computed tomography scans

Lung histopathology is currently based on the analysis of 2D sections of tissue samples. The use of microfocus X-ray-computed tomography imaging of unstained soft tissue can provide high-resolution 3D image datasets in the range of 2-10 μm without affecting the current diagnostic workflow. Important details of structural features such as the tubular networks of airways and blood vessels are contained in these datasets but are difficult and time-consuming to identify by manual image segmentation. Providing 3D structures permits a better understanding of tissue functions and structural interrelationships. It also provides a more complete picture of heterogeneous samples. In addition, 3D analysis of tissue structure provides the potential for an entirely new level of quantitative measurements of this structure that have previously been based only on extrapolation from 2D sections. In this paper, a workflow for segmenting such 3D images semi-automatically has been created using and extending the ImageJ open-source software and key steps of the workflow have been integrated into a new ImageJ plug-in called LungJ. Results indicate an improved workflow with a modular organization of steps facilitating the optimization for different sample and scan properties with expert input as required. This allows for incremental and independent optimization of algorithms leading to faster segmentation. Representation of the tubular networks in samples of human lung, building on those segmentations, has been demonstrated using this approach.</p