36M-pixel synchrotron radiation micro-CT for whole secondary pulmonary lobule visualization from a large human lung specimen

A micro-CT system was developed using a 36M-pixel digital single-lens reflex camera as a cost-effective mode for large human lung specimen imaging. Scientific grade cameras used for biomedical x-ray imaging are much more expensive than consumer-grade cameras. During the past decade, advances in image sensor technology for consumer appliances have spurred the development of biomedical x-ray imaging systems using commercial digital single-lens reflex cameras fitted with high megapixel CMOS image sensors. This micro-CT system is highly specialized for visualizing whole secondary pulmonary lobules in a large human lung specimen. The secondary pulmonary lobule, a fundamental unit of the lung structure, reproduces the lung in miniature. The lung specimen is set in an acrylic cylindrical case of 36 mm diameter and 40 mm height. A field of view (FOV) of the micro-CT is 40.6 mm wide × 15.1 mm high with 3.07 μm pixel size using offset CT scanning for enlargement of the FOV. We constructed a 13,220 × 13,220 × 4912 voxel image with 3.07 μm isotropic voxel size for three-dimensional visualization of the whole secondary pulmonary lobule. Furthermore, synchrotron radiation has proved to be a powerful high-resolution imaging tool. This micro-CT system using a single-lens reflex camera and synchrotron radiation provides practical benefits of high-resolution and wide-field performance, but at low cost

High contrast microstructural visualization of natural acellular matrices by means of phase-based x-ray tomography

Acellular scaffolds obtained via decellularization are a key instrument in regenerative medicine both per se and to drive the development of future-generation synthetic scaffolds that could become available off-the-shelf. In this framework, imaging is key to the understanding of the scaffolds\u2019 internal structure as well as their interaction with cells and other organs, including ideally post-implantation. Scaffolds of a wide range of intricate organs (esophagus, lung, liver and small intestine) were imaged with x-ray phase contrast computed tomography (PC-CT). Image quality was sufficiently high to visualize scaffold microarchitecture and to detect major anatomical features, such as the esophageal mucosal-submucosal separation, pulmonary alveoli and intestinal villi. These results are a long-sought step for the field of regenerative medicine; until now, histology and scanning electron microscopy have been the gold standard to study the scaffold structure. However, they are both destructive: hence, they are not suitable for imaging scaffolds prior to transplantation, and have no prospect for post-transplantation use. PC-CT, on the other hand, is non-destructive, 3D and fully quantitative. Importantly, not only do we demonstrate achievement of high image quality at two different synchrotron facilities, but also with commercial x-ray equipment, which makes the method available to any research laboratory

Imaging intact human organs with local resolution of cellular structures using hierarchical phase-contrast tomography

Imaging intact human organs from the organ to the cellular scale in three dimensions is a goal of biomedical imaging. To meet this challenge, we developed hierarchical phase-contrast tomography (HiP-CT), an X-ray phase propagation technique using the European Synchrotron Radiation Facility (ESRF)’s Extremely Brilliant Source (EBS). The spatial coherence of the ESRF-EBS combined with our beamline equipment, sample preparation and scanning developments enabled us to perform non-destructive, three-dimensional (3D) scans with hierarchically increasing resolution at any location in whole human organs. We applied HiP-CT to image five intact human organ types: brain, lung, heart, kidney and spleen. HiP-CT provided a structural overview of each whole organ followed by multiple higher-resolution volumes of interest, capturing organotypic functional units and certain individual specialized cells within intact human organs. We demonstrate the potential applications of HiP-CT through quantification and morphometry of glomeruli in an intact human kidney and identification of regional changes in the tissue architecture in a lung from a deceased donor with coronavirus disease 2019 (COVID-19)

High contrast microstructural visualisation of natural acellular matrices by means of phase-based x-ray tomography

Acellular scaffolds obtained via decellularization are a key instrument in regenerative medicine both per se and to drive the development of future-generation synthetic scaffolds that could become available off-the-shelf. In this framework, imaging is key to the understanding of the scaffolds’ internal structure as well as their interaction with ells and other organs, including ideally post-implantation. Scaffolds of a wide range of intricate organs (oesophagus, lung, liver and small intestine) were imaged with x-ray phase contrast computed tomography (PC-CT). Image quality was sufficiently high to visualize scaffold micro architecture and to detect major anatomical features, such as the oesophageal mucosal-submucosal separation, pulmonary alveoli and intestinal villi. These results are a long-sought step for the field of regenerative medicine: until now, histology and scanning electron microscopy have been the gold standard to study the scaffold structure. However, they are both destructive: hence, they are not suitable for imaging scaffolds prior to transplantation, and have no prospect for post-transplantation use. PC-CT, on the other hand, is non-destructive, 3D and fully quantitative. Importantly, not only do we demonstrate achievement of high image quality at two different synchrotron facilities, but also with commercially available x-ray equipment, which makes the method instantly available worldwide to any research laboratory

Understanding the Structural and Functional Correlates of Acute Lung Inflammation in Two Murine Models

The outcome of lung inflammation is important to host survival as lungs are necessary for oxygen exchange and fighting pathogens or any injurious stimuli. Thus, diagnosing and understanding the kinetics of lung inflammation is an emerging technological area in the field of imaging research and development. Dr. Aulakh’s lab has two separate established models of neutrophilic murine acute lung injury namely, acute low-dose (0.05 ppm) ozone-induced and intranasal bacterial lipopolysaccharide (LPS)-induced lung inflammation. In order to characterize the dynamics of these models, there are two research hypotheses of my project, which are a) acute low-dose ozone exposure causes lung [18F]F-FDG retention because of increased leukocyte glucose uptake due to inflammation as assessed by sequential micro-Positron Emission Tomography-Computed tomography (microPET-CT) in murine lungs, similar to the effects of intranasal bacterial lipopolysaccharide, LPS and b) acute low-dose ozone exposure induces an increase in ultra-small-angle scatter (USAXS) (due to alveolar recruitment), absorption (due to alveolar edema) and a decrease in refraction (due to peri-bronchiolar edema) comparable to intranasal LPS induced changes in these X-ray optical properties as assessed by Lung Multiple Image X-Radiography (MIR). Thus, the premise of my thesis is to test the utility of longitudinal non-invasive imaging modalities, namely sequential [18F]-fluoro-deoxy glucose ([18F]F-FDG) positron emission tomography-computed tomography (PET-CT) and synchrotron multiple image X-radiography (MIR), to assess the progression of acute murine low-dose ozone or intranasal bacterial lipopolysaccharide (LPS) induced lung inflammation over 24 and 70 h time periods, respectively. Both ozone and LPS induced an increase in murine lung [18F]F-FDG standard uptake ratio (SUR) and a heterogenous lung distribution which was unlike the craniocaudal [18F]F-FDG gradient observed in lungs before any exposure (called as baseline or control [18F]F-FDG). The whole-body distribution profiles revealed that lung [18F]F-FDG activity was higher and prolonged up to 28 h in LPS compared to ozone exposed mice. While [18F]F-FDG is a useful marker to highlight areas with high metabolic uptake of glucose in cells such as neutrophils and macrophages recruited during inflammation, the resolution of PET-CT (hundreds of μm) precludes the evaluation of microscopic histopathologic changes especially in the alveoli. Using lung hematoxylin and eosin stained cryosections, the ratios of total lung tissue to air spaces and specifically alveolar parenchyma to air spaces were assessed in mice lungs exposed to 0.05 ppm ozone for 2 h. Results from the X-ray CT lung tissue volume quantifications as well as the histologically derived percent-stained lung or alveolar area quantifications suggest significant damage that is observed as reduced percentage area as well as variability or standard deviation (S.D.) of binary lung images in mice immediately i.e., at 0 h and 6 h after exposure to 2 h of 0.05 ppm ozone. Alveolar damage was also significant at 0 h as shown by reduction in percentage area and S.D. in the binary image region restricted to alveoli. The synchrotron study aimed at following mice lungs before, immediately i.e., at 0 h, and thereafter at 24, 48 or 70 h after saline, bacterial lipopolysaccharide (LPS, 50 μg), or low dose (0.05 ppm for 2 h) ozone exposure. Our results indicate that the lung ultra-small-angle scatter (USAXS), which is a metric of air-tissue boundaries and refraction (which is due to bending of X-rays across air-tissue conducting airways) reduces, especially in the cranial part of left lung, with a corresponding increase in absorption upon exposure to LPS or ozone and is detectable up to 70 h. The changes in lung X-ray optical properties are indicative of the gross inflammatory changes, in response to LPS or ozone exposure, as indicated by increases in lung absorption but reduction in refraction and USAXS. Overall, the results from my project indicate that for a comprehensive analysis of lung inflammation, a combination of lung histological analysis along with objective lung image analysis as described in the longitudinal microPET-CT and lung MIR experiments form powerful techniques for sensitive delineation of inflammatory changes in gross lung structure and function

Novel infrared and Raman spectroscopic imaging for the elucidation of specific changes in breast microcalcifications

Breast cancer is the second most common cause of death from cancer in women, accounting for more than 1 million deaths globally per year. Current detection is based on X-ray mammographic screening, which involves the use of ionising radiation with potentially detrimental effects, or MRI scans, which have limited spatial resolution. The presence of microcalcifications in breast tissue has been associated with malignant disease. Unfortunately, X-ray mammography and MRI scanning techniques are not able to discriminate between microcalcifications from a benign lesion and those from a malignant lesion. The aim of this project was to use optical techniques based on vibrational spectroscopy, such as Fourier Transform Infrared (FTIR) absorption and Raman scattering, which are non-destructive, label-free and chemically specific, to investigate the composition of microcalcifications in breast tissue for augmented diagnostics and improved outcome for the patient. This work involved the characterisation of mineral standards of the type that can be found in the breast, in order to identify the precise composition of the microcalcifications. A series of calcium hydroxyapatite (Hap) compounds was used for calibration of the micro-FTIR and Raman spectra. The ratio of carbonate-to-phosphate band intensity for each individual Hap powder was determined and the data were used to assess the level of carbonate substitution in each breast tissue biopsy. In parallel, the analysis of potential precursor mineral phases (namely octacalcium phosphate and amorphous calcium phosphate) revealed similar features to Hap in both FTIR and Raman spectra, which can be translated to the biopsy samples. The accessibility to diverse panels of breast tissue sections (frozen and paraffin-embedded) was a great opportunity to test different approaches. A deparaffinisation protocol was applied to a set of samples for Raman analysis and the process was found not to affect the microcalcification composition. The FTIR analysis of the frozen tissues provided information on the carbonate peak in the short wavelength range (1500-1400 cm-1), which normally contains a strong contribution from paraffin in standard histological specimens. The study of breast tissue sections showed the heterogeneity in composition of microcalcifications between different samples from the same stage of pathology in terms of protein, lipid - which is usually not observed in formalin-fixed paraffin-preserved (FFPE) sections - and carbonate content. The mineralisation of the MDA-MB-231 breast cell line induced by two osteogenic agents: inorganic phosphate (Pi) and -Glycerophosphate (G) was investigated using Raman micro-spectroscopy. The uptake of osteogenic agent induced a faster mineralisation for cells cultured with a medium supplemented in Pi (day 3) than G (day 11). A shift (± 3 cm-1) of the phosphate peak at 956 cm-1 in the Raman spectra was apparent when the culture medium was supplemented with G, indicating the presence of precursor phase (octacalcium phosphate) during Hap crystal formation. New IR technologies such as bright laser sources e.g. quantum cascade laser (QCL) open possibilities for the analysis of biological samples. They allowed us to achieve a better signal-to-noise ratio than Globar thermal sources used in traditional FTIR systems, particularly on optically dense samples such as calcifications. The ability of selecting specific incident wavelengths allows significant improvements in the acquisition time. This work illustrates for the first time the identification of microcalcifications using a QCL source in the long wavelength range coupled to an upconversion system with a silicon detector for efficient sensing. The upconverted images showed a good agreement with the micro-FTIR images. Vibrational spectroscopy has been shown to be a powerful tool for discrimination of mineral species in breast calcification. These techniques can provide complementary information for the pathologist to be able to classify breast pathologies - benign, ductal carcinoma in situ (DCIS) and invasive cancer - with higher accuracy.European Commissio

Investigating Perfusion of the Human Placenta

Placental insufficiency is a significant cause of morbidity and mortality, accounting for one third of antenatal stillbirths. It occurs when the maternal spiral arteries fail to remodel normally in early pregnancy, leading to inadequate maternal perfusion of the placenta. The fetus becomes hypoxic and if not delivered prematurely may ultimately die. Assessing the placenta is therefore clinically important, to diagnose placental insufficiency in vivo, and investigate poor pregnancy outcome ex vivo. Ex vivo placental assessment relies on subjective histological analysis of a small proportion of the placenta, looking for features such as oedema, inflammation and the presence of avascular villi. Regional variation and heterogeneity are not defined. In utero clinical assessment is via ultrasound Doppler measurements, looking for increased resistance in the uterine arteries, suggesting poor spiral artery remodeling, and increased resistance within the umbilical artery, suggesting inadequate development of the feto-placental microcirculation. There is therefore an urgent need to develop new ways to evaluate the perfusion of the placenta both in and ex vivo. In this thesis I investigate two imaging modalities with the potential to improve our understanding of placental perfusion. Ex vivo I develop a placental perfusion and micro-CT imaging technique, to directly visualise the feto-placental microcirculation, before applying the technique to investigate heterogeneity within a cohort of normal term placentae. I investigate differences in vascular density through the placenta at multiple scales. In vivo I investigate a novel Magnetic Resonance Imaging model of placental perfusion, that combines diffusion weighted imaging with T2 relaxometry, to estimate maternal and fetal placental perfusion. I develop this technique, exploring MRI parameters relating to perfusion in normally grown and growth restricted pregnancies. This work is important as the techniques developed improve our ability to investigate and understand placental perfusion, and provide potential new parameters of placental function in vivo

High-throughput transgenic mouse phenotyping using microscopic-MRI

With the completion of the human genome sequence in 2003, efforts have shifted towards elucidating gene function. Such phenotypic investigations are aided by advances in techniques for genetic modification of mice, with whom we share ~99% of genes. Mice are key models for both examination of basic gene function and translational study of human conditions. Furthering these efforts, ambitious programmes are underway to produce knockout mice for the ~25,000 mouse genes. In the coming years, methods to rapidly phenotype mouse morphology will be in great demand. This thesis demonstrates the development of non-invasive microscopic magnetic resonance imaging (\muMRI) methods for high-resolution ex-vivo phenotyping of mouse embryo and mouse brain morphology. It then goes on to show the application of computational atlasing techniques to these datasets, enabling automated analysis of phenotype. First, the issue of image quality in high-throughput embryo MRI was addressed. After investigating preparation and imaging parameters, substantial gains in signal- and contrast-to-noise were achieved. This protocol was applied to a study of Chd7+/- mice (a model of CHARGE syndrome), identifying cardiac defects. Combining this protocol with automated segmentation-propagation techniques, phenotypic differences were shown between three groups of mice in a volumetric analysis involving a number of organ systems. Focussing on the mouse brain, the optimal preparation and imaging parameters to maximise image quality and structural contrast were investigated, producing a high-resolution in-skull imaging protocol. Enhanced delineation of hippocampal and cerebellar structures was observed, correlating well to detailed histological comparisons. Subsequently this protocol was applied to a phenotypic investigation of the Tc1 model of Down syndrome. Using both visual inspection and automated, tensor based morphometry, novel phenotypic findings were identified in brain and inner ear structures. It is hoped that a combination of \muMRI with computational analysis techniques, as presented in this work, may help ease the burden of current phenotyping efforts

Pathological Mineralisation

Pathological mineralisation is a well-known phenomenon in the medical field as it relates to a wide range of diseases, including cancer, neurodegenerative dis-eases, aortic valve stenosis, and atherosclerosis. Despite this, the direct study of pathological minerals has been rare, as most research focuses on the study of the organic components of these pathologies and the microenvironment the minerals are observed in. Even though material science methods have been used for the study of biomaterials, hard tissues, and other biological systems; they have not been widely used in the research of pathological mineralisation. This work is, subsequently, con-centrating on the direct study of the minerals found in cardiovascular, breast, and brain tissues aiming to provide a full physicochemical characterization. The presence of in-organic material in these soft tissues has been long observed in relation to several diseases, but the relationship between their properties and specific pathologies is not fully understood. Therefore, through the direct investigation of the minerals present, this study aims to provide new insights into the association of unique mineral proper-ties to specific disease characteristics. In addition, the data on the mineral properties will then be evaluated to gain information on the mineral formation processes, in order to identify proteins, cells, or vesicles, which might be involved. Finally, a range of bio-chemical assays will be used, aiming to directly investigate the presence of biological markers in the inorganic material to give new insights on the mineralisation mecha-nisms