Probing the Unseen Depths of the Hepatic Microarchitecture via Multimodal Microscopy

Multimodal microscopy combines the advantages and strengths of different imaging modalities in order to holistically characterise the organisation of biological organisms and their comprising constituents under healthy and diseased conditions, down to the spatial resolution required to understand the morphology and function of such structures. Given the profound advantages conferred by such an approach, this work broadly aimed to develop and exploit various multimodal and multi-dimensional imaging modalities in a complimentary, combined and/or correlative manner – namely, three-dimensional scanning electron microscopy, transmission electron tomography, bright-field light microscopy, confocal laser scanning microscopy and X-ray micro-computed tomography – in order to characterise and collect new information on the normal and pathological microarchitecture of rodent and human liver tissue in 3-D under various experimental conditions. The data reported in this work includes a comparative analysis of a variety of sample preparation protocols applied to rat liver tissue to determine the suitability of such protocols for the application of serial block-face scanning electron microscopy (SBF-SEM). Next, 3-D modelling and morphometric analysis (utilising the premier SBF-SEM protocol) was performed in order to visualise and quantify key features of the hepatic microarchitecture. We further outline a large-volume correlative light and electron microscopy approach utilising selective molecular probes for confocal laser scanning microscopy (actin, lipids and nuclei), combined with the 3-D ultrastructure of the same structures of interest, as revealed by SBF-SEM (Chapter 2). Development of a straightforward combinatorial sample preparation approach, followed by a swift multimodal imaging approach – combining X-ray micro-computed tomography, bright-field light microscopy and serial section scanning electron microscopy – facilitated the cross correlation of structure-function information on the same sample across diverse length scales (Chapter 3). Next, we outline a novel “silver filler pre-embedding approach” in order to reduce artefactual charging, minimise dataset acquisition time and improve resolution and contrast in rat liver tissue prepared for SBF-SEM (Chapter 4). Next, we employ a complementary imaging approach involving serial section scanning electron microscopy and transmission electron tomography in order to comparatively analyse the structure and morphometric parameters of thousands of normal- and giant mitochondria in human patients diagnosed with non-alcoholic fatty liver disease. In so doing, we reveal functional alterations associated with mitochondrial gigantism and propose a mechanism for their formation (Chapter 5). Finally, the significance of the results obtained, and major scientific advances reported in this work are discussed in-depth against the relevant literature. This is proceeded by the future outlooks and research that remains to be done, followed by the main conclusions of this Ph.D thesis (Chapter 6). In summary, our findings firmly establish the immense importance and value of contemporary multimodal microscopy modalities in modern life science research, for holistically revealing cellular structures along the vast length scales amongst which they exist, under healthy and clinically relevant pathological conditions

Management of Gastric Cancer

Gastric cancer is the fifth most common cancer and the second most common cause of cancer death worldwide. More than 50% of the patients have advanced disease at diagnosis and in this case the disease has a poor outcome. The staging of gastric cancers is based on endoscopic ultrasound, computed tomography, magnetic resonance imaging, positron emission tomography, in addition to the laparoscopic staging. Many improvements in the surgical techniques have been seen in the last decade. Laparoscopic surgery is an emerging approach which offers important advantages: less blood loss, reduced postoperative pain, accelerated recovery, early return to normal bowel function and reduced hospital stay. D1 lymphadenectomy, with a goal of examining 15 or greater lymph nodes is a standard. D2 dissection is considered as a standard in several institutions especially in eastern Asia. Perioperative chemotherapy and adjuvant concurrent radiochemotherapy are recognized as standards treatments. Palliative chemotherapy is the mainstay treatment of advanced stages of the disease (metastatic and non-operable tumors). Despite these treatment advances, the prognosis of gastric cancer remains poor with a 5-year survival ranging from 10 to 15% in all stages combined

New Techniques in Gastrointestinal Endoscopy

As result of progress, endoscopy has became more complex, using more sophisticated devices and has claimed a special form. In this moment, the gastroenterologist performing endoscopy has to be an expert in macroscopic view of the lesions in the gut, with good skills for using standard endoscopes, with good experience in ultrasound (for performing endoscopic ultrasound), with pathology experience for confocal examination. It is compulsory to get experience and to have patience and attention for the follow-up of thousands of images transmitted during capsule endoscopy or to have knowledge in physics necessary for autofluorescence imaging endoscopy. Therefore, the idea of an endoscopist has changed. Examinations mentioned need a special formation, a superior level of instruction, accessible to those who have already gained enough experience in basic diagnostic endoscopy. This is the reason for what these new issues of endoscopy are presented in this book of New techniques in Gastrointestinal Endoscopy

Evaluation of a diffraction-enhanced imaging (DEI) prototype and exploration of novel applications for clinical implementation of DEI

Conventional mammographic image contrast is derived from x-ray absorption, resulting in breast structure visualization due to density gradients that attenuate radiation without distinction between transmitted, scattered, or refracted x-rays. Diffraction-enhanced imaging (DEI) allows for increased contrast with decreased radiation dose compared to conventional mammographic imaging due to monochromatic x-rays, its unique refraction-based contrast mechanism, and excellent scatter rejection. Although laboratory breast imaging studies have demonstrated excellent breast imaging, important clinical translation and application studies are needed before the DEI system can be established as a useful breast imaging modality. This dissertation focuses on several important studies toward the development of a clinical DEI system. First, contrast-enhanced DEI was explored using commercially available contrast agents. Phantoms were imaged at a range of x-ray energies and relevant contrast agent concentrations. Second, we performed a reader study to determine if superior DEI contrast mechanisms preserve image quality as tissue thickness increases. Breast specimens were imaged at several thicknesses, and radiologist perception of lesion visibility was recorded. Lastly, a prototype DEI system utilizing an x-ray tube source was evaluated through a reader study. Breast tissue specimens were imaged on the traditional and prototype DEI systems, and expert radiologists evaluated image quality and pathology correlation. This dissertation will demonstrate proof-of-principle for contrast-enhanced DEI, establishing the feasibility of contrast-enhanced DEI using commercially available contrast agents. Further, it will show that DEI might be able to reduce breast compression, and thus the perception of pain during mammography, without significantly decreasing breast lesion visibility. Finally, this research shows the successful implementation of a DEI prototype, displaying breast features with approximately statistically equivalent visibility to the traditional DEI system. Together, this research is an important step toward the clinical translation of DEI, a technology with the potential to facilitate early breast cancer detection and diagnosis

Goggle Augmented Imaging and Navigation System for Fluorescence-Guided Surgery

Surgery remains the only curative option for most solid tumors. The standard-of-care usually involves tumor resection and sentinel lymph node biopsy for cancer staging. Surgeons rely on their vision and touch to distinguish healthy from cancer tissue during surgery, often leading to incomplete tumor resection that necessitates repeat surgery. Sentinel lymph node biopsy by conventional radioactive tracking exposes patients and caregivers to ionizing radiation, while blue dye tracking stains the tissue highlighting only superficial lymph nodes. Improper identification of sentinel lymph nodes may misdiagnose the stage of the cancer. Therefore there is a clinical need for accurate intraoperative tumor and sentinel lymph node visualization. Conventional imaging modalities such as x-ray computed tomography, positron emission tomography, magnetic resonance imaging, and ultrasound are excellent for preoperative cancer diagnosis and surgical planning. However, they are not suitable for intraoperative use, due to bulky complicated hardware, high cost, non-real-time imaging, severe restrictions to the surgical workflow and lack of sufficient resolution for tumor boundary assessment. This has propelled interest in fluorescence-guided surgery, due to availability of simple hardware that can achieve real-time, high resolution and sensitive imaging. Near-infrared fluorescence imaging is of particular interest due to low background absorbance by photoactive biomolecules, enabling thick tissue assessment. As a result several near-infrared fluorescence-guided surgery systems have been developed. However, they are limited by bulky hardware, disruptive information display and non-matched field of view to the user. To address these limitations we have developed a compact, light-weight and wearable goggle augmented imaging and navigation system (GAINS). It detects the near-infrared fluorescence from a tumor accumulated contrast agent, along with the normal color view and displays accurately aligned, color-fluorescence images via a head-mounted display worn by the surgeon, in real-time. GAINS is a platform technology and capable of very sensitive fluorescence detection. Image display options include both video see-through and optical see-through head-mounted displays for high-contrast image guidance as well as direct visual access to the surgical bed. Image capture options from large field of view camera as well high magnification handheld microscope, ensures macroscopic as well as microscopic assessment of the tumor bed. Aided by tumor targeted near-infrared contrast agents, GAINS guided complete tumor resection in subcutaneous, metastatic and spontaneous mouse models of cancer with high sensitivity and specificity, in real-time. Using a clinically-approved near-infrared contrast agent, GAINS provided real-time image guidance for accurate visualization of lymph nodes in a porcine model and sentinel lymph nodes in human breast cancer and melanoma patients with high sensitivity. This work has addressed issues that have limited clinical adoption of fluorescence-guided surgery and paved the way for research into developing this approach towards standard-of-care practice that can potentially improve surgical outcomes in cancer

Aggressive pituitary tumors and pituitary carcinomas: from pathology to treatment

Aggressive pituitary tumors (APTs) and pituitary carcinomas (PCs) are heterogeneous with regard to clinical presentation, proliferative markers, clinical course, and response to therapy. Half of them show an aggressive course only many years after the first apparently benign presentation. APTs and PCs share several properties, but a Ki67 index greater than or equal to 10% and extensive p53 expression are more prevalent in PCs. Mutations in TP53 and ATRX are the most common genetic alterations; their detection might be of value for early identification of aggressiveness. Treatment requires a multimodal approach including surgery, radiotherapy, and drugs. Temozolomide is the recommended first-line chemotherapy, with response rates of about 40%. Immune checkpoint inhibitors have emerged as second-line treatment in PCs, with currently no evidence for a superior effect of dual therapy compared to monotherapy with PD-1 blockers. Bevacizumab has resulted in partial response (PR) in few patients; tyrosine kinase inhibitors and everolimus have generally not been useful. The effect of peptide receptor radionuclide therapy is limited as well. Management of APT/PC is challenging and should be discussed within an expert team with consideration of clinical and pathological findings, age, and general condition of the patient. Considering that APT/PCs are rare, new therapies should preferably be evaluated in shared standardized protocols. Prognostic and predictive markers to guide treatment decisions are needed and are the scope of ongoing research.Metabolic health: pathophysiological trajectories and therap

Liver Biopsy

Liver biopsy is recommended as the gold standard method to determine diagnosis, fibrosis staging, prognosis and therapeutic indications in patients with chronic liver disease. However, liver biopsy is an invasive procedure with a risk of complications which can be serious. This book provides the management of the complications in liver biopsy. Additionally, this book provides also the references for the new technology of liver biopsy including the non-invasive elastography, imaging methods and blood panels which could be the alternatives to liver biopsy. The non-invasive methods, especially the elastography, which is the new procedure in hot topics, which were frequently reported in these years. In this book, the professionals of elastography show the mechanism, availability and how to use this technology in a clinical field of elastography. The comprehension of elastography could be a great help for better dealing and for understanding of liver biopsy