Probing the Unseen Depths of the Hepatic Microarchitecture via Multimodal Microscopy

Abstract

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