Advances In FIB-SEM Nanotomography of Biological Systems

Abstract

FIB-SEM nanotomography is a powerful 3D nanoscale characterization tool that performs iterative ion milling and electron imaging to produce high-resolution and large volume imaging. However, for biological samples, FIB-SEM nanotomography requires development and optimization to mitigate artifacts, drift and misalignments to ensure accurate volumetric data acquisitions. The research within this thesis seeks to optimize, analyze and evaluate the use of FIB-SEM nanotomography in biological systems consisting of bone, fossilized bone and bone cells. Bone tissue has a hierarchical organization with distinct hard and soft components at the macro- to nanoscale level. The fundamental nanoscale features of bone, mineral ellipsoids and collagen fibrils constitute the building blocks of bone, and how these components organize and change during external processes, including fossilization, is of high interest. FIB-SEM was optimized to visualize the organization of ellipsoidal mineral clusters and collagen fibrils within highly mineralized human bone tissue, where the twisted plywood organization of mineral ellipsoids was unveiled. Similarly, fossilized Albertosaurus sarcophagus bone tissue was imaged using FIB-SEM to analyze the microstructural organization of the bone, which revealed preserved mineral and organic fundamental components but also diagenetic changes in the specimen. Lastly, FIB-SEM was evaluated with a created semiconductor and Saos-2 (osteosarcoma) cell sample. Through post-acquisition analysis, deviations and errors were identified and corrected, thereby providing a customizable and biologically relevant standard that can be used for 3D FIB-SEM data validation. This thesis advances the utilization of FIB-SEM nanotomography as a reliable nanoscale characterization tool for bone imaging by providing in-depth details on the structural organization of key bone components and insight into the FIB-SEM acquisition behaviour during biological acquisitions. These insights advance the broader bioimaging field, where enhanced nanometer-scale 3D imaging over large volumes provides greater understanding of biological building blocks.