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Electron cryo-tomography (cET), in combination with sub‐volume averaging (SVA), is a powerful technique for determining the structure of complex biological specimens that are not amenable to more traditional structure determination methods. In Chapter 1, SVA was used to achieve a sub-nanometre resolution structure of Herpes simplex virus 1 glycoprotein B in its pre-fusion conformation. Nevertheless, several challenges still undermine the routine use of cET for structural cell biology. Imaging with an electron beam damages specimens, leading to localised warping of tomograms and reduced resolution of SVA maps if not compensated for. Using densities of biological structures within tomograms as fiducial markers is one solution to this problem. A software implementation of this concept, termed ‘Flexo’ and its application is demonstrated in Chapter 2. While this improves tomogram quality, other issues persist. The lack of a specific labelling technique for cET often limits the use of SVA to large and recognisable structures; correlation with super‐resolution cryo-fluorescence microscopy (cryo‐FM) is a promising solution. The primary drawback of this technique has been devitrification induced by the high laser intensities typically required. Chapter 3 describes Cryo-SOFI as a general solution to this problem, enabling substantial resolution improvement in cryo‐FM, and allowing cET to be performed in a correlative fashion. Chapter 4, explores the avenues of combining live-cell FM imaging with cET to study the transport of virus particles along cellular protrusions, called virus surfing. Events of surfing and static viruses can look alike in cET snapshots, making it necessary to link these high-resolution observations with their dynamic history. This led to the development of techniques towards live-CLEM: correlative live‐cell light and electron microscopy, which also revealed that cells undergo major structural changes during the preparation for cryogenic preservation. Overall, the work presented in this thesis presents a number of promising solutions to overcome some major obstacles for cET to fulfil its full potential for in situ structural cell biology.

In this thesis, I detail the use of various forms of correlative microscopy to investigate reorganization of mitochondrial architecture during two processes, apoptosis and mitophagy, in an effort to better understand the contribution of membrane remodeling to these biological processes. During apoptosis, Bax/Bak oligomerize and accumulate in and adjacent to the outer mitochondrial membrane, resulting in outer membrane ruptures several hundred nanometers wide. Using correlative microscopy, it was found that Bax clusters accumulated next to outer membrane ruptures and appeared to be amorphous. Electron cryo-tomography of vitreous cells was used to discern a higher-order organization to cluster components. In these near- native conditions, Bax clusters appeared to contain a sponge-like meshwork, suggesting that components of the outer membrane may be bound by Bax and then sequestered in the cytosol. Mitochondria with outer membrane ruptures presented a diverse array of inner membrane morphologies, such as dilution of the matrix and cristae unfolding. This unfolding was found to be linked to a loss of typical ATP synthase dimer organization. A new, comprehensive model of Bax-mediated apoptosis is presented, linking molecular reorganizations of the inner membrane with sequestration of outer membrane components into Bax clusters in an interplay to drive rupture formation. In mitophagy, the PINK1 kinase is stabilized on the outer mitochondrial membrane, where it recruits Parkin. Parkin then ubiquitinates a host of outer membrane proteins, targeting a mitochondrion for autophagy. While most literature details a wholesale removal of all mitochondria from a cell, recent work describes a localized form of mitophagy that removes selective parts of the network. Mitochondria targeted by Parkin during localized mitophagy were observed, and this process was compared between cells intact for Drp1, the mitochondrial fission protein, and those with Drp1 knocked out. Fluorescently-tagged Parkin was found on mitochondrial fragments, mitochondria-containing autophagosomes, and mitochondria-containing lysosomes. In both lines, the fragmentation that led to isolation of a mitochondrion targeted for degradation occurred prior to the recruitment of autophagic machinery. In some cases, mitochondria surrounded by multiple autophagosomes were observed. These results reveal new details on the tight link between mitochondrial membrane dynamics and the functional state of the mitochondrion. Overall, I present a thesis that describes the insights into mitochondrial biology gained from various correlative microscopy approaches.

The ‘missing wedge’ is a long-standing problem in electron tomography, caused by the use of slab-like flat specimens, which increase in thickness when tilted to high angles. Attempts have been made to reduce the undesirable effects caused by the missing wedge, but the problem remains, particularly for the radiation-sensitive frozen-hydrated specimens used for high resolution imaging. Specimens with cylindrical symmetry offer a way to overcome this problem, since the thickness remains constant at all viewing angles. However, while this has been suggested before, it has never been demonstrated for frozen-hydrated specimens. In this work, I present a way to make cylindrical specimens for electron cryo-tomography, using thin-walled carbon tubes as specimen holders. The tubes are made in a multi-step process, involving carbon deposition on glass micropipette templates and subsequent removal of the glass. Tube diameters are typically a few hundred nanometres, with a wall thickness of 10–20 nm. To make frozen-hydrated specimens, the tubes are filled with an aqueous sample and then plunge-frozen in liquid ethane. Electron images acquired from the tubes have equal quality at all viewing angles, with a tilt range restricted only by the physical limits of the microscope. Tomograms from specimens such as gold particles and liposomes show that the effects of the missing wedge are substantially reduced, with much improved resolution along the electron beam axis. Structural features oriented in all directions are visible in the reconstructions, in marked contrast to tomograms acquired over a more restricted angular range. These results are promising, however some technical challenges remain before this method can be used routinely.