Use of Serial Block Face-Scanning Electron Microscopy to Study the Ultrastructure of Vertebrate and Invertebrate Biology

PhD ThesisThe development of Serial Block Face Scanning Electron Microscopy (SBF-SEM) allows for acquisition of serially sectioned, imaged data of ultrastructure at high resolution. In this project, optimisation of both SBF-SEM methodology and 3-D image segmentation analysis was applied to the ultrastructural examination of two types of biological tissues, each requiring a different experimental approach. The first project was a connectomic based study, to determine the relationship between the neurons that synapse upon the Lobula Giant Movement Detector 2 (LGMD 2) neuron, within the optic lobe of the locust. A substantial portion of the LGMD 2 neuron was reconstructed along with the afferent neurons, enabling the discovery of retinotopic mapping from the photoreceptors of the eye onto the LGMD 2 neuron. A sub-class of afferent neurons was also found, most likely vital in the process of signal integration across the large LGMD 2 neuron. For the second project, two types of skeletal muscle (psoas and soleus) obtained from fetal and adult guinea pigs were analysed to assess tissue-specific changes in mitochondrial morphology with muscle maturation. Distinct mitochondrial shapes were found across both muscles and age groups and a classification system was developed. It was found that, in both muscles, by late fetal gestation the mitochondrial network is well developed and akin to that found in the adult. Quantitative and qualitative differences in mitochondria morphology and complexity were found between the two muscles in the adult group. These differences are likely to be related to functional specialisation. All data collected during the experiments have also been made available online on Zenodo, roughly 240GB, which can be used for further studies. Overall SBF-SEM was proven to be a robust method of gaining new insights into the ultrastructure in both models and has wide ranging capabilities for a variety of experimental objectives

Investigation of conserved Flagellum proteins in Trypanosoma brucei

The single celled protozoan parasite Trypanosoma brucei is an excellent model organism to study eukaryotic cilia and flagella as it has a single flagellum that remains assembled throughout the cell cycle. The new flagellum assembles in a known position relative to the old flagellum, therefore creating a model system of identifiable organelle generations. In additional to a sequenced genome, there are many reverse genetics tools developed for T. brucei which makes the functional analysis of proteins possible. More than 300 proteins have been identified as components of the T. brucei flagellum but functional analysis of the majority of these proteins has not been carried out to date. This project used a bioinformatics approach to identify potential flagellum proteins in T. brucei that were also conserved in Homo sapiens, thereby identifying potential ciliopathy candidates. Candidate proteins were confirmed as flagellum components through endogenous localisation techniques and co-localisation studies. Functional analysis was performed using inducible RNAi cell lines. Light and electron microscopy techniques were used for phenotypic analysis. Through bioinformatics analysis a novel family of coiled-coil TPH domain-containing proteins were identified that are highly conserved in flagellated eukaryotes. There are three TPH domain-containing proteins conserved in T. brucei that all have a role in flagellum length control and cell morphogenesis. In all three cases protein ablation has a detrimental effect on cellular motility. This work provides further understanding into the complexities of flagellum biogenesis in T. brucei and the downstream effects on cell motility and morphogenesis

MC 2019 Berlin Microscopy Conference - Abstracts

Das Dokument enthält die Kurzfassungen der Beiträge aller Teilnehmer an der Mikroskopiekonferenz "MC 2019", die vom 01. bis 05.09.2019, in Berlin stattfand