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

Abstract

The 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.