[
International Worm Meeting,
2019]
Electron microscopy (EM) was used to acquire the only organism-wide dataset of neuronal connectivity in 1986. This singular dataset from C. elegans provides an invaluable resource but is unfortunately incomplete, missing a portion of the ventral nerve cord posterior to the vulva. Although EM allows for high resolution there are significant shortcomings to its use, including a small field of view, inability to fluorescently tag proteins, and production of very large datasets. This makes imaging large specimen challenging. We are adapting a novel technique known as Protein Retention Expansion Microscopy (ProExM; Nature Biotechnology (2016) 34, 987-992) to collect neuronal connectivity data from C. elegans. ProExM achieves resolution beyond the diffraction limit by physically expanding biological specimen fivefold linearly, and with minimal spatial distortion. This allows for confocal microscopy imaging of immuno-fluorescently tagged cellular components which would otherwise be irresolvable. To resolve single synapses, we generated transgenic strains that express epitope-tags conjugated to proteins that are localized at synapses and the neuronal membrane. Optimizing ProExM of tags to specific neuronal targets may reveal missing data in the C. elegans connectome and provide a new technique to gather this data in other specimens. Moreover, we intend to produce multiple samples that will allow us to assess the distribution of variability of neuronal connectivity among nearly isogenic animals.