Handling Difficult Cryo-ET Samples: A Study with Primary Neurons from Drosophila melanogaster

Cellular neurobiology has benefited from recent advances in the field of cryo-electron tomography (cryo-ET). Numerous structural and ultrastructural insights have been obtained from plunge-frozen primary neurons cultured on electron microscopy grids. With most primary neurons having been derived from rodent sources, we sought to expand the breadth of sample availability by using primary neurons derived from 3rd instar Drosophila melanogaster larval brains. Ultrastructural abnormalities were encountered while establishing this model system for cryo-ET, which were exemplified by excessive membrane blebbing and cellular fragmentation. To optimize neuronal samples, we integrated substrate selection, micropatterning, montage data collection, and chemical fixation. Efforts to address difficulties in establishing Drosophila neurons for future cryo-ET studies in cellular neurobiology also provided insights that future practitioners can use when attempting to establish other cell-based model systems.

Automated summary

Cellular neurobiology has made significant progress in understanding structural and ultrastructural aspects through cryo-electron tomography. However, establishing a Drosophila neuronal model for cryo-ET encountered ultrastructural abnormalities. Through optimization of sample preparation and addressing difficulties, valuable insights were gained for future studies in establishing other cell-based model systems.