细胞神经生物学受益于低温电子断层摄影术(cryo-ET)领域的最新进展。从电子显微镜网格上培养的冷冻原代神经元获得了许多结构和超微结构的见解。大多数初级神经元都来自啮齿动物,我们试图通过使用来自3龄果蝇幼虫大脑的原代神经元来扩大样本可用性的广度。在建立这个冷冻ET模型系统时遇到了超微结构异常,例如过度的膜起泡和细胞破碎。为了优化神经元样本,我们整合了基质选择,微图案化,蒙太奇数据收集,和化学固定。为细胞神经生物学中的未来冷冻-ET研究解决建立果蝇神经元的困难的努力也提供了未来从业者在尝试建立其他基于细胞的模型系统时可以使用的见解。
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.