Optimizing environmental scanning electron microscopy of spheroidal reaggregated neuronal cultures

Electrophysiological recordings from hen embryo brain spheroidal reaggregates on penetrating 3D multielectrode arrays could be understood more easily if the surface structure was known in more detail. Electrophysiological activity, as grouped spikes in trains, is acquired from spheroids, indicating the inner formation a neuronal network. To this end, spheroids can be observed by environmental scanning electron microscopy. Live spheroids collapse when the supporting water is evaporated. By careful adjustment of the chamber pressure it is possible to observe fully hydrated fixed spheroids. A thin film of water tends to prevent a clear view of the surface detail. This can be evaporated to reveal the surface while taking steps to avoid both inadvertent shrinkage and rewetting. Conventional SEM shows a very different surface that is rich in protruding cell bodies and fibers. The images are compared and interpreted with some images of the surface using transmission electron microscopy.