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We observed neurons cultivated on an indium tin oxide (ITO) substrate using scanning electron microscopy (SEM) whose high spatial resolution allows us to observe neuronal morphological details. First, we optimized the fixation condition of cultivated neurons for SEM observation. The first fixation with paraformaldehyde and glutaraldehyde, and the second fixation with OsO4 were both necessary to avoid cell removal during the preparation before SEM observation. After optimization, we examined the morphological changes of neurons under an apoptotic condition, induced by staurosporine, by using both SEM and an immunochemical technique. Interestingly, the addition of staurosporine induced both apoptosis and a necrotic phenomenon. Immunostaining analysis revealed late-stage apoptosis after early-stage apoptosis, which was observed induced in cortical neurons by staurosporine for the first time. We confirmed that the SEM imaging of neurons is very useful as regards observing the apoptotic process. It is also a promising tool for understanding such neuronal activities as synaptic formation and axonal growth. Further examination of the interaction between neurons and substrates will contribute to the implementation of the artificial neurological devices. [DOI: 10.1380/ejssnt.2014.179]

Observation of Neuronal Death <i>In Vitro </i>by SEM and Optical Microscopy