Protocol for live enhanced resolution confocal imaging of dendritic spinule dynamics in primary mouse cortical neuron culture

Dendritic spinules are fine membranous protrusions of neuronal spines that play a role in synaptic plasticity, but their nanoscale requires resolution beyond conven-tional confocal microscopy, hindering live studies. Here, we describe how to track individual spinules in live dissociated cortical pyramidal neurons utilizing fluores-cence labeling, optimized confocal imaging parameters, and post-acquisition itera-tive 3D deconvolution, employing NIS Elements software. This approach enables investigations of spinule structural dynamics and function without using super -reso-lution microscopy, which involves special fluorophores and/or high laser power. For complete details on the use and execution of this protocol, please refer to Zaccard et al. (2020).