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 conventional confocal microscopy, hindering live studies. Here, we describe how to track individual spinules in live dissociated cortical pyramidal neurons utilizing fluorescence labeling, optimized confocal imaging parameters, and post-acquisition iterative 3D deconvolution, employing NIS Elements software. This approach enables investigations of spinule structural dynamics and function without using super-resolution 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).