The postsynaptic site of excitatory synapses is characterized by an electron-dense region called postsynaptic density (PSD), which is usually located at the tip of dendritic spines. The PSD has been shown to undergo morphological changes in response to behavioral and electrophysiological inputs. This process can be highly dynamic and occurs over different time scales. To bridge the observation gap between short-term plastic events (ranging from minutes to hours) and long-term effects (several hours to days), we engineered a novel on stage microscope-incubator optimized for long-term culturing and two-photon imaging of organotypic hippocampal slice cultures. Using this setup, we measured the size of endogenous PSDs with fluorescently labeled intrabodies in CA1 pyramidal neurons.To analyze the dynamics and interactions of all labeled PSDs simultaneously, we developed a novel automated image analysis workflow, to first process and optimize large 3D multi-view image stacks for subsequent automated PSD detection based on deep learning and tracking over time.Our initial results show that this refined method allows to perform a detailed analysis of neighboring PSD dynamics and their localization along the dendritic arbor as well as large-scale analysis with high temporal and spatial resolution. As expected, increasing or decreasing the overall activity level in the hippocampal slice culture leads to homeostatic changes in the average size of the PSDs. Moreover, this approach can be easily combined with (opto-)genetic and/or pharmacological manipulations.