Imaging neuronal structure dynamics using 2‐photon super‐resolution patterned excitation reconstruction microscopy

: Visualizing fine neuronal structures deep inside strongly light-scattering brain tissue remains a challenge in neuroscience. Recent nanoscopy techniques have reached the necessary resolution but often suffer from limited imaging depth, long imaging time or high light fluence requirements. Here, we present two-photon super-resolution patterned excitation reconstruction (2P-SuPER) microscopy for 3-dimensional imaging of dendritic spine dynamics at a maximum demonstrated imaging depth of 130 μm in living brain tissue with approximately 100 nm spatial resolution. We confirmed 2P-SuPER resolution using fluorescence nanoparticle and quantum dot phantoms and imaged spiny neurons in acute brain slices. We induced hippocampal plasticity and showed that 2P-SuPER can resolve increases in dendritic spine head sizes on CA1 pyramidal neurons following theta-burst stimulation of Schaffer collateral axons. 2P-SuPER further revealed nanoscopic increases in dendritic spine neck widths, a feature of synaptic plasticity that has not been thoroughly investigated due to the combined limit of resolution and penetration depth in existing imaging technologies.