3D STED Super-Resolution Imaging Strategy for Visualizing Synaptic Nano-architecture in Brain Cryosections

Super-resolution imaging of synapses in intact brain tissue remains challenging because light scattering, photobleaching, and limited probe penetration, along with antigen accessibility within the densely packed postsynaptic densities (PSDs), constrain resolution and labeling efficiency. Here, we present a protocol utilizing thin brain cryosections and tau-stimulated emission depletion (STED) nanoscopy to visualize the intricate nano-architecture of excitatory synapses in situ. Slicing the brain into 6 μm sections allows for highly efficient and even penetration of probes throughout sections while ensuring that the resolution is not significantly impacted by the imaging depth of the tissue. We outline step-by-step instructions for labeling pre- and postsynaptic nano-architecture using antibodies and nanobodies, highlighting how fixative choice influences the labeling efficiency of synaptic proteins. While this protocol is compatible with both confocal and super-resolution imaging, when combined with rapid image acquisition times of tau-STED, it enables clear separation of key synaptic features in three dimensions with minimal photobleaching. Thus, this approach enables robust multiplex imaging of fluorescently labeled synaptic proteins in the brain, providing exceptional spatial resolution for visualization and quantification of synaptic nanoarchitecture in its native environment. Key features • Detailed protocol for in situ 3D STED microscopy with ~50 nm XY and ~100 nm Z resolution. • Optimized strategies for labeling pre- and postsynaptic nano-architecture using antibodies and nanobodies, including guidance on fixative choice. • Unified workflow for visualizing synaptic morphology and nanoarchitecture to uncover molecular synaptic diversity in the brain at the nanoscale.