Multiplexed dSTORM Imaging of Neuronal Tissue and Cells

The nervous system consists of neurons interconnected by densely packed synapses, which are composed of distinct sets of synaptic proteins mediating the highly demanding processes of synaptic transmission. Most classical single-molecule localization microscopy (SMLM) methods allow the visualization and identification of molecular nanostructures at approximately 20–30 nm lateral and 60 nm axial resolution but are often limited to simultaneous acquisition of only two labeled proteins. However, to visualize and understand the nano-architecture of complex protein machineries, such as the presynaptic active zone or postsynaptic density, many proteins need to be identified in the same sample at the highest possible resolution. Such highly multiplexed imaging experiments will help identify geometries of protein composition that may finally drive function. To achieve this, we developed maS3TORM (multiplexed automated serial staining stochastic optical reconstruction microscopy), a microscopy approach allowing fully automated re-staining of the sample directly on the microscope by combining 3D dual-channel dSTORM imaging with automated solution exchange using a pipetting robot. Using maS3TORM, we can successfully identify more than 15 targets within the same presynaptic terminal, revealing protein distributions at a new level. Hence, this technique can be used to explore the nano-architecture of synapses or any other cellular protein machines and thereby help gain a better understanding of how complex macromolecular assemblies may drive function.