Development of Nanoscale Imaging Tools for Neural Synapse Investigation

One of the biggest unsolved questions in neuroscience is how molecules and neuronal circuitry create behaviors, and how their misregulation or dysfunction results in neurological disease. Light microscopy is a vital tool for the study of neural molecules and circuits. However, the fundamental optical diffraction limit precludes the use of conventional light microscopy for sufficient characterization of critical signaling compartments and nanoscopic organizations of synapse-associated molecules. Expansion microscopy (ExM) enables nanoimaging with conventional microscopes by physically and isotropically magnifying preserved biological specimens embedded in a cross-linked water-swellable hydrogel. We describe a new ExM advancement called Magnify, which uses a mechanically sturdy gel that retains nucleic acids, proteins, and lipids without the need for a separate anchoring step. Magnify expands biological specimens up to 11× and facilitates imaging of cells and tissues with effectively ~25-nm-resolution, providing insight into nanoscopic subcellular structures including synaptic proteins from mouse brain, podocyte foot processes in formalin-fixed paraffin-embedded human kidney, and defects in cilia and basal bodies in drug-treated human lung organoids. Finally, we will discuss ongoing work towards easing constraints on protein multiplex imaging in expanded samples by restricting gel movement between imaging rounds