Expansion microscopy is an increasingly widespread technology for nanoimaging, because its precise physical magnification of biological specimens enables ordinary microscopes to achieve nanoscale effective resolutions. Fluorescent labels such as antibodies can be applied either before or after expansion, with the latter offering the potential for better access to proteins within densely packed environments. We here assess this possibility of epitope decrowding through physical expansion of proteins away from each other, using a 20x expansion protocol that we call expansion revealing (ExR), by labeling, within intact brain circuits, the same set of synaptic proteins both pre- and post-expansion. This comparison shows that post-expansion labeling introduces minimal spatial error and off-target staining relative to pre-expansion staining, while revealing the presence of proteins that are invisible when stained pre-expansion. Using ExR, we show in intact brain tissue the alignment of presynaptic calcium channels with postsynaptic machinery in nanocolumns, which may facilitate precision synaptic transmission, as well as the existence of periodic amyloid-containing nanoclusters containing ion channel proteins in Alzheimer’s model mice, which may help generate novel hypotheses for Alzheimer’s pathology and neural excitability. Thus, the decrowding power of ExR is able to reveal novel nanostructures within intact brain circuitry, and may find broad use in biology and medicine for unmasking nanostructures of importance in normal functions and disease.

Resources associated with this Publication:
[Expansion microscopy: physical magnification with nanoscale precision]