I will present Expansion Microscopy (ExM), a method in which the optical diffraction limit is circumvented by physically expanding a biological specimen prior to imaging. Expansion brings sub-diffraction limited structures into the size range viewable by a conventional diffraction-limited microscope. In ExM, proteins and visible probes are chemically anchored to an in situ-synthesized swellable polyelectrolyte gel. Proteolytic digestion is used to disrupt native tissue structures and enable uniform, 4-fold expansion of the material with anchored probes.
Known sub-diffraction limited structures are shown to have the expected shape and size, demonstrating that the expansion is isotropic down to the theoretically resolvable size scale of 70nm (pre-expansion). Optical scattering is dramatically reduced, allowing this resolution to be achieved throughout the depth of the specimen, limited only by objective lens working distance and the diffusion of the gel precursor. ExM is compatible with any optical microscope, and is simple to adopt into existing experimental workflows. ExM promises to be a powerful method for imaging neural circuits at sub-synaptic resolution, in addition to other types of biological specimens.
Thesis Supervisor: Professor Ed Boyden