Volumetric super-resolution microscopy approaches for investigating synaptic connectivity in the mammalian visual system

Abstract

Imaging synapses in the brain is difficult due to the diffraction limit of light microscopy, which limits image resolution to ~200nm laterally and ~600nm axially. Super-resolution fluorescence microscopy techniques circumvent this problem, allowing us to visualize subsynaptic molecular interactions. Stochastic Optical Reconstruction Microscopy (STORM) is a single molecule imaging technique that relies on stochastic photoswitching of organic dyes between fluorescent and non-fluorescent to states to produce a resolution of ~20nm laterally and ~50nm axially. Together with ultra-thin serial sectioning, this approach allows for the collection of volumetric super-resolution data. Expansion Microscopy (ExM), on the other hand, is a different super-resolution approach that does not rely on special dyes or instruments. ExM achieves sub-diffraction-limit image resolution by physically expanding the specimen within a swellable polyacrylamide matrix. This technique is exciting in that it offers a simple, fast, and inexpensive method of achieving high image resolution.