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