Two-color STED imaging of synapses in living brain slices

STED microscopy is a novel fluorescence microscopy technique that breaks the classic diffraction barrier of optical microscopy. It offers the chance to investigate dynamic processes inside living cells with a spatial resolution well below 100 nm, possibly even down to a few nanometers, essentially without forgoing the benefits of conventional light microscopy, such as labeling specificity, sensitivity, and contrast. STED microscopy has already been exploited for several important neurobiological experiments. Given the tremendous potential as a transforming technology, it is important to understand how it works, and what its scope and limitations are. Here, we present a primer on STED microscopy, its basic principles and practical implementation, presenting a how-to guide on building and operating a STED microscope.