STED microscopy for nanoscale imaging in living brain slices.

Stimulated emission depletion (STED) microscopy was the first fluorescence microscopy technique to break the classic diffraction barrier of light microscopy. Even though STED was conceived more than 20 years ago and acknowledged with the 2014 Nobel Prize in Chemistry, it has not yet been widely adopted in biological research, which stands to benefit enormously from the potent combination of nanoscale spatial resolution and far-field optics. STED microscopy is an ensemble imaging technique that uses a pair of lasers for controlling the excitation state of fluorescent molecules in a targeted manner over nanoscale distances. STED is commonly a point-scanning technique, where the fluorescence spot from the first laser is sharpened by way of stimulated emission induced by the second laser. However, recent developments have extended the concept to multi-point scanning and to additional photophysical switching mechanisms. This review explains the basic principles behind STED microscopy and the differences with other super-resolution techniques. It provides practical information on how to construct and operate a STED microscope that can be used for nanoscale imaging of GFP and its variants in living brain slices. We conclude by highlighting a series of recent technological innovations that are bound to enhance its scope and performance in the near future.