Two-Photon Excitation STED Microscopy for Imaging Synapses and Glia in Acute Brain Slices

Abstract

STED (stimulated emission depletion) microscopy is a novel fluorescence microscopy technique that provides unprecedented access to dynamic processes of living cells with a spatial resolution well below 100 nm, even down to a few nanometers. By breaking the classical diffraction barrier of optical microscopy, it offers the chance to close the gap in spatial resolution between electron microscopy and conventional light microscopy and is attracting attention as a potential game-changing imaging technology.

The first-generation STED microscopes typically suffered from significant disadvantages over confocal or two-photon microscopy, regarding labeling, live-cell compatibility, color contrast, and depth penetration. However, due to several recent technical developments, these teething problems have been largely overcome. Similar to confocal microscopy, STED microscopy can be readily combined with a powerful array of complementary techniques, such as photo-uncaging, fluorescence recovery after photobleaching (FRAP), and patch-clamp electrophysiology, giving experimental access to a rich set of molecular, structural, and functional data.

Here, we present a primer on two-photon excitation STED microscopy, its basic principles and instrumentation requirements, providing a detailed how-to guide for the construction and operation of a STED microscope for two-color nanoscale imaging of neural morphology deep inside living brain slices.