Abstract
The confluence of innovations in transgenic labeling and light microscopy techniques in recent years has greatly advanced our understanding of dynamic cell biological events underlying neuronal function and plasticity. Increasingly, it has become possible to perform fundamental experiments inside the relevant subcellular compartments of a neuron embedded in three-dimensional living tissues. Overcoming the limiting role of diffraction in far-field light microscopy, nanoscopy is advancing our ability to see and manipulate cellular events well below the diffraction barrier of ∼200 nm. The first concrete and implemented concept of nanoscopy was STED (stimulated emission depletion) microscopy. This article gives an example of the power that STED microscopy holds for neuroscience research. It provides a method for live-cell time-lapse imaging of the dynamic morphology of dendritic spines of pyramidal neurons. Imaging is performed in an organotypic hippocampal slice culture system, with yellow fluorescent protein (YFP) used as a volume marker for the synaptic structures. In addition, the article describes the basic elements needed to assemble a custom-built STED microscope capable of live cell imaging and how to use it for physiology experiments.
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