Stimulated Emission Depletion (STED) Imaging of Dendritic Spines in Living Hippocampal Slices
Cold Spring Harb Protoc. 2012-05-01; 2012(5): pdb.prot069260
DOI: 10.1101/pdb.prot069260
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1. Cold Spring Harb Protoc. 2012 May 1;2012(5). pii: pdb.prot069260. doi:
10.1101/pdb.prot069260.
Stimulated emission depletion (STED) imaging of dendritic spines in living
hippocampal slices.
Willig KI, Nägerl UV.
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.
DOI: 10.1101/pdb.prot069260
PMID: 22550296 [Indexed for MEDLINE]