STED microscopy for nanoscale imaging in living brain slices.
Methods. 2015-10-01; 88: 57-66
DOI: 10.1016/j.ymeth.2015.06.006
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1. Methods. 2015 Oct 15;88:57-66. doi: 10.1016/j.ymeth.2015.06.006. Epub 2015 Jun 9.
STED microscopy for nanoscale imaging in living brain slices.
Chéreau R(1), Tønnesen J(1), Nägerl UV(2).
Author information:
(1)Interdisciplinary Institute for Neuroscience, Université de Bordeaux,
Bordeaux, France; UMR 5297, Centre National de la Recherche Scientifique,
Bordeaux, France.
(2)Interdisciplinary Institute for Neuroscience, Université de Bordeaux,
Bordeaux, France; UMR 5297, Centre National de la Recherche Scientifique,
Bordeaux, France. Electronic address: .
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.
Copyright © 2015 Elsevier Inc. All rights reserved.
DOI: 10.1016/j.ymeth.2015.06.006
PMID: 26070997 [Indexed for MEDLINE]