Two-Color STED Microscopy of Living Synapses Using A Single Laser-Beam Pair

Jan Tønnesen, Fabien Nadrigny, Katrin I. Willig, Roland Wedlich-Söldner, U. Valentin Nägerl
Biophysical Journal. 2011-11-01; 101(10): 2545-2552
DOI: 10.1016/j.bpj.2011.10.011

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1. Biophys J. 2011 Nov 16;101(10):2545-52. doi: 10.1016/j.bpj.2011.10.011. Epub 2011
Nov 15.

Two-color STED microscopy of living synapses using a single laser-beam pair.

Tønnesen J(1), Nadrigny F, Willig KI, Wedlich-Söldner R, Nägerl UV.

Author information:
(1)Interdisciplinary Institute for Neuroscience, Université de Bordeaux,
Bordeaux, France.

The advent of superresolution microscopy has opened up new research opportunities
into dynamic processes at the nanoscale inside living biological specimens. This
is particularly true for synapses, which are very small, highly dynamic, and
embedded in brain tissue. Stimulated emission depletion (STED) microscopy, a
recently developed laser-scanning technique, has been shown to be well suited for
imaging living synapses in brain slices using yellow fluorescent protein as a
single label. However, it would be highly desirable to be able to image
presynaptic boutons and postsynaptic spines, which together form synapses, using
two different fluorophores. As STED microscopy uses separate laser beams for
fluorescence excitation and quenching, incorporation of multicolor imaging for
STED is more difficult than for conventional light microscopy. Although two-color
schemes exist for STED microscopy, these approaches have several drawbacks due to
their complexity, cost, and incompatibility with common labeling strategies and
fluorophores. Therefore, we set out to develop a straightforward method for
two-color STED microscopy that permits the use of popular green-yellow
fluorescent labels such as green fluorescent protein, yellow fluorescent protein,
Alexa Fluor 488, and calcein green. Our new (to our knowledge) method is based on
a single-excitation/STED laser-beam pair to simultaneously excite and quench
pairs of these fluorophores, whose signals can be separated by spectral detection
and linear unmixing. We illustrate the potential of this approach by two-color
superresolution time-lapse imaging of axonal boutons and dendritic spines in
living organotypic brain slices.

Copyright © 2011 Biophysical Society. Published by Elsevier Inc. All rights

DOI: 10.1016/j.bpj.2011.10.011
PMCID: PMC3218326
PMID: 22098754 [Indexed for MEDLINE]

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