Two-photon excitation STED microscopy in two colors in acute brain slices.

Philipp Bethge, Ronan Chéreau, Elena Avignone, Giovanni Marsicano, U. Valentin Nägerl
Biophysical Journal. 2013-02-01; 104(4): 778-785
DOI: 10.1016/j.bpj.2012.12.054

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1. Biophys J. 2013 Feb 19;104(4):778-85. doi: 10.1016/j.bpj.2012.12.054.

Two-photon excitation STED microscopy in two colors in acute brain slices.

Bethge P(1), Chéreau R, Avignone E, Marsicano G, Nägerl UV.

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

Comment in
Biophys J. 2013 Feb 19;104(4):741-3.

Many cellular structures and organelles are too small to be properly resolved by
conventional light microscopy. This is particularly true for dendritic spines and
glial processes, which are very small, dynamic, and embedded in dense tissue,
making it difficult to image them under realistic experimental conditions.
Two-photon microscopy is currently the method of choice for imaging in thick
living tissue preparations, both in acute brain slices and in vivo. However, the
spatial resolution of a two-photon microscope, which is limited to ~350 nm by the
diffraction of light, is not sufficient for resolving many important details of
neural morphology, such as the width of spine necks or thin glial processes.
Recently developed superresolution approaches, such as stimulated emission
depletion microscopy, have set new standards of optical resolution in imaging
living tissue. However, the important goal of superresolution imaging with
significant subdiffraction resolution has not yet been accomplished in acute
brain slices. To overcome this limitation, we have developed a new microscope
based on two-photon excitation and pulsed stimulated emission depletion
microscopy, which provides unprecedented spatial resolution and excellent
experimental access in acute brain slices using a long-working distance
objective. The new microscope improves on the spatial resolution of a regular
two-photon microscope by a factor of four to six, and it is compatible with
time-lapse and simultaneous two-color superresolution imaging in living cells. We
demonstrate the potential of this nanoscopy approach for brain slice physiology
by imaging the morphology of dendritic spines and microglial cells well below the
surface of acute brain slices.

Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights
reserved.

DOI: 10.1016/j.bpj.2012.12.054
PMCID: PMC3576543
PMID: 23442956 [Indexed for MEDLINE]


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