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Valentin Nägerl

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

Le 14 février 2013

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

Philipp Bethge1,2, Ronan Chéreau 1,2, Elena Avignone1,2, Giovanni Marsicano3 and U. Valentin Nägerl1,2 1 Univ. Bordeaux, Interdisciplinary Institute for Neuroscience, UMR 5297, Bordeaux, France 2 CNRS, Interdisciplinary Institute for Neuroscience, UMR 5297, Bordeaux, France 3Univ. Bordeaux, NeuroCentre INSERM U862, Bordeaux, France
Biophysical Journal,  Volume 104, Issue 4, 778-785, 19 February 2013

ABSTRACT

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, including acute brain slices and in vivo. However, the spatial resolution of a two-photon microscope, which is limited to about 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 STED microscopy, have set new standards of optical resolution in imaging living tissue. However, the important goal of superresolution imaging with significant sub-diffraction 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 STED 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 new approach for brain slice physiology by imaging the morphology of dendritic spines and microglia cells well below the surface of acute brain slices.

 

Correspondence: U. Valentin Nägerl, E-mail: valentin.nagerl @ u-bordeaux2.fr

+ See Superresolving dendritic spines by L.M Loew and S.W. Hell, Biophys J: here

+ Link to the publisher: here

Valentin Nägerl


Professeur - PhD, Université Bordeaux 2. Responsable de l'équipe - Plasticité synaptique et microscopie à superrésolution. Institut interdisciplinaire de Neurosciences (IINS) CNRS UMR 5297.  Lien Equipe