Location & dates EMBL Heidelberg, Germany 3 - 6 Oct 2013
Deadlines Registration closed Abstract submission closed
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Leica

Venue: EMBL Advanced Training Centre, Flex Lab A & B

Workshop Session 1: 11:30 – 12:30

GSDIM Super-resolution microscopy in 3D

Tamara Straube, Leica Microsystems, Wetzlar

In the past, light microscopy was restricted by the diffraction limit of light.  Using different localization microscopy techniques imaging beyond the diffraction limit has become possible.

The Leica SR GSD 3D enables super-resolution visualization of molecules and cellular structures beyond the diffraction limit in three dimensions. Based on the localization technology of GSDIM (Ground state depletion followed by individual molecule return) and dSTORM (stochastical optical reconstruction microscopy), individual well separated molecules are temporarily imaged and their localization in three dimensions is determined with nanometer precision (Foelling et al., Nature Methods, 2008). The super-resolution image is reconstructed from the position information of the fluorophores. The localization in z is generated by implementation of an astigmatism using a manipulated PSF.

With this localization-technology, a resolution down to approx. 20 nm in x- and y-dimension and about 70 nm in z-dimension can be achieved.

GSDIM is using standard organic fluorophores or standard fluorescent proteins such as Rhodamine dyes, Alexa and Atto dyes or eYFP,. The sample preparation is based on already established standard protocols.

This workshop will present the basic principles of the SR GSD 3D technology, as well as an overview of possible applications. A recent commercial implementation of the astigmatism approach will be discussed in more detail and used to highlight the special features and requirements of this approach.

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Workshop Session 2: 13:00 – 14:00

SHARPER IMAGES AT LOWER POWER: Gated STED: The Next Milestone in Confocal Super-Resolution

Ulf Schwarz, Leica Microsystems, Mannheim

Leica Microsystems now presents the next milestone in confocal super-resolution.

Leica gated STED is an option for STED CW based on Leica Microsystems NEW highly versatile core confocal: The Leica TCS SP8.

Stimulated emission depletion (STED) microscopy [1] provides fast and easy access to resolutions far beyond the Abbe limit. Gated STED is a new further development of STED microscopy [2]. It combines STED with CW lasers and time gating detection, to significantly increase resolution and photo stability.

During the last few years, STED microscopy has proven to be an excellent super-resolution technology to study nanostructures inside cells and tissues and has generated spectacular results.

STED CW provides easy and intuitive access to dual color super-resolution as well as live cell imaging. It realizes confocal nanoscopy in the visible and allows selection from a wide range of fluorophores. Standard green dyes such as Alexa 488, FITC and Oregon Green 488 can be applied just as easily as fluorescent proteins such as eYFP, Venus and Citrin and many more.

Gated STED maintains all these advantages and resolves details smaller than 50nm. Furthermore, gated STED enables increased image acquisition and drives live cell super-resolution studies to the next level. Impressive results have been obtained even with eGFP.

In this workshop, the principles of gated STED imaging along with the commercial implementation are presented. An overview of the application portfolio, ranging from super-resolution inside live cells to colocalization studies at the nanoscale will be given.

[1] S.W. Hell SW, J. Wichmann, “Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy”, Opt Lett., 19, 780-2 (1994).
[2] G. Vicidomini , G. Moneron, K.Y. Han, V. Westphal , H. Ta, M. Reuss, J. Engelhardt, C. Eggeling, S.W. Hell, “Sharper low-power STED nanoscopy by time gating”, Nat Methods, 8(7), 571-3 (2011).