Localization Microscopy Using GFP

Scientists at Heidelberg University, Germany have developed a new technique for localization microscopy, the “spectral precision distance microscopy” (SPDM). Using visible light, this method allows a single molecule resolution of celullar structures down to the range of few nanometer, about 20 times better than the conventional optical resolution. The researchers invented a new instrument which is a combination of the world’s fastest nano light microscope for 3D cell analysis and the new SPDM technique. Prof. Christoph Cremer of the Kirchhoff Institute of Physics and his team were able to show that SPDM can be realized by common fluorescent dyes, such as the green fluorescent protein (GFP) which can be switched on and off by means of light, as long as certain photophysical conditions are fulfilled. This can be achieved via the so-called “reversible photobleaching” of the dye. So far, only special fluorescent dyes could be used as temporally convertible light signals. According to Cremer there are millions of specimens containing gene constructs with dyes from the GFP group available in biomedical laboratories all over the world. They could be put into immediate use for this new kind of localization microscopy.
www.uni-heidelberg.de

Molecular Light Switches for Higher Resolution

The “Superresolution” research network, founded by the German Ministry of Education and Sciences, demonstrated a new widefield microscopy technology with resolutions better than 20 nanometers. The method is based on special dyes, which’s fluorescence can be optically and reversibly switched on and off in aqueous solutions. The dyes are bond to cellular structures by using a functional group. By switching the dyes on and off, the fluorescence emission is separated in time until only those dye molecules fluoresce that have enough distance to allow their localization as single molecules. After several thousand switching cycles, a total image is constructed (dSTORM – direct stochastic optical reconstruction microscopy). Involved in the project were the work groups of Prof. Dr. M. Sauer and Prof. Dr. J. Mattay (University of Bielefeld, Germany ), Prof. Dr. K.-H. Drexhage (University of Siegen, Germany), Prof. Dr. J. Enderlein (University of Goettingen, Germany), and Prof. Dr. S. Hell (Max Planck Institute of Biophysical Chemistry, Goettingen, Germany).
www.biophotonik.org

Cytoskeleton of a fixed cell. Left: Fluorescence image at standard conditions. Right: dSTORM image using molecular switches.