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Nanoscale Beacons

What blinking molecules reveal about cell structure

Munich, 05/12/2009

There has been a strong drive behind the development of high-resolution microscopic methods in recent years. Developers are aiming at achieving a high spatial resolution right down to the nano scale for making clear, precise images of very small, tightly packed molecules. A research group working with Professor Philip Tinnefeld of the Department of Physics of Ludwig-Maximilians-Universität (LMU) Munich has now managed to develop fluorescent molecules that can be switched on and off on command using two opposite chemical processes. Unlike conventional methods, this mechanism even works in the presence of oxygen, and can be applied to a wide range of dyes. The method, which the developing researchers have dubbed “blink microscopy”, can therefore be used to observe even living cells, in order to discern the arrangements of very closely neighboring molecules. The new method could as well be valuable to nanotechnology, for developing precisely controlled electro-optical switch elements, for example. (PNAS Early Online Edition, 11 May 2009).

The spatial resolution of an optical microscope is limited by the wavelength of light. Structures smaller than 200 to 300 nanometers – or millionths of a millimeter – are too small to be distinguished from one another using a conventional microscope. However, in nanotechnology and cellular biology the particles researchers are interested in are far smaller still. While it is possible to achieve higher resolutions using electron microscopes, they are highly complicated and have certain disadvantages – they don’t allow observation of living systems, for example. Using special techniques, however, even optical microscopy can be improved to achieve resolutions down to the scale of a few nanometers. In a number of steps, the arrangements of individual, color-stained molecules can be discerned.

A research group at Ludwig-Maximilians-Universität (LMU) Munich has now made a crucial contribution to this field of superresolution microscopy. Professor Philip Tinnefeld and his co-workers of the Chair for Applied Physics have found a way to purposely “switch on” and “switch off” the luminescence of a conventional fluorescent molecule using photochemical processes. The duration of the "on" and "off" states can be suitably controlled by the composition of the chemicals. This “switch” has also proven to be extraordinarily long-lasting: it can be switched on and off between 400 and 3000 times before the molecule finally degrades.

In their study, funded by the Deutsche Forschungsgemeinschaft and by the Federal Ministry of Education and Research, Tinnefeld and his group used a dye from the class of oxazines to create these blinking particles. This dye is fluorescent by nature, which means that after light is shone onto it, it will emit light of its own for a short while. The researchers managed to switch this luminescence on and off using a so-called redox reaction. In this chemical process, a substance – the reducer – donates electrons, which in turn are taken up by a second substance – the oxidizer. The researchers first added a reducer to the oxazine so that the dye accepted an electron and the fluorescence was “switched off”. This state then remained stable for a period of several minutes.

When the scientists then added an oxidizer into the mixture, the oxazine surrendered the electron it had previously gained, and thereby “switched” itself back into its original, stable state. “The principle has significant advantages over previously developed chemical switches that involve highly complex chemical processes, and often have only a limited lifetime,” explains Tinnefeld. “Also, it can be used with many different dyes.” Another advantage of the new method is that it even works in the presence of oxygen, which typically destroys such dyes. This means it can be used to study living cells, where oxygen will always be present, playing some part or another.

In the next step of their study, the biophysicists made a successful demonstration that the new method can be applied to ultra-fine structures inside cells. To do this, the researchers applied so-called actin filaments – parts of the cytoskeleton of body cells – onto a glass surface. Next, they adjusted the reducer and oxidizer concentrations in such a way that the individual molecules only lit up in bursts. They then photographed the ensuing “blinking concert” of the molecules with a special camera so that, afterwards, they were able to reconstruct the location of each individual molecule to great precision. “This gave us a resolution of a few nanometers, and made structures visible that we have not been able to see using previous methods,” says Tinnefeld.

In future, the researchers intend to adapt the fluorescent dyes specifically to the environmental conditions of living cells. “We are also planning projects together with biologists from Munich, in which blink microscopy will be used for all kinds of biological problems,” says Tinnefeld. For example, the method could help to observe the activity of molecules artificially introduced into cells. Yet, these new molecular switches could be just as useful in a whole series of other fields – in particular nanotechnology. “The fluorescent molecule can be switched on and off not only chemically, but also electrically,” explains Tinnefeld. “That means it could be used as an electro-optical component in PCs – for data storage or for color displays, for example.” (CA/suwe)

The project was conducted in the scope of the cluster of excellence “Nanosystems Initiative Munich” (NIM). This cluster of excellence has its sights set on developing, researching and bringing into operation functional nanosystems for application in information processing and life sciences.

“Controlling the fluorescence of ordinary oxazine dyes for single-molecule switching and superresolution microscopy”;
Jan Vogelsang, Thorben Cordes, Carsten Forthmann, Christian Steinhauer and Philip Tinnefeld;
PNAS Early Online Edition, 11 May 2009;
DOI: 10.1073_pnas.0811875106

Prof. Dr. Philip Tinnefeld
Applied Physics – Biophysics & Center for NanoScience
Ludwig-Maximilians-Universität, Munich
Tel: +49 89 - 2180 1438
Fax: +49 89 - 2180 2050

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