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On the Way to Molecular Resolution with the Help of Blinking Molecules

To make smallest structures within a cell visible and thus being able to observe them in detail is a crucial aspect of biological research. For this task, fluorescence microscopy is a powerful tool; however, the image resolution is limited because of the diffraction of light: If you want to display a tiny point-like source of light such as for example a light-emitting molecule of about one nanometer (i.e. a billionth of a meter), you will see a broader circular spot with a diameter of at least 200 nanometers (nm). When two molecules are closer to each other than 200 nm, then the lights of both point sources interfere and you will see fuzzy structures instead of two individual objects. This is shown in Figure 1A where actin filaments with a diameter of approximately 7 nm appear to have a width of approx. 500 nm in the image.

Quite recently it was shown that this limitation of image resolution can be overcome with the help of new techniques. One method is to determine the position of many single molecules one after the other and to reconstruct a fluorescence picture from these data. If it can be assumed that the observed light originates from one individual fluorescent molecule, this molecule can be located precisely: it is situated right in the middle of the observed light point that is around 200 nm big. However, in case that one wants to observe a structure which consists of many hundreds of proteins it has to be guaranteed that at each moment only one molecule in the “critical” range of approx. 200 nm x 200 nm is emitting light. This can be achieved e. g. by using dyes whose light emission can be switched on and off with the help of light. Therefore it is possible to locate multiple dye molecules precisely although they are in this “critical” area at the same time by making them light-up individually one after the other. Up to now however, only few of these “switchable” dye molecules have been available, which significantly restricts the applicability of this method. Scientist at the Center for NanoScience (CeNS) in Munich now developed a technique to make many of the dye molecules, already well-established in biology, useable for this new type of microscopy going beyond the classical resolution limit.


Picture: © J. Am. Chem. Soc., 130 (50), pp 16840 (2008)

Figure 1: Image taken by conventional fluorescence microscopy (A) and “blink microscopy” (B) showing actin filaments on a glass surface. The real width of the filaments is about a few nanometer. The image on the right represents reality much closer and shows details which are not visible in the conventional image.


For this purpose, Christian Steinhauer, member of the International Doctorate Program “NanoBioTechnology” and PhD student in the group of Prof. Tinnefeld, uses a usually undesirable characteristic of the dyes: just like a defective contact in a bulb the fluorescence of the dye molecules is consistently interrupted – i.e. the molecules are “blinking”. These dark states are e.g. triggered by the reversible electron transfer reactions taking place in the excited states. For most applications this phenomenon is highly unwanted since one usually needs continuous fluorescence. With a more thorough and exact understanding of the dye molecules, Christian Steinhauer and colleagues have now been successful in adjusting the frequency and duration of the dark states precisely. Thanks to this technique it is now possible to put the majority of the molecules at a specific moment into a dark state. Like that, the position of the few light-emitting molecules left can be determined exactly. Figure 1B shows one fluorescence image that was taken with this method (published in J. Am. Chem. Soc. 130, 16840, 2008). This example clearly illustrates the significant improvement of the resolution of the observed actin filaments compared to the image taken with conventional microscopy shown in Fig. 1A.

This new type of microscopy is called “blink microscopy”.  By performing computer simulations it could be shown that it is possible to achieve an image resolution of 50 nm, i.e. four times higher than before. As many dye molecules emitting light of different colors also show such blinking characteristics it is also possible to investigate several structures with different dyes simultaneously, which is of a great advantage. In addition, because of the fast kinetics of the blinking, this new method constitutes today the fastest superresolution microscopy which is based on the successive localization of individual molecules.


"Superresolution Microscopy on the Basis of Engineered Dark States"
C. Steinhauer, C. Forthmann, J. Vogelsang and P. Tinnefeld
J. Am. Chem. Soc., 130 (50), pp 16840 (2008)

Christian Steinhauer

Since 2007
PhD candidate in the group of
Prof. Philip Tinnefeld, now TU Braunschweig

2002 - 2007
Diploma in Molecular Biotechnology at the Universität Bielefeld and the University of Adelaide, Australien

Selected Publication

J. Vogelsang, R. Kasper, C. Steinhauer, B. Person, M. Heilemann, M. Sauer and P. Tinnefeld:
"A reducing and oxidizing system minimizes photobleaching and blinking of fluorescent dyes
Angew Chem Int Ed, 47(29): 5465-5469 (including cover picture)