STED for biology research

July 12, 2012

Classically the resolution of light microscopy has been limited by the diffraction of light. Resolution of sub 200 nm structures were thought to be impossible. Things have changed with the invention of stimulated emission depletion (STED) microscopy and other superresolution techniques. STED might be especially interesting for cell biology research in the future because cellular structures can be resolved on a molecular level. In the following I will shortly explain how STED works and I will also present a recent paper in which STED has been used to elucidate the molecular features of the centriole, a protein that is important for cell division.

The basis of STED microscopy is the on-off switching of fluorescent molecules within a probe such as a cellular structure. By turning on single or at least only a few fluorescent particles at the same time, imaging them, turning them off again and then moving to the next spot (scanning the sample) drastically increases the available resolution. As only single point-like objects are detected at one point of time at one specific location, summing up all these points in a picture allows for drastic improvements when compared to classic confocal microscopy (Fig. 1). In classic confocal microscopy all fluorescent molecules within the excited spot are detected which means that some spots are overlapping each other and thereby reduce the resolution.

Fig. 1: STED versus classic fluorescence confocal microscopy (Source: Willig et al., New J. Phys., 2006).

An essential question, however, remains: How to rapidly switch fluorophores on and off? This phenomenon is achieved by making use of stimulated emission. As described in the previous post, electrons can rise to an excited state upon absorption of photon. When they fall back fluorescence photons are emitted again. However, this process takes some time. Shining red-shifted at the previously excited spot can stimulate a faster photon emission of a different wavelengths. From now on this second beam will be called STED beam. This “forced emission” is the basic principle of STED microscopy. When the STED beam is modified to become doughnut shaped via a polarizer then only the small inner circle of the previously excited molecules will light up. All surrounding molecules will emit earlier and are therefore unavailable for imaging (Fig. 2). As described above, the size of the inner circle of the STED beam determines the resolution because it selectively allows the detection of single molecules which can be pictured by scanning whole sample and summing up all individual molecules. The diffraction barrier which has been formulated by Ernst Abbe is therefore not only shifted, but broken because theoretically the inner doughnut circle can reach extremely small diameters allowing very detailed images.

Fig. 2: Left: Diffraction limited excitation spot. Middle: Doughnut-shaped STED beam which is applied to the excitation spot and leads to stimulated emission of photons. Right: Stimulated emission of unwanted photons results in a small center containing molecules which emit at the desired wavelength. This spot is not diffraction limited anymore (Source: Marcel Lauterbach).

Now, with the above described features in mind, I shortly want to demonstrate how STED microscopy can be used in a biology related context. In a recent  article Moerner and coworkers from the Department of Chemistry at Stanford University show that the 250 nm sized centriole complex, responsible for the coordination of cell division, is surrounded by nine clusters of the Cep164 protein (Lau et al., 2012). Observations of features of protein complexes were long thought to be impossible with relatively non-invasive light microscopic techniques. However, STED can do the job. Fig. 3 shows how the nine-fold complexes around the centriole actually look like. In addition the obtained results are compared to standard confocal microscope results which of course does not make use of a STED beam. The picture in the bottom right corner also clearly illustrates the advantage of STED microscopy over confocal microscopy in a graph: When displaying the fluorescence intensity as a function of spatial location in nanometers, the observed intensity peak for confocal microscopy is much broader, but not higher.  In STED microscopy two distinct peaks become visible which allow the resolution of two individual objects. The maximum achieved resolution in this study were 60 nm which delivers about four times sharper pictures than could be achieved before Stefan Hell demonstrated the STED principle for microscopy in the 1990s.

Fig. 3: Confocal and STED microscopy of the Cep164 proteins which surround the centriole.

The researchers also note that the observed features are comparable to previously observed structures obtained by transmission electron microscopy (I explained some of its principles in an earlier post). However, electron microscopy exerts high electron forces on the samples which might distort protein conformations. In addition electron microscope pictures are assembled based on “ideal” reference pictures which inevitably leads to a modeling bias. Fig. 4 shows that it is indeed possible to compare light microscopic images to electron microscopic ones. They significantly overlap with each other and underline the strengths of both imaging techniques and especially the combination of the two.

 

Fig. 4: Overlap of a transmission electron microscope with a STED microscope obtained picture. The locations of the nine Cep164 complexes match each other. The scale bar has a size of 200 nm.

Here, the researches made use of fluorescent antibodies and it should be noted that antibody artifacts always distort images. Also the on-off switching right of the used fluorescence molecules determines the capabilities of this technique, next to the brightness. Still, it is very likely that in the future many practical aspects of STED microscopy will be improved which will lead to a number of interesting insights into molecular cell biology.

If you are interested actual proposed functions of the Cep164 complexes, you can find more information in the first listed article. The second article describes the above mentioned research.

Articles:

Graser, S., Stierhof, Y.-D., Lavoie, S.B., Gassner, O.S., Lamla, S., Le Clech, M., and Nigg, E.A. (2007). Cep164, a novel centriole appendage protein required for primary cilium formation. J. Cell Biol. 179, 321–330.

Lau, L., Lee, Y.L., Sahl, S.J., Stearns, T., and Moerner, W.E. (2012). STED Microscopy with Optimized Labeling Density Reveals 9-Fold Arrangement of a Centriole Protein. Biophysical Journal 102, 2926–2935.

Advertisements

Leave a Reply

Fill in your details below or click an icon to log in:

WordPress.com Logo

You are commenting using your WordPress.com account. Log Out / Change )

Twitter picture

You are commenting using your Twitter account. Log Out / Change )

Facebook photo

You are commenting using your Facebook account. Log Out / Change )

Google+ photo

You are commenting using your Google+ account. Log Out / Change )

Connecting to %s

%d bloggers like this: