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.

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In previous posts I tried to explain why light is so important for biology and how its properties can be used in biology research. The process of absorption is especially important for understanding the role of light within the field of biology, which is obvious since light can only have an effect if it interacts with matter. Molecules can absorb light because their electrons become more “energetic” and rise form the so-called ground state to one of the excited states. These different states and their significance for biology will be explained in the following. Molecules that are able to absorb light of the biologically interesting spectrum with wavelengths form approximately 100 to 800 nm are also called chromophores. Such molecules often contain a delocalized π-electron system which means that these molecules can form bonds that occur between electron orbitals (outer electron cloud around the atomic nucleus) called π-orbitals. Many of such π-bonds result in an electron system within the molecule which is flexible once it absorbs the energy of photons which make up light. The electrons within such a flexible system can then become delocalized or spread out across the molecule. Therefore, molecules which contain a delocalized π-electron system are especially sensitive to the absorption of light. In practice a π-electron system can be found in molecules containing aromatic systems and/or a relatively high number of conjugated double bonds. Figure 1 displays how pH changes influence the protonation states of an anthocyanine molecule, a plant colorant which gives flowers and berries their distinct colors in many plants. Of course, one finally only observes the non-absorbed photons. The anthocyanine molecule is a good example for the interesting color properties of molecules that can change once the atomic make-up changes. This molecule is also a good example for a π-electron system (see the aromatic rings).

Figure 1

An electron excitation can, however, only occur if photon energy matches the energy difference between the ground and the excited state. When the electrons of single atoms fall from the excited state back to the ground state they emit the previously absorbed energy again in the form of a photon which matches the wavelength of the previously absorbed photon. Therefore, absorption and emission spectra of single atoms are identical. However, things are different for molecules which of course by definition always contain at least two atoms. Bound atoms can vibrationally interact with each other which costs energy. In a molecule every electron state can be subdivided vibrational states and each vibrational state can again be split into different rotational states. Still, the photon’s energy must match the energy difference between the ground and excited state in order to become absorbed. This results in the fact that molecules can absorb a range of wavelengths and due to vibrations emit longer wavelengths containing less energetic photons. While single atoms can only absorb and emit single spectral lines (Figure 2) molecules can absorb and emit broader band light spectra.

Figure 2

Hopefully, the basics of absorption are now a bit clearer. However, we have not discussed yet how an excited electron can lose its energy again. Depending on the excited state it is in the electrons can choose different paths to fall back into the ground state. As described, during these paths the electrons emit longer wavelengths photons which are responsible for biologically interesting processes such as fluorescence, bioluminescence, or phosphorescence. But also other processes  occur that are not directly visible by eye when the electrons fall back to the ground state. These include internal conversion, intersystem crossing, resonance energy transfer, and photochemical reactions. In the following, all of these effects will be described. A so-called Jablonski diagram (Figure 3) schematically displays the different electron states and their subdivision into vibrational and rotational states. A Jablonski diagram also helps to “visibly understand” what happens to excited and returning electrons. So let’s start.

Figure 3 (Source: Olympus)

First of all electrons become excited by photon absorptions which rises their energy level to the first or second singlet state depending on the available energy and molecular properties. This process is extremely fast is depicted by the green arrows.

Internal conversion (IC) always occurs in molecules when excited electrons fall back to the ground state. During IC the absorbed energy is converted into kinetic energy in the form of vibrations or rotations. No electromagnetic radiation occurs.  Yellow curly arrows indicate this process which logically occurs between the vibrational states, but also within one vibrational state containing more rotational states (not shown).

An observable process is fluorescence. When an electron falls back from the first singlet state into the ground state it emits electromagnetic radiation in the form of a photon. However the emission wavelength is longer because the electron has lost energy due to IC on its way from the second singlet state to the first singlet state or due to IC within just the first singlet state. Fluorescence is indicated by the red down facing arrows from S1 to S0. Bioluminescence is the process of fluorescence within biomolecules such as green fluorescent protein (GFP).

Another important feature of electron states is the process of intersystem crossing. During intersystem crossing an electron moves from the excited first singlet state into the first triplet state. A triplet state is a state in which an electron can only be found once its quantum mechanical spin reverses from -1/2 to +1/2. Quantum mechanically this very unlikely and therefore a triplet state occurs less often than the other two excited states. Intersystem crossing is indicated by the blue curly line.

The process of phosphorescence occurs one an electron has managed to enter the triplet state by intersystem crossing and falls back into the ground state. As in fluorescence a photon is emitted, but it contains less energy and it time delayed with a factor of about one million because a triplet state is not stable state. In research applications (such as the earlier described fluorescence correlation spectroscopy) where only the emitted fluorescence at a specific point in time is required, a certain percentage of phosphorescence signal therefore needs to be subtracted from the total signal. Phosphorescence is indicated by the red arrow facing to the lower left corner.

Zooming out and looking not only at one molecule, but for example two neighboring molecules, makes it possible to observe two other interesting electron effects. The first one is called resonance energy transfer. If the fluorescence spectrum of a donor molecule and the absorption spectrum of a acceptor molecule match the vibrations of the former molecule’s electron can excite the latter molecule’s electron. Then a longer wavelength fluorescent emission of the second molecule can be observed even though it has not been excited directly with photons. This process is the basis of Förster Resonance Energy Transfer (FRET) which can be used to determine whether two molecules are in close proximity. If two proteins are close two each other, the chromophore electrons of the first one therefore might vibrationally excite the electrons of the second one and fluorescence of a distinct wavelength can be observed. A second interesting photo effect between two neighboring molecules is a so-called photochemical reaction. Here, a very strong excitation removes an electron from its original orbital and the molecule therefore becomes oxidized. Another molecule which receives the electron is reduced. Oxidized and reduced molecules can then participate in “regular” chemical reactions. Photochemical reactions are the basis of photosynthesis where chlorophyll electrons power the electron transport chain in plants.

I hope that this short summary of light and biology serves its purpose of demonstrating that understanding a little bit of physics and a little bit of biology and joining both, can lead to some very interesting insights! For people who are interested more in photons and their effects on electrons I want to recommend an extremely appealing Java-animated tutorial created by Olympus.