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High resolution fluorescence microscopy

17.10.2014

To view the cell and its contents, we must take a microscope. Its principle of operation is relatively simple: light rays pass through an object and then enter magnifying lenses, so that we can see both the cell and some of the organelles inside it, such as the nucleus or mitochondria.

But if we want to see a protein or DNA molecule, or look at a large supramolecular complex like a ribosome, or a virus particle, then an ordinary light microscope will be useless. Back in 1873, the German physicist Ernst Abbe deduced a formula that puts a limit on the capabilities of any light microscope: it turns out that it is impossible to see an object smaller than half the wavelength of visible light - that is, less than 0,2 micrometers.

The solution, obviously, is to choose something that can replace visible light. You can use an electron beam, and then we get an electron microscope - you can observe viruses and protein molecules in it, but the observed objects during electron microscopy fall into completely unnatural conditions. Therefore, the idea of Stefan W. Hell from the Institute for Biophysical Chemistry of the Max Planck Society (Germany) turned out to be extremely successful.

The essence of the idea was that an object could be irradiated with a laser beam, which would put biological molecules into an excited state. From this state, they will begin to move into the normal state, freeing themselves from excess energy in the form of light radiation - that is, fluorescence will begin, and the molecules will become visible. But the emitted waves will be of very different lengths, and we will have an indefinite spot before our eyes. To prevent this from happening, together with the excitation laser, the object is treated with a quenching beam, which suppresses all waves except those that have a nanometer length. Radiation with a wavelength of the order of nanometers just makes it possible to distinguish one molecule from another.

The method was called STED (stimulated emission depletion), and it was for this that Stefan Hell received his part of the Nobel Prize. With STED microscopy, the object is not completely covered by laser excitation at once, but is, as it were, drawn by two thin beams of rays (exciter and quencher), because the smaller the area that fluoresces at a given time, the higher the image resolution.

The STED method was subsequently supplemented by so-called single-molecule microscopy, developed independently in the late XNUMXth century by two other current laureates, Eric Betzig of the Howard Hughes Institute and William E. Moerner of Stanford. In most physicochemical methods that rely on fluorescence, we observe the total radiation of many molecules at once. William Merner just proposed a method by which one can observe the radiation of a single molecule. While experimenting with green fluorescent protein (GFP), he noticed that the glow of its molecules can be arbitrarily turned on and off by manipulating the excitation wavelength. By turning on and off the fluorescence of different GFP molecules, they could be observed in a light microscope, ignoring the Abbe nanometer limitation. The whole image could be obtained by simply combining several images with different luminous molecules in the field of view. These data were supplemented by the ideas of Eric Betzig, who proposed to increase the resolution of fluorescence microscopy by using proteins with different optical properties (that is, roughly speaking, multi-colored).

The combination of Hell's excitation-quenching method with the Betzig-Merner sum-imposition method has made it possible to develop nanometer-resolution microscopy. With its help, we can observe not only organelles and their fragments, but also the interactions of molecules with each other (if the molecules are labeled with fluorescent proteins), which, we repeat, is far from always possible with electron microscopy methods. The value of the method can hardly be overestimated, because intermolecular contacts are what molecular biology stands on and without which it is impossible, for example, neither the creation of new drugs, nor the decoding of genetic mechanisms, nor many other things that lie in the field of modern science and technology.

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