Nobel Prize in Chemistry 2014: details
Microscopy without banks
From 1901 to 2013, it was awarded 106 times to 169 scientists (and not all of them were chemists). In 2014, three laureates were added to them, awarded "for the development of ultra-high-resolution fluorescence microscopy". Interestingly, they all hold administrative positions. These are William E. Moerner, head of the Chemistry Department at Stanford University, Eric Betzig, head of the laboratory at the Howard Hughes Medical Institute research campus in Virginia, and Stefan Hell, a native of Romania, director of the Max Planck Society Institute for Biophysical Chemistry in Göttingen and head of the department at the German Cancer Research Center. (DKFZ) in Heidelberg.
Winners of the 2014 Nobel Prize in Chemistry
(from left to right: Eric Betzig, Stefan Hell, William Merner).
Image from the website nobelprize.org
The works of the new laureates lie at the junction of biochemistry, physical optics and molecular biology. They led to the emergence of two new methods of optical microscopy, which made it possible to overcome the so-called diffraction limit of microscopic observations, which in 1870-80 was established (first experimentally, and then theoretically) by the German physicist Ernst Karl Abbe. Abbe showed that the wave nature of light does not allow infinitely improving the resolution of optical devices. In particular, it follows from his work that the minimum size of the details available for observation in a classical optical microscope is equal to the partial of dividing half of the wavelength of light by the refractive index of the medium that fills the space between the microscope lens and the object of observation. In practice, this coefficient usually does not exceed 1.5-1.6, and therefore the limit of the resolution of the microscope corresponds to one third of the wavelength of light. Since the human eye does not perceive waves shorter than 380-400 nanometers, the capabilities of standard optical microscopy are limited to observing objects larger than 130-140 nanometers. This is enough for bacteria, cells, and even large cellular organelles such as mitochondria, but too little for microscopic examination of viruses, not to mention protein molecules.
In 1980-90, scientists found a number of opportunities to improve the resolution of optical devices used to study the microcosm. Confocal and multiphoton (Multiphoton microscopy) systems made it possible to reduce the minimum size of distinguishable objects by about half, and scanning microscopes of the near field – even tenfold. However, near-field microscopy has many limitations and cannot claim to be widely applicable. Two Nobel Prize-winning optical microscopy technologies not only provide ultra-high resolution, but can also be used to observe a wide variety of objects. Thanks to them and other similar methods, optical microscopy is rapidly turning into nanoscopy.
Both technologies use support networks consisting of luminous molecules. Such grids are created and work in different ways, but in both cases their elements are registered independently of each other. Therefore, information from the grids is read without regard to the diffraction limit, which makes the new methods practically universal.
Stefan Hell's method is based on the so-called Stimulated Emission Depletion (STED). The object under study is marked with molecular markers capable of emitting light quanta (fluoresce) under the action of laser radiation (such an object can be a DNA molecule, and the labels are fluorescent antibodies). However, the same molecules can be made to emit photons with a longer wavelength with some delay if they are irradiated with another laser with properly selected characteristics. Let the first laser create a circular light spot on the surface of the sample, and the rays of the second are focused in a ring covering the entire circle except the center. The labels in the central zone will glow at one wavelength, and the labels inside the ring will glow at another, much larger one (this is the depletion of fluorescent emission). If you set up the receiving system of the microscope to register only short-wave photons, the areas with depleted emission will go out, as it were.
This system can be turned into a scanning microscope if you direct laser beams to different parts of the object, register signals from glowing zones and process them on a computer. If the labels tightly cover the surface of the object, then the images obtained during such a scan will reproduce its structure. The degree of resolution of such a device is determined by the size of the zones with undressed emission, which in principle can even be nanometer.
Hell developed the theory of his method in 1993-94, and in 1999 demonstrated it in practice. At first, STED technology was little better than confocal microscopes. Now, on factory devices, it provides a resolution of 30 to 80 nanometers, and in the experiment – two and a half nanometers.
Photo of the same object with a confocal microscope (left) and a STED system (right).
The length of the scale ruler is 1 microns, the length of the scale rulers in the insets is 250 nm
(from an article by Benjamin Harke et al., 2008. Resolution scaling in STED microscopy).
The second method is called PALM, Photoactivated Localization Microscopy. Eric Betzig is recognized as its main developer (although his colleague at the Hughes Institute, Harald F. Hess, made almost the same contribution). This technology was first demonstrated in 2006. The third winner, William Merner, was not engaged in optical microscopy. However, PALM uses proteins that emit a bright green glow under the influence of blue or ultraviolet light. These so-called green fluorescent proteins (GFP) were first isolated from the tissues of jellyfish of the species Aequorea victoria, and later found in other marine invertebrates (their discovery was awarded the 2008 Nobel Prize in Chemistry). In 1989, Merer was the first in the world to find an opportunity to measure the absorption of light by a single molecule, and 8 years later discovered a way to control the fluorescence of individual GFP molecules using laser radiation.
Merner's discovery was used by Betzig and colleagues to develop PALM technology. It is based on the use of laser radiation with the wavelength necessary to excite green fluorescent proteins. The sample is repeatedly irradiated with very weak laser pulses containing a small number of photons. These photons cause protein molecules to glow – again, in small quantities. Since light randomly selects these molecules on the surface of an object of rather large extent, almost all of them are separated from each other by distances exceeding the Abbe limit. The position of each luminous center can be recorded with great accuracy using an optical microscope. Individually, such images are not very informative, but computer analysis of all images, which is done on the basis of probabilistic algorithms, allows you to restore the structure of the original sample. Today, PALM provides a resolution of up to 20 nanometers, and, most likely, this is not the limit.
Image of the actin cytoskeleton of a living cell.
The central part of the image is made using PALM technology.
Drawing from the website cfn.kit.edu
In conclusion, it is worth noting that STED and PALM are by no means the only systems of optical supermicroscopy, but it was on them that the grace of the Nobel Prize fell. Why exactly – this mystery is great.
Portal "Eternal youth" http://vechnayamolodost.ru20.10.2014