Nobel prize winners in chemistry defeat the laws of physics

Nobel Prize in Chemistry for scientists who have developed innovative optical methods that allow observing, among other things, biological molecules

Anthony van Leeuwenhoek, a petty merchant and junior official in the municipality of Delft in the Netherlands, had a hobby in his spare time. He liked to polish glass lenses. In the second half of the 17th century, van Leeuwenhoek was very impressed by the first microscopes built only a few decades earlier The microscopes he built were not more sophisticated than those of his predecessors, but they were equipped with lenses that he polished with love. With their help and with the help of his excellent vision, patience and wisdom, the simple Dutchman was the first person to see bacteria and other microscopic creatures with his own eyes, and in fact founded a new science - microbiology. Van Leeuwenhoek's successors built stronger and more sophisticated microscopes, with higher resolution, and broke the boundaries of human knowledge in medicine, biology, and engineering. However, as science progressed, it became clear that the light microscope had an inherent limitation in terms of the size of the objects that could be seen through it. The German physicist, Ernst Abbe, who studied the optical principles of the microscope at the end of the 19th century, established the rule in the equation he formulated in 1873, according to which it is impossible to see objects smaller than half the visible wavelength in a microscope. The shortest wavelength of visible light is that of violet light , about 400 nanometers (nanometer = millionth of a millimeter), which means that with such a microscope it is possible to distinguish objects whose size is not smaller than 200 nanometers. This makes it possible to observe most types of cells, the organelles inside them, bacteria and even large viruses, but nothing more. Various attempts to develop optical methods that would bypass the limitation of Abbe's law, such as a microscope or near field, provided partial success, but suffered from many limitations. Another development was the electron microscope, which entered the picture as early as the late 30s. In these microscopes (which, of course, have been greatly improved since then), an electron beam is used that hits the sample and is scattered from it, or passes through it. According to the scattering of the electrons on surrounding detectors, it is possible to reconstruct the angle of their impact on the sample and assemble an image at a much higher magnification than that of the optical microscope. However, using an electron microscope requires freezing the examined sample or coating it with metal, which does not allow this method to examine the processes that occur in living cells.

Northern inspiration

Many scientists have searched for methods to circumvent the limitation of the Abe equation, but in vain. The first to come up with an effective idea was Stefan W. Hell, a physicist born in Romania. He did his academic studies in Heidelberg, Germany, where he also completed his doctorate in physics in 1990, where he worked on the improvement of an optical microscope. He continued to work in a European laboratory in Heidelberg, and tried to develop a method to circumvent Abe's law. In 1993 he headed north, and got a research position at the University of Turku in Finland, where he worked on a fluorescence microscope. Fluorescent materials are materials that, in response to light of a certain wavelength, emit a flash of light of a different wavelength (due to the movement of an electron within the material). For example, such a material is illuminated with blue light, and it emits red light. It is possible to mark biological molecules, such as proteins, with such colors. If, for example, an antibody is labeled with a fluorescent dye, you can shine a light on it while the cell is under a microscope, and see where it binds. The fluorescence microscope makes it possible to filter only the wavelengths we are looking for, thus effectively tracking the sample. However, this is a normal optical microscope, which is subject to the limitation of Abe's law, so that it is impossible to distinguish a single antibody, but only a very large cluster that binds to a certain cell or a certain region of the cell. The exact details of the process remain unclear. While working on research in Finland, inspiration struck.

Lasers under a microscope

The idea that Hal came up with, made use of two very thin laser beams - a few nanometers - that would scan the sample being tested one after the other, in a systematic manner. The first beam activates the fluorescent dye, and the one that follows it turns it off. In this way, only a very tiny section of the sample remains illuminated at any given moment. Since the device devised by Hell "knows" at any given time where the laser beam is, the computer connected to the microscope can connect the points of light received by the microscope, and assemble from them - with the help of the position - an image with a greater magnification than the Abbe equation allows. Hell published the idea in 1994, calling the device STED (Stimulated Emission Depletion). The publication did not provoke many echoes in the scientific community, and he decided to build such a microscope himself. In the meantime, he returned to Heidelberg, where in 2000 he succeeded in demonstrating the operation of the device he had developed, and in photographing a bacterium with an optical microscope, at a resolution roughly ten times higher than what Abe's law allows. In other words, the method he developed makes it possible to photograph with a microscope objects whose size is close to 20 nanometers, and to get a much clearer picture of the processes that take place in the living cell.

Glowing proteins

Meanwhile, at an IBM research center in California, William Esco Moerner was engaged in other research in optics. Murner, who is not known by the name William but by the nickname WE (as opposed to him and his father and grandfather, who were also both named William) was born in California in 1953, and at the age of 29 he already completed a doctorate in physics with honors at Cornell University. In his research at IBM he did not deal with the emission of light, but with its absorption, and in 1989 he was the first scientist in the world to measure the light absorption of a single molecule. Even relatively large molecules, such as proteins or DNA, are still too small to be seen with an optical microscope, and even if they are marked with colors, the researchers still only see clusters of molecules, and not single molecules. Mourner's breakthrough did not make it possible to observe such molecules, but it did make it possible to track them through the absorption of light. From the IBM Research Institute, Murner moved to the University of San Diego, where he continued his research, and was engaged in the study of a green fluorescent protein (Green Fluorescent Protein, or GFP for short) that had been isolated a few years before from jellyfish. He discovered that a type of this protein can be turned off and on in response to light of a certain wavelength. The possibility of turning a single molecule on and off using light was published in 1997, and was the solution to the problem published by Eric Betzig two years earlier.

כלי עבודה

Eric Betzig was born in 1960 in Ann Harbor (Michigan, USA) and he also completed his doctorate at Cornell at the age of 28. He then engaged in optical research at Bell Laboratories and was also looking for a way to circumvent the Abbe equation, but in 1995 he despaired of his efforts and resigned. The last article he published before leaving the academy dealt with a theoretical idea that came to his mind during a sports march. Betzig believed that if it were possible to control the light emission of single molecules, it would be possible to achieve a higher separation than is possible according to Abbe's law. The idea works like this: a fluorescence microscope is observed on a sample containing molecules that emit light at different wavelengths, and it is possible to activate each of them separately. When we activate, let's say, all the molecules that emit green light, we know that a stain is only one molecule, which allows the computer to determine its position with great precision (if we didn't know that it was a single molecule, the separation of an ordinary microscope would not allow us to determine this). After that, the different wavelengths are checked, and finally, all the obtained images are placed on top of each other, and the overlap gives us an image with a higher resolution than that of a normal light microscope. Betzig did all the calculations, published them in a scientific journal for the study of optics, put down his resignation letter and went to work at his father's business, a tool manufacturing company in Michigan.

research tool

A few years later in the business world, the scientific bug returned to nest at Betzig. He went back to browsing scientific articles on the Internet, mostly while leisurely sailing on the lake near his home. When he came across the articles on Mourner's luminous proteins, he thought that such proteins might be the practical solution to the theoretical problem he had developed. In 2005 he decided to try to develop the invention in a practical way, and joined the Howard Hughes Research Institute in Virginia. He approached the craft with great vigor, and a year later he already demonstrated the correctness of his idea, when he showed that the overlapping of many images obtained from the photography of molecules of different colors, emitting different wavelengths, indeed makes it possible to obtain a resolution higher than the limit in a light microscope, and to see even objects smaller than 200 nanometers . Of course, there is no need to do the overlap manually - a computer and a camera connected to the microscope do the job automatically. Betzig's paper was published in the journal Science in 2006, and provided an additional method to the one developed by Hell to circumvent the limitation formulated by Abe more than 130 years earlier.

The research tools developed by Hell, Morner and Zieg paved the way for a new observation of materials much smaller than the light microscope could see before, or as the Nobel Prize Committee defined it, they turned the microscope into a nanoscope. Although these are objects larger than those that can be seen with an electron microscope, the great advantage of their methods is the possibility to observe living matter: to follow the movement of substances inside the cell, to check which molecules bind to where, and to try to decipher the processes that are essential to understanding life. Thanks to these methods, scientists are able to better understand disease processes, try to examine the effect of different drugs, and decipher the chain of actions of different proteins, which drive all the processes in the living cell. It is only a matter of time before research based on these methods also wins Nobel Prizes.

* Thanks to Dr. Yaron Bromberg, Dr. Yael Klisman and Dr. Rinat Nebo for the professional enlightenment. If there are still inaccuracies in the article, they are mine alone.

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