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Nobel Prize in Chemistry 2014: How the optical microscope became a nanoscope

With the use of fluorescent molecules, scientists can now monitor the interrelationships between individual molecules within cells: they can observe proteins involved in the development of diseases while they accumulate together and they can follow cell division at the nanometer level.

Figure 1 - Aba's limit (0.2 microns): you can see an ant, a hair, a mammalian cell, a bacterium and mitochondria; Virus, protein and small molecules cannot be seen.
Figure 1 - Aba's limit (0.2 microns): you can see an ant, a hair, a mammalian cell, a bacterium and mitochondria; Virus, protein and small molecules cannot be seen.

Eric Betzig, William Mourner and Stefan Hell are awarded the 2014 Nobel Prize in Chemistry for their contribution to overcoming a scientific limitation that an optical microscope could never produce resolution higher than 0.2 micrometers. With the use of fluorescent molecules, scientists can now monitor the interrelationships between individual molecules within cells: they can observe proteins involved in the development of diseases while they accumulate together and they can follow cell division at the nanometer level.

Red blood cells, bacteria, yeast and sperm cells. When scientists in the 17th century began to examine living things under an optical microscope for the first time, a new and fascinating world was revealed to their eyes. This point in time was the birth of microbiology, and from then until today, the optical microscope has become one of the most important tools in the biologist's toolbox. Other microscopic methods, for example, electron microscopy, required destructive measurements on samples that resulted in cell death.

In 1873, German microscopy expert Ernst Abe published an equation that states how the resolution of a microscope is limited by, among other things, the wavelength of light. For most of the twentieth century scientists concluded following this publication that the resolution of an optical microscope would not be better than 0.2 micrometers (Figure 1). The three researchers won this year's prize for overcoming this limitation. Thanks to their achievements, the optical microscope has now entered the nanometer world. The outlines of some organelles in the cell, for example, the mitochondria, were visible. However, it was not possible to distinguish smaller objects, for example, to follow the interrelationships between individual protein molecules within the cell. It is similar to the situation where we can see the buildings of a city without being able to distinguish the inhabitants of the city and their movements in order to properly and fully understand the functions of the cell, you need to be able to follow the activity of the individual molecules. Although the equation developed by Abe is valid and exists, ways have been found to circumvent it. The winners of the Nobel Prize in Chemistry for 2014, Eric Batzig, William Morner and Stefan Hell, introduced optical microscopy to a new dimension, the nanometer dimension, with the help of fluorescent molecules. Theoretically, there is no longer any structure that is too small to be examined. As a result, microscopy became nanoscopy.

The story of overcoming Abe's limitation moves on two separate axes - the win was awarded for two separate principles that were developed independently of each other. The story begins in 1993 in a student apartment in southwestern Finland, when Stefan Hell came up with a brilliant idea while leafing through a textbook on quantum optics.

The rebellion of the youth against the limit of Abe's probability

Stefan tried to find a way around the limitation established by Ernst Abe more than a century earlier. The willingness to challenge such an established principle was tempting in Stefan's eyes. However, senior scientists in Germany poured cold water on his enthusiasm and so he fled north, to a colder place. A professor at the University of Turku who worked on fluorescence microscopy offered him a position in his research team. Hell was convinced that there must be a way to overcome Abe's probability limit, and when he read the term forced emission in a book on quantum optics, a novel idea came to mind, way back in 2009.

Figure 2: A flash of light excites all the fluorescent molecules, while another flash of light quenches the fluorescence from all molecules except those that are nanometer in size
Figure 2: A flash of light excites all the fluorescent molecules, while another flash of light quenches the fluorescence from all molecules except those that are nanometer in size

His solution: a nanometer "flashlight" that scans the sample

Figure 3: Imaging of Escherichia coli at a resolution that has never been achieved before using optical microscopy
Figure 3: Imaging of Escherichia coli at a resolution that has never been achieved before using optical microscopy

In his new laboratory, Stefan worked in the field of fluorescence microscopy, a method in which scientists use fluorescent molecules to image parts of the cell. For example, they can use fluorescent antibodies that bind selectively to cellular DNA. The scientists electronically stimulate the antibodies with a short flash of light, causing them to glow in the blink of an eye. If the antibodies are bound to the DNA they will be careful of the center of the cell, where the DNA is compacted in the nucleus of the cell. In this way, scientists can see what the exact location of a molecule is. However, at this stage the scientists were only able to place clusters of molecules, such as intertwined strands of DNA. The resolution was too low to differentiate between separate DNA strands - try to imagine being able to see a coil of threads without seeing the separate threads themselves. When Stefan read about forced emission, he realized that a sort of nanometer flashlight could be produced that could scan the sample, nanometer by nanometer. Through the use of forced emission scientists are able to dampen the fluorescence of molecules - they direct a laser beam towards the molecules which immediately lose their energy and turn black. In 1994 Stephen Hell published an article detailing his ideas. In the proposed method, called stimulated emission depletion (STED), a flash of light excites all the fluorescent molecules, while another flash of light suppresses the fluorescence from all molecules except those that are nanometer in size (Figure 2). In the next step, only these molecules are absorbed. By scanning the sample and continuously measuring the light levels, you can get a comprehensive and detailed picture. The smaller the volume emitting the fluorescence at a given moment, the higher the resolution of the final image. Therefore, and in principle, there is no longer any limit to the resolution of optical microscopes.

The development of Stefan Hell's first nanometer flashlight in Germany

His theoretical paper did not cause any immediate uproar, but it was interesting enough that he was offered a position at the Max Planck Institute for Biophysical Chemistry in Göttingen. In the following years, he put his ideas into practice and developed a microscope that works on the STED principle. In 2000, he was able to demonstrate that his ideas actually work as expected in a practical way by, among other things, performing imaging of the bacterium Escherichia coli in a separation capacity that had never been achieved before with the help of optical microscopy (Figure 3).

In contrast to this method, the second principle that the scientists won, single molecule microscopy, involves the use of multiple images, one on top of the other. William Morner and Eric Betzig each contributed, independently, different fundamental insights to the development of this method. The foundation was laid when Morner was able to locate a single small fluorescent molecule.
William Morner - the first to locate a single fluorescent molecule

In most chemical methods, for example during the measurement of absorbance or fluorescence, scientists examine millions of molecules simultaneously. The results of these types of experiments represent a type of average, typical molecule. The scientists had to accept this fact, since no other tool existed, but they continued to dream of measuring a single molecule. This is in light of the fact that the more and richer the details, the better one can understand biological processes, for example - the development of a disease.

Thus, when in 1989 Morner became the first scientist ever to measure the light absorption of a single molecule, it was a groundbreaking achievement. At that stage he worked at the IBM research center. in San Jose, California. His experiment paved the way for a new future and inspired many other chemists to turn their attention to single molecules. One of these scientists was Eric Batzig, whose achievements we will present below.
Eight years later Morner took the next step towards single molecule microscopy, relying on the discovery that earned its inventor the Nobel Prize - the green fluorescent protein (GFP).

Molecular light bulbs turn on and off
In 1997 Morner joined the University of California, San Diego, where Roger Tessien, a future Nobel laureate, was trying to make the green fluorescent protein glow in all the colors of the rainbow. The green protein was isolated from a fluorescent jellyfish and its importance lies in the fact that it is able to make other proteins within living cells clearly visible. Using gene technologies, scientists connect the green fluorescent protein to other proteins. In the next step, the green light reveals exactly where in the cell the labeled protein is.

Morner discovered that the fluorescence of one strain of the green fluorescent protein could be turned off and on at the researcher's will. When he excited the protein with light at a wavelength of 488 nanometers, the protein began to glow, but after a while the glow began to fade. Regardless of the amount of light the researcher directed at the protein, the fluorescence gradually faded until it disappeared. However, it turned out that light with a wavelength of 405 nanometers is able to revive the protein. When the protein was reactivated, it returned to luminescence at a wavelength of 488 nm.

Morner soaked these proteins in a gel, and positioned them so that the distance between them was greater than Abe's threshold of 0.2 micrometers. Since they were sparsely distributed, an ordinary optical microscope could distinguish between the blinking of each of the proteins individually - they acted like tiny flashlights with on and off switches. The results were published in 1997 in the prestigious journal Nature.

With this discovery Morner proved that it is possible to optically control the fluorescence of single molecules. Morner's idea solved the problem that Eric Batzig had formulated two years earlier.

Exhausted the academy - however I remain compulsive about the limit of Abe's probability

Just like Stefan Hell, Eric Betzig was compulsive about overcoming his father's probability limit. In the early 90s, he worked on a new optical microscopy called near-field microscopy, at Bell Laboratories in New Jersey. In near-field microscopy, the light beam is projected from an extremely tiny tip located only a few nanometers above the sample. This microscopy method can also overcome Abe's limitation, but it also has significant weaknesses. For example, the projected light has such a short range that it is difficult to see structures below the surface of the cell.
In 1995 Eric Batzig concluded that this microscopy could not be further improved. In addition, he did not feel comfortable in academia and decided that he was ending his research career; Without knowing where he was going from there, he retired from Bell Labs. However, Abe's probability limit kept nagging at his mind. During a walk on a cold winter's day, a new idea came to his mind - is it possible to overcome the limit of amplification using molecules with different properties, molecules with fluorescence in different colors?

Taking inspiration from Mourner's research, among other scientists, Eric Batzig was already able to detect fluorescence in a single molecule using near-field microscopy. He began to wonder whether an ordinary microscope could produce such a high separation power if different molecules glowed in different colors, for example - red, yellow and green. The idea that came to his mind was that the microscope would record one image per subject. If all the molecules of the same color are dispersed and located at a distance exceeding Abbe's limit of 0.2 micrometers, then their location can be located with great precision. In the next step, when these separate images are placed one on top of the other, the total image will give rise to a separation power that will be much better than Abba's, and it will be impossible to distinguish molecules of different colors even if they are several nanometers apart. However, there were a number of practical problems, for example, the lack of molecules with a sufficient amount of distinct optical properties.

Figure 4 - The principle of single molecule microscopy
Figure 4 - The principle of single molecule microscopy

In 1995 Eric Betzig published his theoretical ideas in the scientific journal Optics Letters, after which he retired from academia and joined his father's business company.
The temptation to return to microscopy following the discoveries of the green fluorescent proteins
For many years Eric Batzig was completely cut off from the research community. However, one bright day his passion for science came to life again and while reading the scientific literature he came across the green fluorescent protein for the first time. Realizing that there was a protein capable of making other proteins appear inside the cells, Batzig began to roll the idea in his mind with the help of which he could overcome Abe's limitation. The real breakthrough came in 2005 when he came across fluorescent proteins that can be activated by the researcher, similar to those found by Morner in 1997 at the level of a single molecule. Betzig realized that such a protein was the tool he needed in order to implement the idea he had arrived at 10 years earlier. The fluorescent molecules don't have to have different colors, they can simply glow at different times.

Figure 5: This is what a cell of a biological molecule looks like in a normal microscope (left), in a high-resolution microscope (middle) and an enlargement of a section that illustrates the resolution (right)
Figure 5: This is what a cell of a biological molecule looks like in a normal microscope (left), in a high-resolution microscope (middle) and an enlargement of a section that illustrates the resolution (right)

Overcoming Abe's limitation with photo stacking
Only one year later, Eric Betzig demonstrated, in collaboration with scientists who worked on the excitation of fluorescent proteins, that his idea worked in practice. Among their other experiments, the scientists bound the luminescent protein to the membrane surrounding the lysosome, the cell's cycle station. With the help of a flash of light, the proteins were activated and became fluorescent, but because the flash was so weak, only some of them began to glow. Due to their small number, almost all proteins were found at a distance that was greater than Abe's detection limit of 0.2 µm. Otherwise, the exact location of each of the proteins could be recorded under a microscope. After a while, the fluorescence fades and then the scientists activate a new subset of proteins. Again, the flash was so weak that only some of the proteins began to glow, and then another image of them is recorded. When Betzig stacked the images one on top of the other, he ended up with a high-resolution superimage of the lysosome membrane. Her level was better than her father's probability limit. An article published in the prestigious scientific journal Science in 2006 eventually presented this groundbreaking work.

Laureates are still mapping life's deepest secrets
The methods developed by the three researchers led to the development of several nanoscopic methods that are used today throughout the world. The three winners are still active researchers in the growing scientific community of researchers in the field of nanoscopy. When they point their powerful nanoscopes at the tiniest components of life they also provide us with the most advanced knowledge at the same time. Stefan Hell poked inside the living nerve cells in order to better understand the synapses in the brain. Morner studied proteins associated with Huntington's disease. Eric Batzig followed cell division inside embryos. These are just a small part of many examples. One thing is certain - the winners of the Nobel Prize in Chemistry for 2014 laid the foundations for the development of knowledge of the greatest importance to the human race.

10 תגובות

  1. Today, the use of bright-field and dark-field microscopes in the visible field also make it possible to observe nanoscale structures with an optical microscope. It is about refraction and reflection of the light rays, and nanometer bodies are clearly visible (we see Si-nano-wires easily). This effect has been known for more than 15-20 years.

  2. elbentzo
    I realised. I'm interested in why a microscope is chemistry, and those who dealt with chemical substances received an award in physics...

  3. That was not the intention. When I wrote the response the title was "Nobel for Physics 2014:...". It has since been changed to chemistry and now everything is fine.

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