The Development of High-Resolution Fluorescence Microscopy

Robert Eric Betzig (January 13, 1960) is an American physicist, winner of the Nobel Prize in Chemistry in 2014 for the development of high-resolution fluorescence microscopy together with Stefan Helle and William E. Moerner. Eric Betzig, William Moerner and Stefan Hell provided techniques that gave a possibility to look inside nerve cells, find proteins which could cause diseases, and track the division of cell into living embryos. According to Sample (2014), “Winners made it possible to see features at the scale of billionths of a metre, smashing a theoretical barrier for optical microscopy”.

Betzig studied physics at the California Institute of Technology. He graduated with a bachelor's degree in 1983. He received master's degree (MS) and a doctorate in Applied and Engineering Physics at Cornell University in 1985 and 1988 respectively. After receiving his doctoral degree, Betzig worked at Bell Laboratories in the Department of Semiconductor Research. In 1996 he became Vice-president of research and development in Ann Arbor Machine Company. His father, Robert Betzig, owned this company. Betzig engaged in developments in photoactivated localization microscopy. Since 2006, he has been working in the Howard Hughes Medical Institute, where he leads a group of development of high-resolution fluorescence microscopy.

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Eric Betzig, Stefan Hell and William Moerner contributed to the creation of a new generation of microscopes. They are fluorescent and allow seeing objects at a higher resolution than previously used optical microscopes. A new generation of microscopes is called nanoscopes. They allow seeing objects of nanoscale (for example, into the cell). Previously, it was thought that this was impossible. This contribution deals with two unrelated developments. In 2000, Stefan Hell used method of Stimulated Emission Depletion with the use of two laser beams, which act on a fluorescent molecule. Eric Betzig and William Moerner, who were working separately from each other, laid the scientific basis for the creation of the second method - Single Molecule Microscopy, which was based on the ability to turn on and off fluorescent glow of each individual molecule.

The advent of the microscope at the turn of the 16-17th centuries has revolutionized biology and medicine. People saw the world of bacteria and cells and found out that a human was multicellular creature. Scientists saw the living cell and hoped to understand what was happening inside it, why it could age and died. It seemed that there were many new discoveries, but a barrier to further progress appeared in the 19th century. It dealt with the physical limit of resolution of optical microscopes.

According to Fernholm (2014), in 1873 an optician Ernst Abbe derived an equation of resolving power of the microscope, on the basis of which it was impossible to see objects that were smaller than half the wavelength of visible light, 0.2 microns. Scientists could see large objects inside the cell (the nucleus and mitochondria) with optical microscopes. However, it was no longer possible to see what was happening inside the nucleus. In the 20th century, an engineering thought created the X-ray and electron microscopes which used radiation with a wavelength of many orders of magnitude smaller than visible light. Everything would be fine if this radiation did not literally kill biological samples.
For live agents, the method of fluorescence microscopy which consists of coloring objects with special non-toxic substances that are capable of shining upon irradiation with ultraviolet rays began to be used. This method gave better resolution than visual. However, the quality of studies was insufficient.

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In 1990, a young scientist at Heidelberg University, Stefan Hell, was obsessed with the idea to find a way to overcome the Abbe limit. However, his senior mentors did not support the ardor of a young colleague. Thus, Helle went to work in Finland, University of Turku, where he began to study fluorescence microscopy. There, while reading a textbook on quantum optics, he came up with the idea of STED-microscopy. His idea was based on using two lasers, one of which stimulated the glow of fluorescent molecules that were deposited on biologics, and the other, which was configured in a special way, extinguished any illumination other than of the highest frequency.

Eric Betzig and William Moerner went a slightly different way, though also using the technique of fluorescence microscopy. Their method is called single-molecule microscopy, which is based on sequential laser irradiation of preparations’ parts, i.e. their scanning. Individual pictures of the same drug are summarized, and result in the qualitative image with the super fine resolution.

Betzig was obsessed with the idea of overcoming the diffraction barrier in optical microscopy. In the early '90s, he unsuccessfully experimented with so-called near-field microscopy at Bell Labs in New Jersey. In 1995, he published theoretical principles on how one would circumvent the diffraction barrier due to manipulation of the molecules of different colors under the microscope and the imposition of appropriate images at each other. Then, however, Betzig dropped out of academic life for almost ten years and returned to active research only in the 2000s, when he found the work on fluorescent protein, glow of which can be controlled. In 2006, Betzig together with a group of colleagues used a scattered group of individual molecules, the distance between which exceed the Abbe’s limits. By combining a large number of shots, he has achieved the image of lysosome membrane of super-resolution.

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The observation of biochemical processes became possible with these discoveries. Fluorescent nanoscopes allow exploring the structure of fixed and living cells with high resolution and obtaining three-dimensional images, which was not possible until now. It is probably to see what happens at the intracellular level: the movement of macromolecules in living cells, the movement of proteins. Particularly, it is realizable to monitor the synapses, i.e. the junctions of brain neurons, track the movement of proteins which are associated with Parkinson's and Alzheimer's diseases, and observe the behavior of a fertilized egg.

There is one more essential thing: in fact, Betzig and Moerner were able to overcome the Abbe’s limit on the basis on the method of fluorescence microscopy and combining it with the latest technical innovations. This gives hope that other unsolvable obstacles will be resolved sooner or later.

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