Nobel Prize for Chemistry has been equally divided among the Laureates Eric Betzig, Stefan W. Hell and William E. Moerner for having bypassed a presumed scientific limitation stipulating that an optical microscope can never yield a resolution better than 0.2 micrometres (half the wavelength of light).
Using the fluorescence of molecules, scientists can now monitor the interplay between individual molecules inside cells; they can observe disease-related proteins aggregate and they can track cell division at the nanolevel.
Living organisms were studied for the first time in the 17th using an optical microscope. The instrument opened a new window to studying living organisms non-invasively.
Despite the advantages, the optical microscope suffers from a major drawback — a physical restriction as to what size of structures is possible to resolve. Ernst Abbe in 1873 said that microscope resolution is limited by, among other things, the wavelength of the light (0.2 micrometres).
While Abbe’s microscope resolution limitation still hold true, the Laureates have successfully demonstrated ways of bypassing the limitation. The three have taken optical microscopy into a new dimension using fluorescent molecules. Two different principles have been able to do this and they developed independently of each other.
Stefan Hell who was a student in South-western Finland was looking at ways of bypassing Abbe’s limitation. In 1993, he got a brilliant idea when reading a textbook on quantum optics. He soon found himself working as a part of research team using fluorescence microscopy — a technique where scientists use fluorescent molecules to image parts of the cell — at the University of Turku.
Hell was convinced that there had to be a way of circumventing Abbe’s diffraction limit, and when he read the words stimulated emission in the book on Quantum Optics a new line of thought took shape in his mind: “At that moment, it dawned on me. I had finally found a concrete concept to pursue – a real thread.”
Using fluorescence microscopy it was possible for scientists to see where a certain molecule was located. But only clusters of molecules, like entangled strands of DNA, could be located. The resolution was too low to discern individual DNA strings.
But Hell realized that it should be possible to devise a kind of nano-flashlight that could sweep along the sample, a nanometre at a time using stimulated emission.
By using stimulated emission scientists can quench fluorescent molecules. They direct a laser beam at the molecules that immediately lose their energy and become dark. In 1994, he published an article outlining his ideas. In the proposed method, so-called stimulated emission depletion (STED), a light pulse excites all the fluorescent molecules, while another light pulse quenches fluorescence from all molecules except those in a nanometre-sized volume. Only this volume is then registered. By sweeping along the sample and continuously measuring light levels, it is possible to get a comprehensive image.
The smaller the volume allowed to fluoresce at a single moment, the higher the resolution of the final image. Hence, there is, in principle, no longer any limit to the resolution of optical microscopes.
He soon shifted to Max Planck Institute for Biophysical Chemistry in Göttingen. In 2000, he was able to demonstrate that his ideas actually work in practice, by, among other things, imaging an E. coli bacterium at a resolution never before achieved in an optical microscope.
Single-molecule microscopyUnlike the STED microscopy, the single-molecule microscopy entails the superposition of several images. Eric Betzig and W. E. Moerner have independently of each other contributed different fundamental insights in its development. The foundation was laid when W. E. Moerner succeeded in detecting a single small fluorescent molecule.
Using absorption and fluorescence, scientists were able to study only average molecules; measuring single molecules was not possible. As a result, it was difficult to get detailed knowledge of things, for instance, how diseases develop.
Therefore, in 1989, when W. E. Moerner as the first scientist in the world was able to measure the light absorption of a single molecule, it was a pivotal achievement. At the time he was working at the IBM research centre in San Jose, California.
Eight years later Moerner took the next step towards single-molecule microscopy, building on the previously Nobel Prize-awarded discovery of the green fluorescent protein (GFP).
In 1997 W. E. Moerner had joined the University of California in San Diego, where Roger Tsien, Nobel Prize Laureate to be, was trying to get GFP to fluoresce in all the colours of the rainbow.
W. E. Moerner discovered that the fluorescence of one variant of GFP could be turned on and off at will. When he excited the protein with light of wavelength 488 nanometres the protein began to fluoresce, but after a while it faded. But he found that light of wavelength 405 nanometres could bring the protein back to life again. When the protein was reactivated, it once again fluoresced at 488 nanometres.
Moerner dispersed these excitable proteins in a gel, so that the distance between each individual protein was greater than Abbe’s diffraction limit of 0.2 micrometres. Since they were sparsely scattered, a regular optical microscope could discern the glow from individual molecules — they were like tiny lamps with switches. The results were published in Nature in 1997.
By this discovery Moerner demonstrated that it is possible to optically control fluorescence of single molecules. This solved a problem that Eric Betzig had formulated two years earlier.
Near-field microscopyEric Betzig was working on near-field microscopy in the early 1990s at the Bell Laboratories in New Jersey. In near-field microscopy the light ray is emitted from an extremely thin tip placed only a few nanometres from the sample. This kind of microscopy can also circumvent Abbe’s diffraction limit, although the method has major weaknesses. For instance, the light emitted has such a short range that it is difficult to visualize structures below the cell surface.
Eric Betzig concluded that near-field microscopy could not be improved much further. He quit Bell Labs.
Inspired by W. E. Moerner, among others, Eric Betzig had already detected fluorescence in single molecules using near-field microscopy. He began to ponder whether a regular microscope could yield the same high resolution if different molecules glowed with different colours, such as red, yellow and green.
The idea was to have the microscope register one image per colour. If all molecules of one colour were dispersed and never closer to each other than the 0.2 micrometres stipulated by Abbe’s diffraction limit, their position could be determined very precisely.
Next, when these images were superimposed, the complete image would get a resolution far better than Abbe’s diffraction limit, and red, yellow and green molecules would be distinguishable even if their distance was just a few nanometres. In this manner Abbe’s diffraction limit could be circumvented.
However, there were some practical problems, for instance a lack of molecules with a sufficient amount of distinguishable optical properties. In 1995 Eric Betzig published his theoretical ideas in the journal Optics Letters, and subsequently left academia
Lured backFor many years Eric Betzig was entirely disconnected from the research community. But one day he came across the green fluorescent protein for the first time. Realizing there was a protein that could make other proteins visible inside cells revived Betzig’s thoughts of how to circumvent Abbe’s diffraction limit.
The real breakthrough came in 2005, when he stumbled across fluorescent proteins that could be activated at will. The fluorescent molecules did not have to be of different colours, they could just as well fluoresce at different times.
Just one year later, Eric Betzig demonstrated, in collaboration with scientists working on excitable fluorescent proteins, that his idea held up in practice. Among other things, the scientists coupled the glowing protein to the membrane enveloping the lysosome, the cell’s recycling station.
Using a light pulse the proteins were activated for fluorescence, but since the pulse was so weak only a fraction of them started to glow. Due to their small number, almost all of them were positioned at a distance from each other greater than Abbe’s diffraction limit of 0.2 micrometres. Hence the position of each glowing protein could be registered very precisely in the microscope. After a while, when their fluorescence died out, the scientists activated a new subgroup of proteins. This procedure was then repeated over and over again.
When Betzig superimposed the images he ended up with a super-resolution image of the lysosome membrane.
Its resolution was far better than Abbe’s diffraction limit. An article published in Science in 2006 subsequently presented the ground-breaking work
( Edited excerpts from popular material available at Nobel Prize website )
