Nobel Prize/Chemistry

Microscopy to nanoscopy

Print edition : November 28, 2014

Eric Betzig. Photo: MICHAELA REHLE/REUTERS

Stefan Hell. Photo: Hubert Jelinek/AP

William Moerner. Photo: Demetrio Araujo/AP

The Diffraction limit on optical microscopy meant that scientists could distinguish whole cells as well as some parts of the cell called organelles. However, they would never be able to discern something as small as a normal-sized virus or single proteins.

(A) The Intensity distribution, called the Airy pattern, of a single fluorophore emitting light onto a CCD camera. The fluorophore is at the centre of the image and can be considered a point source. For a green fluorescent protein (GFP) emitting at a peak of 510 nm, and an objective numerical aperture of 1.4, the width of this intensity profile will be 444 nm. Thus, a point source (the fluorescent protein) is no longer viewed as a point source, but rather as a diffuse, delocalised intensity pattern. (b) The profile of this two-dimensional pattern along the green line.

To the left, an E. coli bacterium imaged using conventional microscopy; to the right, the same bacterium imaged using STED. The resolution of the STED image is three times better. Image from Proc. Natl. Acad. Sci. USA 97: 8206–8210

The centre image shows lysosome membranes. To the left, the same image taken using conventional microscopy. To the right, the image of the membranes has been enlarged. The scale division of 0.2 micrometre is equivalent to Abbe’s diffraction limit. The resolution is many times improved. Image from Science 313:1642–1645.

The Nobel Prize-winning works of Eric Betzig, Stefan W. Hell and William E. Moerner fall under what has come to be called “super-resolution microscopy”, an umbrella term for a number of techniques that achieve sub-diffraction resolution. Theoretically, there is no limit to the resolution that their methods can achieve.

ACCORDING to a fundamental principle of optics, commonly known as the “diffraction limit”, two points of an object cannot be distinguished as separate if they are closer than about half the wavelength of the light being used to observe them. The visible range of the electromagnetic spectrum extends from a wavelength of 700 nanometres (a billionth of a metre, or 10 -9 m) at the red end to 400 nm at the blue end. Therefore, according to the principle, which is quantitatively described by two closely related equations called the Abbe criterion and the Rayleigh criterion, an optical microscope cannot yield a resolution better than about 0.2 micrometre (a millionth of a metre, or 10 -6 m) corresponding to the shortest wavelength in the visible range. While Ernst Abbe (1840-1905), a German physicist, formulated his criterion in 1873, Lord Rayleigh (1842-1919), a British physicist, wrote down his criterion in 1875. There is a small difference between the two criteria—not of any significance for practical applications—arising basically because of the slightly different definitions used by the two scientists in defining what it means to resolve two distinct objects.

For nearly the whole of the 20th century, it was believed that optical microscopy could not do any better because of this theoretical limit on the maximum resolution of 0.2 micrometre that was attainable (Figure 1). So, while the contours of some of a cells’ organelles, such as mitochondria, could be discerned, it was not possible to discern smaller objects such as interactions between individual protein molecules in the cells. If one has to fully understand how a cell functions, one should be able to see how each molecule in the cell behaves.

Eric Betzig, Stefan W. Hell and William E. Moerner have been awarded the year’s Chemistry Nobel Prize for inventing methods that help in circumventing the limit of a conventional microscope to achieve much finer resolutions. The three share the prize money of eight million Swedish kronor ($1.12 million) equally.

The electron microscope, which is another important instrument that biologists use to study organisms, uses an electron beam instead of light as the probe, and the wavelength of electrons (in accordance with quantum theory) in a typical electron microscope is as small as 5 picometres (thousandth of a billionth of a metre, or 10 -12 m). But, owing to distortions at the edges of images caused by the magnetic lenses used, the resolution is far worse than the electron wavelength though it is still three orders of magnitude better than an optical microscope. However, the preparatory measures required in electron microscopy eventually kill the cell itself. So an electron microscope mostly cannot see a working cell. The Nobel Prize-winning works fall under what has come to be called “super-resolution microscopy”, an umbrella term for a number of techniques that achieve sub-diffraction resolution. Theoretically, there is no limit to the resolution that their methods can achieve. Thus, as a result of their techniques, microscopy has become nanoscopy.

diffraction limit

The “diffraction limit” in an optical microscope arises because of the wave nature of light and its interaction with the optical system through which the light passes. The incoming light undergoes diffraction or scattering at the entrance of the microscope objective (the lens system). This results in a loss of information with regard to the exact location of the point from which light is being received into the microscope. Consider, for example, a single green fluorescent protein (GFP) emitting light at a wavelength of 510 nm. It gives rise to an intensity distribution in the image plane of the microscope or the camera (called Airy pattern or disc) that is diffuse, delocalised and symmetric with a radius of about 220 nm (Figures 2a & b). The actual size of a GFP molecule is only 2-4 nm compared with the diffraction-limited diffuse pattern of about 450 nm across. It is clear that if we had tens of such proteins or even just two proteins within this single Airy profile, we could not tell them apart with optical microscopy. Thus, the resolution limit is the physical distance in space between two point sources of light needed for the instrument to distinguish their respective light intensity patterns.

The Nobel Prize has been awarded for two separate principles corresponding to the two main streams of super-resolution microscopy techniques that have emerged in the last decade or so, which have had an enormous impact on research in cell biology, medicine and material science. One, pioneered by the invention of Hell in 2000, is called stimulated emission depletion (STED) microscopy. This relies on manipulating diffuse light intensity patterns with external light to make it as narrow as possible.

The other is what Betzig and Moerner independently developed, which has laid the foundation for a class of techniques collectively called single-molecule microscopy. This includes photo-activated localisation microscopy (PALM), stochastic optical reconstruction microscopy (STORM) and points accumulation for imaging in nanoscale topography (PAINT) microscopy. These techniques make use of the discovery in the last few years of a new generation of “photo-activable” and “photo-switchable” proteins.

Ever since his PhD in 1990 from the University of Heidelberg, Hell had been convinced that there must be some way of surpassing the resolution limit in optical microscopy. But his conviction, which challenged an established principle of optics defined 120 years ago, was only met with scepticism from scientists in Germany and he struggled to obtain a lasting job in German institutions. So, Hell moved to Finland when a professor at the University of Turku working on fluorescence microscopy offered him a position in his research group.

Although he had a rough plan for overcoming the limit, a decisive trick to realise it was lacking. One Saturday morning in the fall of 1993, Hell was sitting in his student apartment in Turku and leafing through a book on quantum optics. Obsessed with the idea, he was scanning through the book in search of a suitable quantum phenomenon. The eureka moment occurred when his eye caught the phrase “stimulated emission”. In stimulated emission, atoms and molecules that are excited to higher quantum energy levels are de-excited to lower levels and they lose energy by emitting photons. If molecules excited to fluoresce are made to switch off temporarily through the process of stimulated emission, he knew he could beat the limit. “At that moment,” he has said, “it dawned on me. I had finally found a concrete concept to pursue—a real thread.”

Over a day and a half, he did some rough calculations and ran a few computer simulations and realised that it would work and that the resolution would drop to at least 30 nm, one-tenth the conventional limit. “But it was also clear to me that, in principle, there was no lower limit,” he commented in 2009. “I had realised that… so much physics happened in the 20th century that it is impossible that there is no… phenomenon that would allow you to overcome the diffraction barrier,” he told the www.nobelprize.org interviewer after the announcement. “So I tried to find something, and eventually I found ways to overcome that limit.”

Fluorescence microscopy

In fluorescence microscopy, the technique that Hell was using in his work at Turku, scientists use fluorescent molecules to image parts of the cell. For instance, fluorescent antibodies are used to couple specifically to cellular DNA (deoxyribonucleic acid). To do this, antibodies are excited with a brief light pulse, making them glow for a while, and when they couple to the DNA they will radiate from where the molecule is located in the cell nucleus. But this technique only allowed them to locate clusters of molecules, such as entangled strands of DNA. The resolution was not good enough to discern the individual strands.

Hell thought that if he could devise some kind of nano-flashlight that could sweep along the sample, a nanometre at a time, he could quench all the fluorescent molecules through stimulated emission except the one of interest in the middle (Figure 3). All the once-fluorescent molecules can be made to lose their energy and become dark except the nanometre volume to be imaged. In 1994, Hell published an article outlining his ideas. In the proposed method, a laser light pulse excites all the fluorescent molecules, while another pulse quenches fluorescence from all molecules except the molecule to be studied. Only light from this volume is registered. By sweeping along the sample and continuously measuring light levels, a comprehensive image is obtained. The smaller the volume allowed to fluoresce, the finer the resolution of the final image. Thus, in principle, there is no limit to the resolution that can be attained with an optical microscope.

The article, however, did not create any immediate flutter in the research community. But the Max Planck Institute for Biophysical Chemistry in Gottingen found it interesting enough to offer him a position, and Germany is where Hell really wanted to live and work. Working there, he could realise his ideas in practice and build a STED microscope. In 2000, he demonstrated that his revolutionary ideas actually worked by imaging an E.coli bacterium at a resolution never before achieved (Figure 4). Today, the 52-year-old Hell is the director of the institute and is also the division head at the German Cancer Research Centre in Heidelberg.

Single-Molecule Microscopy

While STED microscope collects light from hundreds of small nanoscale volumes to create a large whole, the technique of single-molecule microscopy involves superposition of several images. The foundation for the field was laid when Moerner first detected a single small fluorescent molecule.

As mentioned earlier, because of the limiting resolution of conventional optical microscopes, all chemical and biological methods for measuring absorption or fluorescence can only get a representation of a typical molecule, a kind of average of millions of molecules that get observed.

But like the physicist Hell, chemists too had been hoping for a long time to be able measure the behaviour of a single molecule. And that happened when in 1989, working at the IBM Research Centre, San Jose, California, Moerner was the first to measure the light absorption of a single molecule. The experiment unleashed a flurry of activity among chemists in the 1990s to begin studying single molecules.

In 1997, when Moerner joined the University of California, San Diego, he could build on his successful experiment towards single-molecule microscopy. In this, he took advantage of the Nobel Prize-winning discovery of the GFP by Roger Tsien, who was working there ( Frontline, November 21, 2008).

The GFP had been isolated from a fluorescent jelly fish and could be used to study other proteins in the cell. Using gene technology, scientists could couple the GFP to other proteins, and the green light of the GFP revealed exactly where in the cell the marked protein was located. Moerner discovered that a variant of GFP could be switched on and off at will.

He found that when he excited the protein with light of wavelength 488 nm, the protein glowed but after a while the fluorescence died. It could not be made to glow again even if light was directed on it for a sufficiently long time.

But Moerner discovered that light of wavelength 405 nm could reactivate the molecule, which then glowed again at 488 nm. With this discovery, he realised that he could devise an imaging strategy that could bypass the diffraction limit and enable single-molecule microscopy. By dispersing these excitable molecules in a gel, Moerner could keep them at a distance greater than the Abbe/Rayleigh limit of 0.2 micrometre. Individual molecules in this scattered distribution could then be switched on and off at will, and the glow from individual molecules could then be observed with a regular microscope. This discovery demonstrated that it was possible to optically control the fluorescence of single molecules.

Near-field microscopy

What Moerner achieved was actually a solution to a problem that the third laureate, Betzig, had been struggling with earlier. Like Hell, Betzig had also been obsessed with the idea of somehow surpassing the diffraction limit. In the early 1990s, Betzig was working at Bell Labs, New Jersey, with a new technique of optical microscopy called “near-field microscopy” in which light is emitted from an extremely thin tip placed only a few nanometres from the study sample. Near-field microscopy also enables bypassing the diffraction limit as the Abbe and Rayleigh criteria are valid only when the light that is diffracted is detected from far-field as the light propagates without any restrictions. This condition is violated in near-field microscopy and the resolution limit can be circumvented.

But the method has serious weaknesses, chief among them being that it is difficult to visualise structures below the cell surface because the light emitted has a very short range. Around that time, he had also come across Moerner’s work of making single molecules fluoresce and had detected the same phenomenon using near-field microscopy. He wondered whether a regular microscope itself would do the trick of bypassing the diffraction limit if different molecules glowed in different colours.

Betzig’s idea was to make the telescope register one image per colour. He thought that if he could ensure that molecules of one colour were dispersed farther apart from each other than the 0.2 micrometre limit, their position could be determined very accurately.

If these images were superimposed, the complete image would then have a resolution that would be better than the conventional limit and the molecules with red, green or yellow could then be distinguished even if the distances between them were only a few nanometres. In 1995, he published his theoretical ideas in Optics Letters. But he concluded that near-field microscopy could not be improved any further as his idea could not progress because of the lack of molecules with sufficiently differing fluorescence properties. Also, not very comfortable in the prevalent environment within the academia, he gave up research altogether and joined his father in his business. But the thought of surpassing the diffraction limit continued to haunt him.

While leafing through research literature one day, he came across the GFP of which he was not aware before. Realising that there was now a protein that could make other proteins visible inside cells, Betzig came back to research and revived his ideas using the GFP. The real breakthrough came in 2005, when he stumbled upon fluorescent proteins that could be made to fluoresce at will—similar to those Moerner had discovered in 1997. He realised that here was a protein that would enable him to implement his 10-year-old idea (Figure 5).

The fluorescent molecule did not have to be of different colours fluorescing at the same time. They could just as well fluoresce at different times. And just a year later, he could actually build a practical device that realised his idea in collaboration with his colleagues at Janelia Farm Research Campus, Howard Hughes Medical Institute, Virginia, U.S., where he had gone back into research.

It is interesting to look at the way he achieved this. For example, the glowing protein is coupled to the membrane enveloping the lysosome, the part of the cell that contains enzymes that break down complex molecules and digests them. Using a weak light pulse only a fraction of the proteins in the sample is made to glow. Owing to their small number, the positions of all the glowing proteins are such that their distances from one another are greater than 0.2 micrometre. By this, the position of each glowing protein could be registered very precisely.

When the fluorescence dies out after a while, a new subgroup of proteins is activated so that another fraction of them is made to fluoresce this time. This gives rise to another image and this procedure is repeated several times. By superimposing such a series of images, Betzig obtained a super-resolution image of the lysosome membrane with a resolution far better than the diffraction limit (Figure 6). This path-breaking work was published in Science in 2006. At present, the 54-year-old Betzig is the Group Leader at the Janelia centre.

The methods developed by the trio of laureates have led to several nanoscopy or super-resolution microscopy techniques, which are being used all over the world. Their works have resulted in a very large community of researchers in the field in which the three continue to be active.

While the super-resolution techniques have enabled the 61-year-old Moerner to study proteins related to Huntington’s disease, they have enabled Betzig to track down cell division inside embryos. Hell and his group on the other hand are constantly exploring the potential of these microscopes to be able to see not just individual molecules but also what is inside them as the STED technique he developed has no limit to the minutest dimensions that one can see.

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