From the August 2016 issue of The Rotarian

Four hundred years ago, the invention of the microscope gave us a glimpse into an aspect of the world too tiny to be seen by the human eye. The microscope works by capturing light shone on or through an object observed through lenses, which magnify the resulting image so we can see it. But a microscope has its limits. In 1873, German physicist Ernst Abbe discovered that the ability of a microscope to see past a certain size was limited not by the quality of its lens, but by the wavelength of light shining into it. And since the wavelength of visible light falls within a specific range – and can’t be altered – then it was believed the ability of a microscope was limited. It’s a concept called the diffraction barrier.

Because of this, microscopes have been able to create a clear image only up to 200 nanometers (about 500 times smaller than the thickness of a human hair). But that leaves out a world of detail – and even the more advanced microscopes that followed, such as the electron microscope, were unable to show what was going on in a living cell.

It was assumed that the diffraction barrier was impossible to overcome. But physicist Stefan Hell, a member of the Rotary Club of Göttingen, Germany, thought differently. Hell, who grew up under communist rule in Romania, spent his youth seeing a chasm between what officials told him to believe and what was true. Despite an entire scientific community of detractors who did not think advance in this area of microscopy was possible, Hell pursued a career dedicated to breaking past the long-believed barrier with hopes of creating a light microscope capable of illuminating a world that humans had never seen.

Eventually Hell and his colleagues found that by switching the ability of molecules to fluoresce on and off to make them discernible, the diffraction barrier could be broken. His discovery created a new field of science called superresolution microscopy, allowed scientists to see details less than 200 nanometers apart using focused light for the first time, and, in 2014, won him the Nobel Prize in chemistry. Hell, 53, a director at the Max Planck Institute for Biophysical Chemistry, spoke with contributor Erin Biba about what it’s like to break impossible barriers.

THE ROTARIAN: The discovery that led to your Nobel Prize was overcoming a barrier in microscopy that had existed for 130 years. Why did you pursue a discovery that most people believed was impossible to achieve?

HELL: In my childhood, I noticed that the official “truth” conveyed by the communist rulers on TV and in newspapers did not coincide with the reality around me. I must also add that we were well-informed about what was really going on in the world by listening to West German radio stations, including Radio Free Europe. Therefore, as a child I was confronted with the fact that the official truth was not the real truth, which I could see by opening my eyes. I became more skeptical about official and accepted opinions. When I was confronted with the then well-accepted resolution barrier of light microscopy, I wondered if it was really true or just a well-accepted scientific opinion.

TR: Four Romanians have won a Nobel Prize, which seems like a lot for such a small country. What is the attitude toward science and research there?

HELL: George Palade was one of the most influential cell biologists ever. Two laureates were writers, namely Elie Wiesel and Herta Müller. At the time I grew up there, Romania was a communist country and virtually nobody was allowed to build up personal wealth. As a consequence, people were quite keen to educate their children well and early on, because it was the only way to master life reasonably well. It is also fair to state that all Romanian-born Nobel laureates did their decisive work, be it in literature or in science, outside the country – either in Germany or in the United States. I should add that Romania has come a long way since 1989. It is now a democracy and a staunch member of the free world.

TR: Why is it important that we advance the field of microscopy and increase the resolution of the devices we already have?

HELL: The resolution of the light microscope was fundamentally limited to about 200 nanometers. The tiny details in a human cell, such as the spatial arrangements of proteins, are yet smaller by ten- to one-hundredfold. While we have the electron microscope to see such small details, the electron microscope cannot image living cells and also has difficulty identifying proteins and other molecules in the cell specifically. This is highly relevant so that we know which molecules are where to uncover the principles of how, where, and ideally when molecules come together to perform their cellular functions, and under what circumstances such processes go wrong and cause disease – something the light microscope is really good at. For these reasons, it was so important to make the light microscope sharper. Modern so-called superresolution fluorescence light microscopes can do both: They enable us to see proteins at tenfold higher resolution and also in living cells.

TR: When you began the work that led to your groundbreaking microscope, you were alone – in fact, somewhat of an outcast – as the optic community didn’t embrace the ideas you were testing. How did being on your own affect your discovery?

HELL: I think this was a major ingredient in my becoming successful and finding a solution. If you work more or less on your own, you can start thinking about the problem very deeply. Isolation can be critical to going a very different route. Studying the field, I was aware of previous approaches that all turned out to be futile, and I looked into the math of diffraction theory again to convince myself that there really was no loophole to focus light any sharper with a single lens. Two lenses from two sides helped along the axis, as I was able to show, but I concluded that, just like everyone believed, the focusing of light really was not a strategy that, on its own, would support higher spatial resolution. The “problem” in a way was that optical diffraction was an established subject. I had the insight that the solution would not come from thinking about the light alone, but from involving the molecules, which we examine in the microscope themselves. If I had worked with a group of other people initially, I would not have started to do what I did. Actually, many people told me that it would not work. Sometimes, being naive and a bit ignorant about the difficulties of a subject helps to see it from a radically different angle.

TR: Can you explain in simple terms (for those of us who aren’t educated in physics) how you overcame the barrier of one quarter of a thousandth millimeter imaging and allowed for images that had been fuzzy to become clear?

HELL: In the past, microscopes were designed to discern adjacent tiny features by focusing the light as sharply as possible in space. However, this strategy reaches limits by the fact that light propagates as a wave: The sharpest spot of light that can be created in space is just half the wavelength of light, about 200 nanometers. I realized that in order to discern adjacent tiny details, one has to adopt a different strategy: I made sure that not all the features that are illuminated by that finite spot of light are able to send light back to the eye or to the electronic light detector, to be precise. By making sure that the features light up consecutively, one can tell them apart. 

TR: So, you achieved your success in microscopy by making molecules dark rather than the traditional method of illuminating them. But when you shared your theory about this with the community, you received some negative feedback. Why did you continue to pursue your ideas?

HELL: It is important to be realistic and thorough in one’s appraisal of the problems ahead. I realized that my way of thinking about the problem was radically new and definitely correct. I could not find a major physical flaw in my concept. The only challenges that remained were technical, meaning they could be met by improving the instrumentation used for building the microscope. The good thing is that such technical challenges can be overcome by development. This is how I knew that I would be right in the end.

TR: Why do you think the scientific community wasn’t more receptive to your ideas at the time?

HELL: It wasn’t particularly receptive because the diffraction resolution barrier was well-accepted in the scientific community. It was textbook knowledge. The situation was aggravated by the fact that there had been many false prophets before me who claimed that they would be able to overcome the resolution  barrier, but failed. People therefore simply thought: “So many people have tried and failed. Why should this guy succeed?”

TR: What do you think are the most important advances that will result from our ability to look at the world on the small scale?

HELL: Currently, superresolution microscopy techniques are spreading into different realms of application, primarily in the life sciences and biomedicine. We have helped HIV experts understand infection pathways much better, and my colleagues at Stanford have shown how one can advance the understanding of protein aggregation in Huntington’s disease. But these are just two examples. There are many others, and many more will follow.

TR: Are there ways in which your work could help Rotarians tackle humanitarian issues related to health, clean water, or sanitation, for example?

HELL: Making microscopes that can help researchers better understand what goes on inside cells will definitely impact human health in the long run. It enables the discovery of new molecular mechanisms in biology, but also pathology. New hypotheses can be carefully tested in the cell, the great advantage being that one can observe so directly. This is something that does not work overnight, but as the capabilities of observing things grow, so, too, will the ability to interpret what is seen, feeding into new knowledge and understanding that can be used to advance medicine. 

TR: Is there any research going on using your microscope that you find particularly interesting or promising?

HELL: There are over 1,000 laboratories worldwide – and the number is still growing – in which science is being conducted using the new opportunities arising from seeing details much better in living cells. Exciting topics range from the changing organization of the shape- and structure-giving cellular cytoskeleton, for example in neurons, to the highly intricate organization of the cell nucleus and genetic information storage, the division machinery of cells and its potential defects, and new kinds of insights in virology or neurodegenerative disease that I touched on before.

TR: Now that you have overcome one barrier in the level of microscopy, has a new barrier developed? In other words, are there still limits on how much we can see through a microscope? Do you think those limits will one day be overcome as well?

HELL: The resolution barrier has been broken, but the new limits of resolution have not been reached yet. Those new limits are given by the size of the molecules themselves, about 1 nanometer. Scientists working in the field are trying to make the microscopes better and better, so that the maximum level of performance possible will be reached for the benefit of science and medicine, and thus of mankind.