The Nobel Laureate nobody believed


Rebecca Pool

Monday, March 27, 2017 - 15:45
When Professor Stefan Hell first floated the theory that would break the diffraction barrier, he was ignored. Rebecca Pool plots his path from scientific curiosity to worldwide acclaim.
Professor Stefan Hell's Nobel Prize in Chemistry was a long time coming. Rewarded in 2014 for the development of super-resolved fluorescence microscopy, Hell had worked out how to break the diffraction barrier in far-field microscopy as early as 1993 but at the time his approach challenged convention and was dismissed.
Seven tortuous years later with rejection from journals far and wide, he finally published his seminal paper in the Proceedings of the National Academy of Sciences. The results stunned the skeptics, sparked interest in the overlooked topic of far-field light microscopy, and as he tells Microscopy and Analysis, helped to save his career.
"We were able to show that we could overcome the diffraction barrier which made a huge difference," he says.
"A couple of years earlier I had to struggle to find money to basically live and now I was known within the microscopy community and was finally getting job offers," he adds. "It changed everything."
Hell's journey to acceptance and recognition in the world of science began in communist Romania. His parents were passionate about education and filled their family home with as many books as they could get their hands on.
"In those days, information was scarce but my parents tried to buy books that were not biased by communist ideology," he says. "We listened to Western Radio, watched, for example, the moon landings - probably not the live event - and I soon became fascinated by scientific progress as well as nature."
Resolution of confocal versus STED microscopy: proteins of nucleus labelled with fluorescent dyes.
Still, stifled by the communist regime, the family emigrated to Ludwigshafen, West Germany in 1978, when Hell was fifteen years old. As his parents adapted to their new lives, Hell thrived at secondary school, inspired by a physics teacher and new found freedom.
"In Romania, I had already realised that what was said publicly was not necessarily the truth, so in the more liberal West I was prepared to be skeptical at times," he says.
"And I now realise that teachers can play a crucial role; at school I was the best in physics and my teacher really encouraged me to study the subject," he adds.
Hell's education went so well that come 1981, the enthusiastic 18 year old left school a year early to attend the nearby University of Heidelberg to, quite naturally, study physics. Feeling a little daunted, Hell immersed himself in studies and university life.
The university itself had always housed a liberal and free-minded spirit, and exams were kept to a minimum. Hell loved it.
"The atmosphere allowed a lot of freedom of thinking and creativity, and I could basically go to any lecture I wanted to," he recalls.
"I was fascinated by physics and wanted to understand things at a deep level, so spent hours pouring over text books so I really understood the basic points," he adds. "I just wanted to know the essential phenomenon behind the greater picture."
Hell's obsession with understanding the essence of any subject would one day prove crucial to the world of microscopy but at the time and like many other young physicists, he was drawn to theoretical particle physics. However, without firm financial backing and given Germany's surplus of physicists, Hell followed advice from an older colleague and opted for a diploma thesis in the very practical microlithography.
Practical path
Low temperature solid-state physicist, Professor Siegfried Hunklinger, had just moved to Heidelberg and wanted to produce piezoelectric surface-wave transducers lithographically using laser scanning optical systems. Hell, against his better judgment, concurred and several years later found himself still working with Hunklinger on his PhD.
By now, Hunklinger had launched a start-up company to develop laser-scanning systems for myriad applications including confocal microscopy and microlithography inspection. Hell was tasked with finding out if this up and coming microscopy method could be used to accurately measure transparent 3D photoresist microstructures.
"This is how I very reluctantly got acquainted with optical microscopy, and I found it so very very boring," he laughs. "At the time, I thought there was absolutely nothing interesting you could do with optical microscopy so I started trying to work out if there was actually something interesting I could do with this subject of 19th century physics."
"And that's when I thought wouldn't it be cool to break the diffraction barrier?" he says. But the year was 1988, breaking the diffraction barrier was deemed crazy, Hunklinger’s start-up was about to go bankrupt and Hell had a PhD to complete.
Two-colour STED image of a glisoblastoma, the most frequent tumour in adults; confocal image is blurred. [Buckers, Wildanger, Kastrup, Medda/Max Planck Institute for Biophysical Chemistry]
While finishing his thesis, he ruminated over the diffraction barrier, mulling over the Stark and Zeeman effects, and the like, and even hit on the notion of molecule localisation. But in his words: "I couldn't work out how to separate the molecules.... and just couldn't find a concept that would give me an image."
Instead, he developed a two-lens method to drastically boost the axial resolution of confocal microscopy and on completing his doctorate in 1990 headed to the European Molecular Biology Laboratory, Heidelberg, to demonstrate the new concept. Working under the head of microscopy at EMBL, Dr Ernst Stelzer, he proved what would come to be known as the 4Pi microscope, a laser scanning microscopy breakthrough that would later be commercialised by Leica Microsystems.
Crucially, the microscope also showed the world that the resolution of far-field microscopy was ripe for change. And as Hell says: "I had to offer EMBL something concrete so for me, [4Pi] was just a foot in the door. I couldn't have gone to EMBL to speculate overcoming the diffraction barrier, I would have been labelled ‘crazy’."
Come 1993, EMBL funding ran out and Hell headed out to University of Turku, Finland, to set-up a small optics laboratory largely based on 4Pi microscopy with Professor Erkki Soini and former EMBL colleague Pekka Hänninen.
Stefan Hell: "Laboratories had funds to build near-field optical microscopes but I was seen as this funny guy claiming these efforts were ill-fated."
Soini had brought Hell in for his fluorescence microscopy expertise but was also willing to give him 'a little freedom' to think about the resolution barrier. By this time, Hell had well and truly realised the importance of, as he says, 'following your passion', so he made the most of this freedom.
Hell's hunch was that merely changing the way light is focused or re-arranging lenses, as in 4Pi microscopy, would not bring a fundamental leap forward in resolution. Instead, he figured changing the states of the molecules being imaged would deliver a bigger breakthrough.
Quickly his thoughts focused on fluorescent molecules, whose states could be most easily altered, and within months he had conceived stimulation emission depletion - STED- microscopy.
"I had been thinking about this for years and was so excited as I felt STED could work," says Hell. "At that very moment I wasn't one hundred percent sure it would definitely work in practice but I couldn't find a flaw in my thinking and preliminary calculations."
STED Progress
Come 1994, Hell had published his approach for a 'new type of scanning fluorescence microscope capable of resolving 35 nm in the far-field' in Optics Letters. The method was based on stimulated emission to inhibit the fluorescence of molecules and, critically, overcame the diffraction limit.
Hell was pleased but also frustrated. His second paper on a related idea submitted in the same year had been previously rejected by more popular journals as referees with expertise in near-field rather than far-field optical microscopy had requested experimental data.
"Of course, I hadn't got my my own laboratory and didn't have the means to do this. So the theory was now in Optics Letters and Applies Physics B, but I was still a kind of nobody," he says. "Laboratories across the US and Europe had funds to build near-field optical microscopes but I was seen as this funny guy claiming these efforts were ill-fated for the life sciences as super-resolution microscopy should be done in the far-field instead. It was a problem."
Still development continued and come late 1996, Hell received his habilitation from the University of Heidelberg - crucial to access a professorship - moved over to the Max Planck Institute for Biophysical Chemistry in Göttingen, Germany.
Until now, Hell hadn't written up his experimental developments on STED for submission to journals for fear of rejection, but swiftly submitted a grant for the method to the Germany Federal Ministry of Research. It was promptly rejected but granted on appeal against referee opinion. Within months, Hell had produced what he describes as his first 'compelling images' while working alongside his doctoral student Thomas Klar.
"You could clearly see the resolution was better and we showed details that would not have been attainable in a diffraction-limited system," he says. "It was now clear to people that this was going to work."
Actin filaments in cells: STED microscopy provides significant resolution improvement over that possible with confocal microscopy. [Taken on a Leica SP5 2P STED/Howard Vindin]
Come 1999, Hell's research group was expanding and he was ready to publish unequivocal proof that the resolution of far-field fluorescence microscopy could be radically improved via STED.
"STED had put super-resolution on the map and was the first concept that showed you could overcome the diffraction barrier by briefly switching fluorophores off," explains Hell. "I was now getting job offers for chaired positions from several places, including Kings College London, but accepted a counter offer from Max Planck. That paper saved my career."
Post-PNAS, far-field super-resolution imaging progress was rapid. Hell and colleagues continued to develop STED, synthesizing photoswitchable fluorophores and reporting the first nanoscale far-field immunofluorescence images using STED. 
Around the same time, the critical pieces of the super-resolution microscopy puzzle were falling into place. In 2006, former advocate of near-field microscopy, Eric Betzig, had joined the far-field, with photo-activated localization microscopy, PALM, and Professor William E Moerner, had honed single molecule localisation. At the same time, the development of the first commercial STED microscope was underway.
Eight years later, Hell, Betzig and Moerner, were awarded the 2014 Nobel Prize in Chemistry for the development of super-resolved fluorescence microscopy. But for Hell, super-resolution microscopy had already moved on.
Stefan Hell and colleagues with their latest microscope, MINFLUX.
In 2011, Hell came up with a new approach that he reckoned combined the advantages of STED with PALM as well as STORM, pioneered by Professor Xiaowei Zhuang, Harvard University. In his never-ending eagerness to get to the bottom of any phenomenon, the researcher had asked himself 'what is it that really makes these super-resolution methods tick'?
His answer was 'MINFLUX' - MINimal emission FLUXes - a concept that combines the power of the two different superresolution families. In MINFLUX, as in PALM/STORM, individual molecules are switched randomly on and off but at the same time, a doughnut-shaped laser beam - as used in STED - excites the fluorescence so the user can locate molecule position.
Fluorescing molecules: PALM/STORM microscopy (right) delivers a diffuse image of the molecules compared to MINFLUX (middle). [Gwosch/MPIBiophysical Chemistry]
Using the method, Hell has since resolved molecules only 6 nm apart, surpassing the best super-resolution fluorescence microscopy methods by up to twenty times. Results were published results in Science in late 2016, and Hell and his team will now expand the method's field of view and introduce a multi-colour scheme, ready for commercial exploitation.
With MINFLUX, users can follow faster movements than with STED or PALM/STORM microscopy. Left: Movement pattern of 30S ribosomes in E. coli (black-white). Right: Movement pattern of a 30S ribosome (green), enlarged. [MPIBiophysical Chemistry/Eilers]
Hell believes that breakthrough concepts, such as STED and now MINFLUX emerge around every decade, highlighting: "We saw STED emerge in 1994, PALM in 2006 and now we have MINFLUX."
And for him, the secret of success will always lie in tracing anything back to first principles. 
"My inclination of really trying to boil down everything to basics, as well as coming up with explanations that are different from the scientitic mainstream, helped me to develop STED and now MINFLUX," he says. "You have to be able to break it down to very simple language, if you can't do that then you just haven't understood it."
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