It was 1993, on a Saturday morning in Turku, Finland. As Stefan Hell was leafing through a physics book, he suddenly had the idea of how he could overcome the resolution limit of light microscopy. The problem had been occupying him for years: In conventional light microscopes, objects smaller than 200 nanometers appear only as blurry spots.

While it is possible to achieve higher resolutions with electron microscopes, operating such a microscope requires a vacuum. Additionally, the object must be prepared in ultra-thin slices. Consequently, it is not possible to observe biological processes in living cells. Fluorescence microscopy, on the other hand, allows the observation of the interaction of individual proteins in living cells. Selected proteins are coupled with fluorescent molecules, called marker molecules, and illuminated by a light beam. The backscattering of the thus excited marker molecules can be observed with conventional light microscopes – but only up to the mentioned resolution of 200 nanometers. Densely packed proteins appear as a blurred light spot.

With the STED microscope, Stefan Hell found a way to overcome this limit. Simply put, he came up with the idea of covering the round light beam that excites the marker molecules with a ring-shaped "template." In the center of this "template" is a "hole" whose diameter can theoretically be reduced indefinitely. With this "hole lens," the STED microscope can resolve details smaller than 200 nanometers. For this discovery, Stefan Hell received the Nobel Prize in Chemistry in 2014.

How Stimulated Emission Depletion Microscopy Works

When trying to focus light on a point as small as possible, the wave is diffracted, and the point appears as a light spot of about half the wavelength (approximately 200 nanometers). A STED microscope achieves significantly better resolutions. The trick lies in the interplay of excitation and depletion of the fluorescent marker molecules.

A laser beam excites the marker molecules of a specimen in a focus of 200 nanometers in diameter. Simultaneously, a second donut-shaped "depletion beam" with a longer wavelength ensures that no fluorescent dyes are stimulated in the outer area of the excitation focus. Only the marker molecules in the center light up. If the intensity of the depletion beam is greater than that of the exciting laser beam, the central area where the marker molecules are still excited becomes much smaller than the area illuminated by the exciting laser beam. The more intense the depletion beam, the smaller the diameter of the resulting light spot.

The object is scanned with this light spot, and the measured fluorescence signal is evaluated by a computer and displayed on a monitor. The achievable resolution is far beyond the previous diffraction limit of 200 nanometers.

But the path to the prize was long and winding. Stefan Hell had studied physics in Heidelberg. After his diploma in 1987, he worked with Siegfried Hunklinger on his dissertation on "Imaging Transparent Microstructures in the Confocal Microscope." Siegfried Hunklinger, together with Josef Bille – both professors of applied physics – had founded the company Heidelberg Instruments in the Technology Park at Neuenheimer Feld. There, Stefan Hell was to conduct his research.

"I was supposed to examine lithographic structures on silicon wafers using confocal laser scanning microscopy as a doctoral student. I was torn: On the one hand, I found it appealing to write my dissertation in an industrial environment. On the other hand, I wanted to create values that were also of interest to physicists. But my investigations were very technical. At times, I was frustrated and almost gave up. But then I came up with the idea of overcoming the diffraction limit in light microscopy using laser scanning technology. Without the application-oriented environment of the Technology Park Heidelberg, I would never have gotten into microscopy and would not be a Nobel laureate today."

The idea of developing light microscopes with higher resolution than before did not let Stefan Hell go. At EMBL, he was able to take another step towards realization. After his doctorate, Stefan Hell developed the idea for 4Pi microscopy, which uses two objectives to increase resolution perpendicular to the focal plane. A DFG postdoc scholarship allowed Hell to prove the functionality of 4Pi microscopy. In Turku, he finally discovered the principle of STED microscopy. After another stop in Oxford and his habilitation in physics at the University of Heidelberg, he led a junior group at the Max Planck Institute for Biophysical Chemistry in Göttingen. Here, he was able to demonstrate that STED microscopy was also practically applicable.

"Looking back, I think young scientists should be allowed to think and act unconventionally. Unusual characters who want to make a difference must be given freedom beyond standard careers. I became the head of a junior group at the MPI in Göttingen, even though I had hardly any high-profile publications at the time. Unfortunately, such criteria are often overweighted. One must engage more intensively with the content when assessing young scientists, not with superficial parameters. But that takes time."

In 2002, Stefan Hell was appointed director at the MPI Göttingen. He teaches at the universities of Heidelberg and Göttingen. In Heidelberg, Stefan Hell led a small department at the German Cancer Research Center (DKFZ) from 2003 to 2016. Since 2016, he has also been a director at the Heidelberg MPI for Medical Research, heading the "Optical Nanoscopy" department.

In 2011 and 2012, Stefan Hell founded two successful companies in Göttingen, of which Abberior Instruments is also based in Heidelberg. The establishment of the Heidelberg location led to an astonishing coincidence.

"When planning my spin-offs, I remembered my observations in the corporate world. I had set myself three things: First, I wanted to develop products with my companies not just centered around my own technology but directly for customers. Second, I wanted to start my companies without the help of investors to avoid being dependent on external decisions. Third, I learned in Heidelberg that it is wise to establish a company in a technology park close to the university campus. From my own experience at Heidelberg Instruments, I knew that students or postdocs are more likely to write their qualification papers in an industrial company located on campus. This way, a technology company can attract young, motivated people.

Therefore, I insisted on founding my companies on the university campus in Göttingen. In Heidelberg, I also got a location on the Technology Park premises at Neuenheimer Feld – by chance, exactly in the rooms where I wrote my dissertation at Heidelberg Instruments."

by Dr. Stefan Burkhardt

Download: Testimonial from Prof. Dr. Stefan Hell