Researchers from Stefan Hell’s department at the Max Planck Institute for Biophysical Chemistry in Göttingen have achieved yet another breakthrough in light microscopy: With their new MINFLUX microscope they can separate molecules that are only a few nanometres apart, meaning its resolution is 100 times higher than conventional light microscopy, and about 20 times higher than super-resolution light microscopy.
The 2014 Nobel Prize in Chemistry was dedicated to the breaking of the optical diffraction limit. For more than a century, half the wavelength of light – about 200 nanometres – was considered the absolute limit for light microscopes. When Stefan Hell was studying physics in Heidelberg, this limit was still taught – but Hell couldn’t accept it. By developing stimulated emission depletion (STED) microscopy the following years, he was the first researcher to successfully venture beyond this limit in the 1990s, both theoretically and experimentally. Based on these breakthroughs he received the 2014 Nobel Prize in Chemistry, together with the American physicists Eric Betzig and William E. Moerner, “for the development of super-resolved fluorescence microscopy”.
But how does STED work, and how does it lay the foundations for MINFLUX? Hell explains STED in his own words: “If I cannot resolve two points because they are too close together and they are both emitting fluorescence, I need to darken one of them – and suddenly, you’re able to see the other point. If I make sure that all molecules are dark, except the one or the few that I’m interested in, then I can finally resolve this one, or these few.” The darkening is achieved by optical interference: Since a laser beam is used to excite fluorescence in molecules, it’s also possible to darken some molecules in the probe with a second laser beam whose wave properties cancel out the first beam. This second laser beam is doughnut-shaped, leaving only a small centre spot still emitting fluorescence. Thus STED functions by depleting a circular region of the sample, while leaving a small focal point (see graph below).
With the MINFLUX microscope, Hell’s research group combined the advantages of STED microscopy with the principles of PALM (photo-activated localization microscopy) developed by Eric Betzig. In PALM, also called PALM/STORM, single molecules are also excited by switching them on and off, but these molecules light up randomly, they’re not targeted. Betzig and his colleagues would use a very short laser pulse, thus exciting only a few molecules which could then be localised. As these molecules bleach out, the researchers turn the laser on again to see the next batch and so forth. Finally, the entire probe has been activated, seen and plotted. The result is a graphic resolution far beyond the diffraction limit.
With this approach, PALM already operates on the single-molecule level but the exact location of each molecules isn’t easily determined. With STED, the location of an excited molecule is well known, but the laser beam isn’t able to confine its emission to a single molecule. MINFLUX, on the other hand, switches individual molecules randomly on and off. Simultaneously, their exact positions are determined with a doughnut-shaped laser beam, something we know from STED. But in contrast to STED, this doughnut beam excites the fluorescence instead of darkening it. So if a molecule is located on the ring, it will glow; if it is exactly at the dark centre, it will not glow – but its exact position will be known.
Dr. Francisco Balzarotti, a researcher in Hell’s department, developed an algorithm to locate this centre position fast and with high precision. “With this algorithm, it was possible to exploit the potential of the doughnut excitation beam,” the first lead author of the Science paper explains. Klaus Gwosch, a PhD student in Hell’s group and third lead author, obtained the molecular-resolution images: “It was an incredible feeling as we, for the first time, were able to distinguish details with MINFLUX on the scale of a few nanometres”, the young physicist describes the team’s reaction to the potential of studying life at the molecular level.
In addition to the high optical resolution, this new microscope has another advantage over both STED and PALM: high temporal resolution. Stefan Hell: “MINFLUX is much faster in comparison. Since it works with a doughnut laser beam, it requires much lower light signal, i.e. fewer fluorescence photons, per molecule as compared to PALM/STORM for attaining the ultimate resolution.” MINFLUX stands for “MINimal emission FLUXes”, alluding to this reduced light requirement. Already with STED, researchers had been able to record real-time videos from the inside of living cells. But now it is possible to trace the movement of molecules in a cell with a 100 times better temporal resolution.
Yvan Eilers, another PhD student involved and the second lead author of the Science paper, was responsible for ‘filming’ protein actitivity within a living cell. He filmed the movements of ribosome subunits inside a living E. coli bacterium. “The past has shown that major resolution enhancements have led to new insights into the biology of cells, as STED and PALM have demonstrated,” Eilers elaborates. “Now everybody here is optimistic that this will hold true for MINFLUX as well.” The researchers in Hell’s group are convinced that in the future, even extremely fast changes in living cells will be investigated with the help of their new microscope, for instance the folding of proteins.
I asked Klaus Gwosch whether other research groups had already been in touch to acquire a MINFLUX microscope. “The Science publication has of course reached a large international audience,” he replied. “Currently, the department of NanoBiophotonics in Göttingen has the only MINFLUX microscope, but we expect other research groups to adopt and implement our approach.” His boss Stefan Hell agrees: “I am convinced that MINFLUX microscopes have the potential to become one of the most fundamental tools of cell biology. This could revolutionise our knowledge of the molecular processes occurring in living cells.”
Both Yvan Eilers and Klaus Gwosch will participate in the 67th Lindau Nobel Laureate Meeting this summer as young scientists, together with their doctoral adviser Stefan Hell. William Moerner, the third recipient of the 2014 Nobel Prize in Chemistry, will also attend and talk about super-resolution microscopy. We are looking forward to an interesting and inspiring week in Lindau!