Seeing the Invisible, Achieving the Impossible

Scientific breakthroughs tend to occur as short bursts of activity that overturn previous conventions and long-held beliefs. The ingredients that produce rapid advances often include cognitively diverse teams and a high degree of tenacity from the researchers.

Sessions at the 71^st Lindau Nobel Laureate Meeting highlighted excellent examples of such breakthroughs in recent history, focusing on three techniques that allow scientists to see far beyond the capabilities of the human eye. Developments in super-resolution optical microscopy, cryogenic electron microscopy (cryo-EM), and X-ray crystallography have revolutionised aspects of not only chemistry, but also biology, medicine, and even physics.

Pushing the Resolution Limit

Stefan Hell received the 2014 Nobel Prize in Chemistry “for the development of super-resolved fluorescence microscopy,” a radical achievement that overcame a fundamental assumption of physics. The Abbe diffraction limit stated that optical microscopy could never obtain a better resolution than half the wavelength of light. In groundbreaking work that stunned the world, Hell discovered that fluorescent molecules could help him cross this presumed threshold with the invention of a technique he called stimulated emission depletion (STED) microscopy.

In his #LINO22 lecture on Thursday 30 June titled ‘Molecular-Scale Resolution in Fluorescence Microscopy’, the Nobel Laureate described his laboratory’s efforts to further improve the resolution of fluorescence microscopy down to the level of a single molecule.

“In principle, STED… conceptually can have a spatial resolution that goes down to the size of a single molecule. But at the time the Nobel Prize was given in 2014, the resolution went down to about 20 nanometers,” said Hell. “The microscopes crossed the threshold, but did not attain the ultimate limit.”

To attain a molecule-size 3D-resolution of roughly 1 nanometer, only one fluorescent molecule should be allowed to emit at a time, and that molecule must be localised in the imaging area with precision.

“It is clear that you need many photons to define a coordinate in space. If you had just one photon, due to diffraction, it would go anywhere,” he said. “You couldn’t define a coordinate with one photon – it’s ridiculous. The Heisenberg uncertainty principle would not allow you to do so.”

In the end, Hell and his colleagues came up with the concept of minimal photon fluxes (MINFLUX), which uses a donut-shaped pattern of illumination light to locate the molecule. It works by placing the zero-intensity minimum of the donut beam close to a single activated fluorophore and moving it around ever so slightly, so that the position of the molecule is established with high precision.

With MINFLUX, researchers can resolve structures as small as a single molecule, along all three dimensions, with unprecedented speeds. With the help of chemists, Hell foresees a plethora of biological applications including molecular maps in 3D living cells, molecular dynamics like protein folding and interaction, and drug discovery.

“We have reached now the fundamental limits of fluorescence imaging, and many applications are awaiting. For that, we really need very good, dyed-in-the-wool chemists because we have to sort out the question of, how do we attach the fluorophore to the protein of interest or another molecule, such as DNA and so on,” Hell said. “Many huge discoveries can be made once this remaining problem – which is not a physics problem of course – is solved.”

The Cryo-EM Revolution

Electron microscopy was not applied to structural biology until the 1960s, when scientists first began to recognise that it could be used to gather molecular information. Early studies were only on molecules in tight crystalline arrangements, and it was thought impossible to apply the technique to single molecules.

A revolution in the field occurred with the invention of cryo-EM, which preserves the structure of biological specimens by cooling them to cryogenic temperatures in an environment of vitreous water. Joachim Frank received the 2017 Nobel Prize in Chemistry “for developing cryo-electron microscopy for the high-resolution structure determination of biomolecules in solution.”

In his Agora Talk on Monday 27 June titled ‘Biological Molecules Captured in Motion by Cryo-EM’, Frank gave insights into the history of cryo-EM, where the technique stands now, and future directions. He came up with a method for 3D reconstruction of asymmetric molecules by single-particle techniques that merged blurry 2D images into a sharp 3D image.

“The electrons are very strongly damaging, so we need to keep the dose down, which means we have a lot of noise and means we have to average over a very large number of molecules. It’s a very simple idea and very straightforward, but the devil is in the details,” said Frank. “One has to develop programs, and that’s exactly what I got credit for – developing the math and the computation techniques in order to extract all that information.”

The advent of cryo-EM has moved biochemistry into a new era, with researchers now able to routinely produce three-dimensional structures of biomolecules. “Now, if you go on the internet of cryo-EM of macromolecules, you see an unbelievable variety of things that have been solved,” he said. “The best resolution is 1.2 angstrom. That is like a dream situation. We never thought that we would get to that place.”

Frank believes the future of the field lies in time-resolved and state-resolved cryo-EM techniques to get information on the changing of molecular conformation. His laboratory and others are starting to work on this aspect using microfluidic chips to control the timing and follow a reaction between two or three components.

The Century of Vision

The Agora Talk of Robert Huber on Tuesday 28 June titled ‘The Century of Vision in Molecular Biology’ began with a quote from English physicist Robert Hooke: “By the invention of optical glasses and by the means of telescopes, there is nothing so far distant but may be represented to our view and by the help of microscopes, there is nothing so small as to escape our inquiry; Hence there is a new visible world discovered to the understanding.”

Huber, recipient of the 1988 Nobel Prize in Chemistry “for the determination of the three-dimensional structure of a photosynthetic reaction center,” used the quote to demonstrate that the desire of humans to see far and near goes back much further than a mere century ago. His talk served as a brief history of the last 100 years of microscopy and how such advances have enabled revolutions in both biology and medicine.

“We learned in this last century to use the whole spectrum of electromagnetic radiation for imaging,” said Huber, who covered breakthroughs in super-resolution light microscopy, nuclear magnetic resonance spectroscopy, X-ray crystallography, single particle cryo-EM, and other techniques.

Later, he described his Nobel Prize-winning work that allowed the determination of the structure of a photosynthetic reaction center from a purple bacterium with X-ray crystallography. He added that, in general, the study of proteins and their structure can give insights into how the sequence composition of molecules change across generations, a fascinating field known as molecular evolution.

“My first protein structure was in late ’68, an insect heme protein. I didn’t know at the time that it contained heme, so the structural analysis showed that it was a heme protein,” said Huber. “Then we found that this heme protein is related to the classical sperm whale myoglobin – a great example of molecular evolution.”