Imaging has become a common, widespread technology in modern biological laboratories. Rapid advances in imaging techniques and instrumentation over the last several decades have opened a window into aspects of biology once thought impossible to visualise.
At the 70th Lindau Nobel Laureate Meeting, two sets of Agora Talks from four Nobel Laureates – Stefan Hell, Joachim Frank, William E. Moerner, and Steven Chu – gave an overview of the latest biological imaging techniques and their applications. On Monday, Hell and Frank discussed their respective specialties of super-resolution fluorescence microscopy and cryo-electron microscopy (cryo-EM). Moerner spoke on Thursday about combining single-molecule microscopy and cryo-EM, while Chu examined upconversion nanoparticles.
Pushing the Limits of Microscopy
In 1873, the microscopist Ernst Abbe specified a physical limit to the resolution of optical microscopy as a result of diffraction processes and the wave nature of light. The scattering of incoming light at the entrance to the microscope objective results in a loss of information that prevents the instrument from pinpointing the exact location of a light source within the sample.
“In the 20th century, it was widely accepted that the resolution of any lens-based microscope was fundamentally limited by diffraction to about half the wavelength of light, or about 200 nanometers,” said Hell in his Agora Talk. “In fluorescence microscopy – an important technology in the life sciences – the gold standard was the confocal microscope, which was also limited to about 200 nanometers in spatial resolution.”
Hell, along with Eric Betzig and Moerner, discovered an ingenious way around the Abbe diffraction limit that brought optical microscopy into the nanodimension. They were awarded the 2014 Nobel Prize in Chemistry for two separate super-resolution techniques: stimulated emission depletion (STED) microscopy and single-molecule microscopy.
Both techniques rely on the same principle of separating out fluorescent features or molecules in order to shrink the minimum distance between two distinguishable points in the sample. The resolution of STED microscopy had dropped to 20 nanometers – a considerable improvement from 200 nanometers, but still far away from the theoretical limit of 1 nanometer. “The goal in my lab in the last five to seven years has been to get down to that limit – namely, resolution that is at the size of a molecule,” said Hell.
Instead of making tweaks to the instrumentation itself, Hell wanted to achieve better resolution through major conceptual advancements. Specifically, he combined the strengths of the two super-resolution techniques: the injection of a supersharp coordinate through donut illumination from STED microscopy and single-molecule separation with fluorescent proteins that can be activated at will from single-molecule microscopy. The new method, called MINFLUX, has an incredible spatial resolution of 1 to 3 nanometers.
“This is really crazy. I remember the days in the late 80s and early 90s, people warned me not to speak about breaking the diffraction barrier, and definitely not about improving the resolution by more than a factor of two because this is not acceptable and so on,” he said. “But now we are better by a factor of 100, and this barrier is gone in a very fundamental way.”
Next, Frank shifted gears a bit for his Agora Talk by speaking about single-particle cryo-EM, which uses beams of electrons rather than light to visualise biological molecules in their native states. Before the advent of cryo-EM, scientists were having great success determining the structure of biomolecules with X-ray crystallography – so why the need for another method?
Many molecules do not form highly ordered crystals, and crystal packing itself means that molecules are not visualized in all conformations or binding states that are important for function. In fact, many crystal structures depict the molecule in a state that would be incapable of biological function.
However, electron microscopy had issues for many years that prevented it from producing consistent, quality results. “Electrons strongly damage the molecules and there is a need for a very low dose. As a consequence, images are very noisy and normally to get any clear structure, you need to average over many molecules that have the same structure,” said Frank, who received the 2017 Nobel Prize in Chemistry.
In response, Frank developed cryo-EM, a revolutionary electron microscopy technique that cooled molecules to cryogenic temperatures in order to reduce beam-induced radiation damage. It then gathered structural information from images of single, unattached molecules that were free to assume all naturally occurring conformations. A single snapshot could already give hundreds of particle views, and more images result in enough information for a detailed three-dimensional reconstruction.
In 2012, single-electron detecting cameras transformed the entire field by drastically improving recording quality and from there, the popularity of cryo-EM exploded. Cryo-EM signs responsible for more than 4,500 depositions into the Protein Data Bank, a worldwide database for the three-dimensional data of large biological molecules.
New Methods for Biological Imaging
In his Agora Talk, Moerner described efforts to combine the power of super-resolution fluorescence microscopy with that of cryo-EM. Moerner received the 2014 Nobel Prize in Chemistry for the development of single-molecule microscopy, a super-resolution optical technique that uses fluorescent proteins to switch biomolecules on and off.
With single-molecule microscopy, specific proteins can be labeled and identified, while cryo-EM makes it difficult to directly distinguish types of molecules. On the other hand, cryo-EM has a much higher spatial resolution than single-molecule microscopy and can show the surrounding structural and morphological context of a sample. “The strengths of super-resolution fluorescence actually are the weaknesses of electron microscopy and vice versa,” Moerner said.
He discussed new developments and challenges in uniting the two techniques to study the same sample. Labeled cells must be plunge-frozen on an electron microscope grid and kept very cold at all times to avoid the formation of crystalline ice. The sample must also be protected from humidity to avoid the condensation of water. It undergoes single-molecule microscopy before being carried to an electron microscope. Lastly, the two images must be registered together.
One focus of Chu’s recent research is the application of upconversion nanoparticles – nanoscale particles that convert two or more incident photons of low energy into one emitted photon with higher energy – as optical probes to biological imaging. They have a number of advantages such as a high signal-to-noise ratio, photostability and biocompatibility. His laboratory started making them four years ago and, over time, learned to improve their brightness and size.
“We have our nanoparticles, shine in 980 nanometer light and it is upconverted into the visible, which can be emitted in the red, green, or blue depending on what impurities you use,” said Chu, who received the 1997 Nobel Prize in Physics for development of methods to cool and trap atoms with laser light. “These are photostable and go for many, many hours at the highest intensities with no degradation, nor do they show any blinking.”
As an example of their use for biological imaging, Chu has demonstrated that upconversion nanoparticles can successfully track the transport of nerve growth factor along the axon of a live neuron.