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Stefan Hell gathers with his team, Francisco Balzarotti, Yvan Eilers and Klaus Gwosch (from left to right), all first authors on the MINFLUX paper. Photo: © Irene Böttcher-Gajewski/Max Planck Institute for Biophysical Chemistry

The 2014 Nobel Prize in Chemistry was awarded to three researchers who saw past perceived limitations and challenged established dogma to break the optical diffraction limit. For more than a century, half the wavelength of light—about 200 nanometers—was considered the absolute limit for light microscopes. But Nobel Laureate Stefan Hell couldn’t accept that. 

In the 1990s, Hell developed stimulated emission depletion (STED) microscopy, which allowed the imaging and labeling of molecules only 20 nanometers in size—a 10x improvement over conventional microscopy. STED separates molecules not by focusing, but by turning their fluorescence on and off so features residing closer together than 200 nanometers can be distinguished by their sequential emission. 

At Pittcon 2018 in Orlando, Hell, a director at the Max Planck Institute for Biophysical Chemistry in Germany, will deliver the Plenary Lecture, titled “Optical Microscopy: The Resolution Revolution.” Hell will focus on the powerful principles he used to overcome the limiting role of diffraction, as well as MINFLUX, the latest development out of his lab—which sets the ultimate limit (~1 nanometer) in fluorescent nanoscopy. 

But when Hell delivers his lecture on February 27 at 5 p.m., he won’t be the only one speaking at the nanoscale level. There are more than a dozen oral sessions dedicated to nanotechnology, and even more poster sessions. For example, earlier in the day, Alexis Vallée-Bélisle will give an oral session about the DNA-based switches and nanomachines his team at the University of Montreal developed. The nanomachines can be activated by inputs ranging from temperature to large macromolecules, and can be used for rapid medical diagnosis, drug delivery and other point -of-care applications. In a separate session, Sam Rasmussen Nugen, an associate professor at Cornell University, will detail the design of the nanobots his lab created via phage engineering. The nanobots have the potential to significantly enhance the safety of our food by giving farmers, distributors, manufacturers and retailers rapid knowledge about the presence, or absence, of pathogens and other contaminants.

And that’s just three examples. There seems to be endless potential opportunities at the nanoscale, be it in medicine, agriculture, food safety, pharma or a different industry. With the continuing advancement of scientific tools and techniques—some of which will be on display at Pittcon 2018—researchers have been and will be able to delve deeper into the intricacies that lie at the very limitation of the nanoscale.

MINFLUX: the ultimate limit

In December 2016, Hell and his team at Max Planck made yet another nanoscopy breakthrough—they developed a new fluorescence microscope, called MINFLUX, allowing, for the first time, to optically separate molecules resting only nanometers apart from each other.

To accomplish this feat, Hell combined elements of his STED nanoscopy technique with 2014 co-Nobel Laureate Eric Betzig’s PALM/STORM technique.

Both STED and PALM/STORM separate neighboring fluorescing molecules by switching them on and off, one after the other so they emit fluorescence sequentially. However, the methods differ in one essential point: STED microscopy uses a doughnut-shaped laser beam to turn off molecular fluorescence at a fixed location in the sample, i.e. everywhere in the focal region except at the doughnut center. The advantage is that the doughnut beam defines exactly at which point the corresponding glowing molecule is located. The disadvantage is that, in practice, the laser beam is not strong enough to confine the emission to a single molecule at the doughnut center. 

In the case of PALM/STORM, on the other hand, the switching on and off is at random locations and at the single-molecule level. The advantage is that one is already working at the single-molecule level, but a downside is that one does not know the exact molecule positions in space. The positions have to be found by collecting as many fluorescence photons as possible on a camera; more than 50,000 detected photons are needed to attain a resolution of less than 10 nanometers. Therefore, it does not lend itself well to routine molecular (1 nanometer) resolution.

Like PALM/STORM, MINFLUX fluorescence nanoscopy switches individual molecules randomly on and off. However, like STED, their exact positions are determined with a doughnut-shaped laser beam. In contrast to STED, the doughnut beam excites the fluorescence. If the molecule is on the ring, it will emit; if it is exactly at the dark center, it will not emit but one has found its exact position. Essentially, the rate of emission indicates the position of the molecule in space.

Thus, MINFLUX is more than 100 times sharper than conventional light microscopy, and surpasses STED and PALM/STORM by up to 20 times. 

“Already with STED, we could 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,” Hell explained to Laboratory Equipment. “In the original paper [published in Science in December 2016], with MINFLUX, we managed to film the movement of molecules in a living E. coli bacterium, with an unprecedented spatio-temporal resolution.”

But Hell says that’s not MINFLUX’s only advantage: it is also much faster since it requires a lower light signal per molecule as compared to PALM/STORM—and the researchers believe there’s still room for improvement on the speed front. 

“We are convinced that even extremely fast-occurring changes in living cells can be investigated in the future like, for example, the movement of cellular nanomachines or the folding of proteins,” said Hell. “MINFLUX is inherently compatible with optical sectioning and suppressing unwanted background, which will be relevant to imaging and tracking in cellular contexts.”

Of course, Hell and his team still have a few more goals in mind for their research. The scientists are always working to improve the temporal resolution and image contrast of nanoscopy further, having recently pushed STED imaging to millisecond resolution by employing electro-optical scanning. Extending the region of interest in MINFLUX nanoscopy is also at the top of their minds. 

Having already revolutionized cell biology, Hell said he believes improved nanometric resolution will have a profound impact on virology and neurobiology, especially synaptic architecture. 

“For me as a physicist, it is fascinating to develop concepts for better imaging—basic concepts that, in many cases, have drastically improved aspects of methods. Seeing what advances colleagues are then making with these methods in biology is rewarding, and I look forward to collaborating more as our newest approaches mature and capabilities are being pushed to new levels,” said Hell. 

Alexis Vallée-Bélisle’s molecular slingshot is composed of a synthetic DNA strand that can load a drug and then effectively act as the rubber band of a slingshot. Photo: Marco Tripodi/University of Montreal

Nanomachines from nature

Life is based on biomolecular switches, or biomolecules that make life-sustaining decisions depending on chemical inputs. For example, biomolecules will react differently and change their course if light is present, if there’s a change in pH, or even if a different molecule shows up unexpectantly. These are the natural “machines” inside our body, fueling us at the nanoscale level. 

“Life is smart. Life is complex. It’s built on these molecular switches. So if life is built on them, why wouldn’t we use these switches to develop nanotechnology?,” Vallée-Bélisle said. 

So, that’s exactly what his lab has been doing for the past decade—trying to identify some of the natural mechanisms that could be useful in applications like drug delivery and biosensing, and then attempting to mimic those systems using simple DNA chemistry.

Inspired by nature, Vallée-Bélisle and his team of researchers at the University of Montreal have already built several useful DNA-based nanomachines, such as a programmable thermometer that can measure temperature at the nanoscale, as well as rapid, inexpensive point-of-case diagnostic tests that can detect a host of diseases. 

The lab’s newest design is a nanoscale molecular “slingshot” made of DNA that is 20,000 times smaller than a human hair. The slingshot can “shoot” and deliver drugs at precise locations in the human body once triggered by specific disease markers. 

For this research, the team was inspired by one specific natural nanomachine: hemoglobin, the protein responsible for transporting oxygen throughout a human’s body.

“Oxygen is a dangerous molecule, and not only is hemoglobin able to transport it, but it also specifically releases oxygen where the body needs it most, at a location where pH is lower than normal,” Vallée-Bélisle explained to Laboratory Equipment. “We took this idea and designed a DNA transporter for drugs.”

The slingshot is only a few nanometers long and is composed of a synthetic DNA strand that can load a drug and then effectively act as the rubber band of a slingshot. The two ends of this DNA “rubber band” contain two anchoring moieties that can specifically stick to a target antibody, a Y-shaped protein expressed by the body in response to different pathogens such as bacteria and viruses. When the anchoring moieties of the slingshot recognize and bind to the arms of the target antibody, the DNA rubber band is stretched and the loaded drug is released.

This allows a potentially toxic drug to be delivered directly to the infection site, rather than throughout a person’s entire body. For drug delivery purposes, the method could be a game changer.

Vallée-Bélisle used the DNA transporter to deliver siRNA in his proof-of-concept experiments conducted in mid-2017. The team will move forward with cells and mouse models, but are still a few steps away from employing antibodies as they continue to work with a team of clinicians. 

“This is not an easy question,” said Vallée-Bélisle. “You really need a multidisciplinary team to see which specific condition will render the transporter most useable. When you design a nanomachine, obviously the goal is to use it. So we’re still in the process of identifying specific clinically relevant cases with a good drug and a good marker.”

With the slingshot research moving forward, Vallée-Bélisle can focus on the company he started last fall based on his development of nanomachines for point-of-care diagnostics. Vallée-Bélisle believes point-of-care testing is so important due to the lag time between testing and treatment with current healthcare methods. 

The rapid diagnostic tests use steric effect—a repulsion force that arises when atoms are brought too close together—to detect a wide array of protein markers that are linked to various diseases, including allergies, autoimmune diseases, STDs, etc. The diagnostically relevant protein, if present, binds to an electro-active DNA strand, and limits the ability of this DNA to hybridize to its complementary strand located on the surface of a gold electrode. This produces a sufficient enough change in current to be measured using inexpensive electronics, similar to those used in home glucose test meters. During research, the scientists were able to detect multiple protein markers directly in whole blood in less than 10 minutes.

“The goal is to provide people, either at the point-of-care or further [at home], with treatment options,” said Vallée-Bélisle.

He said the company is currently focused on designing switches for specific disease markers as they continue to work with engineers to build clinically relevant nanomachines. 
 

This illustration depicts phages conjugated to magnetic nanoparticles for bacteria separation and detection. Photo: Nugen Research Group/Sam Rasmussen Nugen

Nanobots for food safety

Did you eat leafy greens at all last month? If you did, there’s a chance it could have been contaminated with E.coli bacteria. The CDC linked leafy greens to an E.coli outbreak that sickened 25 and killed one person between December and January. 

Sam Rasmussen Nugen wants to make sure these outbreaks are a thing of the past—and he’s using nanobots to show us how. 

Nugen’s platform to improve rapid food and water safety testing relies on bacteriophages, viruses that can infect and kill specific bacteria. Nugen and his team took the natural role phages play as bacteria’s predators and capitalized on it. They genetically engineered phages to not only find and infect bacteria, but—using an enzyme—programmed the phages to emit an electrochemical signal as a reporter.

But the bottleneck is not always bacteria detection, Nugen said. Once the infection has been found, it’s more about separating the bacteria from the sample so they can be concentrated. 

“We’re engineering the phages to be magnetic so we can actually put them in a solution, they go out and bind, start infecting the bacteria, and then we can yank them all to the side and concentrate them,” Nugen explained to Laboratory Equipment.

Nugen’s multidisciplinary lab at Cornell University is currently working on advancing this nanobot platform, which may end up being commercialized as a portable kit, rather than a test for a traditional lab setting. One part of the lab is focusing on changing the range of bacteria the phages can and will infect, others are working on engineering the phages to attach to these magnetic particles, while still others are working on ways to make detection more sensitive. 

Price and speed are two of the main reasons Nugen is focusing on developing his research into a kit. 

“When I started thinking about it, the resources an average or small dairy/produce farmer would have is not too different from the resources in a remote clinic in sub-Saharan Africa,” said Nugen. “In most cases, the kit you would need would be essentially the same. A farmer can’t have a $50 test they have to run all the time—it has to be a couple dollars. The constraints are similar enough to apply what I learned from making devices for resource-limited areas to farms here in the U.S. as well.”

A rapid nanotech test may have been of use in the most recent E.coli outbreak, as experts believe irrigation water was the most likely culprit in the contamination of the lettuce. Currently, the FDA requires farmers to test their irrigation water, but it takes days for the test to confirm the presence or absence of bacteria. With perishable food, farmers do not have the luxury to wait. Hence, compromised food can make it to a distribution point, such as a grocery store, and even into consumers’ hands. 

“To devise a test with instant results is very, very difficult,” Nugen said. “So we’re trying to make something that at least does it in just a few hours. Essentially, the shorter your assay time, the less compromised a product you would have.”

Beyond food safety, Nugen and his group are looking at using bacteriophages in a medical environment, for phage therapy—or the therapeutic use of bacteriophages to treat pathogenic bacterial infections. Phage therapy was popular decades ago, until the discovery of antibiotics. However, with antibiotic-resistance currently on the rise, some researchers have turned back the clock to reinvestigate phage therapy. 

Bacteriophages are more specific than antibiotics, and typically harmless to the host organism. They do have some of the same limitations of drugs though, as bacteria could eventually become resistant to the phage. 

“The thing is, though, it’s a lot easier to find new phages than it is to find new antibiotics,” Nugen said. 

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