Almost a century ago, a breakthrough in microscopy gave scientists an entirely new way to examine the inside of cells. Known as phase contrast, the technique made previously faint or nearly invisible cellular structures much easier to distinguish. Its importance was recognized with a Nobel Prize in 1953.
Now, physicists at the University of California, Berkeley, have brought that same principle to electron microscopy, a technology capable of magnifying objects roughly 10,000 times more than conventional light microscopes.
The researchers have developed a device called a laser phase plate that could significantly improve cryoelectron microscopy (cryo-EM). This powerful imaging method has transformed the study of protein structures and helped speed up drug discovery over the past decade. Even so, it has a major limitation: Most human proteins are so small that obtaining clear images of them remains extremely difficult.
The new laser technology could help overcome that obstacle, potentially allowing scientists to image proteins as small as one-third the size of those that challenge current microscopes.
An even greater impact could come from improvements to cryoelectron tomography (cryo-ET). This newer approach combines images captured from multiple angles to construct three-dimensional views of proteins and other molecules. Unlike techniques that study isolated molecules in a solution, cryo-ET can reveal how proteins are arranged and interact within their natural cellular surroundings.
“Cryo-EM has become the new, fastest-growing method for resolving the structure of biological macromolecules, and cryo-ET is expected to show how these molecules work together in their natural, cellular context,” said Holger Müller, a UC Berkeley professor of physics and faculty scientist at Lawrence Berkeley National Laboratory who led the development effort. “But because of signal-to-noise limitations, the majority of human and animal proteins are too small to be analyzed by these methods. The increase in signal-to-noise ratio provided by this laser phase plate is expected to overcome these important limitations.”
An Extraordinary Laser Reveals Hidden Cellular Structures
The breakthrough relies on what the researchers describe as the world’s most intense focused continuous-wave laser. Rather than simply illuminating a specimen, the laser interacts directly with the microscope’s electron beam, altering its phase to increase image contrast.
This adjustment makes it easier to distinguish small molecules, including hemoglobin, from their surroundings. It could also improve the visibility of structures inside cells, such as the nucleus and mitochondria.
“With cryo-ET, we’re looking at small, very complicated cellular material that’s incredibly crowded inside the cell,” said Bridget Carragher, founding technical director of imaging at Biohub in Redwood City, California. “It’s like a forest of trees, and you’re trying to find one leaf on one tree in there. Cryo-ET needs a dramatic step forward in contrast, so we can start to see what’s going on inside the cell. That’s what the laser phase plate promises to give us.”
With financial support from Biohub, Müller acquired an advanced cryo-EM instrument and equipped it with the new laser phase plate. He named the resulting microscope Theia, after the ancient Greek Titaness associated with light and radiance.
Meanwhile, Carragher is directing the construction of a related microscope at Biohub’s imaging facility in Redwood City. Her team’s instrument uses two lasers rather than one, building on theoretical research conducted by Müller and his collaborators.
The two laser beams are arranged at right angles to one another and each operates at approximately half the power. This configuration helps prevent components from burning out while also reducing optical distortions that could interfere with image quality.
Both research teams are working with Thermo Fisher Scientific, the leading manufacturer of cryo-EM instruments.
“Theia is the Formula 1 microscope,” Müller said. “It has extra electron optics that give it better resolution than the standard cryo-EM, even without the laser. With the addition of the laser phase plate, we hope that it really becomes the world’s best instrument overall.”
The Berkeley team’s latest images and technical findings are presented in the journal Science. Details of Biohub’s two-laser design are available in a recently posted preprint.
How a Nobel Prize-Winning Discovery Changed Microscopy
To understand why the new technology matters, it helps to look at a longstanding challenge in biological imaging.
Because plant and animal cells consist largely of water, they are relatively transparent when viewed under an ordinary light microscope. Although this transparency might seem beneficial, it creates a problem. Small cellular structures, including mitochondria and nuclei, scatter very little light and therefore appear only slightly darker than the surrounding material.
Scientists have traditionally addressed this problem by adding stains to create greater contrast. However, staining also kills the cells.
In 1930, Dutch physicist Frits Zernike realized that light passing through biological material changes in more ways than simply becoming brighter or dimmer. When light scatters inside a cell, it also experiences a tiny delay that changes its phase, or the timing of its wave peaks.
Although the human eye cannot directly detect this phase shift, Zernike discovered that it could be converted into a visible difference in brightness. By shifting the phase of the unscattered light by 90 degrees, he made subtle cellular features stand out more clearly against their background when the light reached the viewer’s eye.
This innovation became the foundation of phase-contrast microscopy, earning Zernike the 1953 Nobel Prize in Physics.
By the early 1940s, scientists had already recognized how useful phase contrast was for studying cells. They began exploring whether the same idea might improve electron microscopes, which use electrons instead of light to examine structures as small as individual proteins.
Unfortunately, early attempts encountered serious technical difficulties. Devices designed to alter the phase of electron beams often weakened the beam too much, introduced image instability, or reduced resolution.
A possible solution emerged in 2010, when Müller and Robert Glaeser, now a UC Berkeley professor emeritus of molecular and cell biology, proposed using an intense laser to produce the necessary phase shift. Their approach offered a way to improve contrast without sacrificing electron beam intensity.
Overcoming the Limits of Electron Microscopy
Glaeser has played an important role in developing cryo-EM, which offers a potentially simpler way to determine molecular structures than traditional X-ray crystallography.
X-ray crystallography requires researchers to grow crystals of the molecules they want to study and obtain access to an intense X-ray source. Cryo-EM avoids the need to crystallize molecules, but it presents difficulties of its own.
One of the biggest problems is that an electron beam can heat and eventually damage the specimen being examined. This limits how much information can be collected and how detailed the resulting images can become. Covering specimens with metal can reduce damage and improve contrast, but it also makes the images less sharp.
During the 1960s, researchers began investigating whether freezing biological samples could protect them from radiation damage.
Glaeser demonstrated that cooling specimens helped preserve them during imaging. He also proposed using a less intense electron beam to examine thousands of frozen molecules at once, further limiting the damage.
Because the molecules would be positioned at different angles, computers could analyze and combine their images to reconstruct a highly detailed molecular structure.
These advances helped establish the foundations of modern cryo-EM, whose developers received the 2017 Nobel Prize in Chemistry. During their acceptance remarks, the Nobel laureates acknowledged Glaeser’s contributions.
The Nobel Committee praised the technology, explaining that cryo-EM “both simplifies and improves the imaging of biomolecules. This method has moved biochemistry into a new era.”
A 15-Year Quest to Harness an Exceptionally Powerful Laser
Turning the laser phase plate concept into working technology required years of engineering.
Following their 2010 proposal, Müller spent 15 years pursuing the idea, with part of the research supported by the National Institutes of Health.
The first major challenge was generating an extraordinarily intense laser beam concentrated into a tiny area. The researchers needed enough light intensity to change the phase of an electron beam by 90 degrees.
It took a decade to develop the necessary system.
Their solution was to confine a continuous laser beam inside a spherical chamber lined with mirrors. As the light repeatedly reflected through the cavity more than 10,000 times, its intensity increased dramatically while the beam became focused into an extremely small spot.
“It’s 75 kilowatts focused to a few microns,” Müller said. “That’s more powerful than what you use for welding. It’s more power than a military laser. It builds up the brightest continuous laser focus ever.”
The team first demonstrated that the concept worked by installing a laser phase plate in one of Glaeser’s older electron microscopes.
Additional funding from Biohub then made it possible to acquire a customized, state-of-the-art Thermo Fisher Krios cryo-EM microscope and modify it to accommodate the laser technology.
In their latest research, the scientists tested the upgraded microscope using six different samples that varied in size and preparation method. The laser consistently improved image resolution, with particularly important gains for specimens that are difficult to image using standard methods.
“For the most challenging cases — small particles, bad specimens — the laser produces a very considerable advantage,” Müller said.
Sharper Images of Proteins Once Too Small to Study
To demonstrate the new microscope’s capabilities, the researchers reconstructed images of two different proteins.
The first was aldolase, a protein found in muscle that modern cryo-EM systems can already image relatively easily. The second was hemoglobin, the protein responsible for carrying oxygen through the bloodstream.
Hemoglobin is much smaller and lies near the lower size limit of proteins that conventional cryo-EM instruments can reliably analyze.
Using the laser phase plate, the researchers improved the resolution of both protein structures. The improvement was especially pronounced for hemoglobin, highlighting the technology’s potential to overcome the difficulties associated with smaller molecules.
“The bottom line is, if you have a large protein and a really good sample — a fresh one or one frozen without bubbles, for example — you may not need the phase plate to get a single, high-quality image. But for a small protein and a bad sample, laser-on is best,” Müller said. “This could fill an enormous gap in our knowledge of protein structures that can’t be crystallized or are too small for today’s cryo-EM. And it will be revolutionary for cryo-ET.”
Protein mass is typically expressed in daltons, a unit named after English chemist John Dalton. One dalton is defined as one-twelfth the mass of a carbon-12 atom.
Conventional cryo-EM struggles to image proteins smaller than approximately 70 kilodaltons. That limitation is particularly significant because proteins below this threshold account for about 90% of the human proteome, the complete collection of proteins produced by the human body.
With the laser phase plate, researchers can now image proteins as small as 50 kilodaltons, although doing so remains challenging. Such proteins are even smaller than hemoglobin.
The Next Goal Is to Reveal Even Smaller Proteins
Müller believes the technology could eventually allow scientists to study proteins as small as 17 kilodaltons (the size of the protein myoglobin).
Reaching that goal may require another improvement: using a tightly focused electron beam rather than the defocused beam currently needed to produce sufficient contrast without the laser phase plate.
According to Müller, the laser system should make it possible to obtain contrast directly from phase changes in a focused electron beam.
If successful, this adjustment could double the contrast and signal-to-noise ratio again, adding to the improvement already achieved with the laser phase plate.
Such progress could make it possible to study a far larger proportion of the body’s proteins and provide unprecedented insight into how they interact within the complex environment of cells.
“This technology is a step function change for biology,” said Stephani Otte, Biohub’s Vice President of Imaging Science. “We are going to be able to see how molecular machines operate inside the living cell, in context, for the first time. What was once invisible will become visible — and that changes everything about how we understand disease.”
The Berkeley research team also includes Glaeser; postdoctoral fellows and co-first authors Petar Petrov and Jessie Zhang; staff scientist Jonathan Remis; postdoctoral fellow Hang Cheng; and current and former physics graduate students Jeremy Axelrod, Eric Cooper, Ian Hicklin, Shahar Sandhaus, and Cooper Schnurr.
At Biohub, Carragher and David Agard, the organization’s founding scientific director of imaging, jointly lead the Dynamic Structural Cell Biology group. Both are co-corresponding authors of the Biohub preprint, alongside Biohub engineer Pavel Olshin.
