Almost a century ago, a monumental breakthrough in microscopy gave the scientific community an entirely new way to examine the intricate architecture hidden inside biological cells. Known as phase contrast, the technique transformed previously faint, nearly invisible cellular structures into distinct, highly observable features. The sheer importance of this innovation was formally recognized on the global stage when it was awarded the Nobel Prize in Physics in 1953.
Now, a team of pioneering physicists at the University of California, Berkeley, has successfully adapted that exact same foundational principle for electron microscopy—a remarkably sophisticated technology capable of magnifying objects roughly 10,000 times more powerfully than conventional light microscopes.
The researchers have developed a groundbreaking device known as a laser phase plate, an invention poised to significantly enhance cryoelectron microscopy (cryo-EM). Over the past decade, cryo-EM has fundamentally transformed the study of protein structures and dramatically accelerated the pace of modern drug discovery. Even with its massive success, however, the technology has labored under a major limitation: the vast majority of human proteins are simply too small, meaning that obtaining sharp, clear images of them remains an extraordinarily difficult task.
This new laser technology promises to effectively clear that formidable hurdle. By boosting contrast and signal clarity, the system could potentially allow scientists to image proteins as small as one-third the size of those that currently challenge even the most advanced microscopes.
An even greater impact could soon materialize through improvements to cryoelectron tomography (cryo-ET). This newer, highly sophisticated imaging approach combines multiple images captured from a wide variety of angles to construct detailed, three-dimensional views of proteins and other vital molecules. Unlike traditional techniques that must study isolated molecules dissolved in a solution, cryo-ET possesses the unique capacity to reveal precisely how proteins are arranged and interact dynamically within their natural, bustling 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 the Lawrence Berkeley National Laboratory who led the rigorous 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 technological breakthrough relies on what the research team describes as the world’s most intense focused continuous-wave laser. Rather than merely illuminating a physical specimen from the outside, the laser interacts directly and dynamically with the microscope’s electron beam, precisely altering its phase to dramatically increase image contrast.
This precise optical adjustment makes it markedly easier to distinguish small, elusive molecules—including oxygen-carrying hemoglobin—from their complex surrounding environments. Furthermore, it could substantially improve the real-time visibility of critical structures residing deep inside cells, such as the nucleus and energy-producing mitochondria.
"With cryo-ET, we’re looking at small, very complicated cellular material that’s incredibly crowded inside the cell," noted 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 backing from Biohub, Müller acquired an advanced cryo-EM instrument and equipped it directly with the newly engineered laser phase plate. He named the resulting custom-built microscope Theia, paying homage to the ancient Greek Titaness historically associated with light, sight, and radiance.
Meanwhile, Carragher is directing the construction of a related, highly specialized microscope at Biohub’s prominent imaging facility in Redwood City. Her team’s instrument utilizes an innovative dual-laser configuration rather than a single beam, directly building upon advanced theoretical research previously conducted by Müller and his academic collaborators.
In this dual-laser system, the two separate laser beams are meticulously arranged at right angles to one another, with each operating at roughly half the total power. This clever engineering configuration helps prevent delicate optical components from burning out while simultaneously minimizing optical distortions that could otherwise compromise ultimate image quality.
Both active research teams are closely collaborating with Thermo Fisher Scientific, the undisputed global market leader in the manufacture of high-end cryo-EM instruments.
"Theia is the Formula 1 microscope," Müller remarked. "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 physics team’s latest experimental images and comprehensive technical findings have been published in the prestigious journal Science. Meanwhile, specific details regarding Biohub’s innovative two-laser design have been made publicly available through a recently posted preprint.
How a Nobel Prize-Winning Discovery Changed Microscopy
To fully grasp why this new technological leap matters so profoundly to modern science, it is helpful to examine a longstanding, foundational challenge in biological imaging.
Because plant and animal cells consist overwhelmingly of water, they are naturally quite transparent when viewed beneath an ordinary light microscope. Although this transparency might initially seem beneficial for observation, it actually creates a significant obstacle. Small cellular structures, including nuclei and mitochondria, scatter very little light as it passes through them, causing them to appear only fractionally darker than the surrounding intracellular material.
Historically, scientists attempted to solve this persistent problem by introducing chemical stains to create sharper contrast. Unfortunately, the chemical staining process invariably kills the living cells, halting dynamic biological observation.
Everything changed in 1930, when Dutch physicist Frits Zernike realized that light passing through a biological sample changes in more ways than simply becoming brighter or dimmer. When light scatters internally within a cell, it also experiences an infinitesimal delay that alters its phase—specifically, the timing of its wave peaks.
Although the human eye cannot directly detect this subtle phase shift, Zernike discovered that it could be cleverly converted into a visible difference in brightness. By shifting the phase of the unscattered background light by exactly 90 degrees, he made delicate cellular features stand out with striking clarity against their background when the light finally reached the viewer’s eye.
This profound innovation laid the permanent foundation for phase-contrast microscopy, ultimately earning Zernike the 1953 Nobel Prize in Physics.
By the early 1940s, visionary scientists already recognized how immensely useful phase contrast could be for studying cellular biology. They quickly began exploring whether the exact same optical concept could be adapted to improve electron microscopes, which substitute traditional light waves with focused beams of electrons to examine structures as unimaginably small as individual proteins.
Regrettably, those early investigative attempts encountered severe technical roadblocks. Devices engineered to alter the phase of high-energy electron beams frequently weakened the beam too drastically, introduced unacceptable image instability, or severely degraded overall resolution.
A promising solution finally emerged in 2010, when Müller and Robert Glaeser—now a UC Berkeley professor emeritus of molecular and cell biology—published a seminal proposal suggesting the use of an exceptionally intense laser to produce the necessary phase shift without sacrificing beam integrity.
Overcoming the Limits of Electron Microscopy
Glaeser has played a monumental role in the historical development of cryo-EM, a technology that offers a significantly more streamlined path to determining molecular structures than traditional X-ray crystallography.
X-ray crystallography requires researchers to grow near-perfect crystals of the target molecules they wish to study—a notoriously difficult bottleneck—and gain access to an intensely powerful X-ray source. Cryo-EM elegantly circumvents the requirement to crystallize molecules entirely, but it introduces its own distinct set of engineering hurdles.
Among the most persistent obstacles is the fact that a high-energy electron beam can heat, disrupt, and eventually destroy the biological specimen under examination. This thermal damage inherently limits the total amount of data that can be collected and caps the ultimate resolution of the resulting images. While coating biological specimens in thin layers of metal can reduce structural damage and boost visual contrast, it unfortunately renders the final images noticeably less sharp.
During the 1960s, researchers began investigating whether ultra-fast freezing of biological samples could shield them from severe radiation damage.
Glaeser subsequently demonstrated that rapidly cooling specimens to cryogenic temperatures successfully preserved their native structural integrity during imaging. He also proposed utilizing a significantly less intense electron beam to examine thousands of frozen molecules simultaneously, further minimizing radiation damage.
Because individual molecules naturally settle at randomized angles within the frozen suspension, advanced computer algorithms can analyze and mathematically combine their individual projections to reconstruct a remarkably detailed three-dimensional molecular structure.
These foundational advances laid the groundwork for modern cryo-EM, whose core pioneers were awarded the 2017 Nobel Prize in Chemistry. During their acceptance speeches in Stockholm, the Nobel laureates explicitly acknowledged Glaeser’s visionary early contributions.
The Nobel Committee praised the transformative 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 theoretical laser phase plate concept into a functional, highly reliable laboratory instrument required nearly a decade and a half of relentless engineering.
Following their initial 2010 theoretical proposal, Müller spent 15 years doggedly pursuing the idea, with crucial portions of the exploratory research funded by the National Institutes of Health.
The primary engineering hurdle involved generating an extraordinarily intense laser beam and concentrating it securely into an infinitesimal area. The researchers required a massive concentration of light energy to successfully induce a 90-degree phase shift in an electron beam.
Developing the necessary optical architecture took an entire decade of trial and error.
Their ultimate solution involved confining a continuous laser beam inside a specialized spherical chamber lined with ultra-reflective mirrors. As the light repeatedly bounced and reflected through the optical cavity more than 10,000 times, its cumulative intensity surged dramatically while the beam was simultaneously focused down to an extremely minute focal spot.
"It’s 75 kilowatts focused to a few microns," Müller emphasized. "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 research team first demonstrated the viability of the concept by retrofitting a laser phase plate into one of Glaeser’s older, legacy electron microscopes.
Subsequent financial support from Biohub then enabled the acquisition of a customized, state-of-the-art Thermo Fisher Krios cryo-EM microscope, which was meticulously modified to house the newly developed laser hardware.
In their most recent round of experiments, the scientists tested the heavily upgraded microscope using six distinct biological samples varying widely in physical size and preparation methods. The integrated laser consistently improved image resolution, delivering critically important visual gains for specimens that have historically proven nearly impossible to image using standard techniques.
"For the most challenging cases — small particles, bad specimens — the laser produces a very considerable advantage," Müller noted.
Sharper Images of Proteins Once Too Small to Study
To clearly demonstrate the enhanced microscope’s capabilities, the researchers successfully reconstructed high-resolution images of two different biological proteins.
The first target was aldolase, a common muscle protein that modern cryo-EM systems can already image relatively easily. The second target was hemoglobin, the vital protein responsible for transporting oxygen throughout the human bloodstream.
Hemoglobin is substantially smaller than aldolase, resting precariously near the lower size limit of proteins that conventional cryo-EM instruments can reliably analyze.
By employing the new laser phase plate, the research team successfully sharpened the resolution of both protein structures. The performance improvement was especially pronounced for hemoglobin, highlighting the technology’s immense potential to conquer the longstanding analytical barriers associated with smaller biological 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 explained. "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 customarily measured in daltons, a standard scientific unit named after English chemist John Dalton, where one dalton is defined as one-twelfth of the mass of a carbon-12 atom.
Conventional cryo-EM routinely struggles to capture clear images of proteins smaller than approximately 70 kilodaltons. This limitation is particularly crippling for biomedical research because proteins falling below this specific threshold account for roughly 90% of the entire human proteome—the complete catalog of proteins produced by the human body.
With the deployment of the new laser phase plate, researchers have demonstrated that they can now image proteins as small as 50 kilodaltons, though analyzing molecules of this scale remains technically demanding. Such proteins are significantly smaller even than hemoglobin.
The Next Goal Is to Reveal Even Smaller Proteins
Looking forward, Müller believes the technology can be further refined to allow scientists to study proteins as small as 17 kilodaltons, roughly the size of myoglobin, an oxygen-binding protein found in muscle tissue.
Achieving that ambitious milestone will likely require an additional technical enhancement: utilizing a tightly focused electron beam rather than the defocused beam currently required to maintain adequate contrast without the active assistance of the laser phase plate.
According to Müller, perfecting the laser system should eventually make it feasible to obtain high contrast directly from native phase changes within a tightly focused electron beam.
If successful, this upcoming adjustment could theoretically double the visual contrast and signal-to-noise ratio once again, stacking additional improvements on top of the gains already secured by the laser phase plate.
Such sweeping progress could ultimately make it possible for scientists to study an unprecedented proportion of the human body’s proteins, granting researchers an extraordinary, real-time window into how these molecular machines interact within the crowded, chaotic environment of living 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 UC 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 serve as co-corresponding authors of the Biohub preprint, working alongside Biohub engineer Pavel Olshin.
