Almost a century ago, a monumental breakthrough in microscopy gave scientists an entirely new way to examine the intricate inner workings of living cells. Known as phase contrast, the technique made previously faint, transparent, or nearly invisible cellular structures much easier to distinguish by converting tiny phase shifts in light waves into visible differences in brightness. Its profound importance to the biological and physical sciences was ultimately recognized with a Nobel Prize in 1953, forever altering how researchers visualize the microscopic world.
Now, physicists at the University of California, Berkeley, have successfully brought that exact same fundamental principle into the realm of modern electron microscopy, a sophisticated technology capable of magnifying objects roughly 10,000 times more powerfully than conventional light microscopes.
The research team has developed an ingenious device called a laser phase plate that could dramatically improve cryoelectron microscopy, widely known as cryo-EM. This powerful imaging method has fundamentally transformed the study of complex protein structures and helped accelerate drug discovery pipelines over the past decade. Even so, the technology has long suffered from a major limitation: most human and animal proteins are so exceptionally small that obtaining clear, high-resolution images of them remains extremely difficult.
The newly developed laser technology could soon help scientists overcome that stubborn obstacle, potentially allowing researchers to image proteins as small as one-third the size of those that currently challenge even the most advanced microscopes today.
An even greater impact could stem from future improvements to cryoelectron tomography, or cryo-ET. This newer, highly sophisticated approach combines images captured from multiple distinct angles to construct comprehensive, three-dimensional views of proteins and other essential biological molecules. Unlike traditional techniques that must study isolated molecules suspended in a solution, cryo-ET can reveal how proteins are actually arranged and how they dynamically interact directly 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 the Lawrence Berkeley National Laboratory who spearheaded 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 proudly describe as the world’s most intense focused continuous-wave laser. Rather than simply illuminating a biological specimen from the outside, the specialized laser interacts directly with the microscope’s high-energy electron beam, subtly altering its phase to dramatically increase image contrast at the molecular level.
This precise adjustment makes it significantly easier to distinguish small molecules, including oxygen-carrying hemoglobin, from their complex surrounding environments. Furthermore, it could improve the visibility of vital structures deep inside cells, such as the nucleus and energy-producing mitochondria, which have traditionally been obscured by poor optical contrast.
“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 substantial financial backing from Biohub, Müller acquired an advanced cryo-EM instrument and equipped it with the new laser phase plate. He named the resulting custom-built microscope Theia, paying homage to the ancient Greek Titaness associated with light, radiance, and the glowing sky.
Meanwhile, Carragher is directing the construction of a related, highly specialized microscope at Biohub’s imaging facility in Redwood City. Her team’s instrument utilizes two separate lasers rather than just one, building directly upon foundational theoretical research conducted by Müller and his academic collaborators.
In this multi-laser configuration, the two laser beams are arranged at precise right angles to one another, with each operating at approximately half the total power. This clever engineering helps prevent delicate optical components from burning out under extreme stress while simultaneously reducing optical distortions that could otherwise interfere with final image quality.
Both independent research teams are working closely with Thermo Fisher Scientific, the undisputed leading global manufacturer of cryo-EM instruments, to explore future applications and broader accessibility.
“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 have been published in the journal Science, while comprehensive details concerning Biohub’s innovative two-laser design are available in a recently posted preprint.
How a Nobel Prize-Winning Discovery Changed Microscopy
To truly understand why this new technological leap matters so much, it helps to look closely at a longstanding, fundamental challenge in biological imaging.
Because plant and animal cells consist largely of water, they are remarkably transparent when viewed under an ordinary light microscope. Although this transparency might initially seem beneficial for observing live specimens, it creates a severe technical problem. Small cellular structures, including mitochondria and nuclei, scatter very little light as it passes through them and therefore appear only slightly darker than the surrounding material, rendering them practically invisible.
Scientists traditionally addressed this persistent problem by adding chemical stains to create greater visual contrast. However, the harsh staining process invariably kills the living cells, destroying the very dynamic processes researchers hope to observe.
Everything changed in 1930, when Dutch physicist Frits Zernike realized that light passing through transparent biological material changes in more ways than simply becoming brighter or dimmer. When light scatters inside a cell, it also experiences a tiny, nearly imperceptible delay that alters its phase—specifically, the precise 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 subtle cellular features stand out with striking clarity against their background when the light finally reached the viewer’s eye or photographic plate.
This brilliant innovation became the absolute foundation of phase-contrast microscopy, ultimately earning Zernike the prestigious 1953 Nobel Prize in Physics.
By the early 1940s, scientists had already recognized how immensely useful phase contrast was for studying biological cells. Naturally, they began exploring whether the exact same conceptual framework might be applied to improve electron microscopes, which use concentrated beams of electrons instead of light to examine minuscule structures as small as individual proteins.
Unfortunately, those early attempts encountered severe technical difficulties. Devices designed to alter the phase of high-energy electron beams often weakened the beam far too much, introduced unacceptable image instability, or severely compromised overall resolution.
A promising path forward finally emerged in 2010, when Müller and Robert Glaeser, now a UC Berkeley professor emeritus of molecular and cell biology, formally proposed using an exceptionally intense laser beam to produce the necessary phase shift without the destructive side effects of earlier physical phase plates. Their theoretical approach offered an elegant way to dramatically improve contrast without sacrificing vital electron beam intensity.
Overcoming the Limits of Electron Microscopy
Glaeser has played a critically important role in the historical development of cryo-EM, which offers a potentially simpler and more direct way to determine molecular structures compared to traditional X-ray crystallography.
X-ray crystallography requires researchers to grow high-quality physical crystals of the molecules they want to study and gain access to an intensely powerful X-ray source, such as a synchrotron. Cryo-EM successfully avoids the notoriously difficult need to crystallize biological molecules, but it presents a unique set of technical hurdles of its own.
One of the most persistent problems is that an electron beam can easily heat, degrade, and eventually destroy the fragile biological specimen being examined. This radiation damage strictly limits how much data can be collected and how detailed the resulting structural images can ultimately become. While covering specimens with thin layers of metal can reduce damage and improve contrast, it simultaneously makes the final images much less sharp.
During the 1960s, researchers began investigating whether flash-freezing biological samples could effectively protect them from radiation damage during electron exposure.
Glaeser demonstrated that rapidly cooling biological specimens helped preserve their native structures during imaging. He also proposed using a much less intense electron beam to examine thousands of frozen molecules simultaneously, further limiting cumulative radiation damage.
Because the individual molecules would naturally be positioned at random angles within the ice, powerful computers could later analyze and combine their individual two-dimensional projections to mathematically reconstruct a highly detailed, three-dimensional molecular structure.
These pioneering advances helped establish the core foundations of modern cryo-EM, whose primary developers were honored with the 2017 Nobel Prize in Chemistry. During their acceptance remarks, the Nobel laureates explicitly acknowledged Glaeser’s foundational contributions to the field.
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 working, reliable technology required nearly fifteen years of relentless engineering and optical refinement.
Following their initial 2010 proposal, Müller spent a decade and a half pursuing the idea, with crucial portions of the fundamental research receiving financial support from the National Institutes of Health.
The first major hurdle was generating an extraordinarily intense laser beam concentrated into an unimaginably tiny area. The researchers needed a tremendous amount of optical light intensity to successfully alter the phase of a high-speed electron beam by the required 90 degrees.
It took a full decade of painstaking experimentation just to develop a functional system capable of achieving this feat.
Their ultimate solution was to confine a continuous-wave laser beam inside a specialized spherical optical chamber lined with highly reflective mirrors. As the light repeatedly reflected back and forth through the resonant cavity more than 10,000 times, its internal power density increased dramatically while the beam became tightly focused into an extremely small focal 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 research team first demonstrated that the radical concept actually worked by installing an early prototype laser phase plate into one of Glaeser’s older, legacy electron microscopes at UC Berkeley.
Subsequent funding from Biohub then made it possible to acquire a customized, state-of-the-art Thermo Fisher Krios cryo-EM microscope and heavily modify it to seamlessly accommodate the high-power laser technology.
In their latest round of rigorous research, the scientists tested the upgraded microscope using six distinct biological samples that varied widely in physical size and preparation method. The laser consistently improved image resolution, delivering particularly important gains for delicate specimens that have historically proven difficult or impossible 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 clearly demonstrate the newly upgraded microscope’s advanced capabilities, the researchers successfully reconstructed high-resolution images of two different proteins.
The first was aldolase, a well-known structural protein found in muscle tissue that modern, conventional cryo-EM systems can already image relatively easily. The second was hemoglobin, the vital protein responsible for efficiently carrying oxygen throughout the human bloodstream.
Hemoglobin is substantially smaller than aldolase and lies perilously near the lower size limit of proteins that standard cryo-EM instruments can reliably analyze.
Using the innovative laser phase plate, the researchers significantly improved the resolution of both protein structures. The improvement was especially pronounced for hemoglobin, highlighting the technology’s immense potential to overcome the persistent difficulties associated with analyzing smaller, elusive 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 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 standard scientific unit named after English chemist John Dalton. One dalton is defined precisely as one-twelfth of the mass of an unbound carbon-12 atom at rest.
Conventional cryo-EM traditionally struggles to resolve proteins smaller than approximately 70 kilodaltons. That technical limitation is particularly significant because proteins falling below this mass threshold account for roughly 90% of the entire human proteome—the complete collection of proteins produced and expressed by the human body.
With the advent of the laser phase plate, researchers can now begin imaging proteins as small as 50 kilodaltons, although doing so remains a technically challenging endeavor. Such proteins are even smaller and lighter than hemoglobin.
The Next Goal Is to Reveal Even Smaller Proteins
Müller and his colleagues believe the technology could eventually allow scientists to study proteins as small as 17 kilodaltons, which is roughly the size of myoglobin, the oxygen-storing protein found in muscle tissues.
Reaching that ambitious goal may require yet another technical refinement: utilizing a tightly focused electron beam rather than the defocused beam currently required to produce sufficient optical contrast without the laser phase plate.
According to Müller, the advanced laser system should eventually make it possible to obtain direct contrast enhancements purely from phase changes occurring within a focused electron beam.
If successful, this upcoming adjustment could double the optical contrast and overall signal-to-noise ratio once again, building upon the substantial improvements already achieved with the current laser phase plate setup.
Such continuous progress could eventually make it possible for scientists to study a far larger proportion of the human body’s vast protein network, providing unprecedented insight into how these essential molecular machines function and interact within the crowded, complex 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 multidisciplinary 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 research preprint, alongside Biohub engineer Pavel Olshin.
