You know when you are at the eye doctor getting an updated prescription, and suddenly the world snaps into sharper focus? Physicists at the University of California (UC), Berkeley, have now done something similar for electron microscopy. By introducing phase contrast into a cryo‑electron microscope, they have delivered dramatically sharper images of some of biology’s smallest and most elusive proteins.

The advance comes from a new laser phase plate (LPP), described in the paper “Laser phase plate improves structure determination of small proteins by cryo‑EM,” which was published recently in Science. Led by physicist Holger Mueller, PhD, of UC Berkeley and Lawrence Berkeley National Laboratory, the team demonstrated that a laser‑driven phase plate can overcome one of cryo‑EM’s most persistent limitations: poor contrast for small proteins.

Cryo‑EM has transformed structural biology over the past decade, earning a Nobel Prize in 2017 for enabling high‑resolution structures without crystallization. But despite its impact, the technique still struggles with proteins below ~70 kilodaltons—a size range that includes about 90% of the human proteome. “Because of signal-to-noise limitations, the majority of human and animal proteins are too small to be analyzed by these methods [cryo-EM and cryoelectron tomography]. The increase in signal-to-noise ratio provided by this laser phase plate is expected to overcome these important limitations.”

The new LPP begins to address that problem. The LPP uses an intense, continuous‑wave laser to shift the phase of the electron beam itself. This produces true phase contrast without dimming or destabilizing the beam. Mueller described the laser focus as “75 kilowatts focused to a few microns… That’s more powerful than what you use for welding. It has more power than a military laser. It builds up the brightest continuous laser focus ever.”

Installed in a custom Thermo Fisher Titan Krios, the LPP immediately improved the clarity and resolvability of small proteins, including hemoglobin, which sits at the lower limit of what today’s cryo‑EM instruments can handle. As the authors wrote in the abstract: “Here, we show that the laser phase plate (LPP)… enhances the resolution in single-particle reconstruction of small proteins by improving specimen-motion correction, recovery of information from the early frames, as well as particle visualization, 3D classification, and alignment.”

These improvements were achieved using standard defocus ranges and reconstruction workflows. “For the most challenging cases—small particles, bad specimens—the laser produces a very considerable advantage,” Mueller said.

The impact extends beyond single‑particle analysis. Cryo‑electron tomography (cryo‑ET), which assembles multiple angular views of a molecule or protein into a three-dimensional image, stands to benefit even more. “With cryo-ET, we’re looking at small, very complicated cellular material that’s incredibly crowded inside the cell,” said Bridget Carragher, PhD, founding technical director of imaging at Biohub. “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.”

Biohub is developing a dual‑laser version of the system, designed to reduce component wear and minimize aberrations. Meanwhile, Mueller’s team is pushing toward imaging proteins as small as 17 kilodaltons, a threshold that would open access to vast regions of the human proteome previously invisible to cryo‑EM.

“This technology is a step function change for biology,” said Stephani Otte, PhD, Biohub’s vice president of imaging science. “What was once invisible will become visible—and that changes everything about how we understand disease.”

“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,” Mueller 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.”

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