A new laser-based phase plate developed by researchers at UC Berkeley, Lawrence Berkeley National Laboratory, and the Chan Zuckerberg Biohub has overcome a key limitation in cryo-electron microscopy, dramatically improving the imaging of small proteins.
The technology, integrated into a custom microscope system named Theia, aims to solve a persistent challenge: imaging proteins below approximately 70 kilodaltons. The findings were published in Science on June 11, 2025.
Core Technology
The laser phase plate adapts the principle of phase-contrast microscopy—a technique pioneered by Frits Zernike in the 1930s—for electron microscopy. In standard light microscopy, contrast is enhanced by shifting the phase of non-scattered light relative to scattered light.
To achieve this effect with electrons, the device uses an intense continuous-wave laser focused within a mirrored cavity. This laser generates a localized electromagnetic field that shifts the phase of the electron beam passing through it.
Unlike previous attempts to create electron phase plates, this laser-based method enhances contrast without significantly reducing beam intensity, causing instability, or degrading resolution. The laser system achieves a focused power of 75 kilowatts. The development follows a theoretical proposal made by Holger Müller and Robert Glaeser in 2010, leading to over 15 years of development.
System and Performance
The laser phase plate is paired with a custom Thermo Fisher Scientific microscope called Theia, designed to maximize the benefit of the laser's brightness. Theia was installed at UC Berkeley and has been operational since 2025.
The system was tested on two proteins: aldolase and hemoglobin (64 kilodaltons).
"The laser phase plate improved the resolution of images for both samples. According to the researchers, the improvement was more significant for hemoglobin, which is smaller and at the lower size limit for standard cryo-EM."
The phase plate also improved imaging of samples with suboptimal preparation.
Background on Cryo-EM Limitations
Cryo-EM is a method for determining biomolecular structures by imaging frozen samples. It emerged as a key technique in structural biology, but it faces challenges with proteins smaller than approximately 70 kilodaltons. This size range includes an estimated 90% of human proteins.
The technique relies on averaging many images of individual molecules to build a structure, and the low signal-to-noise ratio for small proteins makes this process difficult. Cryo-electron tomography (cryo-ET), which constructs 3D structures by combining images of molecules in their native cellular environment, also requires high contrast to visualize structures within crowded cells.
Future Applications
The researchers plan to extend the technology from single-particle analysis to cryo-electron tomography. A dual-laser version of the system is being developed at the Chan Zuckerberg Biohub in Redwood City, California.
The team has stated that the laser phase plate may enable imaging of proteins down to approximately 17 kilodaltons in the future.
Collaboration and Funding
The work involves UC Berkeley, the Chan Zuckerberg Biohub, and Thermo Fisher Scientific. Co-authors on the study include Petar Petrov, Jessie Zhang, Jonathan Remis, Hang Cheng, Jeremy Axelrod, Eric Cooper, Ian Hicklin, Shahar Sandhaus, Cooper Schnurr, and Robert Glaeser. At the Biohub, Bridget Carragher and David Agard lead the Dynamic Structural Cell Biology group.
The research received funding from the National Institutes of Health (NIH), the National Science Foundation (NSF), the Gordon and Betty Moore Foundation, the Chan Zuckerberg Initiative, and the Berkeley Lab's Laboratory Directed Research and Development (LDRD) program.