Researchers at the University of California (UC), Berkeley report developing a contrast-boosting cryo-EM system that incorporates powerful beams of laser light to make tiny proteins stand out from the surrounding haze. “At the moment, we can see just a snifter of what we’re interested in,” says Michael Grange, a structural biologist at the Rosalind Franklin Institute who was not involved in the study, published online today in Science. With the laser, he says, “we can see more clearly.”
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Despite a decade of spectacular advances in detectors, software and microscope design, cryo-EM still struggles with one fundamental problem: Below a certain size, proteins become nearly invisible to the microscope. Rather than strongly scattering electrons, creating dark and light patterns that directly reveal the proteins’ structures, they mostly shift the timing—or phase—of passing electron waves, encoding information that detectors struggle to capture.
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In the new study, the UC Berkeley team used the laser-enhanced system to sharpen reconstructions of hemoglobin, the oxygen-carrying protein in blood. Hemoglobin sits near the lower size limit of what conventional cryo-EM can reliably resolve, making it a useful benchmark. Comparing experiments performed with and without the laser, they showed the added contrast transformed a blurry 4.46-angstrom hemoglobin structure into a crisp 3.09-angstrom reconstruction, with sharper detail and spatial resolution throughout.
Not content to stop there, Carragher and her Biohub colleagues, with guidance from Müller, built a more powerful variant, adding a second, orthogonal laser to create a dual, criss-crossing phase plate. Described this week in a companion preprint posted to bioRxiv, the design spreads the lasers’ load more evenly, reducing aberrations and minimizing heating that could destabilize the mirrors.
Last month, Carragher says her team combined images from dozens of exposures at different angles to create as-yet-unpublished 3D images of the mélange of structures within frozen yeast cells. “We know so little really about the molecular structure inside of cells,” says preprint co-author David Agard, a structural biologist at Biohub and UC San Francisco. “The phase plate, we’re really hoping, gets us over the hump.”
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The challenge is that laser phase plates are not yet commercially available. Thermo Fisher Scientific, which holds the license for the technology, has not yet said when it will sell phase plates or how much they will cost.
After spending 15 years working on the engineering and optics of laser phase plates, Müller hopes the company will hurry. “There would be nothing better,” he says, “than if one day somebody discovers a cure for something, and the laser phase plate has helped with that.”
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