UC Berkeley and Biohub enhance electron microscopy with laser phase plate

A laser-based means of enhancing cryo-electron microscopy imaging should offer "unprecedented views into our cells," according to researchers from UC Berkeley and Biohub.

In what is described as a landmark achievement in biological imaging, a laser phase plate has been used to improve the contrast of images produced by cryo-EM platforms, several years after the approach was conceived but thought not to be possible in practice.

The team included researchers from the Holger Müller Group at UC Berkeley and the Chan Zuckerberg Biohub, a hub for collaboration between Berkeley, Stanford and UC San Francisco. Results were released via three coordinated papers, in Science, Nature Communications and an arXiv preprint.

Cryo-EM has become widely used in studies of structural biology, but the technique has been hampered by the inability to generate enough contrast to clearly image small molecules. More than 90 percent of the proteins found inside human cells are too small for cryo-EM to capture clearly, according to the researchers.

"The cell is just filled with everything that you could ever want to know, but we can't see it, and we can't find it," said David Agard, Founding Scientific Director of Imaging at Biohub. "To see all those interactions has been the dream of structural cell biologists for decades, and we're on the brink of being able to see that. In my view, the laser phase plate is integral to making this happen."

Phase-contrast microscopy in optical microscopes involves inserting an optical component into the light path to convert differences in wave phase into differences in brightness. Although this can hamper the signal-to-noise performance, the new increase in image contrast this brings can allow samples that do not give rise to strong inherent contrast, such as several kinds of transparent biological tissue, to be successfully imaged. 

The smaller the sample, the greater the improvement

A laser phase plate replaces this optical component with light, by focusing a continuous-wave laser at the spot where the electron beam passes through the diffraction plane. Interaction at that location can alter the electrons' phase and produce the desired improvement.

In two of the new papers, Biohub and UC Berkeley researchers describe installing a laser phase plate in custom versions of a commercial Thermo Scientific Krios microscope, requiring a bespoke optical cavity design and alignment between the laser and electron beams accurate to within 50 nanometers. 

Biohub's third paper describes a further advance, a dual-laser architecture using two beams oriented perpendicular to each other, each in its own cavity and operating at about half the power required by the single-cavity system. At lower power, components are less likely to burn and aberrations are reduced, said Biohub, making the system easier to operate.

In UC Berkeley's trials the laser phase plate provided higher resolution views of six different biological samples, covering different sizes and different sample preparation. The team noted that the smaller the sample, the greater the improvement, as when the approach was able to image both aldolase protein, already relatively easy to handle with conventional cryo-EM; and hemoglobin, which is at the lower limit for current machines. 

"This technology is a step function change for biology," said Stephani Otte, Biohub 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."