New Developments in Cryo-EM Materials Could Progress Cancer and Biomedical Research

Cryogenic-Electron Microscopy (cryo-EM) has been a turning point in the domain of medical research, but the substrate, used to freeze and observe samples under a microscope, has not progressed much in many years. Now, owing to a partnership between Penn State scientists and the applied science company Protochips, Inc., this situation has been changed.

"The traditional type of grid hasn't changed much since the inception of cryo-EM, while materials science has changed vastly," said Deb Kelly, a professor of biomedical engineering at Penn State and director of the Center for Structural Oncology (CSO). "Our team, along with other colleagues in the field, had the idea to try new materials as a means to improve upon current practices."

Issues with traditional carbon grids with holes include irregular surfaces when ice forms across the grid, which necessitates modifying imaging procedures many times; the grid materials expanding at various thermal rates; and failure of the specimens to discover their way into the grid holes, wasting what is frequently limited samples.

"Only having to set initial focus parameters saves a tremendous amount of time during data acquisition," says Cameron Varano, research assistant professor in the CSO and the co-lead author on a new paper recently published online in the journal Small. "The Protochips substrates are made from silicon nitride, a more rigid material than the carbon grids, which makes them less apt to have local deformities. And the wells in the chips can be customized for various ice thicknesses and applications."

With the new substrates, known as Cryo-Chips, the scientists have the potential to obtain all their data on the samples within an hour, contrary to what would presently take days.

This major technical advancement allows us to tackle more challenging questions. It's turning cryo-EM from an art into a science.

Cameron Varano, Co-Lead Author and Research Assistant Professor, CSO.

In their paper titled "Cryo-EM-on-a-Chip: Custom-designed Substrates for the 3D Analysis of Macromolecules," the scientists selected three case studies for which this kind of imaging could be beneficial. The first research was a comparison of the carbon grid with holes and the Cryo-Chip using rotavirus particles, a basic model in cryo-EM studies due to its large size and symmetrical shape. They observed improved contrast with the Cryo-Chip substrate, as well as additional specimen retention in the custom wells.

The second research, using a lot smaller and asymmetrical BRCA1 protein assemblies taken from breast cancer cells, also revealed improved contrast with stronger edge boundaries, making them much better contenders for automated imaging processing procedures.

For our third example, we decided to look at something more unknown, and that's derived from another type of cancer, P53, from brain cancer cells. P53 is the most mutated molecule in nearly all cancers throughout the body. Yet no one has put together what its full 3D structure looks like in cancer. Using our new microchip approach, we were able to see features in these important p53 assemblies that give this cancer an advantage for survival.

Deb Kelly, Professor of Biomedical Engineering (Penn State) and Director of the CSO.

Kelly and Varano, who both of late moved to Penn State from Virginia Tech, are hoping to take these biomedically critical samples to the next level as part of the assignment for the new CSO, part of the Huck Institutes of the Life Sciences.

With the newly-built microscope at the University Park campus and the Cryo-Chip tools in hand, we expect to transition our imaging work from high throughput to intelligent throughput. What's really nice about our collaboration with Protochips is that it emphasizes the company/academic partnership. In that way, we can all grow together.

Deb Kelly, Professor of Biomedical Engineering (Penn State) and Director of the CSO.

Co-lead author Nick Alden, was Kelly's graduate student at Virginia Tech, and he will be joining the doctoral program in biomedical engineering at Penn State this fall. Other authors include William Dearnaley and Maria Solares of Penn State;Yanping Liang and Zhi Sheng, of Virginia Tech; Sarah McDonald of Wake Forest University; and John Damiano, Jennifer McConnell and Madeline Dukes from Protochips, Inc. William Luqiu, a graduating senior at the Roanoke Valley Governor's School for Science and Technology, also participated in the computing phases of the study.

The National Institutes of Health and the National Cancer Institute aided this research. Extra support was given by the University of Virginia-Virginia Tech Carilion Seed Fund Award and the Cartledge Charitable Foundation.