“The spray device at LCLS allows researchers to look at the movements of atoms in microcrystals,” Dahlberg said. “But to my mind, spraying microcrystal samples and spraying cell samples are the same thing.”
“We wanted to combine light source and cryo-ET techniques as much as possible,” SLAC and Stanford professor and senior co-author Soichi Wakatsuki said. “We knew that doing so would be fruitful for microbiology and medicine development.”
With their new approach, researchers sprayed and froze cell samples that had been mixed with a stimulant in milliseconds, rather than 10 seconds, the time hand-mixing takes. This allowed researchers to take images of the cell sample every 25 milliseconds and see changes on that time scale.
“Our new approach helped to identify and characterize some of the interesting morphological changes in the cells that we began to see over the course of our time-resolved experiments,” Stanford University graduate student and paper co-author Jacob Summers said.
From jet to mist
Researchers from Cornell University re-engineered LCLS’s spray nozzle to work for the cryo-ET experiment. But it wasn’t as simple as walking the spray device from LCLS over to a cryo-ET machine. The problem was that, at LCLS, the samples are sprayed in a powerful jet formation – like a garden hose set on jet. This force and pressure would not work for cryo-ET experiments because samples are sprayed onto a thin, fragile grid surface, which would probably break under the force of a jet stream.
Therefore, researchers adjusted the gas flow rate through the nozzle – kind of like changing the setting on a garden hose from jet to mist. With this adjustment, they created a fine spray rather than a powerful stream.
Since this was a relatively new technique, the correct conditions for creating a misty spray were virtually unexplored, said Kara Zielinski, a researcher at Cornell and paper co-author. They had to test a lot of different experimental conditions, like the liquid flow rate, gas flow rate, distance of the sprayer to the grid, and even the grid type to find the optimal conditions for high quality grids and data collection, she said.
The researchers also varied the flow rates of cell and stimulant solutions within the spray device, in effect controlling how fast a sample mixed together and starting the clock for the cellular reactions researchers want to study.
Now that the researchers have proven this new technique, it could be applied to a wide variety of questions about structural dynamics on the cellular level, Zielinski said.
“It is always exciting to be part of the beginning of a new method because it often means opening up entirely new avenues of biological questions,” she said. “The opportunities are endless as we can now trigger cellular dynamics by mixing in small molecules and capture direct structural evidence of its effects.”
“I’m most excited about what this method could lead to in the future,” said Joey Yoniles, paper co-author and Stanford University graduate student. “Even if we just consider bacteria like we did in this study, we could look at the interaction between bacteria and drugs at extremely high resolution.”
The work was supported by grants from the National Institutes of Health and the National Science Foundation and the U.S. Department of Energy, Offices of Basic Energy Sciences and Biology and Environmental Research.
Citation: Yoniles et al., Molecular Biology of the Cell, 8 May 2024 (10.1091/mbc.E24-01-0042)