Cryogenic electron microscopy (Cryo-EM) is an extremely powerful imaging tool that has allowed researchers to view cells and pathogens down to the near-atomic or even atomic level, and has been used to visualize the structure of the spike protein of SARS-CoV-2. Cryo-EM uses ethane as a coolant to flash freeze samples without forming ice crystals, but this powerful coolant comes with disadvantages including being extremely flammable in gas form and being a factor in beam-induced motion due to the stress caused by rapid freezing. Researchers at Cornell University have now developed a new technique that removes ethane from the cryo-EM equation by enabling liquid nitrogen alone to act as an effective coolant for the method.
Liquid nitrogen typically cools at a rate roughly 50 times slower than ethane, which is why ethane is typically used to prevent ice crystals from forming and distorting the image. However, a research group led by Cornell professor Robert Thorne had previously discovered that the main factor that slowed down nitrogen cooling was the presence of cold gas hovering above the surface of the liquid. Thorne’s company MiTeGen later developed an automated cooling instrument for X-ray crystallography that removes the cold gas just before the sample is plunged into the nitrogen, finding that the method increased the cooling rate to be just six times slower than ethane. This technique was eventually tested with cryo-EM samples, which were analyzed in collaboration with the Cornell Center for Materials Research.
The researchers found that using this technique, liquid nitrogen cooled samples at just the right speed to avoid both significant ice crystal formation and beam-induced motion. The resulting images did not require the level of computer processing usually needed to correct motion blur when using ethane, simplifying the cryo-EM workflow. Additionally, the liquid nitrogen method is safer and amenable to the development of automated cooling instruments that meet laboratory safety standards, the researchers said. This research was published in IUCrJ, a journal of the International Union of Crystallography.
“Ethane is overkill. For speed you don’t need, you’re getting blurry images with beam-induced motion, and that is more problematic than any ice crystals that form from slightly slower cooling,” said Thorne. “This is a nice illustration of how basic academic science – looking into how small objects cool and how ice forms within them – can lead to practical solutions and commercial products.”