Glow discharge apparatus for high quality and reproducible cryo-electron microscopy specimen preparation
Cryogenic electron microscopy (cryo-EM) has become the preferred method for determining the atomic-resolution structures of macromolecules 1. The preparation of cryo-EM specimens is a multi-step procedure, typically divided into four main stages: (i) glow-discharging the cryo-EM grids, (ii) applying the macromolecular sample to the grid, (iii) blotting off excess protein solution, and (iv) vitrifying the specimen in a cryogenic liquid. Specialized equipment is employed in steps (iii) and (iv) to meticulously control these processes. The importance of vitrification in cryo-EM grid preparation was highlighted when the Swiss researcher Jacques Dubochet received the Nobel Prize in Chemistry in 2017 for his pioneering work in this area (https://www.nobelprize.org/prizes/chemistry/2017/summary/).
Cryo-EM grids used for high-resolution protein structure determination are typically made of copper or gold with a holey carbon film attached on one side. Chemically, this surface is hydrophobic and would naturally repel hydrophilic protein samples. Therefore, cryo-EM grids must be rendered hydrophilic through a process called glow-discharge. Since the glow-discharging procedure significantly impacts protein absorption behavior on cryo-EM grids, precise control of this process is essential. With the funding acquired through the “UniBern Forschungsstiftungs” grant, we obtained a Dual-Chamber PELCO easiGlow™ glow-discharging device, which allows for controlled glow-discharging and ensures a more reliable and reproducible production of cryo-EM specimens (Figure 1A).

The PELCO easiGlow™ glow-discharging device was installed in the sample preparation room of the structural biological branch of the Microscopy Imaging Center of the University of Bern (MIC, https://www.mic.unibe.ch/). Since its installation, this device has been used to produce cryo-EM specimens that are analyzed on the MIC’s high-end electron microscope Titan KRIOS G4. The use of the PELCO easiGlow™ device has already proven invaluable to our research, enabling our group to recently publish the high-resolution structures of (i) the bacterial green-light-absorbing proton pump proteorhodopsin (GPR) 2 (Figure 1B) and (ii) the bacterial glucose transporter IIC(B) 3 (Figure 1C). Moreover, the PELCO easiGlow™ glow-discharging device holds significant value for all MIC users at the University of Bern.
Jean-Marc Jeckelmann, PhD
Institute of Biochemistry and Molecular Medicine
Links:
1 Guaita, M., Watters, S. C. & Loerch, S. Recent advances and current trends in cryo-electron microscopy. Curr. Opin. Struct. Biol. 77, 102484 (2022). https://doi.org/10.1016/j.sbi.2022.102484
2 Hirschi, S. et al. Structural insights into the mechanism and dynamics of proteorhodopsin biogenesis and retinal scavenging. Nature Commun. 15, 6950 (2024). https://doi.org/10.1038/s41467-024-50960-3
3 Roth, P. et al. Structure and mechanism of a phosphotransferase system glucose transporter. Nature Commun. 15, 7992 (2024). https://doi.org/10.1038/s41467-024-52100-3