Abstract
The increasing demand for cryo-electron microscopy (cryo-EM) reveals drawbacks in current sample preparation protocols, such as sample waste and lack of reproducibility. Here, we present several technical developments that provide efficient sample preparation for cryo-EM studies. Pin printing substantially reduces sample waste by depositing only a sub-nanoliter volume of sample on the carrier surface. Sample evaporation is mitigated by dewpoint control feedback loops. The deposited sample is vitrified by jets of cryogen followed by submersion into a cryogen bath. Because the cryogen jets cool the sample from the center, premounted autogrids can be used and loaded directly into automated cryo-EMs. We integrated these steps into a single device, named VitroJet. The device’s performance was validated by resolving four standard proteins (apoferritin, GroEL, worm hemoglobin, beta-galactosidase) to ~3 Å resolution using a 200-kV electron microscope. The VitroJet offers a promising solution for improved automated sample preparation in cryo-EM studies.
Highlights
The increasing demand for cryo-electron microscopy reveals drawbacks in current sample preparation protocols, such as sample waste and lack of reproducibility
Autogrids were initially developed for increased robustness in order to allow automated handling of grids in cryo-transmission electron microscopes (TEMs), such as the Titan Krios, Glacios, and Arctica (Thermo Fisher Scientific)
Samples applied to pre-mounted autogrids are difficult to blot and even more difficult to vitrify using the existing leading commercial devices or even the next-generation blotless commercial device Chameleon[31]. We overcame both problems by using pin printing, which does not require blotting, and jet vitrification, which yields superior cooling rates starting from the center of the grid where the sample is located
Summary
The increasing demand for cryo-electron microscopy (cryo-EM) reveals drawbacks in current sample preparation protocols, such as sample waste and lack of reproducibility. Our method consists of (i) an integrated glow-discharge module to control and minimize the time between plasma cleaning and sample deposition; (ii) pin-printing for sample application, which requires only sub-nanoliter sample volumes and eliminates sample blotting; and (iii) jet vitrification, which allows for the handling of autogrids. We integrated these features into a single setup, termed the VitroJet, and used it to prepare four standard proteins to obtain high-resolution single-particle reconstructions
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