title: Spotiton: a prototype for an integrated inkjet dispense and vitrification system for cryo-TEM.
abstract: Over the last three decades, Cryo-TEM has developed into a powerful technique for high-resolution imaging of biological macromolecules in their native vitrified state. However, the method for vitrifying specimens onto EM grids is essentially unchanged - application of ∼3 μL sample to a grid, followed by blotting and rapid plunge freezing into liquid ethane. Several trials are often required to obtain suitable thin (few hundred nanometers or less) vitrified layers amenable for cryo-TEM imaging, which results in waste of precious sample and resources. While commercially available instruments provide some level of automation to control the vitrification process in an effort to increase quality and reproducibility, obtaining satisfactory vitrified specimens remains a bottleneck in the Cryo-TEM pipeline. We describe here a completely novel method for EM specimen preparation based on small volume (picoliter to nanoliter) dispensing using inkjet technology. A first prototype system (Spotiton v0.5) demonstrates feasibility of this new approach for specimen vitrification. A piezo-electric inkjet dispenser is integrated with optical real-time cameras (100 Hz frame rate) to analyze picoliter to nanoliter droplet profiles in-flight and spreading dynamics on the grid, and thus provides a method to optimize timing of the process. Using TEM imaging and biochemical assays we demonstrate that the piezo-electric inkjet mechanism does not disrupt the structural or functional integrity of macromolecules. These preliminary studies provide insight into the factors and components that will need further development to enable a robust and repeatable technique for specimen vitrification using this novel approach.
authors.affiliation: The National Resource for Automated Molecular Microscopy, Department of Cell Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA. tjain@scripps.edu
authors.forename: Tilak
authors.initials: T
authors.lastname: Jain
authors.forename: Patrick
authors.initials: P
authors.lastname: Sheehan
authors.forename: John
authors.initials: J
authors.lastname: Crum
authors.forename: Bridget
authors.initials: B
authors.lastname: Carragher
authors.forename: Clinton S
authors.initials: CS
authors.lastname: Potter
mesh_terms: D020285:Cryoelectron Microscopy / Q000295:instrumentation / Q000379:methods*; D055032:Electron Microscope Tomography; D005615:Freezing; D046911:Macromolecular Substances / Q000737:chemistry / Q000648:ultrastructure; D015203:Reproducibility of Results; D013048:Specimen Handling / Q000295:instrumentation / Q000379:methods; D058989:Vitrification
chemical_list: D046911:Macromolecular Substances