CryoNET
October 4-5, 2023, Stockholm, Sweden
October 4th, 16:40 CET: VitroJet poster presentation
While we are continuing the international roll out of VitroJet software 1.5, we are looking forward to sharing the latest VitroJet cryo-EM sample preparation knowledge, experiences and results with the Nordics. We hope to meet you all again live during this year’s poster presentation on Tuesday afternoon October 4th at 16:40 CET.
Application Engineer Vasiliki Angelidi will talk you up to speed about the fully automated VitroJet sample preparation technology and the latest capabilities of VitroJet software 1.5, like the autocycle for fully automated grid vitrification, and how you can determine ice thickness. Preparation is key to advance, so stay ahead and read the abstract below.
VitroJet: Moving Cryo-EM Sample Preparation into the New Era
Cryo-electron microscopy (cryo-EM) is increasing in popularity as a method for biomolecular structure determination. [1,2] Once a good quality grid is prepared for the microscope, data collection and biomolecular processing are streamlined. Within the cryo-EM workflow, the main bottleneck is sample preparation. Optimizing the combination of biochemistry and grid preparation needs many and long iterations. The number of required iterations on the microscope can be reduced significantly with reproducible grids of which the quality can be determined before usage. This makes the cryo-EM infrastructures more efficient, enabling structure determination in a shorter timeframe. For this reason, the VitroJet was developed. The VitroJet focuses on controlling the grid preparation process, enabling to investigate protein behavior in a structured manner.[3] An integrated plasma treatment makes grids reproducibly hydrophilic. Sample deposition through pin-printing decreases shear forces in comparison to the traditional blotting method and reduces the required volume extensively. Furthermore, it allows for reproducible control of layer thickness, on which the obtainable resolution of the protein of interest is highly dependent.[4] Visual feedback from two implemented cameras enables sample quality inspection and layer thickness estimation on a nanoscale before electron microscope screening. The visual feedback of the grid camera correlates with the grid atlas taken on the electron microscope, giving a clear indication of the number of usable holes before electron microscopy imaging. Jet vitrification enables vitrification of autogrids, reducing manual handling and removing the need for the tedious postclipping process. Since any grid type and only sub-nanoliter volumes are used, the VitroJet can be used for all single particle analysis applications. In this manner, the electron microscopes can be fed with high quality samples to investigate protein behavior. In collaboration with different labs over the world, we have obtained results varying from protein complexes, membrane 16 proteins, and cellular like applications. By adapting the parameter settings of the VitroJet, ice thickness and gradients can be adjusted. Measuring of ice thicknesses with the implemented VitroJet camera saves microscope screening time. Ice thicknesses were determined by using the energy filter method in the electron microscope [5], and the outcome was correlated with VitroJet settings and camera feedback. We observed that by changing the velocity, as well as the distance between the grid and the pin during writing, the layer thickness can be adapted in a controlled manner. We show high reproducibility when using the same protocol settings. Furthermore, a high number of different samples have been processed together with our customers, of which some results will be shown. Overall, the VitroJet enables high control and reproducibility of a broad range of cryo-EM samples.
Applicants Vasiliki and Erin Leahy will be available live in Stockholm to answer your questions to move your cryo-EM Sample Preparation into the new era of full automation! Visit the CryoNET event page for additional info