The Pittsburgh Center for CryoEM includes four Thermo Fisher Scientific (previously FEI) microscopes and ancillary equipment for grid preparation. The principal instrument for high resolution data collection is a Krios 3Gi microscope equipped with a Selectris energy filter, Volta phase plate and a Falcon 4i direct electron detecting camera. A Tecnai TF20 microscope is used for screening cryo-grids and also for data collection using SerialEM and TVIPS software and XF416 CMOS camera. A Gatan 910 cryoholder allows three cryo-grids to be imaged together for more rapid screening. Cryo-grids are plunge-frozen with a Mk 4 Vitrobot. A Tecnai T12 microscope is used for negative stain EM to check sample status and optimize concentration. It is equipped with an LaB6 electron source and a Gatan Orius CCD camera. An Aquilos 2 is used for FIB-milling thick cellular samples so that they are compatible with cryo-EM, and particularly tomography.
Ancillary equipment includes:
- Vitrobot Mk 4 (www.thermofisher.com)
- EmiTech K100X glow discharger (www.quorumtech.com – in “not supported”)
- Fischione 1020 plasma cleaner (https://www.fischione.com/products/m1020)
- Cryoholder pumping station (Gatan 655 top & Pfeiffer HiCube 80)
- Denton DV-502 evaporator (www.dentonvacuum.com)
Acknowledgements. Please use one of the following statements in publications and presentations to acknowledge funding for the Center for CryoEM:
- If images were collected on the Krios microscope:
The Pittsburgh Center for CryoEM (RRID:SCR_025216) used for data collection in this project was supported, in part, by the University of Pittsburgh, the School of Medicine, the Department of Structural Biology, and the National Institutes of Health (grants S10-OD-019995 and S10-OD-025009). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. - Other data not using the Krios:
The Pittsburgh Center for CryoEM (RRID:SCR_025216) used for data collection in this project was supported, in part, by the University of Pittsburgh, the School of Medicine, and the Department of Structural Biology.
Please acknowledge Center personnel who assist with your work, and consider extending co-authorship(s) if their involvement is significant.
Equipment
| Equipment Name | Description |
|---|---|
| Thermo Fisher Scientific Aquilos 2 | The Aquilos 2 is a cryo-FIB (focused ion beam) mill used for preparing thin, electron-transparent lamellae for high-resolution cellular cryo-electron tomography on the Krios. |
| Thermo Fisher Scientific Krios 3Gi | The Titan Krios 3Gi is used for collecting large datasets of high-resolution images of protein complexes from which structures can be solved to 2 Ångstroms and better. |
| Thermo Fisher Scientific Tecnai T12 | The Tecnai T12 operates at 120 kVolts and is equipped with an LaB6 electron source and a Gatan Orius CCD camera. |
| Thermo Fisher Scientific Tecnai TF20 | The Tecnai TF20 operates at 200 kVolt and is equipped with a field emission gun and a TVIPS XF416 CMOS camera. |
| Thermo Fisher Scientific Vitrobot Mk 4 | The Thermo Scientific Vitrobot Mark IV System offers semi-automated vitrification to provide fast, easy, and reproducible sample preparation for cryo-EM. |