Cryo-TEM

Krios G4

The ThermoFisher 300 kV X-FEG Krios G4 transmission electron cryo-microscope (cryo-TEM) is coupled to a Gatan K3 (5,760 x 4,092 pixels) direct electron detector paired with a BioContinuum Imaging Filter. The system also includes a phase plate, a pre-filter Falcon III (4,096 × 4,096 pixels) direct electron detector, and a Ceta-D (4,096 × 4,096 pixels) CMOS camera optimized for low-dose diffraction data collection. Thanks to its state-of-the-art imaging technologies such as aberration-free image shift (AFIS) and fringe free imaging, the Krios G4 can collect hundreds of movies per hour. The autoloader can store and manipulate up to 12 grids and the cryo-stage with double axis holder allows ± 70 degree tilts.

The Krios G4 is ideally suited for automated applications such as cryo-electron tomography (cryo-ET), single-particle analysis (SPA), and micro-electron diffraction (MicroED).

Cryo-FIB-SEM

Aquilos 2 plus

The ThermoFisher Aquilos 2 plus is a focused ion beam scanning electron cryo-microscope (cryo-FIB-SEM) for the preparation of thin, electron-transparent, lamellae for high-resolution cryo-electron tomography (cryo-ET) and in situ structural biology. The Aquilos 2 plus features an Integrated Fluorescence Light Microscope (iFLM) for correlative light and electron microscopy (CLEM).

The microscope has a fully-rotatable cryo-stage that protects specimens from contamination and keeps them at vitrified temperatures for extended unattended runtime operation. It also features an in-chamber retractable sputter coater to render cryo-lamellae conductive and prevent their charge-up, and a gas injection system to apply protective layers over the specimen.

Relevant software available include Maps 2 for correlation and lamella preparation, AutoTEM for milling automation, and AutoSlice&View for 3D volume imaging.

Grid preparation

ELMO glow discharge system

The Cordouan ELMO glow discharger modifies the surface properties of grids and TEM support films to optimize the adsorption and spread of the specimen. A glow discharge treatment with air makes the carbon film surface negatively charged and hydrophilic which allows adsorption of aqueous solutions. The system includes a second bell jar for glow discharge with Amylamine for positively charged surface preparation.

Quorum Q150V ES Plus

The Quorum Q150V ES Plus is a combined sputter coater/evaporator system capable of both carbon coating and sputtering of oxidising metals with ultra-fine grain sizes. The lower background pressure removes oxygen, nitrogen and water vapour from the chamber avoiding chemical reactions during the sputtering process. The Q150V ES Plus uses easy-change, 57 mm diameter, disc-style targets and it is fitted as standard with a chromium (Cr) sputter target. Other targets options include: Au, Au/Pd, Pt/Pd, Pd, Pt, Cu, Ir, W, ITO and Al. Also included is an automated system for controlled carbon rod evaporation, which allows precise control of carbon film thickness. The quality of the resulting film is enhanced by the eradication of “sparking”, a common feature of less advanced coaters. The equipment features a Film Thickness Monitor (FTM) and is also fitted with a Glow Discharge insert to make the carbon film hydrophilic and negatively charged.

Vitrification

Leica EM GP2

The Leica EM GP2 automatic plunge freezer allows single, double and multiple sensor controlled blotting. Prior to freezing the specimen is kept under controlled temperature and humidity (between 4 and 60ºC and up to 99% humidity. The system allows precise pre-blot, blot and post-blot time control.

Vitrobot Mark IV

The ThermoFisher Vitrobot Mark IV system offers semi-automated vitrification to provide fast, easy and reproducible specimen preparation for cryo-EM. It can perform the cryo-fixation process at constant temperature, relative humidity, blotting force and freezing velocity.

Leica EM ICE

The Leica EM ICE high pressure freezer can cryo-immobilize aqueous specimens under high pressure with the possibility of light and electrical stimulation. Aqueous specimens thinner than 200 microns are simultaneously pressurized under 2100 bars and vitrified with liquid nitrogen at -196ºC. The sample storage dewar can hold up to 3 different specimens to perform up to 9 consecutive freezing cycles.

Computational Resources

On-the-fly data processing and storage

A 4 GPU server featuring 1 TB of RAM allows on-the-fly single-particle analysis (SPA) data processing. A 130 TB SSD hot storage server and a 500 TB HDD warm storage server are used for data management and short-term storage.