Equipment

Cryo-Transmission Electron Microscope

1. Titan Krios G3 300kV Cryo-Electron Microscope

The FEI Titan Krios G3 300kV Cryo-Transmission Electron Microscope is the premier cryo-TEM that is available for both single particle averaging and 3D tomography of vitrified samples. Equipped with a high-brightness field emission gun (X-FEG) and a high-stability 300kV high-tension (HT) generator, the system easily facilitates the high-resolution, sub-nanometer scale imaging of frozen biological specimens. The electron optical column is equipped with ConstantPower™ lenses, which have optimized magnetic circuitry designed to reduce cross talk and keep beam hysteresis to a minimum. This platform is also equipped with an image corrector that removes the increased spherical aberration present due to the larger pole-piece gap of the C-TWIN objective lens, which is required to accommodate large angle specimen tilts in tomographic data acquisition. In addition, the system is equipped with a unique, one-of-a-kind cryogenically cooled sample handling and auto-loading system. The Titan Krios G2 platform has been configured with two detection cameras, a scintillated CMOS camera (FEI Ceta), which will be used for regular TEM/STEM imaging and diffraction applications and an energy-filtered Direct Electron Detection camera (Gatan K2 Summit on a GIF energy filter) for the low dose imaging of frozen biological specimens. In addition, the platform is equipped with two other capabilities, phase contrast imaging and STEM imaging. The phase contrast system almost entirely removes the necessity to acquire images of low contrast objects under beam defocus conditions. Finally, the STEM imaging functionality is essential in high-resolution studies of cellular macromolecules in situ.

System configuration:

  • Sample Auto-loading System - allows twelve independent samples to be loaded into a cassette, which can be transferred into the auto-loading system for delivery into the Titan Krios for imaging. The auto-loader is maintained at liquid nitrogen temperatures to protect the samples from any environmental factors, and each individual sample holder (AutoGrid) is robust and protects the sample against deformation and / or damage.
  • Ceta CMOS camera - The Ceta is a 16 Megapixel digital camera designed specifically for regular TEM/STEM imaging. The large field of view and small pixel size (a characteristic of CMOS sensors) allows a fast read-out for dynamic experiments and is capable, of recording full-frame 4k x 4k images at 30 fps and a reduced field of 512 x 512 at > 250 fps. The camera can also easily switch between this fast mode and high-resolution mode with ease.
  • Gatan K2 Summit Direct Electron Detector with GIF Energy Filter - The combined Gatan K2 Summit Direct Electron Detection camera and GIF energy filter – the “BioQuantum 967” allows for the direct detection of electrons at high sensitivity. In addition, the presence of the GIF energy filter allows the preferential detection of zero-loss electrons, thus serving as an additional mechanism to enhance the contrast of unstained, vitrified biological specimens.
  • Cs Aberation Corrector - The C-TWIN objective lens with its larger pole-piece gap is essential for high tilt angle tomographic acquisitions. Its symmetric design allows for the changing of the accelerating voltage without adjusting the stage z-position. Additionally, it ensures that there is no performance compromise, or any re-alignment between TEM and STEM imaging modes. However, whilst important for tomography, the larger pole-piece gap in the C-TWIN objective lens increases spherical aberration. The addition of an image corrector removes this added spherical aberration (Cs) thus restoring the TEM/STEM resolution to that of a Titan microscope platform with a smaller pole-piece separation.
  • Volta Phase Contrast System - A major issue in the imaging of unstained, vitrified samples is the lack of electron density, and thus contrast in the acquired images. A new, and cutting-edge approach that almost entirely removes the need for the addition of beam defocus is phase contrast imaging. By separating the phases of elastically and inelastically scattered electrons that pass through the sample by 90o, the in-focus contrast in the imaging plane can be drastically improved, which is beneficial in the imaging of weak phase objects, such as vitrified biological specimens.
  • STEM detector - The ability to scan the electron beam whilst acquiring the transmitted electron signal, a so-called “STEM” image is essential in high-resolution studies of cellular macromolecules. The incorporation of STEM functionality onto the Titan Krios G2 platform increases its versatility without compromising any of the other imaging modes of the microscope (TEM, EFTEM etc).

 

Sample Preparation

1. FEI Vitrobot Mk IV

As a part of the Cryo-TEM workflow, it is necessary to create high-quality vitrified samples for single particle analysis or cryo-tomography applications. The FEI Vitrobot offers fully automated vitrification, fast and easy. It performs the cryo-fixation process at constant physical and mechanical conditions like temperature, relative humidity, blotting conditions and freezing velocity. This ensures high quality cryo-fixation results and a high sample preparation throughput prior to cryo-TEM observation. The Vitrobot's controlled environmental technology prevents cooling and concentration of artifacts that are inevitable in other freezing protocols. The transfer from the vitrification medium into the liquid nitrogen atmosphere has been automated, ensuring consistent sample quality at high sample throughput.

Benefits for Cryo-EM:

  • User-defined settings ensure maximum flexibility
  • Fully automated and reproducible vitrification of suspensions
  • High vitrification quality through controlled environment
  • High sample throughput
  • An effective user interface assures easy and flexible instrument control

2. Gatan Solarus 950 Advanced Plasma Cleaner

Solarus is a new generation plasma system engineered on new concepts and the latest technology resulting in better performance, simplified use, and consistent cleaning results. It is radically different from any plasma system available in the market and offers the most effective cleaning for removal of hydrocarbon contamination on transmission electron microscope (TEM) and scanning electron microscope (SEM) samples.

System Features:

  • Exclusive H2/O2 gas chemistry (patent pending)—this unique chemistry provides superior cleaning with less sputter damage of all samples including holey carbon films, and further enables samples to remain significantly cooler than if cleaned with the traditional Ar/O2 gas recipe.
  • Interactive touch screen interface offers seven pre-programmed recipes with optimized process parameters for greater consistency and repeatability.
  • Real time RF auto-match and variable RF power supply (10 – 65 W) ensure optimum plasma power and low loss under any cleaning conditions.
  • Mass flow controllers (MFC) provide accurate gas control for long term plasma stability and support up to three independent process gases H2, O2 and Ar.
  • Multi-functional chamber accepts two TEM holders through front ports while multiple SEM samples are loaded through the top opening lid.
  • Full time turbo drag pumping system enables overall faster cycle times—faster pump down (50 s vs. the competition's 3 min); faster venting (5 s vs. the competition's 6 min).

3. Gatan 626 Cryo Transfer Holder for JEOL JEM-1400 TEM

Cryo-transfer holders are used in applications that require low-temperature transfer and observation of frozen-hydrated specimens for cryo-electron microscopy (cryo-EM). The 626 from Gatan has a minimum operating temperature of -170C. An associated cryo-workstation provides a protected environment for loading a frozen specimen into the holder. A cryo-shield encapsulates the frozen-hydrated specimen to protect it from contamination during atmospheric transfers from the cryo-workstation to the transmission electron microscope (TEM).

4. EMS GloQube™ Glow Discharge System

The EMS GloQube™ is the first-of-its-kind compact, easy to use, stand-alone glow discharge system. The GloQube™ has two independent vacuum chambers: a clean chamber, designed for applications requiring hydrophobic/hydrophilic conversion, typically using air as the process gas; and a vapor chamber, designed for use with reagents such as methanol and alkylamine. With operator safety firmly in mind, reusable septum-sealed reagent vials are used. Loading and removing reagents is convenient and reliable – the vial, located in its holder, is inserted into a shielded needle using a simple bayonet fitting. To prevent accidental damage, the high voltage lead is shielded. The plasma current is variable by adjustment of the vacuum level using an argon leak valve with the plasma voltage being preset. For maximum sputter coating efficiency, the gas injector system ensures that argon gas enters the chamber close to the plasma discharge. Venting is to argon.

The primary application of the EMS GloQube™ is the hydrophilization (wetting) of carbon-coated TEM support films and grids which otherwise have the tendency to be hydrophobic. Glow discharge treatment with air will make film surfaces negatively charged and hydrophilic and allow the easy spread of aqueous solutions.

Easy sample loading, fast turnaround times
Each chamber can accommodate two 25 x 75 mm glass microscopes slides. Loading could not be easier using draw-style chamber doors and specimen stages. The stages are height adjustable and fitted with removable glass slide holders. For additional convenience – and to allow easy access for chamber cleaning – the stages can be completely removed.

Touch screen control – rapid data input, simple operation
The intuitive touch screen allows multiple users to rapidly input and store preferred process "recipes". Typical default glow discharge protocols are loaded as standard. Additionally, help files and useful maintenance data such as system on time and time since last clean are readily available to the operator. An Ethernet communications port is included for software updates.

Vacuum, automatic valving and controlled venting
The GloQube™ has automatic valving between chambers which maintains cleanliness by preventing cross-contamination. At the end of a process run, automatic soft venting to atmosphere through filtered inlets ensures TEM grids are not disturbed. The GloQube™ requires a single vacuum pump working in the 0.1 to 1 mbar range. A typical pump time to operational vacuum is 60 seconds.

Key System Features:

  • Dual independent chambers
  • Hydrophilic/hydrophobic and negative/positive modes
  • Fully automatic, short process times
  • Intuitive touch screen control
  • Safe vapor delivery using septum-sealed vials
  • Automatic valving between chambers to prevent cross-contamination
  • Quick and easy sample loading
  • Controlled venting to prevent sample disturbance
  • Consistent, reliable results