Cryo-Electron Microscopy

About 

Advances in the detector technology and image processing have revolutionized cryo-electron microscopy. It is now a method of choice for structural characterization of biological macromolecules. The combination of high-end electron microscopes coupled to direct electron detectors allows users to directly image macromolecules in vitrified ice and compute their three-dimensional structure. The approach is also well suited to study the inherent dynamics of biomolecules on molecular scale. In cell biology, tomography-based approaches can be applied to visualize subcellular structures with unprecedented resolution. The aim of the facility is to provide researchers in Dundee with access to these enabling technologies.

Equipment

Microscope

We host a state-of-the-art ThermoFisher Glacios, 200kV X-FEG cryogenic transmission electron microscope (CryoTEM) optimized for Single Particle Analysis (SPA), electron tomography, and micro-electron diffraction (microED). The Glacios is equipped with industry-leading Autoloader grid manipulation technology allowing users to assess sample quality in a contamination-free environment. It allows users to pre-screen up to 12 grids, making it easy to optimize grid preparation parameters before extended data collection.

Capabilities

Our facility is primarily aimed to provide range of techniques.

• Single particle analysis of biomolecules. In this case a sample, which is typically a protein or protein complex with a molecular weight greater than 75kDa, is frozen in solution. Many 1000's of images are collected and processed computationally to obtain a high resolution map.
• Electron diffraction (MicroED) is used to collect the electron diffraction pattern from samples. It is similar to X-ray diffraction and can be used to compute structures of small molecules and proteins. The sample can be in the form of powder or small crystals.
• Tomography provides a means to collect 3-dimensional information by rotating the sample stage instead of collecting many images. The short wavelength of electron beams means that higher resolution is possible than imaging using visible light. This provides a powerful means of characterising subcellular structures.

Our microscope is equipped with a Falcon4i Direct Electron Detector, making the Glacios both a stand-alone system for high-resolution SPA data acquisition, and a tool for pre-optimization of sample quality before transfer to a 300keV system. The microscope is also fitted with robust Ceta-D detector. This has a large dynamic range which Is suited for collection of electron diffraction data and continuous mode Imaging.

Access

New users are requested to contact the facility manager in the first instance. Established users can book time for sample preparation, microscope sessions, and consultations via the bookit system. Charges are available from the facility manager. A repository of instructional material is available via the facility SharePoint page.

Additional microscopes

To facilitate early sample optimisation through negative staining and pre-screening proteins we can also arrange access to JEOL 1100 and 1400 side entry microscopes.

Sample preparation

We have sample prep lab that has been established to provide users with the tools they need for successful sample preparation. We provide access to our glow discharge system (Quorum technologies), a FEI Mark IV Vitrobot for plunge freezing, liquid nitrogen Dewars for short-term sample storage.

Data Management and Computing

Data collected in our Glacios is transferred to centralised network file system (NFS) where internal users can access to their data. 

To facilitate image processing the facility has worked with UoD IT to install a RELION based pipeline that can be run on the high-performance computing cluster.  We are currently working to add additional capability. Users wishing to gain access should in the first instance request an account by logging a ticket via help4U. Further guidance will be provided in consultation with the facility

Access to 300 kV Cryo-EM Microscopes

Our Glacios microscope is designed for rapid screening of samples, but can also be used to collect larger datasets that can be used to generate high resolution data. In commissioning, our microscope was used to obtain a structural envelope for Apoferritin at 2.5Å resolution. In some cases, collection of data on 300 keV microscopes may provide additional information. The Dundee EM facility provides a route to submit screened samples to 300Kv microscopes at National facilities. These facilities include:

• Scottish national facility, SCMI located in Glasgow
• UK national facility at eBIC 

Facility Management

Our facility director and academic lead welcome constructive feedback and suggestions for improvement. These can also be channelled via members of the management committee.

External users are encouraged to get in touch. We can advise on how to progress your project.

Supporting your research

If you are internal to the University, you can find out more details, including booking information and pricing, on our internal SharePoint site.

If you are an external client and you’d like to find out more about how we can support your research, please get in touch to discuss your project further.

All work with external clients is undertaken in accordance with our standard Terms & Conditions.  

Funders

Wellcome, Medical Research Council