Cryo-EM Core

Primary use: Data collection

The Titan Krios is a revolutionary, state-of-the-art transmission electron microscope that visualizes cells, proteins and other crucial structures through atomic-resolution 3-D microscopy and cellular tomography. Unlike traditional methods, it does not require samples to be crystallized prior to imaging, saving time and resources while also capturing the target in its natural state.

Capabilities

• 3-D cryo-electron microscopy
• 2-D electron crystallography
• Single particle analysis
• Dual-axis cellular tomography

Specifications

• 80–300 kV operating range
• Gatan Quantum 967 LS imaging filter
• Post-GIF Gatan K3 direct detection camera
• Pre-GIF Ceta 16M camera
• Automated loading for up to 12 samples
• Ultra-stable Schottky field emitter gun
• Three-lens condenser system for parallel sample illumination
• Digital system for remote operation
• Volta phase plate, live image processing

Primary use: Cryo-EM grid screening and data collection

Considered to be the workhorse of VAI’s Cryo-EM Core, the Talos Arctica is a transmission electron microscope that provides high-resolution and high-throughput 2-D and 3-D imaging of frozen, hydrated biological components, including cells, organelles and proteins.

Capabilities

• 3-D cryo-electron microscopy
• 2-D electron crystallography
• Single-particle analysis

Specifications

• 200 kV FEG transmission electron microscope
• Automated loading for up to 12 samples
• Falcon 3EC direct electron detector
• Ceta 16M camera
• K2 direct detection camera

Primary use: Biological sample imaging and protein screening

The Tecnai Spirit G2 BioTWIN is a flexible 80–120 kV microscope mainly used for life science biological specimen imaging.

Capabilities

• Life science TEM imaging
• Negative-stain imaging
• High-contrast, high-resolution imaging

Specifications

• 80–120 kV operational range
• Gatan Orius 832 CCD camera
• Auto-Gun and automatic tuning
• Smart tracking position system

Linux workstations are an indispensable part of the cryo-EM image processing pipeline. The Core has three Linux workstations (Silicon Mechanics Workform 2000.v6) dedicated to cryo-EM analysis that are reserved for core users.

Specifications

• CPU: Intel Xeon E5-2620. 8 cores, hyperthreading, 20 MB cache, 14 nm, 85W
• Ram: 128GB DDR4-2400 ECC registered
• GPU: NVIDIA GeForce GTX 1080 Founders Edition with 8 GB GDDR5X
• Disk: Two 6TB drives in a RAID1 mirror

Software

• Linux Centos 7.3 OS with GNOME
• Relion
• EMAN2
• Pymol

• Automatic EM and LM tissue processor: Leica EM TP
  • Primary use: Automated tissue processing
  • Capabilities: Preparation of tissue for embedding

• Ultramicrotome: Leica EM UC7
  • Primary use: Preparation of ultrathin sections
  • Capabilities: Preparation of high-quality, consistent ultrathin sections with a thickness feed range from 1 nm–15 µm
  • Specifications: Cutting speed 0.05–100 mm/s, feed 1nm–15mm

• Automatic contrast system: Leica EM AC20
  • Primary use: Staining of ultrathin section
  • Capabilities: Automatic double staining of ultrathin sections
  • Specifications: Adjustable stain time and reagent consumption,  capable of  staining 20 grids at the same time, up to 10 prestored programs

• Free cryo-EM project consultation
• Training in all aspects of cryo-EM, including sample preparation, microscope operation, image processing
• Cryo-grid freezing and screening
• Single particle cryo-EM data collection
• Cryo-electron tomography data collection

Training and access to services

Internal users should log in to CrossLab and schedule service times through the system. New users who need login information should contact Lori Moon. Once an account is opened, new users could submit a training request in iLab.

For all Cryo-EM Microscopy inquiries, please contact Dr. Gongpu Zhao.

• Free consultation at the beginning of any new project
• Optimization of experimental procedures and develop methods to meet investigator’s need
• Processing of biological specimens for TEM
• Negative stain TEM imaging
• Immuno-EM
• Ultramicrotome thin sectioning for TEM
• Semi-thin section glass slide preparation and staining for light microscopy
• Production of high-quality ready-to-image samples and high-quality micrographs
• Data interpretation and image analysis 

Biological EM workflow

• Typical biological specimens may include tissue blocks, vibratome tissue slices, cell pellets, a monolayer of cells, cells on transwell membranes, etc.
• We can process up to six different types of samples (six different experiments/treatments) per processing run, for the same cost. Multiple specimens per the same type of sample (duplicates) are allowed.
• We do not accept unfixed specimens. We recommend fixing specimens using a fixative that contains both paraformaldehyde and glutaraldehyde (e.g., 2% PFA and 2.5% GA, in 0.1M NaPO₄ buffer, pH 7.2-7.4). If perfusion fixation is not possible, get the sample into a fixative solution as quickly as possible once removed from a live source. You can further dice to the appropriate size in fixative.
• Ideally, tissue dimensions for TEM should be ~1mm³. But 1 mm in only one dimension is also acceptable (e.g., 1 x 1 x 2mm). The size limit is due to penetration capability of fixatives for good TEM ultrastructure.
• For cell pellets, we prefer a minimum of 1 million cells per sample. The typical thickness of vibratome tissue sections should be 10–300 um. For negative stain TEM, 1–5 million particles per 100 uL would be acceptable.
• Core staff will perform the sample preparation, ultramicrotomy sectioning, post-staining, and TEM imaging. Once you receive full training on Tecnai G2 Spirit TEM from us, you can perform TEM imaging by yourself.
• Immuno-EM requires special fixation and techniques. Please reach out to us if you are interested in this service.

Core staff will provide a better estimate based on the investigator’s needs. For biological EM services, please contact Dr. Gongpu Zhao and Dr. Ishara Ratnayake.

Cryo-EM is short for cryo-electron microscopy, a technique that allows scientists to see tiny molecules down to 1/10,000^th the width of a human hair. Although cryo-EM techniques have been around since the mid-1980s, recent advances in technology have led to a scientific revolution, giving scientists the ability to obtain more detailed images of cells, viruses and molecules in their natural state than ever before. In 2015, cryo-EM was named Nature’s Method of the Year.

1. An introduction to electron microscopy
2. Biological Electron Microscopy books

• • Biological Electron Microscopy: Theory, Techniques, and Troubleshooting
    Michael J. Dykstra and Laura E. Reuss

• • Biological Specimen Preparation for Transmission Electron Microscopy
    Audrey M. Glauert and P. R. Lewis

• • Electron Microscopy: Principles and Techniques for Biologists
    J. Bozzola and L.D. Russell

3. Virtual learning tool
4. Electron Microscopy Atlas

Structural biology focuses on the shape of the molecules that make up a living organism. As many in the field say, structure is function, meaning that the architecture of a molecule directly informs its role and how it does its job.

Cryo-EM allows scientists to more precisely see proteins and other important structures without many of the constraints posed by traditional methods.

To put its impact in context, in 2007 fewer than 10 molecular structures at less than 5 angstroms (a very tiny unit of measurement) were reported in the literature. Thanks to improvements in cryo-EM technology, by 2015 that number had shot up to nearly 200 (Egelman EH et al; 2016).

Remember, structure informs function. A better understanding of the architecture of cells and molecules gives scientists important insight that allows for the development of new preventative measures, diagnostics and therapies for an untold number of diseases and health problems.

A classic example is that of the key and the lock. If scientists know what the lock looks like, they can develop a key to fit it. In much the same way, if they know what a virus looks like, they are much better equipped to develop a drug that can combat it.