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Cryo Electron Microscopy

Vanderbilt Cryo Electron Microscopy Facility

Overview

The V-CEM facility provides expertise and resources for the structural analysis of biological molecules using single particle analysis, tomography, and two-dimensional (2D) crystallization. The facility houses high-resolution transmission electron microscopes (TEMs) and cameras needed to collect data for single particle analysis and tomography.

The facility houses a 300 kV FEI Polara equipped with a Gatan K2 Summit Direct Electron Detector (DED) camera, a 200 kV FEI F20 equipped with a Gatan Ultrascan 4K x 4K CCD, two FEI vitrobots (mark II\III), and the accompanying instrumentation needed for both negative stain and cryo-EM analysis. Biological samples are prepared for Molecular EM using either negative stain or vitrification (i.e. cryo-EM). For negative stain EM, the sample is embedded in a layer of dried heavy metal solution. For cryo-EM, the sample is suspended across a holey carbon grid and then rapidly plunged into cryogen, trapping the molecules in a thin layer of vitrified ice composed of the purification buffer. Negative stain provides high-contrast structural information mainly of the outer envelope of a molecule, while cryo-EM allows for the structural determination of near-atomic to atomic resolution 3D structures.

Mission Statement

The goal of V-CEM is to make molecular EM accessible to all interested researchers at Vanderbilt. Molecular-EM is one of the fastest developing, cutting-edge structural techniques for exploring the organization of biological macromolecules. The unique strength of molecular EM is its versatility. Structures determined using molecular EM can span resolution ranges from very low- to atomic-levels (>30–1.8Å) and do not require crystalline samples for analysis.

In pursuit of its goal, the V-CEM enables molecular EM training and outreach by

  1. Providing extensive training to new users.
  2. Providing advise and support including specimen preparation, data collection and image processing, computational image analysis, and structure determination.

Equipment

FEI Tecnai F20 cryo-transmission electron microscope

  • 200keV Field Emission Gun (FEG) electron source
  • 4096 x 4096 pixel CCD camera
  • Side-entry Gatan 626 cryo-holders

 

FEI Tecnai TF30 Polara cryo-transmission electron microscope

  • 300keV Field Emission Gun (FEG) electron source
  • 4096 x 4096 pixel CCD camera
  • Ultra-stable Polara liquid nitrogen or helium stage

 

FEI Vitrobot: Climate-controlled robotic grid preparation

  • Two carbon evaporators and glow discharge units
  • Automated microscopy software packages for both single particle and tilt series (tomographic) data acquisition