Cryo-EM (Electron Microscopy)

The MRL Cryo-EM (cryogenic electron microscopy) laboratory suite comprises rooms MRL0026 (entrance), MRL0025, MRL0026, MRL0026a and MRL0027, which are certified for Biological Safety Level 2 (BSL2) work and part of the open-access, shared facilities. The lab suite includes (see lab layout below):

• The ThermoFisher Glacios Cryo-TEM system, in addition to high-end computational resources with access to our campus computer clusters for data processing. This system is configured for single particle analysis, tomography, micro-electron diffraction (micro-ED) and general biological imaging.
• The MFP-3D-Bio atomic force microscope from Asylum Research with an inverted optical brightfield and fluorescence microscope. The tool covers a maximum lateral scan 90 µm x 90 µm, and maximum vertical range is 28 µm (closed loop, or 40 µm open loop).
• Two ThermoFisher Vitrobot Mark III and Mark IV for deep plunge and vitrification process for cryo-EM sample preparation.
• Autogrid assembling and cassette loading area.
• Negative staining area.
• Auxiliary equipment such as a fume hood, a glow discharge unit, optical microscope, and 4^oC and -22^oC fridges for storage.

The Cryo-EM lab is adjacent to the MRL Carver BioMaker, also part of the open-access, shared facilities, which includes several tools for sample preparation and processing of biological and soft materials under BS2 environment, in addition to related 3D printing.

The ThermoFisher Glacios is a high-end, 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 controlled by the ThermoFisher EPU software, allowing users to assess sample quality in a contamination-free environment. The microscope is also equipped with a Volta Phase Plate and Falcon4 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 Krios CryoTEM system.

The ThermoFisher Glacios is a high-end, 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 controlled by the ThermoFisher EPU software, allowing users to assess sample quality in a contamination-free environment. The microscope is also equipped with a Volta Phase Plate and Falcon4 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 Krios CryoTEM system.

The Glacios fills the gap in high-end electron imaging for various research areas, including structural biology, medicine, chemistry and biochemistry. Specifically, techniques such as SPA and microED are designed to determine structures of proteins, protein complexes, and small molecules at near-atomic resolution, while eliminating exclusionary sample preparation steps. For example, vitrification of biological samples permits users to sidestep the crystallization process, allowing analysis of a wide range of targets, from small, single proteins to large protein complexes. Further, the complete automation of samples manipulation allows users to pre-screen up to 12 grids at once, making it easy to optimize experimental parameters before extended data collection. Additionally, because electrons have much stronger interactions with small crystals compared to X-rays, microED application allows users to obtain solvable structures from nanocrystals (~100 nm) that would be inadequate for standard X-ray techniques. The ±70° alpha tilt capabilities of the Glacios sample stage along with the added contrast of the Volta Phase Plate also provides state-of-the-art cryo-electron tomography capabilities for ion-milled or cryo-sectioned specimens.

The Glacios sample prep lab has been constructed to provide users will the tools they need for successful sample preparation and imaging, all in one space. We provide access to our CryoTEM glow discharge system, a new FEI Vitrobot for plunge freezing, and liquid nitrogen dewars for short-term sample storage. The Glacios CryoTEM instrument room and sample preparation facilities have been specifically designed to reduce humidity and temperature fluctuations to ensure a high-quality environment for our users.

This instrument was purchased with partial financial support from the National Institutes of Health.