Cryogenic Electron Microscopy Techniques for Soft-Matter Materials Liquid Crystal Institute, Kent State University, Kent, OH 44242 In this poster, we present an overview of cryogenic electron microscopy (cryo-EM) techniques established at LCI Characterization Facility for soft-matter materials (e.g., bio-materials, liquid crystals, oils, and polymers) . These materials often impose great challenges for electron microscopy studies due to the weak contrast, difficulties in specimen preparations and radiation damage. As in situ electron microscopy techniques are still not practical at the current stage, cryo-EM techniques are often considered to be a desired choice for many soft-matter materials. Excellence in Action Introduction Other techniques @ LCI Characterization Facility FEI Vitrobot (Mark IV) Cryo-ultramicrotome (Leica UC7/FC7) Freeze fracture (BalTec/Leica BAF060) Lipid Vesicles • Dr. Min Gao: [email protected] or 330-672-7999 • Lab website: Just Google “LCI TEM ” UV exposure The self-assembly of a light-driven supramolecular polymer. See J. Am. Chem. Soc., 2013, 135, p 5990 for details. Contact Plunge freezing High pressure freezing Cryo-TEM RT-TEM (cryo-) Ultra- microtome Freeze fracture Freeze substitution Generally available Cryo - techniques for soft - matter materials * Some techniques can also be used to prepare cryo - SEM specimens . * Cryo - FIB can be a promising alternative to cryo - ultramicrotome . Specific specimen preparation routines established for liquid crystals Thin film approach: plunge freezing Bulk approach: cryosectioning Bulk approach: freeze fracture Mainly for thermotropic LCs (TLCs) Mainly for Lyotropic LCs (LLCs) Thin film approach: Part of the specimen is electron-transparent before freezing. No further processing is required. Bulk approach: A thick (> a few microns) material is frozen, and goes through further processing to get thin TEM specimen. Plunge Freezing • Basic idea : preserve the native structure of a soft matter sample by throwing it rapidly into cryogen (e . g . , liquid nitrogen or liquid ethane) manually or using a machine . • For aqueous samples (for example, many biological materials and lyotropic liquid crystals), > 10 4 degree/second cooling rate is required for vitrification . • For very thin specimens, the above cooling rate can be achieved routinely using liquid ethane as cryogen . • For non - aqueous samples, a slower cooling rate may still lead to good preservation of the native structure . It is possible to freeze thick samples to make TEM specimens using cryo - sectioning or freeze fracture . High Pressure F reezing • Basic idea : To slow down the crystallization of water in aqueous samples by applying a high pressure (~ 2000 times the atmosphere pressure) . So a much slower cooling rate can be used for vitrification of thick aqueous samples . • The frozen samples can then be cryo - sectioned or freeze - fractured to obtained TEM specimens . High pressure freezer (Leica EM Pact2) FEI Tecnai F20 equipped with STEM, EELS, EDS, etc. Freeze Fracture • Basic idea : A frozen sample is fractured inside a vacuum chamber at low temperature . Supposedly, the tomography of the fractured surface may represent the native structure . The fractured surface is shadowed and replicated by depositing Pt /C (or other heavy metals) at an angle (for example, 45 ° ), and carbon from the top . The real material is then dissolved/removed . The replica is studied by room temperature TEM (RT - TEM) . • Freeze fracture can work for a variety of materials . Cryo - Ultramicrotomy • Basic idea : Use a diamond knife to section frozen samples into electron transparent slices which are then collected on carbon coated TEM grids . • The cryo - ultramicrotome at LCICF TEM lab is equipped with a discharge device and a micro - manipulator for challenging materials . Application examples Microvesicles produced during blood macrophage differentiation. Ismail, et al., Blood 121 (2013) 984. Suspended lyotropic chromonic liquid crystal (nematic phase): side-view (a) and top-view (b) of the of the aggregates. See J. Mater. Chem. C, 2014,2, 8780-8788 for details. Carbon Suspended 5CB STEM “Bulk” Replica FFTEM of CB7CB in twist- bend nematic phase. See Nature Comm 4: 2635, 2013 for details. Thin film plunge - freezing and cryo - TEM Cryo - ultramicrotomy and cryo - TEM TEM Thin film plunge-freezing (for comparison) “Bulk” cryo-ultramicrotomy (Plunge-frozen bulk sample) Liquid Crystal-nanoparticles composite: Au nanoparticles doped 5CB. High-pressure frozen lyotropic chromonic DSCG: side-view (a) and top-view (b) of the aggregates. Microscopy Research and Technique, 77, p.754-772 (2014) Freeze fracture and RT - TEM E-beam writer on a environmental SEM Atomic force microscopes Website: http://www.lcinet.kent.edu/organization/facility/characterization/index.php • High Speed Camera (HS-camera) • FTIR microscope • Confocal scanning laser microscope • Abbe Refractometer • Differential Scanning Calorimeter (DSC) • … Room temperature heliconical twist-bend nematic liquid crystal. CrystEngComm, 17, 2778-2782, 2015. Version: 05/2015 @ JCU
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Cryogenic Electron Microscopy Techniques for Soft-Matter Materials
Liquid Crystal Institute, Kent State University, Kent, OH 44242
In this poster, we present an overview of cryogenic electron microscopy (cryo-EM) techniques established at LCI Characterization Facility forsoft-matter materials (e.g., bio-materials, liquid crystals, oils, and polymers) . These materials often impose great challenges for electronmicroscopy studies due to the weak contrast, difficulties in specimen preparations and radiation damage. As in situ electron microscopytechniques are still not practical at the current stage, cryo-EM techniques are often considered to be a desired choice for many soft-mattermaterials.
Excellence in Action
Introduction
Other techniques @ LCI Characterization Facility
FEI Vitrobot (Mark IV)
Cryo-ultramicrotome(Leica UC7/FC7)
Freeze fracture (BalTec/Leica BAF060)
Lipid Vesicles
• Dr. Min Gao: [email protected] or 330-672-7999• Lab website: Just Google “LCI TEM”
UV exposure
The self-assembly of a light-drivensupramolecular polymer. See J. Am. Chem.Soc., 2013, 135, p 5990 for details.
Contact
Plunge freezing
High pressure freezing
Cryo-TEM RT-TEM
(cryo-) Ultra-microtome Freeze
fracture
Freeze substitution
Generally available Cryo-techniques for soft-matter materials
* Some techniques can also be used to prepare cryo-SEM specimens.
* Cryo-FIB can be a promising alternative to cryo-ultramicrotome.
Specific specimen preparation routines established for liquid crystals
Thin film approach: plunge freezing
Bulk approach: cryosectioning
Bulk approach: freeze fracture
Mainly for thermotropic LCs (TLCs) Mainly for Lyotropic LCs (LLCs)
Thin film approach: Part of the specimen is electron-transparent before freezing. No further processing is required.
Bulk approach: A thick (> a few microns) material is frozen, and goes through further processing to get thin TEM specimen.
Plunge Freezing
• Basic idea: preserve the native structure of a soft matter sample by throwing it rapidly into cryogen (e.g.,liquid nitrogen or liquid ethane) manually or using a machine.
• For aqueous samples (for example, many biological materials and lyotropic liquid crystals), >104 degree/secondcooling rate is required for vitrification.
• For very thin specimens, the above cooling rate can be achieved routinely using liquid ethane as cryogen.• For non-aqueous samples, a slower cooling rate may still lead to good preservation of the native structure. It is
possible to freeze thick samples to make TEM specimens using cryo-sectioning or freeze fracture.
High Pressure Freezing
• Basic idea: To slow down the crystallization of water in aqueous samples by applying a high pressure (~2000times the atmosphere pressure). So a much slower cooling rate can be used for vitrification of thick aqueoussamples.
• The frozen samples can then be cryo-sectioned or freeze-fractured to obtained TEM specimens.
High pressure freezer (Leica EM Pact2)
FEI Tecnai F20 equipped with STEM, EELS, EDS, etc.
Freeze Fracture
• Basic idea: A frozen sample is fractured inside a vacuum chamber at low temperature . Supposedly, thetomography of the fractured surface may represent the native structure. The fractured surface is shadowedand replicated by depositing Pt/C (or other heavy metals) at an angle (for example, 45°), and carbon from thetop. The real material is then dissolved/removed. The replica is studied by room temperature TEM (RT-TEM).
• Freeze fracture can work for a variety of materials.
Cryo-Ultramicrotomy
• Basic idea: Use a diamond knife to section frozen samples into electron transparent slices which are thencollected on carbon coated TEM grids.
• The cryo-ultramicrotome at LCICF TEM lab is equipped with a discharge device and a micro-manipulator forchallenging materials.
Application examples
Microvesicles produced during blood macrophage differentiation. Ismail, et al., Blood 121 (2013) 984.
Suspended lyotropic chromonic liquid crystal (nematic phase): side-view (a) and top-view (b) of the of the aggregates. See J. Mater. Chem. C, 2014,2, 8780-8788 for details.
CarbonSuspended 5CB
STEM
“Bulk” Replica FFTEM of CB7CB in twist-bend nematic phase. See Nature Comm4: 2635, 2013 for details.
Thin film plunge-freezing and cryo-TEM
Cryo-ultramicrotomy and cryo-TEM
TEM
Thin film plunge-freezing (for comparison)
“Bulk” cryo-ultramicrotomy (Plunge-frozen bulk sample)
Liquid Crystal-nanoparticles composite: Au nanoparticles doped 5CB.
High-pressure frozen lyotropic chromonic DSCG: side-view (a) and top-view (b) of the aggregates. Microscopy Research and Technique, 77, p.754-772 (2014)
Freeze fracture and RT-TEM
E-beam writer on a environmental SEM
Atomic force microscopes
Website: http://www.lcinet.kent.edu/organization/facility/characterization/index.php
• High Speed Camera (HS-camera)
• FTIR microscope
• Confocal scanning laser microscope
• Abbe Refractometer
• Differential Scanning Calorimeter (DSC)
• …
Room temperature heliconical twist-bend nematic liquid crystal. CrystEngComm, 17, 2778-2782, 2015.
Version: 05/2015 @ JCU
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