Cryogenic Transmission Electron Microscopy: Aqueous Suspensions of Nanoscale Objects

Abstract Direct imaging of nanoscale objects suspended in liquid media can be accomplished using cryogenic transmission electron microscopy (cryo-TEM). Cryo-TEM has been used with particular success in microbiology and other biological fields. Samples are prepared by plunging a thin film of sample into an appropriate cryogen, which essentially produces a snapshot of the suspended objects in their liquid medium. With successful sample preparation, cryo-TEM images can facilitate elucidation of aggregation and self-assembly, as well as provide detailed information about cells and viruses. This work provides an explanation of sample preparation, detailed examples of the many artifacts found in cryo-TEM of aqueous samples, and other key considerations for successful cryo-TEM imaging.

[1]  A. Bangham,et al.  NEGATIVE STAINING OF PHOSPHOLIPIDS AND THEIR STRUCTURAL MODIFICATION BY SURFACE-ACTIVE AGENTS AS OBSERVED IN THE ELECTRON MICROSCOPE. , 1964, Journal of molecular biology.

[2]  David B. Williams,et al.  Transmission Electron Microscopy , 1996 .

[3]  J. L. Zakin,et al.  Cryo-TEM Imaging the Flow-Induced Transition from Vesicles to Threadlike Micelles , 2000 .

[4]  P. Burns.  Nanoscale uranium-based cage clusters inspired by uranium mineralogy , 2011, Mineralogical Magazine.

[5]  V. Yuwono,et al.  Oriented aggregation: formation and transformation of mesocrystal intermediates revealed. , 2010, Journal of the American Chemical Society.

[6]  Samuel I Stupp,et al.  Advances in cryogenic transmission electron microscopy for the characterization of dynamic self-assembling nanostructures. , 2012, Current opinion in colloid & interface science.

[7]  R. L. Penn,et al.  Size-dependent anatase to rutile phase transformation and particle growth , 2013 .

[8]  T. Lodge,et al.  Equilibrium vs Metastability: High-Temperature Annealing of Spherical Block Copolymer Micelles in an Ionic Liquid , 2009 .

[9]  J. Dubochet,et al.  Beam damage to organic material is considerably reduced in cryo-electron microscopy. , 1980, Journal of molecular biology.

[10]  Robert M Glaeser,et al.  Retrospective: radiation damage and its associated "information limitations". , 2008, Journal of structural biology.

[11]  G. Murphy,et al.  Electron cryotomography sample preparation using the Vitrobot , 2006, Nature Protocols.

[12]  R. L. Penn,et al.  Effect of pH on the Kinetics of Crystal Growth by Oriented Aggregation , 2013 .

[13]  R. L. Penn,et al.  Controlled growth of alpha-FeOOH nanorods by exploiting-oriented aggregation , 2006 .

[14]  L. Issman,et al.  Cryo‐SEM specimen preparation under controlled temperature and concentration conditions , 2012, Journal of microscopy.

[15]  J. Dubochet,et al.  Electron beam radiation damage to organic inclusions in vitreous, cubic, and hexagonal ice , 1986 .

[16]  R. Wyckoff.  Frozen-dried preparations for the electron microscope. , 1946, Science.

[17]  D. Pochan,et al.  Cryogenic Transmission Electron Microscopy for Direct Observation of Polymer and Small-Molecule Materials and Structures in Solution , 2010 .

[18]  R. L. Penn,et al.  Size dependent kinetics of oriented aggregation , 2007 .

[19]  Jolene L. Johnson,et al.  Biophysical analysis of HTLV-1 particles reveals novel insights into particle morphology and Gag stoichiometry , 2010, Retrovirology.

[20]  Wei Zhou,et al.  True solutions of single-walled carbon nanotubes for assembly into macroscopic materials , 2009, Nature Nanotechnology.

[21]  P. Frederik,et al.  Phospholipid, Nature's own slide and cover slip for cryo‐electron microscopy , 1989, Journal of microscopy.

[22]  Y. Talmon.  Electron Beam Radiation Damage to Organic and Biological Cryospecimens , 1987 .

[23]  E. Thomas,et al.  Electron Beam Heating Temperature Profiles in Moderately Thick Cold Stage STEM/SEM Specimens , 1978 .

[24]  J. Dubochet,et al.  Cryo‐EM—the first thirty years , 2012, Journal of microscopy.

[25]  Y. Talmon.  Transmission Electron Microscopy of Complex Fluids: The State of the Art , 1996 .

[26]  F. J. Esselink,et al.  Cryoelectron microscopy of block-copolymers in an organic solvent , 1995 .

[27]  J. Zasadzinski,et al.  Microstructure of Complex Fluids by Electron Microscopy , 1994 .

[28]  D. Chiappetta,et al.  Colloids and Surfaces B: Biointerfaces , 2013 .

[29]  J. Dubochet,et al.  Electron microscopy of frozen water and aqueous solutions , 1982 .

[30]  Sandeep Kumar,et al.  A structural resolution cryo-TEM study of the early stages of MFI growth. , 2008, Journal of the American Chemical Society.

[31]  T. Lodge,et al.  Polymersomes with ionic liquid interiors dispersed in water. , 2010, Journal of the American Chemical Society.

[32]  B. Rybtchinski,et al.  Noncovalent self-assembly in aqueous medium: Mechanistic insights from time-resolved cryogenic electron microscopy , 2012 .

[33]  Y. Talmon.  Staining and drying-induced artifacts in electron microscopy of surfactant dispersions , 1983 .

[34]  R. Glaeser,et al.  Hydrophilic support films of controlled thickness and composition. , 1973, The Review of scientific instruments.

[35]  F. Hofer,et al.  Electron microscopy of nanoemulsions: an essential tool for characterisation and stability assessment. , 2012, Micron.

[36]  Honggang Cui,et al.  Elucidating the assembled structure of amphiphiles in solution via cryogenic transmission electron microscopy. , 2007, Soft matter.

[37]  D. Branton,et al.  Fracture Planes in an Ice-Bilayer Model Membrane System , 1967, Science.

[38]  L E Scriven,et al.  Controlled environment vitrification system: an improved sample preparation technique. , 1988, Journal of electron microscopy technique.

[39]  Sidney R. Cohen,et al.  Self-assembly of light-harvesting crystalline nanosheets in aqueous media. , 2013, ACS nano.

[40]  V. Yuwono,et al.  Aggregation of ferrihydrite nanoparticles in aqueous systems , 2012 .

[41]  J. Dubochet,et al.  Cryo-electron microscopy of viruses , 1984, Nature.

[42]  R. Horne,et al.  A negative staining method for high resolution electron microscopy of viruses. , 1959, Biochimica et biophysica acta.

[43]  Sandeep Kumar,et al.  Aggregative growth of silicalite-1. , 2007, The journal of physical chemistry. B.

[44]  A. Engel,et al.  What transmission electron microscopes can visualize now and in the future. , 2008, Journal of structural biology.

[45]  Heiner Friedrich,et al.  Imaging of self-assembled structures: interpretation of TEM and cryo-TEM images. , 2010, Angewandte Chemie.

[46]  P. Frederik,et al.  Cryoelectron microscopy of liposomes. , 2005, Methods in enzymology.

[47]  R. Glaeser,et al.  Limitations to significant information in biological electron microscopy as a result of radiation damage. , 1971, Journal of ultrastructure research.

[48]  C. Böttcher,et al.  High-Yield Preparation of Oligomeric C-Type DNA Toroids and Their Characterization by Cryoelectron Microscopy , 1998 .

[49]  F. Bates,et al.  Giant wormlike rubber micelles , 1999, Science.

[50]  Direct observation of dipolar chains in ferrofluids in zero field using cryogenic electron microscopy , 2003 .

[51]  R. Epand,et al.  The mechanism of lamellar-to-inverted hexagonal phase transitions in phosphatidylethanolamine: implications for membrane fusion mechanisms. , 1997, Biophysical journal.

[52]  Bjoern Sander,et al.  Visualization of bionanostructures using transmission electron microscopical techniques , 2011, Microscopy research and technique.

[53]  A. J. Koster,et al.  Electron Tomography: a tool for 3D structural probing of heterogeneous catalysts at the nanometer scale , 2004 .

[54]  H. Zeng.  Synthesis and self-assembly of complex hollow materials , 2011 .

[55]  R. L. Penn,et al.  Effect of Ionic Strength on the Kinetics of Crystal Growth by Oriented Aggregation , 2012 .

[56]  K. Landfester,et al.  Incorporation of nanoparticles into polymersomes: size and concentration effects. , 2012, ACS nano.

[57]  C. Boettcher,et al.  Comparative Cryo-Electron Microscopy of Noncovalent N-Dodecanoyl- (d- and l-) serine Assemblies in Vitreous Toluene and Water , 2001 .

[58]  R. Koole,et al.  Dipolar structures in colloidal dispersions of PbSe and CdSe quantum dots. , 2007, Nano letters.

[59]  E. Wachtel,et al.  Solubilization of simvastatin and phytosterols in a dilutable microemulsion system. , 2013, Colloids and surfaces. B, Biointerfaces.

[60]  M. Vos,et al.  The development of a glove-box/Vitrobot combination: air-water interface events visualized by cryo-TEM. , 2008, Ultramicroscopy.

[61]  Heike Bunjes,et al.  Cryogenic transmission electron microscopy (cryo-TEM) for studying the morphology of colloidal drug delivery systems. , 2011, International journal of pharmaceutics.

[62]  J. Banfield,et al.  A portable cryo‐plunger for on‐site intact cryogenic microscopy sample preparation in natural environments , 2012, Microscopy research and technique.