Charged nanoparticle dynamics in water induced by scanning transmission electron microscopy

Using scanning transmission electron microscopy we image ~4 nm platinum nanoparticles deposited on an insulating membrane, where the membrane is one of two electron-transparent windows separating an aqueous environment from the microscope's high vacuum. Upon receiving a relatively moderate dose of ~10(4) e/nm(2), initially immobile nanoparticles begin to move along trajectories that are directed radially outward from the center of the field of view. With larger dose rates the particle motion becomes increasingly dramatic. These observations demonstrate that, even under mild imaging conditions, the in situ electron microscopy of aqueous environments can produce electrophoretic charging effects that dominate the dynamics of nanoparticles under observation.     

Direct cryogenic-temperature transmission electron microscopy imaging of phospholipid aggregates in soybean oil

Cryogenic-temperature transmission electron microscopy (cryo-TEM) was fine-tuned to make it applicable to the study of a natural oil-based system. While liquid ethane, the cryogen of choice for specimen preparation, was found to dissolve the oil, we observed that the triglycerides found in many natural oils serve as cryoprotectants that allow vitrification of such oil systems in liquid nitrogen, a cryogen that provides relatively low cooling rates. Using the modified technique combined with digital imaging by a cooled CCD camera, we were able to directly visualize inverse micellar and liquid-crystalline aggregates formed in the soybean oil/soybean phospholipids/water/hexane system, which is commercially important in soybean oil processing. The method developed here should be widely applicable to other organic solvent-based systems.     

Voltammetric monitoring of gold nanoparticle formation facilitated by glycyl-L-tyrosine: relation to electronic spectra and transmission electron microscopy images

Voltammetric techniques have been introduced to monitor the formation of gold nanoparticles produced via the reaction of the amino acid glycyl-L-tyrosine with Au(III) (bromoaurate) in 0.05 M KOH conditions. The alkaline conditions facilitate amino acid binding to Au(III), inhibit the rate of reduction to Au(0), and provide an excellent supporting electrolyte for voltammetric studies. Data obtained revealed that a range of time-dependent gold solution species are involved in gold nanoparticle formation and that the order in which reagents are mixed is critical to the outcome. Concomitantly with voltammetric measurements, the properties of gold nanoparticles formed are probed by examination of electronic spectra in order to understand how the solution environment present during nanoparticle growth affects the final distribution of the nanoparticles. Images obtained by the ex situ transmission electron microscopy (TEM) technique enable the physical properties of the nanoparticles isolated in the solid state to be assessed. Use of this combination of in situ and ex situ techniques provides a versatile framework for elucidating the details of nanoparticle formation.     

Characterization of NOM-colloid aggregates in surface waters: Coupling transmission electron microscopy staining techniques and mathematical modelling

The role of inorganic colloids and natural organic macromolecules in aquatic and soil systems is essential for our understanding of contaminant and nutrient transport. The submicron organic fraction, although important in terms of total surface area, is poorly electron-dense and thus not susceptible to observation by transmission electron microscopy. Several staining techniques were therefore developed to enable the observation of the submicron aggregate fraction in natural waters. Lead and silver based stains were especially successful in increasing the contrast of previously unobservable organic macromolecules. Computer simulations were developed for the interpretation of observed aggregate structures in natural waters. Based on the TEM observations, modelling was employed to examine the formation kinetics and structural characteristics of aggregates under different physicochemical conditions. The modelling results offered insight into the underlying mechanisms and important factors influencing the aggregation processes.Dedicated to Professor Dr. Dieter Klockow on the occasion of his 60th birthday     

Polymerizable surfactant design for transmission electron microscopy

Polymerized liposomes and vesicles are under close scrutiny as long-lived, stable substitutes for their natural and synthetic unpolymerized counterparts. The monomer surfactant, which contains one or more polymerizable groups, is dispersed in water at the proper temperature and concentration to form the lyotropic liquid crystalline phase of interest and polymerized while in the liquid crystalline state. In addition to their applications to slow-release and site-specific drug delivery, membrane-mediated chemistry, artificial photosynthesis, etc., polymerized surfactant liposomes and vesicles hold great promise as model systems for TEM investigations of lamellar liquid crystal structure. One such model polymerizable surfactant is DBPAl, or N,N-dimethyl-N,N-bis(1,3-pentadecadienyl-carbonyloxyethyl) ammonium iodide. Polarized light microscopy and differential scanning calorimetry (DSC) confirm that DBPAI forms lamellar liquid crystalline liposomes in water. The DBPAI liposomes were polymerized while in the liquid crystalline state by ultraviolet (UV) irradiation. The DBPAI liposomes were shown to be identical in structure before and after polymerization by a combination of X-ray diffraction and freeze-fracture TEM. However, turbidity measurements showed that the polymerized DBPAI liposomes were much more stable in acetone and ethanol than the monomer DBPAI liposomes, demonstrating that the chemical nature of the surfactant in the liposome had changed. The combination of structural preservation and enhanced chemical stability makes DBPAI a natural choice for TEM thin-sections. A method of preparing DBPAI liposomes for thin-section TEM is outlined and bilayer resolution images of the DBPAI liposomes are presented. Polymerized bilayers in thin-section TEM promise the enhanced resolution required to answer many important structural questions left unresolved by freeze-fracture TEM.     

Energy-filtered transmission electron microscopy: an overview

This paper aims to give an overview of the technique of energy-filtered transmission electron microscopy (EFTEM). It explains the basic principles of the technique and points to the relevant literature for more detailed issues. Experimental examples are given to show the power of EFTEM to study the chemical composition of nanoscale samples in materials science. Advanced EFTEM applications like imaging spectroscopy and EFTEM tomography are briefly discussed.     

Ex situ transmission electron microscopy: a fixed-bed reactor approach.

A fixed-bed reactor has been designed and constructed for ex situ transmission electron microscopy (TEM) studies of heterogeneous catalysts. The ex situ facility exposes a fully prepared TEM sample on a grid to actual process conditions (e.g., temperature, pressure, gas composition, etc.) by placing the grid at the exit section of a conventional fixed-bed reactor. A unique reactor design allows grid transfer into the electron microscope and back into the reactor again under a controlled (inert) environment, thus allowing time-resolved monitoring of catalyst morphology changes under realistic, well-controlled conditions. This facility stands completely independent of the TEM. Thus, no special TEM modifications are required and long-term ex situ studies do not impact microscope utilization. The utility of the facility is demonstrated via the oxidation of intermediate size ( approximately 20- approximately 80 nm) supported copper particles.     

Object oriented database and electronic notebook for transmission electron microscopy.

As high-resolution biological transmission electron microscopy (TEM) has increased in popularity over recent years, the volume of data and number of projects underway has risen dramatically. A robust tool for effective data management is essential to efficiently process large data sets and extract maximum information from the available data. We present the Electron Microscopy Electronic Notebook (EMEN), a portable, object-oriented, web-based tool for TEM data archival and project management. EMEN has several unique features. First, the database is logically organized and annotated so multiple collaborators at different geographical locations can easily access and interpret the data without assistance. Second, the database was designed to provide flexibility to the user, so it can be used much as a lab notebook would be, while maintaining a structure suitable for data mining and direct interaction with data-processing software. Finally, as an object-oriented database, the database structure is dynamic and can be easily extended to incorporate information not defined in the original database specification.     

Electron microscopy methods for studying polymer blends—comparison of scanning electron microscopy and transmission electron microscopy

The possibility of using a scanning electron microscope (SEM) for studying the morphology of mechanical polymer blends was investigated. Compounds of SBS/EPDM, and both filled and unfilled NBR/EPDM were tested. OsO₄-stained thin-sections were also examined in a transmission electron microscope (TEM) and the results were compared.It seemed to be quite possible to use atomic number contrast detection in combination with OsO₄ staining for visualizing the morphology of the blends in SEM. Domains as small as 0·1 μm were clearly seen. This was done by means of a Robinson backscattered electron detector. Sample preparation was easy, 2 mm thick rubber plates were cut on dry ice to obtain a smooth surface. After staining, the samples were coated with a thin conductive carbon layer.The inner structures of SBS and the carbon black particles were not resolved in SEM but were easily seen in TEM.     

High-resolution transmission electron microscopy using negative spherical aberration.

A novel imaging mode for high-resolution transmission electron microscopy is described. It is based on the adjustment of a negative value of the spherical aberration C S of the objective lens of a transmission electron microscope equipped with a multipole aberration corrector system. Negative spherical aberration applied together with an overfocus yields high-resolution images with bright-atom contrast. Compared to all kinds of images taken in conventional transmission electron microscopes, where the then unavoidable positive spherical aberration is combined with an underfocus, the contrast is dramatically increased. This effect can only be understood on the basis of a full nonlinear imaging theory. Calculations show that the nonlinear contrast contributions diminish the image contrast relative to the linear image for a positive-C S setting whereas they reinforce the image contrast relative to the linear image for a negative-C S setting. The application of the new mode to the imaging of oxygen in SrTiO3 and YBa2Cu3O7 demonstrates the benefit to materials science investigations. It allows us to image directly, without further image processing, strongly scattering heavy-atom columns together with weakly scattering light-atom columns.     

Imaging modes for scanning confocal electron microscopy in a double aberration-corrected transmission electron microscope.

Aberration correction leads to reduced focal depth of field in the electron microscope. This reduced depth of field can be exploited to probe specific depths within a sample, a process known as optical sectioning. An electron microscope fitted with aberration correctors for both the pre- and postspecimen optics can be used in a confocal mode that provides improved depth resolution and selectivity over optical sectioning in the scanning transmission electron microscope (STEM). In this article we survey the coherent and incoherent imaging modes that are likely to be used in scanning confocal electron microscopy (SCEM) and provide simple expressions to describe the images that result. Calculations compare the depth response of SCEM to optical sectioning in the STEM. The depth resolution in a crystalline matrix is also explored by performing a Bloch wave calculation for the SCEM geometry in which the pre- and postspecimen optics are defocused away from their confocal conditions.     

Contrast transfer and resolution limits for sub-angstrom high-resolution transmission electron microscopy.

The optimum imaging of an object structure at the sub-angstrom length scale requires precise adjustment of the lens aberrations of a high-resolution instrument up to the fifth order. A least-squares optimization of defocus aberration C1, third-order spherical aberration C3, and fifth-order spherical aberration C5 yields two sets of aberration coefficients for strong phase contrast up to the information limit: one for variable C1 and C3, at fixed C5, another for variable C1, C3, and C5. An additional correction to the defocus aberration, dependent on object thickness, is described, which becomes important for the use of image simulation programs in predicting optimum high-resolution contrast from thin objects at the sub-angstrom scale. For instruments with a sub-angstrom information limit the ultimate structure resolution, the power to resolve adjacent atom columns in a crystalline object, depends on both the instrumental pointspread and an object pointspread due to finite width of the atomic column potentials. A simulation study on a simple double-column model yields a range for structure resolutions, dependent on the atomic scattering power, from 0.070 nm down to 0.059 nm, for a hypothetical 300-kV instrument with an information limit of 0.050 nm.     

High-resolution transmission electron microscopy: the ultimate nanoanalytical technique

To be able to determine the elemental composition and morphology of individual nanoparticles consisting of no more than a dozen or so atoms that weigh a few zeptograms (10(-21) g) is but one of the attainments of modern electron microscopy. With slightly larger specimens (embracing a few unit cells of the structure) their symmetry, crystallographic phase, unit-cell dimension, chemical composition and often the valence state (from parallel electron spectroscopic measurements) of the constituent atoms may also be determined using a scanning beam of electrons of ca. 0.5 nm diameter. Nowadays electron crystallography, which treats the digital data of electron diffraction (ED) and high-resolution transmission electron microscope (HRTEM) images of minute (ca. 10(-18)g) specimens in a quantitatively rigorous manner, solves hitherto unknown structures just as X-ray diffraction does with bulk single crystals. In addition, electron tomography (see cover photograph and its animation) enables a three-dimensional picture of the internal structure of minute objects, such as nanocatalysts in a single pore, as well as structural faults such as micro-fissures, to be constructed with a resolution of 1 nm from an angular series of two-dimensional (projected) images. Very recently (since this article was first written) a new meaning has been given to electron crystallography as a result of the spatio-temporal resolution of surface phenomena achieved on a femtosecond timescale.     

Side chain liquid crystal polymers observed by transmission electron microscopy: Microstructure of polymethacrylates and a polyacrylate

The smectic structures of three side chain liquid crystal polymethacrylates (PMA) with different terminal groups and a polyacrylate (P₈) were analysed by transmission electron microscopy. Using low electron doses, we were able to obtain diffraction patterns and high resolution (HREM) images from highly oriented undamaged specimens. The results are compared with those from previous X-ray diffraction experiments. All the polymers have a smectic A structure. The PMA and P₈, each with a CN terminal group, are the least ordered. Their diffraction patterns show a smectic A_d structure. PMA-OC₄H₉ and PMA-φ) have smectic A₁ structures. The smectic layers, seen by high resolution images, are very well oriented with only a few defects such as notches or kinks. In the A_d structure, the well-ordered domains are smaller, and the defects are more numerous. The differences are due to the influence of the polarization of the side chain terminated with CN.     

Amyloidosis of Alzheimer's Abeta peptides: solid-state nuclear magnetic resonance, electron paramagnetic resonance, transmission electron microscopy, scanning transmission electron microscopy and atomic force microscopy studies

Aggregation cascade for Alzheimer's amyloid-beta peptides, its relevance to neurotoxicity in the course of Alzheimer's disease and experimental methods useful for these studies are discussed. Details of the solid-phase peptide synthesis and sample preparation procedures for Alzheimer's beta-amyloid fibrils are given. Recent progress in obtaining structural constraints on Abeta-fibrils from solid-state NMR and scanning transmission electron microscopy (STEM) data is discussed. Polymorphism of amyloid fibrils and oligomers of the 'Arctic' mutant of Abeta(1-40) was studied by (1)H,(13)C solid-state NMR, transmission electron microscopy (TEM) and atomic force microscopy (AFM), and a real-time aggregation of different polymorphs of the peptide was observed with the aid of in situ AFM. Recent results on binding of Cu(II) ions and Al-citrate and Al-ATP complexes to amyloid fibrils, as studied by electron paramagnetic resonance (EPR) and solid-state (27)Al NMR techniques, are also presented.     

Characterization of magnetic nanoparticles using energy-selected transmission electron microscopy.

Fe, Co, and Ni magnetic nanoparticles have been characterized using energy-selected imaging in a high-resolution transmission electron microscope. The samples comprised Fe/FeO x and Co/CoO x nanoparticles synthesized by inert gas evaporation and a Ni/C nano-composite prepared by a sonochemical method. All of the particles examined were found to be between 5 and 30 nm in size, with the Fe and Co crystals coated in 5-10 nm of metal oxide layer and the Ni metallic crystallites embedded in an amorphous carbon spherical matrix.     

Thin dielectric film thickness determination by advanced transmission electron microscopy.

High-resolution transmission electron microscopy (HR-TEM) has been used as the ultimate method of thickness measurement for thin films. The appearance of phase contrast interference patterns in HR-TEM images has long been confused as the appearance of a crystal lattice by nonspecialists. Relatively easy to interpret crystal lattice images are now directly observed with the introduction of annular dark-field detectors for scanning TEM (STEM). With the recent development of reliable lattice image processing software that creates crystal structure images from phase contrast data, HR-TEM can also provide crystal lattice images. The resolution of both methods has been steadily improved reaching now into the sub-Angstrom region. Improvements in electron lens and image analysis software are increasing the spatial resolution of both methods. Optimum resolution for STEM requires that the probe beam be highly localized. In STEM, beam localization is enhanced by selection of the correct aperture. When STEM measurement is done using a highly localized probe beam, HR-TEM and STEM measurement of the thickness of silicon oxynitride films agree within experimental error. In this article, the optimum conditions for HR-TEM and STEM measurement are discussed along with a method for repeatable film thickness determination. The impact of sample thickness is also discussed. The key result in this article is the proposal of a reproducible method for film thickness determination.