Environmental Cell Research Articles

Depth sectioning using environmental and atomic-resolution STEM.

(Scanning) transmission electron microscopy (TEM) images of samples in gas and liquid media are acquired with an environmental cell (EC) via silicon nitride membranes. The ratio of sample signal against the background is a significant factor for resolution. Depth-sectioning scanning TEM (STEM) is a promising technique that enhances the signal for a sample embedded in a matrix. It can increase the resolution to the atomic level, thereby enabling EC-STEM applications in important areas. This review introduces depth-sectioning STEM and its applications to high-resolution EC-STEM imaging of samples in gases and in liquids.

Time-Resolved Measurement of the ATP-Dependent Motion of the Group II Chaperonin by Diffracted Electron Tracking.

Previously, we demonstrated the ATP-dependent dynamics of a group II chaperonin at the single-molecule level by diffracted X-ray tracking (DXT). The disadvantage of DXT is that it requires a strong X-ray source and also perfect gold nano-crystals. To resolve this problem, we developed diffracted electron tracking (DET). Electron beams have scattering cross-sections that are approximately 1000 times larger than those of X-rays. Thus, DET enables us to perform super-accurate measurements of the time-resolved 3D motion of proteins labeled with commercially available gold nanorods using a scanning electron microscope. In this study, we compared DXT and DET using the group II chaperonin from Methanococcus maripaludis (MmCpn) as a model protein. In DET, the samples are prepared in an environmental cell (EC). To reduce the electron beam-induced protein damage, we immobilized MmCpn on the bottom of the EC to expose gold nanorods close to the carbon thin film. The sample setup worked well, and the motions of gold nanorods were clearly traced. Compared with the results of DXT, the mobility in DET was significantly higher, which is probably due to the difference in the method for immobilization. In DET, MmCpn was immobilized on a film of triacetyl cellulose. Whereas proteins are directly attached on the surface of solid support in DXT. Therefore, MmCpn could move relatively freely in DET. DET will be a state-of-the-art technology for analyzing protein dynamics.

A simple method for environmental cell depressurization for use with an electron microscope.

With the aid of the environmental cell (EC) in electron microscopy, hydrated specimens have been observed at high resolutions that optical microscopy cannot attain. Due to the ultra-high vacuum conditions of the inner column of the electron microscope, the EC requires sealing films that are sufficiently thin to allow electron transmission and that are sufficiently tough to withstand the pressure difference between the inside and outside of the EC. However, most hydrated specimens can be observed at low vacuum because the saturated vapor pressure of water is known to be 0.02 atm at room temperature. These concepts have been used in the differential pumping system, but it is complicated and relatively expensive. In this work, we propose a simple method for depressurization of the EC using a 'balloon structure' and demonstrate the theoretical benefits and practical improvement for specimen observations in low-vacuum conditions.

A Controllable Environmental-Cell (EC) for Wet Environmental TEM (WETEM)

A Controllable Environmental-Cell (EC) for Wet Environmental TEM (WETEM)

Evaluation of Interfacial Fracture Strength in Micro-Scale Components Combined with High-Voltage Environmental Electron Microscopy

A novel experimental method that enables evaluation of interfacial strength of micro-components subjected to well-controlled various environments is proposed. The method combines a high-voltage transmission electron microscope (HVEM) equipped with an open type environmental cell (EC) and a quantitative nano-mechanical holder. The experiments were conducted by using focused-ion-beam-processed micro-cantilever specimens containing interfaces of dissimilar materials (silicon (Si) and copper (Cu)). The in situ observation of fracture phenomenon along a target interface (Si/Cu) and the simultaneous measurement of fracture load were successfully implemented under vacuum and gaseous environments (N2 gas and N2/H2 mixed gas) whose maximum pressure was controlled at 5 × 103 Pa. The stress distribution at the onset of fracture was calculated in detail by finite element analysis. The strength against interfacial debonding was then evaluated by the local normal stress value at the interface free-edge. The proposed technique, which can also be combined with simultaneous electron probe analyses, is highly versatile when compared to the conventional nano-mechanical strength evaluations under limited environments.

A combined environmental straining specimen holder for high-voltage electron microscopy

A novel specimen holder that enables in situ observation of crack-tip deformation and/or fracture under a controlled environment is developed for a high-voltage electron microscope (HVEM). A window-type environmental cell (EC) that incorporates a uniaxial straining apparatus is built into a side-entry-type single-tilt specimen holder. The gas control in EC, straining apparatus design, limited field of view for crack-tip observation, and specimen preparation for the specimen holder are presented in detail. Experimental results successfully demonstrate that the developed specimen holder is quite useful for the dynamic observation of crack-tip deformation and/or fracture subjected to a hostile environment, such as hydrogen gas.

Characterization of Microbially Fe(III)-Reduced Nontronite: Environmental Cell-Transmission Electron Microscopy Study

AbstractMicrostructural changes induced by the microbial reduction of Fe(III) in nontronite by Shewanella oneidensis were studied using environmental cell (EC)-transmission electron microscopy (TEM), conventional TEM, and X-ray powder diffraction (XRD). Direct observations of clays by EC-TEM in their hydrated state allowed for the first time an accurate and unambiguous TEM measurement of basal layer spacings and the contraction of layer spacing caused by microbial effects, most likely those of Fe(III) reduction. Non-reduced and Fe(III)-reduced nontronite, observed by EC-TEM, exhibited fringes with mean d001 spacings of 1.50 nm (standard deviation, σ = 0.08 nm) and 1.26 nm (σ = 0.10 nm), respectively. In comparison, the same samples embedded with Nanoplast resin, sectioned by microtome, and observed using conventional TEM, displayed layer spacings of 1.0–1.1 nm (non-reduced) and 1.0 nm (reduced). The results from Nanoplast-embedded samples are typical of conventional TEM studies, which have measured nearly identical layer spacings regardless of Fe oxidation state. Following Fe(III) reduction, both EC- and conventional TEM showed an increase in the order of nontronite selected area electron diffraction patterns while the images exhibited fewer wavy fringes and fewer layer terminations. An increase in stacking order in reduced nontronite was also suggested by XRD measurements. In particular, the ratio of the valley to peak intensity (v/p) of the 1.7 nm basal 001 peak of ethylene glycolated nontronite was measured at 0.65 (non-reduced) and 0.85 (microbially reduced).

Series in Microscopy in Materials Science: Electron Microscopy in Heterogeneous Catalysis. P.L. Gai and E.D. Boyes. Series Editors: B. Cantor, M.J. Goringe, and J.A Eades. Institute of Physics Publishing, Bristol, England; 2003, 233 pages (Hardback). ISBN 0-7503-0809-5

This book deals with in situ dynamic observation and analysis of heterogeneous catalysis using environmental cells (EC) in transmission (TEM) and scanning electron microscopes (SEM). In general, it is based on outstanding and unique works carried out by the authors themselves over the past three decades, who pioneered this key enabling area of materials science.

Electron energy loss spectroscopy techniques for the study of microbial chromium(VI) reduction

Electron energy loss spectroscopy (EELS) techniques were used to determine oxidation state, at high spatial resolution, of chromium associated with the metal-reducing bacteria, Shewanella oneidensis, in anaerobic cultures containing Cr(VI)O 4 2−. These techniques were applied to fixed cells examined in thin section by conventional transmission electron microscopy (TEM) as well as unfixed, hydrated bacteria examined by environmental cell (EC)-TEM. Two distinct populations of bacteria were observed by TEM: bacteria exhibiting low image contrast and bacteria exhibiting high contrast in their cell membrane (or boundary) structure which was often encrusted with high-contrast precipitates. Measurements by EELS demonstrated that cell boundaries became saturated with low concentrations of Cr and the precipitates encrusting bacterial cells contained a reduced form of Cr in oxidation state +3 or lower.

Microbial Reduction of Chromium(VI): In-Situ Environmental Cell Transmission Electron Microscopy Study Using Electron Energy Loss Spectroscopy

The geochemistry and toxicity of chromium is controlled by its valence state. Chromium is a redox active 3d transition metal with a wide range (− to +6) of possible oxidation states; however only Cr(III) and Cr(VI) are stable. Hexavalent Cr(VI) can be readily reduced to the trivalent state by Fe2+, S2−, organic compounds, wetland plants, and several species of microorganisms. The associated mechanisms of Cr(VI) reduction are technologically and biologically important because they convert a toxic, mobile element into a less toxic, immobile form.Reduction of Cr(VI) by the bacterium, Shewanella oneidensis (previously classified Shewanella putrefaciens strain MR-1), was studied by absorption spectrophotometry and in-situ, environmental cell (EC) - transmission electron microscopy (TEM) coupled with electron energy loss spectroscopy (EELS). Shewanella oneidensis (S. oneidensis), a gram-negative, facultative bacterium is capable of respiring aerobically and anaerobically using a variety of compounds, including O2, Fe(III), Mn(IV), NO2−, NO3−, SO2, SO32−, thiosulfate (S2O32−), trimethyamine oxide, fumarate, U(VI), and Cr(VI) as terminal electron acceptors.

Designer Nanostructures Of Catalysts in the Environmental-HREM

Abstract Major advances in the field of in situ environmental high resolution electron microscopy (EHREM) probe selective oxidation catalysis directly on the atomic scale. Dynamic gas-solid surface interactions are studied in real-time and under realistic reaction conditions to unravel atomic level insights into active sites and structure-property relationships in vital chemical and technological processes [1-3]. The recent developments include a pioneering approach with the controlled environmental cell (ECELL) facilities permanently mounted inside the EHREM [2]. Accessories have been added for simultaneous structural and compositional analyses of the reactor contents in real-time, and using atomic resolution imaging with transmission electron diffraction and parallel electron energy loss spectroscopy (PEELS). We are now developing innovative experimental methods that include very high temperature studies and combining in the same instrument facilities for both EHREM and environmental-SEM (ESEM), (EHREM-ESEM), with attractive possibilities for studying the science of selective oxidation catalysis. Alkane Catalysis, Chlorofluorocarbons and Nanotubes: In the domain of transition metal based oxides, discoveries of fundamental mechanisms underlying selective catalyzation have come from EHREM studies.

Controlled Environment [Ecell] Tem for Dynamic In-Situ Reaction Studies with Hrem Lattice Imaging

AbstractWith a controlled environment electron microscope it is possible to remove the effect of another atmosphere, such air, to which the sample may have become exposed; to study in considerable detail the continuous development of a series of microstructures in a dynamic process, to identify important intermediate phases and defects in them which may only be metastable under specific reaction conditions of atmosphere and temperature, and to establish whether they are the origin, or merely the consequence, of the reaction. In this paper we report the extensive modification of a high resolution transmission electron microscope (Philips CM30T HRTEM) to add facilities for dynamic in-situ gas-solid reaction studies in a controlled atmosphere of gas or vapor at pressures of 0–50mbar which replaces the regular high vacuum TEM environment. The new environmental cell (ECELL) capability is combined with the original 0.23nm TEM resolution, STEM (BF/ADF) imaging and chemical and crystallographic analyses. Regular specimen holders are used; including hot stages capable of temperatures >1000°C. The full TEM resolution has been demonstrated at temperatures above 700°C and with limited (mbar) amounts of gas flowing through the integral 4-aperture differentially pumped ECELL (and with the additional pumps for the system operating). The ECELL system can be used to preserve, or if necessary to recreate, a continuous chain of evidence in a chemical reaction process.

Evaluation of the Critical Electron Dose on the Contractile Activity of Hydrated Muscle Fibers in the Film-Sealed Environmental Cell

By using the film-sealed environmental cell (EC) having a liquid-injection function, we observed a contraction reaction of hydrated myofibrils isolated from crab muscle under the transmission electron microscope. Our purpose in this work was to evaluate the critical electron dose at which the hydrated fibers lose the contractile activity. The decision of whether the fibers contracted was done by the existence of contraction band after a contraction reaction. From the observations, it was concluded that its critical dose was 2 x 10(-4) C/cm2 or 12 electrons/nm2 at a protein level.

In-Situ Observation of Clay Minerals Hydrated and Intercalated With Liquid Chemicals Using Film-Sealed Environmental Cell

A film-sealed high resolution environmental cell(E.C) for observing hydrated materials had been developed by us(l). Main specification of the E.C. is as follows: 1) Accelerated voltage; 100 kV. 2) Gas in the E.C.; saturated water vapour with carrier gas of 50 Torr. 3) Thickness of gas layer; 50 μm. 4) Sealing film; evaporated carbon film(20 nm thick) with plastic microgrid. 5) Resolving power; 1 nm. 6) Transmittance of electron beam; 60% at 100 kV. The E.C. had been successfully applied to the study of hydrated halloysite(2) (3). Kaolin minerals have no interlayer water and are basically non-expandable but form intercalation compounds with some specific chemicals such as hydrazine, formamide and etc. Because of these compounds being mostly changed in vacuum, we tried to reveal the structure changes between in wet air and in vacuum of kaolin minerals intercalated with hydrazine and of hydrated state of montmori1lonite using the E.C. developed by us.

Observation of the Hydrated form of Tubular Halloysite by An Electron Microscope Equipped with An Environmental Cell

AbstractThe hydrated form of tubular halloysite [halloysite (10 Å)] was observed by a conventional electron microscope equipped with an environmental cell (E.C.), by which the “natural” form was revealed without dehydration of the interlayer water. This study mainly reports the selected area electron diffraction (SAED) analysis of the halloysite (10 Å) and its morphological changes by dehydration. The SAED pattern showed halloysite (10 Å) has two-layer periodicity in a monoclinic structure with the unit cell parameters of a = 5.14 Å, b = 8.90 Å, c = 20.7 Å, β = 99.7°, in space group Cc, and almost the same structure as the dehydrated form of halloysite [halloysite (7 Å)]. This means that the dehydration of the interlayer water did not greatly change or affect the structure of halloysite (10 Å). Accompanying the dehydration of the interlayer water, there appeared along the halloysite tube axis clear stripes that were about 50–100 Å in width. The diameters of the tubular particles also increased about 10%. From the results of various experiments, such as a focussing series, observation of the surface structure by the replica method, observation of end-views of the tubular particles, and others, these two phenomena were explained as follows: Halloysite crystals have “domains” along the c-axis direction, the thicknesses of the “domains” vary ca. 50–100 Å. They are tightly connected with each other when the halloysite is hydrated, but are separated from each other by the dehydration of the interlayer water, whereupon the stripes come into existence along the tube axis. Taking these considerations into account, a model of dehydration is proposed. Moreover, a new method of calculating the β-angle is proposed in the Appendix.

Hydrated form of some clay minerals observed using a film sealed environmental cell

Since the interlayer or adsorbed water of some clay minerals are quite easily dehydrated in dried air, in vacuum, or at moderate temperatures even in the atmosphere, the hydrated forms have not been observed by a conventional electron microscope(TEM). Recently, specific specimen chambers, “environmental cells(E.C.),” have been developed and confirmed to be effective for electron microscopic observation of wet specimen without dehydration. we observed hydrated forms of some clay minerals and their morphological changes by dehydration using a TEM equipped with an E.C..The E.C., equipped with a single hole copper-microgrid sealed by thin carbon-film, attaches to a TEM(JEM 7A) with an accelerating voltage 100KV and both gas pressure (from 760 Torr to vacuum) and relative humidity can be controlled. The samples collected from various localities in Japan were; tubular halloysite (l0Å) from Gumma Prefecture, sperical halloysite (l0Å) from Tochigi Pref., and intermediate halloysite containing both tubular and spherical types from Fukushima Pref..