Lipids and Proteins Differentiation in Membrane Fouling Using Heavy Metal Staining and Electron Microscopy at Cryogenic Temperatures.

The detailed characterization of fouling in membranes is essential to understand any observed improvement or reduction on filtration performance. Electron microscopy allows detailed structural characterization, and its combination with labeling techniques, using electron-dense probes, typically allows for the differentiation of biomolecules. Developing specific protocols that allow for differentiation of biomolecules in membrane fouling by electron microscopy is a major challenge due to both as follows: the necessity to preserve the native state of fouled membranes upon real filtration conditions as well as the inability of the electron-dense probes to penetrate the membranes once they have been fouled. In this study, we present the development of a heavy metal staining technique for identification and differentiation of biomolecules in membrane fouling, which is compatible with cryofixation methods. A general contrast enhancement of biomolecules and fouling is achieved. Our observations indicate a strong interaction between biomolecules: A tendency of proteins, both in solution as well as in the fouling, to surround the lipids is observed. Using transmission electron microscopy and scanning electron microscopy at cryogenic conditions, cryo-SEM, in combination with energy-dispersive X-ray spectroscopy, the spatial distribution of proteins and lipids within fouling is shown and the role of proteins in fouling discussed.

Application of a Cryo-FIB-SEM-µRaman Instrument to Probe the Depth of Vitreous Ice in a Frozen Sample.

The development of instruments combining multiple characterization and imaging tools drove huge advances in material science, engineering, biology, and other related fields. Notably, the coupling of SEM with micro-Raman spectrometry (µRaman) provides the means for the correlation between structural and physicochemical properties at the surface, while dual focused ion beam (FIB)-scanning electron microscopes (SEMs) operating under cryogenic conditions (cryo-FIB-SEM) allow for the analysis of the ultrastructure of materials in situ and in their native environment. In cryo-FIB-SEM, rapid and efficient methods for assessing vitrification conditions in situ are required for the accurate investigation of the original structure of hydrated samples. This work reports for the first time the use of a cryo-FIB-SEM-µRaman instrument to efficiently assess the accuracy of cryo-fixation methods. Analyses were performed on plunge-freezed highly hydrated calcium phosphate cement (CPC) and a gelatin composite. By making a trench of a defined thickness with FIB, µRaman analyses were carried out at a specific depth within the frozen material. Results show that the µRaman signal is sensitive to the changes in the molecular structures of the aqueous phase and can be used to examine the depth of vitreous ice in frozen samples. The method presented in this work provides a reliable way to avoid imaging artifacts in cryo-FIB-SEM that are related to cryo-fixation and therefore constitutes great interest in the study of vitreous materials exhibiting high water content, regardless of the sample preparation method (i.e., by HPF, plunge freezing, and so on).

Nanoscale Chemical Heterogeneity in Aromatic Polyamide Membranes for Reverse Osmosis Applications.

Reverse osmosis membranes are used within the oil and gas industry for seawater desalination on off-shore oilrigs. The membranes consist of three layers of material: a polyester backing layer, a polysulfone support and a polyamide (PA) thin film separating layer. It is generally thought that the PA layer controls ion selectivity within the membrane but little is understood about its structure or chemistry at the molecular scale. This active polyamide layer is synthesized by interfacial polymerization at an organic/aqueous interface between m-phenylenediamine and trimesoyl chloride, producing a highly cross-linked PA polymer. It has been speculated that the distribution of functional chemistry within this layer could play a role in solute filtration. The only technique potentially capable of probing the distribution of functional chemistry within the active PA layer with sufficient spatial and energy resolution is scanning transmission electron microscopy combined with electron energy-loss spectroscopy (STEM-EELS). Its use is a challenge because organic materials suffer beam-induced damage at relatively modest electron doses. Here we show that it is possible to use the N K-edge to map the active layer of a PA film using monochromated EELS spectrum imaging. The active PA layer is 12 nm thick, which supports previous neutron reflectivity data. Clear changes in the fine structure of the C K-edge across the PA films are measured and we use machine learning to assign fine structure at this edge. Using this method, we map highly heterogeneous intensity variations in functional chemistry attributed to N-C═C bonds within the PA. Similarities are found with previous molecular dynamics simulations of PA showing regions with a higher density of amide bonding as a result of the aggregation process at similar length scales. The chemical pathways that can be deduced may offer a clearer understanding of the transport mechanisms through the membrane.

Transmission Electron Microscopy Reveals Deposition of Metal Oxide Coatings onto Metal-Organic Frameworks.

Postsynthetic strategies for modifying metal-organic frameworks (MOFs) have proven to be an incredibly powerful approach for expanding the scope and functionality of these materials. Previously, we reported on the postsynthetic exchange (PSE) of metal ions and ligands in the University of Oslo (UiO) series of MOFs. Detailed characterization by several analytical methods, most notably inductively coupled plasma mass spectrometry and transmission electron microscopy reveal that metal ion deposition on the surface of these MOFs occurs in the form of nanoscale metal oxides, rather than yielding exchanged metal sites within the MOFs, as was previously reported. By contrast, these combined analytical methods do confirm that ligand-based PSE can occur in these MOFs. These findings provide new insight into the postsynthetic manipulation of MOF materials, highlight the importance of rigorously characterizing these materials to correctly assign their composition and structure, and provide a new route to making hybrid solids with a MOF@metal oxide architecture.

Mapping VIS-terahertz (≤17 THz) surface plasmons sustained on native and chemically functionalized percolated gold thin films using EELS.

Heterogeneous assemblies of molecules (Rhodamine B) adsorbed onto a nano-corrugated metallic surface (a percolated Au network) are investigated using electron energy loss spectroscopy in the scanning transmission electron microscope (STEM-EELS). Our first measurements target the native metallic substrate, which consists of a commercial Au thin film atop an ultrathin carbon membrane. The Au film displays a percolated morphology with nanostructures of estimated thickness ≤10 nm approximately. We observe a rich plasmonic response from the metallic substrate; one which varies nanometrically and spans the VIS-terahertz region. Multiple localized plasmons are detected at individual nanometric integrated areas, while an analysis of their spatial distribution reveals that for each integrated energy range (50 meV integration window) resonances are simultaneously supported at different locations within the film. We record subsequent EEL spectrum images of the hybrid molecular-metallic construct after deposition of Rhodamine B molecules onto the substrate, where plasmons, molecular vibrations and electronic excitations might all be simultaneously detected. A comparison of average signals for both systems is performed and spectral variations within the three spectral regions where molecular signatures may be observed are discussed. Our measurements and their analysis, if applied to the same location before and after molecular deposition, may be used to rationalize optical microscopic and spectroscopic measurements that take advantage of the interplay between molecules and plasmons.

Electron Microscopy Reveals Structural and Chemical Changes at the Nanometer Scale in the Osteogenesis Imperfecta Murine Pathology.

Alternations of collagen and mineral at the molecular level may have a significant impact on the strength and toughness of bone. In this study, scanning transmission electron microscopy (STEM) and electron energy-loss spectroscopy (EELS) were employed to study structural and compositional changes in bone pathology at nanometer spatial resolution. Tail tendon and femoral bone of osteogenesis imperfecta murine (oim, brittle bone disease) and wild type (WT) mice were compared to reveal defects in the architecture and chemistry of the collagen and collagen-mineral composite in the oim tissue at the molecular level. There were marked differences in the substructure and organization of the collagen fibrils in the oim tail tendon; some regions have clear fibril banding and organization, while in other regions fibrils are disorganized. Malformed collagen fibrils were loosely packed, often bent and devoid of banding pattern. In bone, differences were detected in the chemical composition of mineral in oim and WT. While mineral present in WT and oim bone exhibited the major characteristics of apatite, examination in EELS of the fine structure of the carbon K ionization edge revealed a significant variation in the presence of carbonate in different regions of bone. Variations have been also observed in the fine structure and peak intensities of the nitrogen K-edge. These alterations are suggestive of differences in the maturation of collagen nucleation sites or cross-links. Future studies will aim to establish the scale and impact of the modifications observed in oim tissues. The compositional and structural alterations at the molecular level cause deficiencies at larger length scales. Understanding the effect of molecular alterations to pathologic bone is critical to the design of effective therapeutics.

Gaining Control over Radiolytic Synthesis of Uniform Sub-3-nanometer Palladium Nanoparticles: Use of Aromatic Liquids in the Electron Microscope.

Synthesizing nanomaterials of uniform shape and size is of critical importance to access and manipulate the novel structure-property relationships arising at the nanoscale, such as catalytic activity. In this work, we synthesize Pd nanoparticles with well-controlled size in the sub-3 nm range using scanning transmission electron microscopy (STEM) in combination with an in situ liquid stage. We use an aromatic hydrocarbon (toluene) as a solvent that is very resistant to high-energy electron irradiation, which creates a net reducing environment without the need for additives to scavenge oxidizing radicals. The primary reducing species is molecular hydrogen, which is a widely used reductant in the synthesis of supported metal catalysts. We propose a mechanism of particle formation based on the effect of tri-n-octylphosphine (TOP) on size stabilization, relatively low production of radicals, and autocatalytic reduction of Pd(II) compounds. We combine in situ STEM results with insights from in situ small-angle X-ray scattering (SAXS) from alcohol-based synthesis, having similar reduction potential, in a customized microfluidic device as well as ex situ bulk experiments. This has allowed us to develop a fundamental growth model for the synthesis of size-stabilized Pd nanoparticles and demonstrate the utility of correlating different in situ and ex situ characterization techniques to understand, and ultimately control, metal nanostructure synthesis.

Observing the growth of metal-organic frameworks by in situ liquid cell transmission electron microscopy.

Liquid cell transmission electron microscopy (LCTEM) can provide direct observations of solution-phase nanoscale materials, and holds great promise as a tool for monitoring dynamic self-assembled nanomaterials. Control over particle behavior within the liquid cell, and under electron beam irradiation, is of paramount importance for this technique to contribute to our understanding of chemistry and materials science at the nanoscale. However, this type of control has not been demonstrated for complex, organic macromolecular materials, which form the basis for all biological systems and all of polymer science, and encompass important classes of advanced porous materials. Here we show that by controlling the liquid cell membrane surface chemistry and electron beam conditions, the dynamics and growth of metal-organic frameworks (MOFs) can be observed. Our results demonstrate that hybrid organic/inorganic beam-sensitive materials can be analyzed with LCTEM and, at least in the case of ZIF-8 dynamics, the results correlate with observations from bulk growth or other standard synthetic conditions. Furthermore, we show that LCTEM can be used to better understand how changes to synthetic conditions result in changes to particle size. We anticipate that direct, nanoscale imaging by LCTEM of MOF nucleation and growth mechanisms may provide insight into controlled MOF crystal morphology, domain composition, and processes influencing defect formation.

Tip-enhanced Raman nanographs: mapping topography and local electric fields.

We report tip-enhanced Raman imaging experiments in which information on sample topography and local electric fields is simultaneously obtained using an all-optical detection scheme. We demonstrate how a Raman-active 4,4'-dimercaptostilbene (DMS)-coated gold tip of an atomic force microscope can be used to simultaneously map the topography and image the electric fields localized at nanometric (20 and 5 nm wide) slits lithographically etched in silver, all using optical signals. Bimodal imaging is feasible by virtue of the frequency-resolved optical response of the functionalized metal probe. Namely, the probe position-dependent signals can be subdivided into two components. The first is a 500-2250 cm(-1) Raman-shifted signal, characteristic of the tip-bound DMS molecules. The molecules report on topography through the intensity contrast observed as the tip scans across the nanoscale features. The variation in molecular Raman activity arises from the absence/formation of a plasmonic junction between the scanning probe and patterned silver surface, which translates into dimmed/enhanced Raman signatures of DMS. Using these molecular signals, we demonstrate that sub-15 nm spatial resolution is attainable using a 30 nm DMS-coated gold tip. The second response consists of two correlated sub-500 cm(-1) signals arising from mirror-like reflections of (i) the incident laser field and (ii) the Raman scattered response of an underlying glass support (at 100-500 cm(-1)) off the gold tip. We show that both the reflected low-wavenumber signals trace the local electric fields in the vicinity of the nanometric slits.

Electric field enhancement in a self-assembled 2D array of silver nanospheres.

We investigate the plasmonic properties of a self-assembled 2D array of Ag nanospheres (average particle diameter/inter-particle separation distance of 9/3.7 nm). The structures of the individual particles and their assemblies are characterized using high-resolution transmission electron microscopy (HR-TEM). The plasmonic response of the nanoparticle network is probed using two-photon photoemission electron microscopy (TP-PEEM). HR-TEM and TP-PEEM statistics reveal the structure and plasmonic response of the network to be homogeneous on average. This translates into a relatively uniform surface-enhanced Raman scattering (SERS) response from biphenyl,4-4(')-dithiol (BPDT) molecules adsorbed onto different sites of the network. Reproducible, bright, and low-background SERS spectra are recorded and assigned on the basis of density functional theory calculations in which BPDT is chemisorbed onto the vertex of a finite tetrahedral Ag cluster consisting of 20 Ag atoms. A notable agreement between experiment and theory allows us to rigorously account for the observable vibrational states of BPDT in the ∼200-2200 cm(-1) region of the spectrum. Finite difference time domain simulations further reveal that physical enhancement factors on the order of 10(6) are attainable at the nanogaps formed between the silver nanospheres in the 2D array. Combined with modest chemical enhancement factors, this study paves the way for reproducible single molecule signals from an easily self-assembled SERS substrate.

In-situ electrochemical transmission electron microscopy for battery research.

The recent development of in-situ liquid stages for (scanning) transmission electron microscopes now makes it possible for us to study the details of electrochemical processes under operando conditions. As electrochemical processes are complex, care must be taken to calibrate the system before any in-situ/operando observations. In addition, as the electron beam can cause effects that look similar to electrochemical processes at the electrolyte/electrode interface, an understanding of the role of the electron beam in modifying the operando observations must also be understood. In this paper we describe the design, assembly, and operation of an in-situ electrochemical cell, paying particular attention to the method for controlling and quantifying the experimental parameters. The use of this system is then demonstrated for the lithiation/delithiation of silicon nanowires.

Probing the degradation mechanisms in electrolyte solutions for Li-ion batteries by in situ transmission electron microscopy.

Development of novel electrolytes with increased electrochemical stability is critical for the next generation battery technologies. In situ electrochemical fluid cells provide the ability to rapidly and directly characterize electrode/electrolyte interfacial reactions under conditions directly relevant to the operation of practical batteries. In this paper, we have studied the breakdown of a range of inorganic/salt complexes relevant to state-of-the-art Li-ion battery systems by in situ (scanning) transmission electron microscopy ((S)TEM). In these experiments, the electron beam itself caused the localized electrochemical reaction that allowed us to observe electrolyte breakdown in real-time. The results of the in situ (S)TEM experiments matches with previous stability tests performed during battery operation and the breakdown products and mechanisms are also consistent with known mechanisms. This analysis indicates that in situ liquid stage (S)TEM observations could be used to directly test new electrolyte designs and identify a smaller library of candidate solutions deserving of more detailed characterization. A systematic study of electrolyte degradation is also a necessary first step for any future controlled in operando liquid (S)TEM experiments intent on visualizing working batteries at the nanoscale.

Demonstration of an electrochemical liquid cell for operando transmission electron microscopy observation of the lithiation/delithiation behavior of Si nanowire battery anodes.

Over the past few years, in situ transmission electron microscopy (TEM) studies of lithium ion batteries using an open-cell configuration have helped us to gain fundamental insights into the structural and chemical evolution of the electrode materials in real time. In the standard open-cell configuration, the electrolyte is either solid lithium oxide or an ionic liquid, which is point-contacted with the electrode. This cell design is inherently different from a real battery, where liquid electrolyte forms conformal contact with electrode materials. The knowledge learnt from open cells can deviate significantly from the real battery, calling for operando TEM technique with conformal liquid electrolyte contact. In this paper, we developed an operando TEM electrochemical liquid cell to meet this need, providing the configuration of a real battery and in a relevant liquid electrolyte. To demonstrate this novel technique, we studied the lithiation/delithiation behavior of single Si nanowires. Some of lithiation/delithation behaviors of Si obtained using the liquid cell are consistent with the results from the open-cell studies. However, we also discovered new insights different from the open cell configuration-the dynamics of the electrolyte and, potentially, a future quantitative characterization of the solid electrolyte interphase layer formation and structural and chemical evolution.

A (S)TEM gas cell holder with localized laser heating for in situ experiments.

The advent of aberration correction for transmission electron microscopy has transformed atomic resolution imaging into a nearly routine technique for structural analysis. Now an emerging frontier in electron microscopy is the development of in situ capabilities to observe reactions at atomic resolution in real time and within realistic environments. Here we present a new in situ gas cell holder that is designed for compatibility with a wide variety of sample type (i.e., dimpled 3-mm discs, standard mesh grids, various types of focused ion beam lamellae attached to half grids). Its capabilities include localized heating and precise control of the gas pressure and composition while simultaneously allowing atomic resolution imaging at ambient pressure. The results show that 0.25-nm lattice fringes are directly visible for nanoparticles imaged at ambient pressure with gas path lengths up to 20 µm. Additionally, we quantitatively demonstrate that while the attainable contrast and resolution decrease with increasing pressure and gas path length, resolutions better than 0.2 nm should be accessible at ambient pressure with gas path lengths less than the 15 µm utilized for these experiments.

Photoemission electron microscopy study of sub-200 nm self-assembled La0.7SrLa0.3MnO3 epitaxial islands.

The chemical composition and the magnetic structure of individual La0.7Sr0.3MnO3 (LSMO) ferromagnetic manganite epitaxial nanostructures less than 200 nm in width are explored using Photoemission Electron Microscopy (PEEM). X-ray absorption spectra (XAS) provide separate information on the surface and the bulk composition of the nanoislands and give evidence of Mn(2+) present on the surface of otherwise stoichiometric nanostructures. Ferromagnetic domains less than 70 nm are resolved using X-ray magnetic circular dichroism (XMCD), which allows for the detection of magnetic vortex states in both (001)LSMO square and (111)LSMO triangular manganite nanoislands. The evolution of single nanostructures under an in-plane magnetic field is seen to depend on the specific nanoisland size and geometry. In particular, PEEM XMCD imaging allows detecting opposite chiralities as well as a variety of magnetization behaviors for different nanoislands.

Self-organized Ce(1-x)Gd(x)O(2-y) nanowire networks with very fast coarsening driven by attractive elastic interactions.

Assembling arrays of ordered nanowires is a key objective for many of their potential applications. However, a lack of understanding and control of the nanowires' growth mechanisms limits their thorough development. In this work, an appealing new path towards self-organized epitaxial nanowire networks produced by high-throughput solution methods is reported. Two requisites are identified to generate the nanowires: a thermodynamic driving force for an unrestricted elongated equilibrium island shape, and a very fast effective coarsening rate. These requirements are met in anisotropically strained Ce(1-x)Gd(x)O(2-y) nanowires with the (011) orientation grown on the (001) surface of LaAlO(3) substrates. Nanowires with aspect ratios above ≈100 oriented along two mutually orthogonal axes are obtained leading to labyrinthine networks. A very fast effective nanowire growth rate (≈60 nm min(-1)) for ex-situ thermally annealed nanostructures derives from simultaneous kinetic processes occurring in a branched network. Ostwald ripening and anisotropic dynamic coalescence, both promoted by strain-driven attractive nanowire interaction, and rapid recrystallization, enabled by fast atomic diffusion associated with a high concentration of oxygen vacancies, contribute to such an effective growth rate. This bottom-up approach to self-organized nanowire growth has a wide potential for many materials and functionalities.

Orientational ordering of solution derived epitaxial Gd-doped ceria nanowires induced by nanoscratching.

When one-dimensional nanostructures are epitaxially grown on a substrate a key goal is to control the nanowire's position and orientation. Nanoscratching of single crystalline (001)- LaAlO(3) substrates is demonstrated to be extraordinarily effective in directing the self-assembly of Ce(0.9)Gd(0.1)O(2-y) epitaxial nanowires grown by chemical solution deposition. The local anisotropic elastic strain field imposed by the indentation lines is responsible for the breaking of the pre-existing orientation energy degeneracy and selects the nanowires' orientation parallel to the lines to an extent that can reach 100%.