3D test sample for the calibration and quality control of stimulated emission depletion (STED) and confocal microscopes.

Multiple samples are required to monitor and optimize the quality and reliability of quantitative measurements of stimulated emission depletion (STED) and confocal microscopes. Here, we present a single sample to calibrate these microscopes, align their laser beams and measure their point spread function (PSF) in 3D. The sample is composed of a refractive index matched colloidal crystal of silica beads with fluorescent and gold cores. The microscopes can be calibrated in three dimensions using the periodicity of the crystal; the alignment of the laser beams can be checked using the reflection of the gold cores; and the PSF can be measured at multiple positions and depths using the fluorescent cores. It is demonstrated how this sample can be used to visualize and improve the quality of STED and confocal microscopy images. The sample is adjustable to meet the requirements of different NA objectives and microscopy techniques and additionally can be used to evaluate refractive index mismatches as a function of depth quantitatively.

Topographic features of nano-pores within the osteochondral interface and their effects on transport properties -a 3D imaging and modeling study.

Recent insights suggest that the osteochondral interface plays a central role in maintaining healthy articulating joints. Uncovering the underlying transport mechanisms is key to the understanding of the cross-talk between articular cartilage and subchondral bone. Here, we describe the mechanisms that facilitate transport at the osteochondral interface. Using scanning electron microscopy (SEM), we found a continuous transition of mineralization architecture from the non-calcified cartilage towards the calcified cartilage. This refurbishes the classical picture of the so-called tidemark; a well-defined discontinuity at the osteochondral interface. Using focused-ion-beam SEM (FIB-SEM) on one osteochondral plug derived from a human cadaveric knee, we elucidated that the pore structure gradually varies from the calcified cartilage towards the subchondral bone plate. We identified nano-pores with radius of 10.71 ± 6.45 nm in calcified cartilage to 39.1 ± 26.17 nm in the subchondral bone plate. The extracted pore sizes were used to construct 3D pore-scale numerical models to explore the effect of pore sizes and connectivity among different pores. Results indicated that connectivity of nano-pores in calcified cartilage is highly compromised compared to the subchondral bone plate. Flow simulations showed a permeability decrease by about 2000-fold and solute transport simulations using a tracer (iodixanol, 1.5 kDa with a free diffusivity of 2.5 × 10-10 m2/s) showed diffusivity decrease by a factor of 1.5. Taken together, architecture of the nano-pores and the complex mineralization pattern in the osteochondral interface considerably impacts the cross-talk between cartilage and bone.

Bridging the gap: 3D real-space characterization of colloidal assemblies via FIB-SEM tomography.

Insight in the structure of nanoparticle assemblies up to a single particle level is key to understand the collective properties of these assemblies, which critically depend on the individual particle positions and orientations. However, the characterization of large, micron sized assemblies containing small, 10-500 nanometer, sized colloids is highly challenging and cannot easily be done with the conventional light, electron or X-ray microscopy techniques. Here, we demonstrate that focused ion beam-scanning electron microscopy (FIB-SEM) tomography in combination with image processing enables quantitative real-space studies of ordered and disordered particle assemblies too large for conventional transmission electron tomography, containing particles too small for confocal microscopy. First, we demonstrate the high resolution structural analysis of spherical nanoparticle assemblies, containing small anisotropic gold nanoparticles. Herein, FIB-SEM tomography allows the characterization of assembly dimensions which are inaccessible to conventional transmission electron microscopy. Next, we show that FIB-SEM tomography is capable of characterizing much larger ordered and disordered assemblies containing silica colloids with a diameter close to the resolution limit of confocal microscopes. We determined both the position and the orientation of each individual (nano)particle in the assemblies by using recently developed particle tracking routines. Such high precision structural information is essential in the understanding and design of the collective properties of new nanoparticle based materials and processes.

Shaping Silica Rods by Tuning Hydrolysis and Condensation of Silica Precursors.

We present the synthesis of colloidal silica particles with new shapes by manipulating the growth conditions of rods that are growing from polyvinylpyrrolidone-loaded water-rich droplets containing ammonia and ethanol. The silica rods grow by ammonia-catalyzed hydrolysis and condensation of tetraethoxysilane (TEOS). The lengthwise growth of these silica rods gives us the opportunity to change the conditions at any time during the reaction. In this work, we vary the availability of hydrolyzed monomers as a function of time and study how the change in balance between the hydrolysis and condensation reactions affects a typical synthesis (as described in more detail by our group earlier1). First, we show that in a "standard" synthesis, there are two silica growth processes occurring; one in the oil phase and one in the droplet. The growth process in the water droplet causes the lengthwise growth of the rods. The growth process in the oil phase produces a thin silica layer around the rods, but also causes the nucleation of 70 nm silica spheres. During a typical rod growth, silica formation mainly takes place in the droplet. The addition of partially hydrolyzed TEOS or tetramethoxysilane (TMOS) to the growth mixture results in a change in balance between the hydrolysis and condensation reaction. As a result, the growth also starts to take place on the surface of the water droplet and thus from the oil phase, not only from inside the droplet onto a silica rod sticking out of the droplet. Carefully tuning the growth from the droplet and the growth from the oil phase allowed us to create nanospheres, hollow silica rods, hollow sphere rod systems (colloidal matchsticks), and bent silica rods.

Electron crystallography with the EIGER detector.

Electron crystallography is a discipline that currently attracts much attention as method for inorganic, organic and macromolecular structure solution. EIGER, a direct-detection hybrid pixel detector developed at the Paul Scherrer Institut, Switzerland, has been tested for electron diffraction in a transmission electron microscope. EIGER features a pixel pitch of 75 × 75 µm2, frame rates up to 23 kHz and a dead time between frames as low as 3 µs. Cluster size and modulation transfer functions of the detector at 100, 200 and 300 keV electron energies are reported and the data quality is demonstrated by structure determination of a SAPO-34 zeotype from electron diffraction data.

Coke Formation in a Zeolite Crystal During the Methanol-to-Hydrocarbons Reaction as Studied with Atom Probe Tomography.

Understanding the formation of carbon deposits in zeolites is vital to developing new, superior materials for various applications, including oil and gas conversion processes. Herein, atom probe tomography (APT) has been used to spatially resolve the 3D compositional changes at the sub-nm length scale in a single zeolite ZSM-5 crystal, which has been partially deactivated by the methanol-to-hydrocarbons reaction using (13) C-labeled methanol. The results reveal the formation of coke in agglomerates that span length scales from tens of nanometers to atomic clusters with a median size of 30-60 (13) C atoms. These clusters correlate with local increases in Brønsted acid site density, demonstrating that the formation of the first deactivating coke precursor molecules occurs in nanoscopic regions enriched in aluminum. This nanoscale correlation underscores the importance of carefully engineering materials to suppress detrimental coke formation.

FIB-SEM Tomography Probes the Mesoscale Pore Space of an Individual Catalytic Cracking Particle.

The overall performance of a catalyst particle strongly depends on the ability of mass transport through its pore space. Characterizing the three-dimensional structure of the macro- and mesopore space of a catalyst particle and establishing a correlation with transport efficiency is an essential step toward designing highly effective catalyst particles. In this work, a generally applicable workflow is presented to characterize the transport efficiency of individual catalyst particles. The developed workflow involves a multiscale characterization approach making use of a focused ion beam-scanning electron microscope (FIB-SEM). SEM imaging is performed on cross sections of 10.000 µm2, visualizing a set of catalyst particles, while FIB-SEM tomography visualized the pore space of a large number of 8 µm3 cubes (subvolumes) of individual catalyst particles. Geometrical parameters (porosity, pore connectivity, and heterogeneity) of the material were used to generate large numbers of virtual 3D volumes resembling the sample's pore space characteristics, while being suitable for computationally demanding transport simulations. The transport ability, defined as the ratio of unhindered flow over hindered flow, is then determined via transport simulations through the virtual volumes. The simulation results are used as input for an upscaling routine based on an analogy with electrical networks, taking into account the spatial heterogeneity of the pore space over greater length scales. This novel approach is demonstrated for two distinct types of industrially manufactured fluid catalytic cracking (FCC) particles with zeolite Y as the active cracking component. Differences in physicochemical and catalytic properties were found to relate to differences in heterogeneities in the spatial porosity distribution. In addition to the characterization of existing FCC particles, our method of correlating pore space with transport efficiency does also allow for an up-front evaluation of the transport efficiency of new designs of FCC catalyst particles.

Repulsive van der Waals forces enable Pickering emulsions with non-touching colloids.

Emulsions stabilized by solid particles, called Pickering emulsions, offer promising applications in drug delivery, cosmetics, food science and the manufacturing of porous materials. This potential stems from their high stability against coalescence and 'surfactant-free' nature. Generally, Pickering emulsions require that the solid particles are wetted by both phases and as a result, the adsorption free energy is often large with respect to the thermal energy (kBT). Here we provide the first experimental proof for an alternative scenario: non-touching (effectively non-wetting), charged, particles that are completely immersed in the oil phase through a balance of charge induced attractions and repulsions caused by van der Waals forces. These particles nonetheless stabilize the emulsion. The main advantage of this novel adsorption mechanism is that these particles can easily be detached from the interface simply by adding salt. This not only makes the finding fundamentally of interest, but also enables a triggered de-emulsification and particle recovery, which is useful in fields like enhanced oil recovery, heterogeneous catalysis, and emulsion polymerization.

Directed Self-Assembly of Micron-Sized Gold Nanoplatelets into Oriented Flexible Stacks with Tunable Interplate Distance.

A growing demand for control over the interparticle spacing and the orientation of anisotropic metallic particles into self-assembled structures is fuelled by their use in potential applications such as in plasmonics, catalysis, sensing, and optoelectronics. Here, we present an improved high yield synthesis method to fabricate micron- and submicron-sized gold nanoplatelets with a thickness less than 20 nm using silver nanoplatelets as seeds. By tuning the depth of the secondary minimum in the DLVO interaction potential between these particles, we are able to assemble the platelets into dynamic and flexible stacks containing thousands of platelets arranged face-to-face with well-defined spacing. Moreover, we demonstrate that the length of the stacks, and the interplate distance can be controlled between tens and hundreds of nm with the ionic strength. We use a high frequency external electric field to control the orientation of the stacks and direct the stacks into highly organized 2D and 3D assemblies that strongly polarize light.

Rock mechanics. Superplastic nanofibrous slip zones control seismogenic fault friction.

Understanding the internal mechanisms controlling fault friction is crucial for understanding seismogenic slip on active faults. Displacement in such fault zones is frequently localized on highly reflective (mirrorlike) slip surfaces, coated with thin films of nanogranular fault rock. We show that mirror-slip surfaces developed in experimentally simulated calcite faults consist of aligned nanogranular chains or fibers that are ductile at room conditions. These microstructures and associated frictional data suggest a fault-slip mechanism resembling classical Ashby-Verrall superplasticity, capable of producing unstable fault slip. Diffusive mass transfer in nanocrystalline calcite gouge is shown to be fast enough for this mechanism to control seismogenesis in limestone terrains. With nanogranular fault surfaces becoming increasingly recognized in crustal faults, the proposed mechanism may be generally relevant to crustal seismogenesis.

In-situ integrity control of frozen-hydrated, vitreous lamellas prepared by the cryo-focused ion beam-scanning electron microscope.

Recently a number of new approaches have been presented with the intention to produce electron beam transparent cryo-sections (lamellas in FIB-SEM terminology) from hydrated vitreously frozen cryo samples with a Focused Ion Beam (FIB) system, suitable for cryo-Transmission Electron Microscopy (cryo-TEM). As the workflow is still challenging and time consuming, it is important to be able to determine the integrity and suitability (cells vs. no cells; vitreous vs. crystalline) of the lamellas. Here we present an in situ method that tests both conditions by using the cryo-Scanning Electron Microscope (cryo-SEM) in transmission mode (TSEM; Transmission Scanning Electron Microscope) once the FIB-made lamella is ready. Cryo-TSEM imaging of unstained cells yields strong contrast, enabling direct imaging of material present in the lamellas. In addition, orientation contrast is shown to be suitable for distinguishing crystalline lamellas from vitreous lamellas. Tilting the stage a few degrees results in changes of contrast between ice grains as a function of the tilt angle, whereas the contrast of areas with vitreous ice remains unchanged as a function of the tilt angle. This orientation contrast has subsequently been validated by cryo-Electron BackScattered Diffraction (EBSD) in transmission mode. Integration of the presented method is discussed and the role it can play in future developments for a new and innovative all-in-one cryo-FIB-SEM life sciences instrument.

Defect engineering in sedimentary colloidal photonic crystals.

In this paper, lithographic methods are successfully employed to create growth templates for colloidal self-assembly, enabling the inclusion of crystallographic defects at predetermined positions. It is shown that through smart template design stacking faults can be grown predictably into face centered cubic structures. More interestingly, by precise guiding of the stacking faults hollow intergrowth channels can be grown at predetermined lateral and vertical positions. The mechanisms involved in defect growth are promising for extension of this technique to more complex crystal structures, such as the diamond structure, as well as to more complex faults, including corners and t-junctions.

The porosity, acidity, and reactivity of dealuminated zeolite ZSM-5 at the single particle level: the influence of the zeolite architecture.

A combination of atomic force microscopy (AFM), high-resolution scanning electron microscopy (HR-SEM), focused-ion-beam scanning electron microscopy (FIB-SEM), X-ray photoelectron spectroscopy (XPS), confocal fluorescence microscopy (CFM), and UV/Vis and synchrotron-based IR microspectroscopy was used to investigate the dealumination processes of zeolite ZSM-5 at the individual crystal level. It was shown that steaming has a significant impact on the porosity, acidity, and reactivity of the zeolite materials. The catalytic performance, tested by the styrene oligomerization and methanol-to-olefin reactions, led to the conclusion that mild steaming conditions resulted in greatly enhanced acidity and reactivity of dealuminated zeolite ZSM-5. Interestingly, only residual surface mesoporosity was generated in the mildly steamed ZSM-5 zeolite, leading to rapid crystal coloration and coking upon catalytic testing and indicating an enhanced deactivation of the zeolites. In contrast, harsh steaming conditions generated 5-50 nm mesopores, extensively improving the accessibility of the zeolites. However, severe dealumination decreased the strength of the Brønsted acid sites, causing a depletion of the overall acidity, which resulted in a major drop in catalytic activity.

The making of frozen-hydrated, vitreous lamellas from cells for cryo-electron microscopy.

There has been a long standing desire to produce thick (up to 500 nm) cryo-sections of fully hydrated cells and tissue for high-resolution analysis in their natural state by cryo-transmission electron microscopy. Here, we present a method that can successfully produce sections (lamellas in FIB-SEM terminology) of fully hydrated, unstained cells from high-pressure frozen samples by focused ion beam (FIB) milling. The samples are therefore placed in thin copper tubes and vitrified by high-pressure freezing. For transfer, handling and subsequent milling, the tubes are placed in a novel connective device (ferrule) that protects the sample from devitrification and contamination and passes through all operation steps. A piezo driven sample positioning stage (cryo-nano-bench, CNB) with three degrees of freedom was additionally developed to enable accurate milling of frozen-hydrated lamellas. With the CNB, high-pressure frozen samples can be milled to produce either thin lamellas (<100 nm), for direct imaging by high-resolution cryo-TEM or thicker lamellas (300-500 nm) for cryo-electron tomography. The sample remains vitreous throughout the process by using the presented tools and methods. The results are an important step towards investigating larger cells and even tissue in there natural state which in the end will enable us to gain better insights into cellular processes.

Morphology-dependent zeolite intergrowth structures leading to distinct internal and outer-surface molecular diffusion barriers.

Zeolites play a crucial part in acid-base heterogeneous catalysis. Fundamental insight into their internal architecture is of great importance for understanding their structure-function relationships. Here, we report on a new approach correlating confocal fluorescence microscopy with focused ion beam-electron backscatter diffraction, transmission electron microscopy lamelling and diffraction, atomic force microscopy and X-ray photoelectron spectroscopy to study a wide range of coffin-shaped MFI-type zeolite crystals differing in their morphology and chemical composition. This powerful combination demonstrates a unified view on the morphology-dependent MFI-type intergrowth structures and provides evidence for the presence and nature of internal and outer-surface barriers for molecular diffusion. It has been found that internal-surface barriers originate not only from a 90 degrees mismatch in structure and pore alignment but also from small angle differences of 0.5 degrees-2 degrees for particular crystal morphologies. Furthermore, outer-surface barriers seem to be composed of a silicalite outer crust with a thickness varying from 10 to 200 nm.

Tomography of insulating biological and geological materials using focused ion beam (FIB) sectioning and low-kV BSE imaging.

Tomography in a focused ion beam (FIB) scanning electron microscope (SEM) is a powerful method for the characterization of three-dimensional micro- and nanostructures. Although this technique can be routinely applied to conducting materials, FIB-SEM tomography of many insulators, including biological, geological and ceramic samples, is often more difficult because of charging effects that disturb the serial sectioning using the ion beam or the imaging using the electron beam. Here, we show that automatic tomography of biological and geological samples can be achieved by serial sectioning with a focused ion beam and block-face imaging using low-kV backscattered electrons. In addition, a new ion milling geometry is used that reduces the effects of intensity gradients that are inherent in conventional geometry used for FIB-SEM tomography.