Transmission Electron Microscopy Sample Preparation Research Articles

Ultrastructural observation of natural field phytoplankton cells by using rapid freezing and freeze substitution

Transmission electron microscopy (TEM) is a basic technique used to study the ultrastructures of phytoplankton. However, the use of chemical fixation to prepare phytoplankton samples for TEM is laborious and time-consuming. Furthermore, chemical fixation often causes artificial structural changes (artifacts) in phytoplankton cells. In this study, we induced and optimized a rapid freezing and freeze substitution (RFS) method, accompanied by a filter dehydration system, to prepare TEM samples of marine microalgae, including Heterosigma akashiwo, Chattonella antiqua (Raphidophyceae), Heterocapsa circularisquama (Dinophyceae), Chaetoceros tenuissimus (Bacillariophyceae), and Teleaulax amphioxeia (Cryptophyceae). The whole procedure was much more efficient than the traditional method. In addition, the fine structure of each microalgal cell was well preserved. For example, the fine structures of the nuclear membrane, nuclear pores, and several vesicles were clearly observed, which were comparatively difficult to observe using the chemical fixation method. In addition, this new technique was applicable to the natural blooming cells of H. akashiwo, and high-resolution ultrastructural images were successfully observed. This is the first report on the application of the modified-RFS method for natural phytoplankton assemblages. This method can be used for diverse phytoplankters in natural waters, and the high-quality observations are expected to provide useful information toward understanding phytoplankter physiology and ecology.

Improved FIB milling process for TEM preparation of NiAlPt bulk alloy samples containing residual stress

NiAlPt alloys which undergo complex martensitic and twinning transformations during thermal cycling were prepared for Transmission Electron Microscopy (TEM) analysis using the Focused Ion Beam (FIB) milling process. Coupons of alloys that underwent cyclic heating, with no prior isothermal heat treatment contained a build-up of residual stress that caused sections of the foil to crack or buckle during the thinning process, rendering them useless. The solution was to mill strain isolation trenches perpendicular to the area of interest. These trenches acted as reservoirs for the released residual stress. This step was conducted in conjunction with reducing the beam current much earlier in the thinning process, thus ensuring minimum damage to the section being thinned. These two modifications of the standard FIB milling process, with emphasis on the strain isolation trenches, allowed for successful preparation of TEM samples from a bulk alloy containing residual stress.

Microwave Assisted Rapid Diagnosis of Plant Virus Diseases by Transmission Electron Microscopy

Investigations of ultrastructural changes induced by viruses are often necessary to clearly identify viral diseases in plants. With conventional sample preparation for transmission electron microscopy (TEM) such investigations can take several days1,2 and are therefore not suited for a rapid diagnosis of plant virus diseases. Microwave fixation can be used to drastically reduce sample preparation time for TEM investigations with similar ultrastructural results as observed after conventionally sample preparation3-5. Many different custom made microwave devices are currently available which can be used for the successful fixation and embedding of biological samples for TEM investigations5-8. In this study we demonstrate on Tobacco Mosaic Virus (TMV) infected Nicotiana tabacum plants that it is possible to diagnose ultrastructural alterations in leaves in about half a day by using microwave assisted sample preparation for TEM. We have chosen to perform this study with a commercially available microwave device as it performs sample preparation almost fully automatically5 in contrast to the other available devices where many steps still have to be performed manually6-8 and are therefore more time and labor consuming. As sample preparation is performed fully automatically negative staining of viral particles in the sap of the remaining TMV-infected leaves and the following examination of ultrastructure and size can be performed during fixation and embedding.

Microwave assisted rapid diagnosis of plant virus diseases by transmission electron microscopy.

Investigations of ultrastructural changes induced by viruses are often necessary to clearly identify viral diseases in plants. With conventional sample preparation for transmission electron microscopy (TEM) such investigations can take several days1,2 and are therefore not suited for a rapid diagnosis of plant virus diseases. Microwave fixation can be used to drastically reduce sample preparation time for TEM investigations with similar ultrastructural results as observed after conventionally sample preparation3-5. Many different custom made microwave devices are currently available which can be used for the successful fixation and embedding of biological samples for TEM investigations5-8. In this study we demonstrate on Tobacco Mosaic Virus (TMV) infected Nicotiana tabacum plants that it is possible to diagnose ultrastructural alterations in leaves in about half a day by using microwave assisted sample preparation for TEM. We have chosen to perform this study with a commercially available microwave device as it performs sample preparation almost fully automatically5 in contrast to the other available devices where many steps still have to be performed manually6-8 and are therefore more time and labor consuming. As sample preparation is performed fully automatically negative staining of viral particles in the sap of the remaining TMV-infected leaves and the following examination of ultrastructure and size can be performed during fixation and embedding.

Tem analyses of the local crystal and domain structures in (na1-xkx)0.5bi0.5tio3perovskite ceramics

The local crystal and domain structures of the ((Na(1-x)K(x))(0.5)Bi(0.5)TiO(3) (NBT-KBT) solid solutions were studied because of their influence on the enhanced electromechanical properties of ceramics. Based on X-ray diffraction, the morphotropic phase boundary (MPB) was determined for the composition x = 0.20, in which the rhombohedral and the tetragonal structures were observed to coexist. However, detailed domain-structure analyses using transmission electron microscopy (TEM), performed on the NBT, KBT, and five NBT-KBT solid-solution compositions, revealed some structural changes at/near the MPB. In the samples on the tetragonal side of the MPB, the grains showed a lamellar domain structure with 90° orientations of the individual domains, separated by straight domain boundaries, i.e., (011)/(101) twin planes. The rhombohedral samples on the other side of the MPB showed a typical square-net pattern with needle-like or lamellar ~71°/109° rhombohedral domains with (001) and/or (110) twin planes separating the individual domains. The domain structure at the MPB showed well-defined lamellar domains. Based on the occurrence of the superstructure reflections in the SAED patterns of various crystallographic zones, on the characteristic splitting of the reflections, and on the domain morphology observations, the crystal structure in/near the boundary region was determined to be a tetragonal structure with an in-phase oxygen octahedral tilt system (probably a(0)a(0)c(+)). It is suggested that the tetragonal polar order is partly induced from the rhombohedral structure at the MPB as a result of mechanical loading during TEM sample preparation.

A Mechanical Method for Preparing TEM Samples from Brittle Films on Compliant Substrates

Abstract Preparing transmission electron microscopy (TEM) samples from thin films is technically challenging and traditional preparation routes can sometimes introduce unacceptable artefacts or even prove impossible. A novel method of preparing plan view TEM samples from thin films by a purely mechanical method is assessed. Two examples of films prepared by this route are briefly presented, a Cr film on PET and an amorphous AlxOy film on Cu. The application of this method allows for TEM analysis of the Cr film without the problems associated with a polymer such as PET disintegrating under the electron beam. For the AlxOy films it is demonstrated that this purely mechanical preparation prevents crystallisation of the film resulting from conventional ion milling preparation routes. The technique also allows for an upper bound of thickness approximation for these films.

Oxidation of Rolled and Flash Anodized 3000 Aluminum in Air, Nitrogen, Oxygen, and Carbon Dioxide Atmospheres

Thermal oxidation of a 3000 Al sheet rolled alloy was carried out in various atmospheres with a thermogravimetric analyzer (TGA). The effect of a flash anodized surface versus an untreated rolled surface is investigated. All samples were oxidized for 4 hours at 800°C. Samples oxidized in air, 100 % CO2 and 50% CO2–50% air, showed no difference in total oxidation. The flash anodized samples oxidized in 10% CO2–90% air had approximately half of the mass gain of the rolled samples. Oxidation of anodized samples in pure oxygen and nitrogen showed a drastic decrease in the amount of mass gained compared to the other atmospheres. Flash anodized samples gave consistent results, and the oxidation behavior tended to be linear or S-shaped for the 4 hours investigated. Rolled samples gave less consistent data and tended to be decelerating curves. Transmission electron microscope (TEM) analysis of the oxide layer before and after showed magnesium diffusion toward the surface of the sample, with a steep gradient over a distance of 400 µm. The flash anodized oxide layer was often difficult to find, having been removed during of the TEM sample preparation, but was found to double in size from 200 to 400 µm when located.

Two Distinct Amyloid β-Protein (Aβ) Assembly Pathways Leading to Oligomers and Fibrils Identified by Combined Fluorescence Correlation Spectroscopy, Morphology, and Toxicity Analyses

Nonfibrillar assemblies of amyloid β-protein (Aβ) are considered to play primary roles in Alzheimer disease (AD). Elucidating the assembly pathways of these specific aggregates is essential for understanding disease pathogenesis and developing knowledge-based therapies. However, these assemblies cannot be monitored in vivo, and there has been no reliable in vitro monitoring method at low protein concentration. We have developed a highly sensitive in vitro monitoring method using fluorescence correlation spectroscopy (FCS) combined with transmission electron microscopy (TEM) and toxicity assays. Using Aβ labeled at the N terminus or Lys(16), we uncovered two distinct assembly pathways. One leads to highly toxic 10-15-nm spherical Aβ assemblies, termed amylospheroids (ASPDs). The other leads to fibrils. The first step in ASPD formation is trimerization. ASPDs of ∼330 kDa in mass form from these trimers after 5 h of slow rotation. Up to at least 24 h, ASPDs remain the dominant structures in assembly reactions. Neurotoxicity studies reveal that the most toxic ASPDs are ∼128 kDa (∼32-mers). In contrast, fibrillogenesis begins with dimer formation and then proceeds to formation of 15-40-nm spherical intermediates, from which fibrils originate after 15 h. Unlike ASPD formation, the Lys(16)-labeled peptide disturbed fibril formation because the Aβ(16-20) region is critical for this final step. These differences in the assembly pathways clearly indicated that ASPDs are not fibril precursors. The method we have developed should facilitate identifying Aβ assembly steps at which inhibition may be beneficial.

Measurement of strain and strain relaxation in free-standing Si membranes by convergent beam electron diffraction and finite element method

Bridge-shaped free-standing Si membranes (FSSM), strained by low-pressure (LP) Si x N y , plasma-enhanced (PE) Si x N y and Si x Ge 1− x stressors, were measured by convergent beam electron diffraction (CBED) and the finite element method (FEM). The results of CBED show that, while the strain along the length of the FSSM is compressive in an LPSi x N y /Si sample, those along the length of the FSSM are tensile in PESi x N y /Si and Si x Ge 1− x /Si samples. The average absolute values of strains are different in FSSM with LPSi x N y , PESi x N y and Si x Ge 1− x as stressors. The FEM was used to compensate the results of CBED taking into account the strain relaxation in transmission electron microscopy (TEM) sample preparation. The FEM results give the strain properties in three dimensions, and are in good agreement with the results of CBED. There is approximately no strain relaxation along the length of FSSM, and the elastic strains along the other two axes in FSSM are partially relaxed by thinning down for the preparation of TEM samples.

Design and focused ion beam fabrication of single crystal diamond nanobeam cavities

We present the design and fabrication of nanobeam photonic crystal cavities in single crystal diamond for applications in cavity quantum electrodynamics. First, we describe three-dimensional finite-difference time-domain simulations of a high quality factor (Q∼106) and small mode volume [V∼0.5(λ/n)3] cavity whose resonance corresponds to the zero-phonon transition (637 nm) of the nitrogen-vacancy color center in diamond. This high Q/V structure, which would allow for strong light-matter interaction, is achieved by gradually tapering the size of the photonic crystal holes between the defect center and the mirror regions of the nanobeam. Next, we demonstrate two different focused ion beam (FIB) fabrication strategies to generate thin diamond membranes and nanobeam photonic crystal resonators from a bulk crystal. These approaches include a diamond crystal “side-milling” procedure as well as an application of the “lift-out” technique used in transmission electron microscopy sample preparation. Finally, we discuss certain aspects of the FIB fabrication routine that are a challenge to the realization of the high Q/V designs.

μ-EXAFS and μ-XRD study of selected regions of a Al–Fe–Cr–Ti complex alloy

Al3Ti, Al13Cr2 and Al13Fe4 are important intermetallics in a number of Al alloy systems including complex ultra-high-strength systems with excellent elevated-temperature performance. A full knowledge of their properties and crystallographic structures is a key factor for the understanding of these complex alloys. In the present study samples of the three pure intermetallics were prepared and regions of interest identified in a billet of Al93Fe3Cr2Ti2 alloys and 20 × 10 × 2 µm samples extracted utilizing a Focussed Ion Beam Transmission Electron Microscopy (FIB TEM) sample preparation technique. Using the microfocus spectroscopy beamline I18 at Diamond Light Source we were able to examine 5 µm sections of the samples using X-ray Diffraction (μ-XRD) and Extended X-ray Absorption Fine Structure (μ-EXAFS) in an attempt to describe the local structure of the second-phase particles and characterized the microstructure of the FIBed samples to selectively illuminate the different phases.

Minimization of focused ion beam damage in nanostructured polymer thin films

Focused ion beam (FIB) instruments have proven to be an invaluable tool for transmission electron microscopy (TEM) sample preparation. FIBs enable relatively easy and site-specific cross-sectioning of different classes of materials. However, damage mechanisms due to ion bombardment and possible beam heating effects in materials limit the usefulness of FIBs. Materials with adequate heat conductivity do not suffer from beam heating during FIB preparation, and artifacts in materials such as metals and ceramics are primarily limited to defect generation and Ga implantation. However, in materials such as polymers or biological structures, where heat conductivity is low, beam heating can also be a problem. In order to examine FIB damage in polymers we have undertaken a systematic study by exposing sections of a PS-b-PMMA block copolymer to the ion beam at varying beam currents and sample temperatures. The sections were then examined by TEM and scanning electron microscopy (SEM) and analyzed using electron energy loss spectroscopy (EELS). Our empirical results show beam heating in polymers due to FIB preparation can be limited by maintaining a low beam current (≤100 pA) during milling.

Transmission electron microscopy study of damage layer formed through ion beam induced deposition of platinum on silicon substrate

Ion beam induced deposition is used for nanofabrication operations (e.g., transmission electron microscopy sample preparation) but is still not perfectly understood. Here, the authors describe the use of a wide variety of analytical instruments to develop such an understanding. Pt-films were deposited on native oxide films on silicon (Si) by decomposing trimethyl platinum C5H4CH3Pt(CH3)3 with focused electron or gallium ion beams. Unlike electron beam induced deposition, ion beam induced deposition produces a deep and complex damage layer below the deposition. It is revealed that the damaged area can be divided into three layers: (1) a “white-band” with a lower Pt-concentration and concurrent higher carbon (C)-concentration; (2) a-Pt–Si region where amorphized silicon crystal is mixed with Pt, and Pt-particle formation starts somewhere in the middle area; and (3) a-Si, which is silicon amorphized by Ga ions. The change in the diffusion rate of platinum into the amorphized silicon layer plays a crucial role in the formation of the distinct layers. The diffusions are affected by interatomic chemical affinity as well as by concentration, so that the Pt–Si combination initially dominates Pt agglomeration and the combination of C–Si, resulting in the creation of the novel white-band.

Method of improving the quality of nanopatterning in atomic image projection electron-beam lithography

The authors demonstrated successful nanopatterning using a high resolution lattice image obtained by transmission electron microscopy (TEM) as a mask signal [H. S. Lee et al., Adv. Mater. (Weinheim, Ger.) 19, 4198 (2007)]. In this process, the quality of the patterning result is critically dependent on the lattice image, which, in turn, is critically dependent on the quality of the mask. It is often noted that the quality of the mask prepared by the conventional TEM sample preparation technique is far from perfect, which critically limits the quality of patterning. In this work, they first discuss the various origins of the noise signal generated by the mask and then introduce a special type of objective aperture (noise reduction aperture) to remove the noise signal. The effect of the noise reduction aperture, designed for the Si [110] zone axis, is experimentally demonstrated.

The Focused Ion Beam Fold-Out: Sample Preparation Method for Transmission Electron Microscopy

We have developed the focused ion beam (FIB) fold-out technique, for transmission electron microscopy (TEM) sample preparation in which there is no fine polishing or dimpling, thus saving turnaround time. It does not require a nanomanipulator yet is still site specific. The sample wafer is cut to shape, polished down, and then placed in a FIB system. A tab containing the area of interest is created by ion milling and then "folded out" from the bulk sample. This method also allows a plan-view of the sample by removing material below the wafer's surface film or device near the polished edge. In the final step, the sample is thinned to electron transparency, ready to be analyzed in the TEM. With both a cross section and plan-view, our technique gives microscopists a powerful tool in analyzing multiple zone axes in one TEM session. The nature of the polished sample edge also includes the ability to sample many areas, allowing the user to examine a very large device or sample. More importantly, this technique could make multiple site-specific e-beam transparent specimens in one polished sample, which is difficult to do when prepared by other methods.

Low-Energy Focused Ion Beam Milling Provides Reduced Damage During TEM Sample Preparation

Abstract The combined focused ion beam (FIB) and scanning electron microscope (SEM), known as the DualBeam, is well-known for its unique ability to produce site-specific thin samples starting from bulk and then attaching the section to a transmission electron microscope (TEM) grid, all in-situ. It has been reported that producing a thin sample using a 30 kV gallium FIB creates surface damage several tens of nanometers deep. However, recent DualBeam technology improvements now enable the FIB to produce thin samples with a thickness well below 50 nanometers and deliver a tightly focused ion beam at an energy of 2 kV and below, which dramatically reduces the damage depth to as low as 1 to 2 nanometers in typical materials, such as silicon.

Rapid diagnosis of plant virus diseases by transmission electron microscopy

A clear and rapid diagnosis of plant virus diseases is of great importance for agriculture and scientific experiments in plant phytopathology. Even though negative staining and transmission electron microscopy (TEM) are often used for detection and identification of viral particles and provide rapid and reliable results, it is necessary to examine ultrastructural changes induced by viruses for clear identification of the disease. With conventional sample preparation for TEM it can take several days to obtain ultrastructural results and it is therefore not suitable for rapid diagnosis of virus diseases of plants. The use of microwave irradiation can reduce the time for sample preparation for TEM investigations. Two model virus–plant systems [ Nicotiana tabacum plants infected with Tobacco mosaic virus (TMV), Cucurbita pepo plants infected with Zucchini yellow mosaic virus (ZYMV)] demonstrate that it is possible to diagnose ultrastructural alterations induced by viruses in less than half a day by using microwave irradiation for preparation of samples. Negative staining of the sap of plants infected with TMV and ZYMV and the examination of ultrastructure and size were also carried out during sample preparation thus permitting diagnosis of the viral agent by TEM in a few hours. These methods will contribute towards a rapid and clear identification of virus diseases of plants and will be useful for diagnostic purposes in agriculture and in plant phytopathology.

Localisation and identification of recombination‐active extended defects in crystalline silicon by means of focused ion‐beam preparation and transmission electron microscopy

AbstractThis paper reports on the localisation of extended defects related to the co‐precipitation of transition metal impurities in silicon and their preparation for subsequent analysis using techniques of transmission electron microscopy (TEM). Two approaches are described, i.e. (i) localisation of recombination‐active defects by light‐beam induced current followed by preferential chemical etching and TEM sample preparation using focused‐ion beam (FIB), and (ii) the replacement of the chemical etching by in situ Ga+ irradiation in the FIB system. The former technique is successfully applied to copper‐rich precipitate colonies in silicon whereas the latter proofs to be the superior approach for nickel‐rich particles. Structural and chemical analyses using TEM techniques are described for both cases (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

TEM sample preparation by FIB for carbon nanotube interconnects

A powerful method to study carbon nanotubes (CNTs) grown in patterned substrates for potential interconnects applications is transmission electron microscopy (TEM). However, high-quality TEM samples are necessary for such a study. Here, TEM specimen preparation by focused ion beam (FIB) has been used to obtain lamellae of patterned samples containing CNTs grown inside contact holes. A dual-cap Pt protection layer and an extensive 5 kV cleaning procedure are applied in order to preserve the CNTs and avoid deterioration during milling. TEM results show that the inner shell structure of the carbon nanotubes has been preserved, which proves that focused ion beam is a useful technique to prepare TEM samples of CNT interconnects.

Cross-Sectional Observation of Yttrium and Nickel Oxide Doped Ceria Powder

In this paper, microstructures of ceria powder, synthesized using spray pyrolysis method, were characterized by focused ion beam technique and transmission electron microscopy. Dependence of the shape of powder particle on the size was found. It appears a spherical shape with the diameter less than approximately 500 nm and shows cup-like shape when their diameters become larger as a result of concaving. This suggests that the spherical particle has a hollow structure as proved by focused ion beam cross-sectioning. It was also found that both the inner and the outer shells of particles are crystalline but comprised of different phases with the inner shell being NiO and the outer shell CeO2. These results benefit from a transmission electron microscopy sample preparation method to produce a cross-section of the particle. The detail of the method was described in the paper.