Combined ultramicrotomy for AFM and TEM using a novel sample holder

A new sample holder that allows combined microtomy for atomic force microscopy (AFM) and transmission electron microscopy (TEM) is described. The main feature of this sample holder is a small central part holding the sample. This central part fits into the head of an atomic force microscope. AFM measurements can be performed with a sample mounted in this central part of the sample holder. This makes the alignment of a microtomed bulk sample unnecessary, and offers the opportunity of an easy and fast combined sample preparation for AFM and TEM.     

Supramolecular structure of humic acids by TEM with improved sample preparation and staining

Understanding the role of the dissolved organic matter in the environment will benefit from the characterization of its components at the supramolecular scale. With its very high spatial resolution the transmission electron microscope (TEM) allows the determination of colloidal size, structure, and shape on aggregated substances as well as single particles. It also allows the determination of the chemical composition if coupled with an X-ray detector. However, TEM preparation artifacts may hamper the relevance of such observations. This study demonstrates the capacity of TEM for the size and shape analysis of humic substances. Three sample preparation techniques (ultramicrotomy sectioning after embedding into a hydrophilic resin, drop deposition, and absorption) were evaluated with the Fluka humic acid (HA). Additionally, several staining agents (cesium hydroxide, lead citrate, uranyl acetate, and ruthenium tetraoxide) were used to increase the humic acid contrast for TEM observations. An improved simple preparation method with selective staining of the HA is proposed which permits the imaging of the humic acid macromolecules. The results show that the Fluka humic acid is composed mainly of fibrils generated by the interconnection of basic molecules of about 20 nm diameter with some isolated larger macromolecules (30-200 nm).     

A novel sample holder allowing atomic force microscopy on transmission electron microscopy specimen grids: repetitive, direct correlation between AFM and TEM images

A novel sample holder that allows atomic force microscopy (AFM) to be performed on transmission electron microscope (TEM) grids is described. Consequently, AFM and TEM images were repeatedly obtained on exactly the same sample area. For both techniques, a thin carbon film was used as the imaging substrate. Although these techniques have been previously used in conjunction, AFM and TEM images on exactly the same area have not been repeatedly obtained for any system. Correlation of AFM and TEM images is useful for work where the three‐dimensional topographical information provided by the AFM could be used to better interpret the two‐dimensional images provided by the TEM and vice versa. To demonstrate the applicability of such correlation, new results pertaining to a fibrillar collagen system are summarized.     

Assessment of collagen fibril spacing in relation to selected region of interest (ROI) on electron micrographs--application to the mammalian corneal stroma

AIMS: To evaluate measurements of collagen fibril spacing using different shaped regions of interest (ROI) on transmission electron micrograph (TEM) images of rabbit corneal stroma. METHODS: Following glutaraldehyde fixation and phosphotungstic acid staining, TEM images of collagen fibrils in cross section were projected at a final magnification close to 250,000 × to obtain overlays. Interfibril distances (IFDs; center‐to‐center spacing) were measured within different ROIs of the same nominal area (0.25 μm²) but different shape (with the length to width, L:W, ratio from 1:1 to 6:1). The IFD distribution was analyzed, and the 2D organization assessed using a radial distribution analysis. RESULTS: The fibrils had an average diameter of 35.3 ± 3.8 (SD) nm, packing density of 393 ± 4 fibrils / μm² and a fibril volume fraction of 0.39 ± 0.02. IFDs ranged from 29 to 1400 nm depending on the shape of the ROI, with average values ranging from 263 to 443 nm. By artificially selecting IFD data only to a radial distance of 250 nm, the average IFDs were just 145–157 nm. The radial distributions, to 250 nm, all showed a nearest neighbors first peak which shifted slightly from predominantly at 45–54 nm with more rectangular ROIs. The radial distribution profiles could be shown to be statistically different if the ROI L:W ratio was 2:1 or greater. CONCLUSION: Selection of an ROI for assessment of packing density and interfibril distances should be standardized for comparative assessments of TEMs of collagen fibrils. Microsc. Res. Tech., 2011. © 2011 Wiley‐Liss, Inc.     

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.     

Facility for rapid preparation of silicon and silicide TEM specimens

A simple chemical jet polishing arrangement, for the thinning of semiconductors or metals for transmission electron microscopy (TEM), is described for the specific case of silicon and silicides. The effect of variation in three mechanical parameters on the profile and quality of the specimen is described, and the optimum conditions are determined. A proposed polishing solution is one part 48% HF mixed with one part fuming HNO₃. Reproducibility is only achieved in the presence of a relatively large concentration of nitrous acid in the polishing solution. The solution must also be relatively concentrated, as the reaction rate falls off rapidly with decreasing concentration.     

Complementary TEM characterization of a squeeze-cast metal-matrix composite by dissolution of the sample in butanol

Transmission electron microscopy observations of ion-beam thinned samples and samples extracted using a butanol dissolution technique gave information regarding the interface microstructure which could not be obtained from the ion-beam thinned samples, and that therefore both these sample preparation routes should be considered when observing aluminium metal-matrix composites in the transmission electron microscope.     

Novel sample preparation method of polymer emulsion for SEM observation

The aim of this study was to design a simple and reliable method for obtaining the detailed information about the average size, size distribution, and the surface morphology of particles with variation of the sample preparation of a polymer emulsion. In this work, the characteristic features of the particles of rosin size with high viscosity were first described by scanning electron microscopy (SEM). The morphologies of polymer emulsion of solid lipid nanoparticles and of the microspheres were observed. The advantage of the method is that not only the true size and shape of emulsion particles can be shown, but the problem of high-viscosity emulsion that prevents there study with SEM is solved. Using this new method, the micromorphology and size distribution of the emulsion particles with different viscosities have been clearly observed.     

Microwave-assisted rapid plant sample preparation for transmission electron microscopy

The preparation of plant leaf material for transmission electron microscopical investigations can be a very time- and labour-consuming task as the reagents infiltrate the samples quite slowly and as usually most steps have to be performed manually. Fixation, buffer washes, dehydration, resin infiltration and polymerization of the resin-infiltrated leaf samples can take several days before the specimen can be cut ultrathin and used for ultrastructural investigations. In this study, we present a microwave-assisted automated sample preparation procedure that reduces preparation time from at least 3 days to about 5 h – with only a few steps that have to be performed manually – until the plant sample can be ultrathin sectioned and observed with the transmission electron microscope. For studying the efficiency of this method we have compared the ultrastructure of different leaf material ( Arabidopsis thaliana , Nicotiana tabacum and Picea abies ) which was prepared with a conventional, well-established chemical fixation and embedding protocol and a commercially available automated microwave tissue processor. Despite the massive reduction in sample preparation time no negative effects on cutting properties of the blocks, stability of the sections in the electron beam, contrast and ultrastructure of the cells were observed under the transmission electron microscope when samples were prepared with the microwave-assisted protocol. Additionally, no negative effects were detected on the dimensions of fine structures of grana stacks (including membranes, inter- and intrathylakoidal spaces), the nuclear envelope and the plasma membrane as the diameter of these structural components did not differ between leaf samples (of the same species) that were processed with the automated microwave tissue processor or by conventional fixation and embedding at room temperature.     

A procedure to prepare cross-sectional samples for TEM

Cross-sectional transmission electron microscopy (XTEM) is an imaging technique particularly suited to the study of layered structures. For integrated electronic devices it has become a common practice to use XTEM to assess the shape and crystallinity of component layers as well as defect structures introduced by processing. A procedure for preparing samples to be viewed by XTEM is described.     

A new method for cell culture on an electron-transparent melamine foil suitable for successive LM,TEM and SEM studies of whole cells

A new cell culture technique is described which is based on the observation that foils cast from the melamine resin hexamethylol-melamine-ether are suitable for the cultivation of beating heart muscle cells and fibroblasts of the rat. This foil can be flamed for sterilization, is about 80 nm in thickness, homogeneous and smooth, withstands dehydration and critical point-drying, can be removed from glass and permits the imaging of whole cells successively by light microscopy, transmission and scanning electron microscopy. The method is capable of narrowing the gap between light and electron microscopy, yielding excellent whole cell preparations in various kinds of microscopic studies to be performed on one and the same cell.     

Irregular Shaped,Assumably Semi‐Crystalline Calciumphosphate Platelet Deposition at the Mineralization Front of Rabbit Femur Osteotomy: A HR‐TEM Study

Although bone minerals have been widely studied by various techniques in previous studies, crystal structures, morphology of bone minerals and its building pathway remained still controversy. In this work, the ultrastructure of the mineralization front of rabbit femur has been studied by conventional and high‐resolution (HR) transmission electron microscopy (TEM). In order to induce a healing and demineralization process the animals were subjected to a standardized osteotomy stabilized with titan screws and sonic pins. After 84 days follow‐up time the newly build bone was investigated. The mineralization front of rabbit femur osteotomy contains partly mineralized collagen fibrils with a pronounced striped pattern together with a large number of agglomerated apatite platelets. The striation is caused by mineralization in the hole zones of the collagen fibrils, corresponding to the early stage of mineralization. In the TEM micrographs, the mineralization zone appears denser and compact when compared with fully mineralized bone, although most of the collagen fibrils are completely mineralized in the latter (higher concentration of interfibrillar apatite platelets within the mineralization zone). In bone some partly mineralized collagen fibrils are also observed, revealing the same arrangement, regular shape, and size of apatite platelets as collagen fibrils in the mineralization zone. Apatite platelets with irregular shapes are observed at the vortex‐shaped outer boundary of the mineralization zone, i.e. at the interfaces with nonmineralized collagen or osteoblasts. HR TEM micrographs reveal that the platelets are assumably semicrystalline and that within the platelet nanocrystalline domains of apatite are embedded in an amorphous calciumphosphate matrix. SCANNING 35: 169‐182, 2013. © 2012 Wiley Periodicals, Inc.     

Developing 3D SEM in a broad biological context

When electron microscopy (EM) was introduced in the 1930s it gave scientists their first look into the nanoworld of cells. Over the last 80 years EM has vastly increased our understanding of the complex cellular structures that underlie the diverse functions that cells need to maintain life. One drawback that has been difficult to overcome was the inherent lack of volume information, mainly due to the limit on the thickness of sections that could be viewed in a transmission electron microscope (TEM). For many years scientists struggled to achieve three‐dimensional (3D) EM using serial section reconstructions, TEM tomography, and scanning EM (SEM) techniques such as freeze‐fracture. Although each technique yielded some special information, they required a significant amount of time and specialist expertise to obtain even a very small 3D EM dataset. Almost 20 years ago scientists began to exploit SEMs to image blocks of embedded tissues and perform serial sectioning of these tissues inside the SEM chamber. Using first focused ion beams (FIB) and subsequently robotic ultramicrotomes (serial block‐face, SBF‐SEM) microscopists were able to collect large volumes of 3D EM information at resolutions that could address many important biological questions, and do so in an efficient manner. We present here some examples of 3D EM taken from the many diverse specimens that have been imaged in our core facility. We propose that the next major step forward will be to efficiently correlate functional information obtained using light microscopy (LM) with 3D EM datasets to more completely investigate the important links between cell structures and their functions.     

TEM study of chirality in MoS2 nanotubes

The dark-field diffraction contrast of helical nanotubes (NTs) is shown to be asymmetric when an NT is tilted at appropriate angle with respect to the incident electron beam. This phenomenon was used for the chirality determination of multi-shell NTs observed in MoS₂ layered compound. Both kinds of NT — helical and non-helical — were found. In the case of helical NTs only right-hand chirality was observed.     

EBSD and TEM investigation of the hot deformation substructure characteristics of a type 316L austenitic stainless steel

The evolution of crystallographic texture and deformation substructure was studied in a type 316L austenitic stainless steel, deformed in rolling at 900 °C to true strain levels of about 0.3 and 0.7. Electron backscatter diffraction (EBSD) and transmission electron microscopy (TEM) were used in the investigation and a comparison of the substructural characteristics obtained by these techniques was made. At the lower strain level, the deformation substructure observed by EBSD appeared to be rather poorly developed. There was considerable evidence of a rotation of the pre‐existing twin boundaries from their original orientation relationship, as well as the formation of highly distorted grain boundary regions. In TEM, at this strain level, the substructure was more clearly revealed, although it appeared rather inhomogeneously developed from grain to grain. The subgrains were frequently elongated and their boundaries often approximated to traces of {111} slip planes. The corresponding misorientations were small and largely displayed a non‐cumulative character. At the larger strain, the substructure within most grains became well developed and the corresponding misorientations increased. This resulted in better detection of sub‐boundaries by EBSD, although the percentage of indexing slightly decreased. TEM revealed splitting of some sub‐boundaries to form fine microbands, as well as the localized formation of microshear bands. The substructural characteristics observed by EBSD, in particular at the larger strain, generally appeared to compare well with those obtained using TEM. With increased strain level, the mean subgrain size became finer, the corresponding mean misorientation angle increased and both these characteristics became less dependent on a particular grain orientation. The statistically representative data obtained will assist in the development of physically based models of microstructural evolution during thermomechanical processing of austenitic stainless steels.     

Combining Ar ion milling with FIB lift-out techniques to prepare high quality site-specific TEM samples

Focused ion beam (FIB) techniques can prepare site‐specific transmission electron microscopy (TEM) cross‐section samples very quickly but they suffer from beam damage by the high energy Ga⁺ ion beam. An amorphous layer about 20–30 nm thick on each side of the TEM lamella and the supporting carbon film makes FIB‐prepared samples inferior to the traditional Ar⁺ thinned samples for some investigations such as high resolution transmission electron microscopy (HRTEM) and electron energy loss spectroscopy (EELS). We have developed techniques to combine broad argon ion milling with focused ion beam lift‐out methods to prepare high‐quality site‐specific TEM cross‐section samples. Site‐specific TEM cross‐sections were prepared by FIB and lifted out using a Narishige micromanipulator onto a half copper‐grid coated with carbon film. Pt deposition by FIB was used to bond the lamellae to the Cu grid, then the coating carbon film was removed and the sample on the bare Cu grid was polished by the usual broad beam Ar⁺ milling. By doing so, the thickness of the surface amorphous layers is reduced substantially and the sample quality for TEM observation is as good as the traditional Ar⁺ milled samples.