Transmission electron microscopy with a liquid flow cell

The imaging of microscopic structures at nanometre-scale spatial resolution in a liquid environment is of interest for a wide range of studies. Recently, a liquid flow transmission electron microscopy (TEM) holder equipped with a microfluidic cell has been developed and shown to exhibit flow of nanoparticles through an electron transparent viewing window. Here we demonstrate the application of the flow cell system for both scanning and conventional transmission electron microscopy imaging of immobilized nanoparticles with a resolution of a few nanometres in liquid water of micrometre thickness. The spatial resolution of conventional TEM bright field imaging is shown to be limited by chromatic aberration due to multiple inelastic scattering in the water, and we demonstrate that the liquid in the cell can be displaced by a gas phase that forms under intense electron irradiation. Our data suggest that under appropriate conditions, TEM imaging with a liquid flow cell is a promising method for understanding the in situ behaviour of nanoscale structures in a prescribed and dynamically changing chemical environment.     

Comparative study of a block copolymer morphology by transmission electron microscopy and scanning force microscopy

Using transmission electron microscopy (TEM) and scanning force microscopy (SFM) together, it was possible to verify important structural features of a nanostructured bulk material such as the kp‐morphology in an ABC triblock copolymer. By applying suitable imaging techniques during the SFM measurements it was possible to determine the morphology without additional manipulation steps in between. In comparison, TEM investigations on this type of material usually require selective staining procedures prior to the measurement. Also electron beam damage is often encountered during TEM measurements especially if components such as poly(methacrylates) are present. In contrast, SFM measurements can be assumed not to significantly change the phase dimensions of the components.     

Scanning electron microscopy observation of the substructural evolution of aluminium alloys during cold rolling and partial annealing

Classical etching techniques for revealing cold deformation and partial recrystallisation in metals have been optimised for optical microscopy, which is limited by its resolution. Detailed studies of the mechanisms involved in recovery and recrystallisation during heat treatment are generally made by transmission electron microscopy. The limitation of this technique, with a few exceptions, is its small field of view and the small fraction of the sample available for inspection. The present article departs from the statement that etching, which is a surface alteration technique, must have effects that are detectable by scanning electron microscope (SEM). It was found that carefully adapted polishing and etching procedures allow for substructural investigations by SEM, resulting in various advantages compared with both optical microscopy and TEM.     

Double-layer coating for high-resolution low-temperature scanning electron microscopy

Specimen damage caused by mass loss due to electron beam irradiation is a major limitation in low-temperature scanning electron microscopy of bulk specimens. At high primary magnifications (e.g. 100 000x) a hydrated sample is usually severely damaged after one slow scan (about 3000 e^— nm^—2). The consequences of this beam damage are significantly reduced by coating the frozen-hydrated sample with a 5–10-nm-thick carbon layer. Since this layer covers up surface details, the sample is first unidirectionally shadowed with a thin heavy metal layer (e.g. 2 nm of platinum) that is in close contact with the biological surface (double layer coating). This heavy metal layer can be visualized in field-emission scanning electron microscopy with the material-dependent backscattered electron signal. The method allows for routine observation of large frozen-hydrated samples. By use of an in-lens field-emission SEM and a sensitive backscattered electron detector, structural information comparable to that obtained with the transmission electron microscopy freeze-fracture replica technique can be achieved.     

Free-standing graphene by scanning transmission electron microscopy

Free-standing graphene sheets have been imaged by scanning transmission electron microscopy (STEM). We show that the discrete numbers of graphene layers enable an accurate calibration of STEM intensity to be performed over an extended thickness and with single atomic layer sensitivity. We have applied this calibration to carbon nanoparticles with complex structures. This leads to the direct and accurate measurement of the electron mean free path. Here, we demonstrate potentials using graphene sheets as a novel mass standard in STEM-based mass spectrometry.     

Differential contrast imaging of secondary electron signals in cryo-field-emission scanning electron microscopy

Low-temperature scanning electron microscopy (LTSEM) is limited in resolution and image quality by charging of frozen hydrated samples and collection deficiencies of secondary electron signal contrasts. We measured and corrected both effects using differential hysteresis processing (DHP) of LTSEM images, scanned at 15-bit from 5×4 inch Polaroid negatives. Bulk charging produced a major contrast component equal to 44–87% of the intensity range of the image. The strong charging contrast reduced the local high-resolution signal contrasts to an unrecognizable level. Segmentation and imaging of the unaffected surface contrasts produced high-quality images of high contrast from metal-coated samples as well as from uncoated samples. The differential contrast imaging can be used for control of the sequential etching of ice from the non metal-coated sample as well as improved LTSEM imaging of the finally coated sample.     

Surface damage formation during ion-beam thinning of samples for transmission electron microscopy

All techniques employed in the preparation of samples for transmission electron microscopy (TEM) introduce or include artifacts that can degrade the images of the materials being studied. One significant cause of this image degradation is surface amorphization. The damaged top and bottom surface layers of TEM samples can obscure subtle detail, particularly at high magnification. Of the techniques typically used for TEM sample preparation of semiconducting materials, cleaving produces samples with the least surface amorphization, followed by low-angle ion milling, conventional ion milling, and focused ion beam (FIB) preparation. In this work, we present direct measurements of surface damage on silicon produced during TEM sample preparation utilizing these techniques. The thinnest damaged layer formed on a silicon surface was measured as 1.5 nm thick, while an optimized FIB sample preparation process results in the formation of a 22 nm thick damaged layer. Lattice images are obtainable from all samples.     

Comparison of phase contrast transmission electron microscopy with optimized scanning transmission annular dark field imaging for protein imaging

Henderson has already shown that electron microscopy should be superior to X-ray and neutron diffraction for determining protein structure with minimum radiation damage. Since the contrast for a molecule embedded in vitreous ice is very low, it is conceivable that dark field imaging would be superior to bright field phase contrast microscopy. A detailed analysis of contrast and signal/noise for both imaging modes is presented. Annular dark field scanning transmission microscopy gives improved contrast and equivalent signal/noise to phase contrast TEM when the molecule is the same thickness as a vitreous ice embedding medium. For a constant embedding medium thickness of 200 A the contrast is equivalent to phase contrast TEM but the signal/noise is 5 times worse. Even with an efficient detector that only excludes scattering less than 5 mrad there is insufficient signal at a dose of 5 electrons/A(2) to produce an image with more than 1 electron/per pixel. For larger molecules (>100 A thick which corresponds to 420 kDa for spherical molecules) the weak phase object approximation used to analyse a phase contrast image no longer applies at 100 kV. This limit could be extended to about 200 A (about 3 MDa) if a 400 kV microscope were used.     

Scanning electron microscopy imaging of dislocations in bulk materials, using electron channeling contrast

The imaging and characterization of dislocations is commonly carried out by thin foil transmission electron microscopy (TEM) using diffraction contrast imaging. However, the thin foil approach is limited by difficult sample preparation, thin foil artifacts, relatively small viewable areas, and constraints on carrying out in situ studies. Electron channeling imaging of electron channeling contrast imaging (ECCI) offers an alternative approach for imaging crystalline defects, including dislocations. Because ECCI is carried out with field emission gun scanning electron microscope (FEG-SEM) using bulk specimens, many of the limitations of TEM thin foil analysis are overcome. This paper outlines the development of electron channeling patterns and channeling imaging to the current state of the art. The experimental parameters and set up necessary to carry out routine channeling imaging are reviewed. A number of examples that illustrate some of the advantages of ECCI over thin foil TEM are presented along with a discussion of some of the limitations on carrying out channeling contrast analysis of defect structures.     

Application of Nomarski interference contrast microscopy as a thickness monitor in the preparation of transparent,SiC-based,cross-sectional TEM samples

Reflected light optical microscopy using a Nomarski prism and a differential interference contrast filter have been employed in concert to achieve a technique that provides an accurate color reference for thickness during the dimpling and ion milling of transparent transmission electron microscopy samples of 6H-SiC(000 1) wafers. The samples had thin films of AIN, GaN, and Au deposited on the SiC substrate. A sequence of variously colored primary and secondary interference bands was observed when the SiC was thinner than 20 microm using an optical microscope. The color bands were correlated with the TEM sample thickness as measured via scanning electron microscopy. The interference contrast was used to provide an indication of the dimpling rate, the ion milling rate, and also the most probable location of perforation, which are useful to reduce sample breakage. The application of pressure during the initial cross-sectional preparation reduced the separation of the two halves of the sample sandwich and resulted in increased shielding of the film surface from ion milling damage.     

The observation of intact hepatic endothelial cells by cryo-electron microscopy

Liver sinusoidal endothelial cells (LSECs) can optimally be imaged by whole mount transmission electron microscopy (TEM). However, TEM allows only investigation of vacuum‐resistant specimens and this usually implies the study of chemically fixed and dried specimens. Cryo‐electron microscopy (cryo‐EM) can be used as a good alternative for imaging samples as whole mounts. Cryo‐EM offers the opportunity to study intact, living cells while avoiding fixation, dehydration and drying, at the same time preserving all solubles and water as vitrified ice. Therefore, we compared the different results obtained when LSECs were vitrified using different vitrification conditions. We collected evidence that manual blotting at ambient conditions and vitrification by the guided drop method results in the production of artefacts in LSECs, such as the loss of fenestrae, formation of gaps and lack of structural details in the cytoplasm. We attribute these artefacts to temperature and osmotic effects during sample preparation just prior to vitrification. By contrast, by using an environmentally controlled glove box and a vitrification robot (37 °C and 100% relative humidity), these specific structural artefacts were nearly absent, illustrating the importance of controlled sample preparation. Moreover, data on glutaraldehyde‐fixed cells and obtained by using different vitrification methods suggested that chemical prefixation is not essential when vitrification is performed under controlled conditions. Conditioned vitrification therefore equals chemical fixation in preserving and imaging cellular fine structure. Unfixed, vitrified LSECs show fenestrae and fenestrae‐associated cytoskeleton rings, indicating that these structures are not artefacts resulting from chemical fixation.     

Investigation of quantitative secondary electron imaging of semiconducting polymer materials using environmental scanning electron microscopy

The development of environmental scanning electron microscopy has opened the way for the examination of a wide variety of new sample types that were previously inaccessible to conventional scanning electron microscope techniques. With the advent of such a new methodology comes also the potential for new contrast mechanisms. We investigated the use of environmental scanning electron microscopy on semiconducting organic polymer materials. We observed contrast from these materials in secondary electron images, this contrast being attributed to differences in electron yield due to the polymer's electronic structure. Further study of these materials, and specifically the influence of film thickness on signal, has indicated a significant effect as the secondary electrons move through the sample. Systematic studies such as these are needed for a full understanding of the relationship between electronic properties and signal and, hence, the ability to probe structure–property relationships in greater depth.     

Comparison of reflection contrast microscopy and electron microscopy on the histopathological diagnosis of various kidney diseases

Our aim in this study was to compare reflection contrast microscopy (RCM) with transmission electron microscopy (TEM) to understand whether RCM could be used in the histopathological diagnosis of various kidney diseases as a less expensive and an easier alternative to TEM. The diagnoses of kidney pathologic lesions included Alport syndrome, thin membrane disease, Ig A nephropathy. RCM is a form of light microscope that works in the reflected mode, suitable to observe ultrathin (50-100 nm) plastic sections that is also used in TEM. Our findings showed that RCM showed similar results compared with TEM on these lesions described earlier.     

In situ measurements and transmission electron microscopy of carbon nanotube field-effect transistors

We present the design and operation of a transmission electron microscopy (TEM)-compatible carbon nanotube (CNT) field-effect transistor (FET). The device is configured with microfabricated slits, which allows direct observation of CNTs in a FET using TEM and measurement of electrical transport while inside the TEM. As demonstrations of the device architecture, two examples are presented. The first example is an in situ electrical transport measurement of a bundle of carbon nanotubes. The second example is a study of electron beam radiation effect on CNT bundles using a 200 keV electron beam. In situ electrical transport measurement during the beam irradiation shows a signature of wall- or tube-breakdown. Stepwise current drops were observed when a high intensity electron beam was used to cut individual CNT bundles in a device with multiple bundles.     

Automated electron tomography with scanning transmission electron microscopy

We report the successful implementation of a fully automated tomographic data collection system in scanning transmission electron microscopy (STEM) mode. Autotracking is carried out by combining mechanical and electronic corrections for specimen movement. Autofocusing is based on contrast difference of a focus series of a small sample area. The focus gradient that exists in normal images due to specimen tilt is effectively removed by using dynamic focusing. An advantage of STEM tomography with dynamic focusing over TEM tomography is its ability to reconstruct large objects with a potentially higher resolution.     

Nanometric crystal defects in transmission electron microscopy

Transmission electron microscopy (TEM) is revisited in order to define methods for the identification of nanometric defects. Nanometric crystal defects play an important role as they influence, generally in a detrimental way, physical properties. For instance, radiation-induced damage in metals strongly degrades mechanical properties, rendering the material stronger but brittle. The difficulty in using TEM to identify the nature and size of such defects resides in their small size. TEM image simulations are deployed to explore limits and possible ways to improve on spatial resolution and contrast. The contrast of dislocation loops, cavities, and a stacking fault tetrahedra (SFT) are simulated in weak beam, interfering reflections (HRTEM), and scanned condensed electron probe (STEM) mode. Results indicate that STEM is a possible way to image small defects. In addition, a new objective aperture is proposed to improve resolution in diffraction contrast. It is investigated by simulations of the weak beam imaging of SFT and successfully applied in experimental observations.     

Bias in bacteriophage morphological classification by transmission electron microscopy due to breakage or loss of tail structures

Virtually every study that has used transmission electron microscopy (TEM) to estimate viral diversity has acknowledged that loss of phage tails during sample preparation may have biased the results. However, the magnitude of this potential bias has yet to be constrained. To characterize biases in virus morphological diversity due to tail loss, six phage strains representing the order Caudovirales were inoculated into sterile sediments and soils. Phage particles were then extracted using standard methods. Morphologies of extracted phage particles were compared to those of unmanipulated control samples to determine the extent of tail breakage incurred by extraction procedures. Podoviruses exhibited the smallest frequency of tail loss during extraction (1.2-14%), myoviruses were moderately susceptible to tail breakage (15-40%), and siphoviruses were highly susceptible (32-76%). Thus, TEM assessments of viral diversity in soils or sediments by distribution of tail morphologies may be biased toward podoviruses and virions lacking tails, while simultaneously underestimating the abundance of siphoviruses. However, since the majority of viral capsids observed under TEM were intact, estimates of viral diversity based on the distribution of capsid diameters may provide a more reliable basis for morphological comparisons within and across ecosystems.     

Rationale for the application of thin,continuous metal films in high magnification electron microscopy

Various metal films of different thicknesses were deposited on to a particle test specimen and their effects on topographic contrast generation and specimen preservation were determined. Tobacco mosaic virus adsorbed on to thin carbon supports or silicon chips was imaged in TEM or high resolution SE-I SEM at a magnification of 350,000×. Tantalum films of 1–2 nm (average mass) thickness produced best contrasts and prevented volume loss of the particles from electron beam damage. Excessively thick films of 5–10 nm thickness blanketed fine structures and caused severe volume losses. Discontinuous 2 nm thick films of gold or platinum decorated the surfaces, caused a loss in topographic contrasts and induced very high volume losses. Thin continuous metal films were necessary to generate high topographic contrast and to prevent volume loss from beam damage by providing sufficient mechanical stability for small topographic features and increased thermal conductivity of the specimen surface.     

Effect of sample thickness, energy filtering, and probe coherence on fluctuation electron microscopy experiments

We have explored experimentally the effects of the TEM sample thickness, zero-loss energy filtering, and probe coherence on fluctuation electron microscopy (FEM) experiments implemented using nanodiffraction. FEM measures the variance V of spatial fluctuations in nanodiffraction. We find that V is inversely proportional to the sample thickness, as predicted by earlier models. Energy filtering increases V at all thicknesses we measured. V increases as the coherence of the probe increases. All of these factors must be carefully controlled to obtain quantitatively reliable FEM data.