Reduction of Cr(VI) by the bacterium, Shewanella oneidensis (previously classified Shewanella putrefaciens strain MR-1), was studied by absorption spectrophotometry and in situ, environmental cell–transmission electron microscopy (EC-TEM) coupled with electron energy loss spectroscopy (EELS). Bacteria from rinsed cultures were placed directly in the environmental cell of the transmission electron microscope and examined under 100 Torr pressure. Bright field EC-TEM images show two distinct populations of S. oneidensis in incubated cultures containing Cr(VI)O42−: those that exhibit low image contrast and heavily precipitateencrusted cells exhibiting high image contrast. Several EELS techniques were applied to determine the oxidation state of Cr associated with encrusted cells. The encrusted cells are shown to contain a reduced form of Cr in oxidation state +3 or lower. These results demonstrate the capability to determine the chemistry and valence state of reduction products associated with unfixed, hydrated bacteria in an environmental cell transmission electron microscope.

We review our studies of the initial oxidation stages of Cu(001) thin films as investigated by in situ ultra-high vacuum transmission electron microscopy. We present our observations of surface reconstruction and the nucleation to coalescence of copper oxide during in situ oxidation in O2. We have proposed a semi-quantitative model, where oxygen surface diffusion is the dominant mechanism of the initial oxidation stages of Cu. We have also investigated the effect of water vapor on copper oxidation. We have observed that the presence of water vapor in the oxidizing atmosphere retards the rate of Cu oxidation and Cu2O is reduced when exposed directly to steam.

The environmental transmission electron microscopy (E-TEM) is a budding technique for in situ study of gas–solid chemical reactions with numerous applications. Recent improvements in the design have made it possible not only to obtain atomic level information but also the chemical information during the reaction by incorporating an imaging filter or electron energy-loss spectrometer to an E-TEM. We have been involved in modifying a couple of microscopes to incorporate environmental cells in order to convert them into E-TEMs. These microscopes have been used to obtain atomic level information of the structural and chemical changes during dynamic processes by in situ electron diffraction, high-resolution imaging, and electron energyloss spectroscopy. The applications include, but are not limited to, oxidation, reduction, polymerization, nitridation, dehydroxylation, hydroxylation, chemical vapor deposition, etc. We report recent developments in the design and application along with the limitations of an E-TEM.

In situ transmission electron microscopy is an established experimental technique that permits direct observation of the dynamics and mechanisms of dislocation motion and deformation behavior. In this article, we detail the development of a novel specimen goniometer that allows real-time observations of the mechanical response of materials to indentation loads. The technology of the scanning tunneling microscope is adopted to allow nanometer-scale positioning of a sharp, conductive diamond tip onto the edge of an electron-transparent sample. This allows application of loads to nanometer-scale material volumes coupled with simultaneous imaging of the material’s response. The emphasis in this report is qualitative and technique oriented, with particular attention given to sample geometry and other technical requirements. Examples of the deformation of aluminum and titanium carbide as well as the fracture of silicon will be presented.

The depth profile of thin film layers on bulk substrate, avoiding the cross-sectioning of samples, is commonly performed by techniques such as X-ray photoelectron spectroscopy (XPS), Auger electron spectroscopy (AES), and secondary ion mass spectroscopy (SIMS). Techniques based on X-ray emission intensity measurements by energy dispersive spectroscopy (EDS), with conventional matrix or ZAF correction, are normally applied to cross-sectioned samples. This article compares XPS with surface X-ray intensity measurements by EDS, carried out with a more realistic X-ray generation and absorption model, known as the ϕ(ρ Z) model. The ϕ(ρ Z) approach has been adopted together with Monte Carlo simulation for the proper selection of SEM accelerating voltages, in conjunction with the analysis of SEM morphological images for thin film density correction. The method discussed hereafter and compared with the XPS technique, has advantages of higher lateral resolution, non-destructive elemental analyses, morphological visualization, low cost, and faster performance. This methodology has been followed to verify the layered structure of SnO2/metal-based gas sensors. X-ray intensities were measured using an EDS ultra-thin window detector. Two different porous layers, 25-nm thick of SnO2 and 10-nm thick of Cu, were detected, showing better agreement with their nominal thickness compared to results obtained using XPS measurements where porosity affects XPS data. If confirmed to be reliable and as effective as XPS depth profiling, this technique may be adopted for process quality control purposes.

Both scanning electron microscopy (SEM) and contact mode imaging via atomic force microscopy (AFM) have been utilized to elucidate the ultrastructure of mung bean seed surfaces. The results indicate: 1) that AFM is useful in the examination of seed surface ultrastructure ex-vaccuo without the need for additional complex preparative procedures; and 2) that both the cotyledon and seed coat of different strains of mung beans bear specific ultrastructural details unique to each strain. To our knowledge, these are the first AFM images of seed surfaces.

Yeast cells represent a powerful model system in cell biology mainly due to their amenability to genetic manipulations. Increasingly, studies focus on mutant genes resulting in alterations of cellular structures and organelles. To ascertain the phenotypic changes involved, it is often desirable to use the resolving power of electron microscopy. In contrast to higher eukaryotic cells, yeast cells are particularly difficult to preserve mainly due to the presence of a thick cell wall that acts as a barrier against diffusion of fixatives. Although several procedures are targeted to overcome these difficulties, none of them have become established as a standard procedure. As a consequence, electron microscopy is still not used routinely as a tool in yeast cell biology. This prompted us to develop an easy-to-follow protocol for yeast transmission electron microscopy that should be useful in all cases where membrane integrity and organelle morphology is emphasized. One means of making the yeast cytoplasm more attainable to fixation and staining solutions is by enzymatic digestion of the cell wall. Following this approach, we were able to reliably preserve yeast cells and their cellular organelles. Enzymatic treatment with zymolyase 20T to partially remove the yeast cell wall allowed the fixation, preservation, and visualization of the yeast cytoplasm revealing detailed ultrastructure. The advancement of this technique is demonstrated with mitochondria as a model organelle. Our studies on various yeast mutants clearly show the power of the enzymatic digestion technique in visualizing subtle changes of membrane structure and organelle morphology.