Extended abstract of a paper presented at Microscopy and Microanalysis 2005 in Honolulu, Hawaii, USA, July 31--August 4, 2005

Lowering the electron energy in the transmission electron microscope allows for a significant improvement in contrast of light elements and reduces knock-on damage for most materials. If low-voltage electron microscopes are defined as those with accelerating voltages below 100 kV, the introduction of aberration correctors and monochromators to the electron microscope column enables Ångstrom-level resolution, which was previously reserved for higher voltage instruments. Decreasing electron energy has three important advantages: (1) knock-on damage is lower, which is critically important for sensitive materials such as graphene and carbon nanotubes; (2) cross sections for electron-energy-loss spectroscopy increase, improving signal-to-noise for chemical analysis; (3) elastic scattering cross sections increase, improving contrast in high-resolution, zero-loss images. The results presented indicate that decreasing the acceleration voltage from 200 kV to 80 kV in a monochromated, aberration-corrected microscope enhances the contrast while retaining sub-Ångstrom resolution. These improvements in low-voltage performance are expected to produce many new results and enable a wealth of new experiments in materials science.

Micropropagation techniques allow producing large numbers of clones of genetically identical plants. However, there is evidence of disorders in internal structures due to sophisticated in vitro conditions. Such variations are responsible for the mortality of plantlets in the field and cause huge loss to the tissue culture industry. Anatomical evaluation at different growth conditions allows for understanding structural repair of in vitro raised plantlets. Therefore, the present study was aimed to identify the structural changes that occurred in micropropagated plants of Vitex negundo under heterotrophic, photomixotrophic, and photoautotrophic conditions. To achieve this, structural variations were analyzed in the plantlets obtained from in vitro, greenhouse and field transferred stages using light microscopy. Underdeveloped dermal tissues, palisade cells, intercellular spaces, mechanical tissues, vascular bundles, and ground tissues were observed with the plants growing under in vitro conditions. The self-repairing of structural disorders and transitions in vegetative anatomy was observed during hardening under the greenhouse environment. Field transferred plantlets were characterized by well-developed internal anatomy. These findings showed that the micropropagated plantlets of V. negundo were well-adapted through a series of self-repairing the in vitro induced structural abnormalities at the subsequent stages of plant development.

Piezoresponse force microscopy (PFM) provides a novel opportunity to detect picometer-level displacements induced by an electric field applied through a conducting tip of an atomic force microscope (AFM). Recently, it was discovered that superb vertical sensitivity provided by PFM is high enough to monitor electric-field-induced ionic displacements in solids, the technique being referred to as electrochemical strain microscopy (ESM). ESM has been implemented only in multi-frequency detection modes such as dual AC resonance tracking (DART) and band excitation, where the response is recorded within a finite frequency range, typically around the first contact resonance. In this paper, we analyze and compare signal-to-noise ratios of the conventional single-frequency method with multi-frequency regimes of measuring surface displacements. Single-frequency detection ESM is demonstrated using a commercial AFM.

Three-dimensional (3D) datasets were recorded of gold nanoparticles placed on both sides of silicon nitride membranes using focal series aberration-corrected scanning transmission electron microscopy (STEM). Deconvolution of the 3D datasets was applied to obtain the highest possible axial resolution. The deconvolution involved two different point spread functions, each calculated iteratively via blind deconvolution. Supporting membranes of different thicknesses were tested to study the effect of beam broadening on the deconvolution. It was found that several iterations of deconvolution was efficient in reducing the imaging noise. With an increasing number of iterations, the axial resolution was increased, and most of the structural information was preserved. Additional iterations improved the axial resolution by maximal a factor of 4 to 6, depending on the particular dataset, and up to 8 nm maximal, but also led to a reduction of the lateral size of the nanoparticles in the image. Thus, the deconvolution procedure optimized for the highest axial resolution is best suited for applications where one is interested in the 3D locations of nanoparticles only.

The spatial resolution of aberration-corrected annular dark field scanning transmission electron microscopy was studied as function of the vertical position z within a sample. The samples consisted of gold nanoparticles (AuNPs) positioned in different horizontal layers within aluminum matrices of 0.6 and 1.0 µm thickness. The highest resolution was achieved in the top layer, whereas the resolution was reduced by beam broadening for AuNPs deeper in the sample. To examine the influence of the beam broadening, the intensity profiles of line scans over nanoparticles at a certain vertical location were analyzed. The experimental data were compared with Monte Carlo simulations that accurately matched the data. The spatial resolution was also calculated using three different theoretical models of the beam blurring as function of the vertical position within the sample. One model considered beam blurring to occur as a single scattering event but was found to be inaccurate for larger depths of the AuNPs in the sample. Two models were adapted and evaluated that include estimates for multiple scattering, and these described the data with sufficient accuracy to be able to predict the resolution. The beam broadening depended on z1.5 in all three models.

A method involving a graphite substrate has been developed for the mounting and analysis of sparse, fine particles from a liquid suspension to enable improved study of volcanic ash (tephra) and atmospheric dust preserved in glacial ice. Unpolished grains may be studied by scanning electron microscope–energy dispersive spectrometry (SEM-EDS) at full vacuum without the need for a conductive coating due to the close proximity of the underlying graphite. The same grains in the same relative positions may be subsequently examined in polished mounts by a variety of methods including SEM-EDS, electron probe microanalysis, laser ablation–inductively coupled plasma–mass spectroscopy, secondary ion mass spectrometry, and optical microscopy. Particles as small as 3–5 μm may be routinely and easily prepared for analysis as polished grains, and particles of significantly different sizes may be exposed simultaneously. The general approach also offers significant flexibility, including both single- and multiple-sample mounts, and may be adjusted to suit a variety of samples and analytical methods.

A study of screw dislocations in hydride-vapor-phase-epitaxy (HVPE) template and molecular-beam-epitaxy (MBE) overlayers was performed using transmission electron microscopy (TEM) in plan view and in cross section. It was observed that screw dislocations in the HVPE layers were decorated by small voids arranged along the screw axis. However, no voids were observed along screw dislocations in MBE overlayers. This was true both for MBE samples grown under Ga-lean and Ga-rich conditions. Dislocation core structures have been studied in these samples in the plan-view configuration. These experiments were supported by image simulation using the most recent models. A direct reconstruction of the phase and amplitude of the scattered electron wave from a focal series of high-resolution images was applied. It was shown that the core structures of screw dislocations in the studied materials were filled. The filed dislocation cores in an MBE samples were stoichiometric. However, in HVPE materials, single atomic columns show substantial differences in intensities and might indicate the possibility of higher Ga concentration in the core than in the matrix. A much lower intensity of the atomic column at the tip of the void was observed. This might suggest presence of lighter elements, such as oxygen, responsible for their formation.

Giardia lamblia is a flagellated protozoan of great medical and biological importance. It is the causative agent of giardiasis, one of the most prevalent diarrheal disease both in developed and third-world countries. Morphological studies have shown that G. lamblia does not present structures such as peroxisomes, mitochondria, and a well-elaborated Golgi complex. In this review, special emphasis is given to the contribution made by various microscopic techniques to a better knowledge of the biology of the protozoan. The application of video microscopy, immunofluorescence confocal laser scanning microscopy, and several techniques associated with transmission electron microscopy (thin section, enzyme cytochemistry, freeze-fracture, deep-etching, fracture-flip) to the study of the cell surface, peripheral vesicles, endoplasmic reticulum–Golgi complex system, and of the encystation vesicles found in trophozoites and during the process of trophozoite-cyst transformation are discussed.

A growing problem in cardiac drug toxicity has been blamed on the lack of adequate testing prior to authorization for prescription use. This study offers an effective alternative to the current method of in vivo pharmaceutical testing, which is time and cost prohibitive. We have accomplished this by developing the novel three-dimensional heart tube model. At the “heart” of our model lies our patented collagen scaffold that enables the cardiac myocytes to display an in vivo–like architecture. The cardiac myocytes were cocultured with the collagen tube for a period of 5 weeks, resulting in the heart tubes. Our heart tubes were treated with specific drugs (nifedipine, isoproterenol, and lidocaine) at varying concentrations. The percent of apoptotic cells was calculated based on observing the number of cells that labeled positive for caspase-3 via confocal microscopy. All three drugs exhibited negative effects at high concentrations in that the number of living cells decreased. Lidocaine showed an increase in apoptosis at concentrations of 75 μM and above. This may indicate that certain drugs have a minimum concentration level that must be reached before the cells experience apoptosis from the toxic levels.

The effects of betaine on hepatocytes chromatin architecture changes were examined by using fractal and gray-level co-occurrence matrix (GLCM) analysis in methionine/choline-deficient (MCD) diet-induced, nonalcoholic fatty liver disease (NAFLD). Male C57BL/6 mice were divided into groups: (1) Control: standard diet; (2) BET: standard diet and betaine supplementation through drinking water (solution 1.5%); (3) MCD group: MCD diet for 6 weeks; (4) MCD+BET: fed with MCD diet + betaine for 6 weeks. Liver tissue was collected for histopathology, immunohistochemistry, and determination of fractal dimension and GLCM parameters. MCD diet induced diffuse micro- and macrovesicular steatosis accompanied with increased Ki67-positive hepatocyte nuclei. Steatosis and Ki67 immunopositivity were less prominent in the MCD+BET group compared with the MCD group. Angular second moment (ASM) and inverse difference moment (IDM) (textural homogeneity markers) were significantly increased in the MCD+BET group versus the MCD group (p<0.001), even though no difference between the MCD and the control group was evident. Heterogeneity parameters, contrast, and correlation were significantly increased in the MCD group versus the control (p<0.001). On the other hand, betaine treatment significantly reduced correlation, contrast, and entropy compared with the MCD group (p<0.001). Betaine attenuated MCD diet-induced NAFLD by reducing fat accumulation and inhibiting hepatocyte proliferation. Betaine supplementation increased nuclear homogeneity and chromatin complexity with reduction of entropy, contrast, and correlation.

Extended abstract of a paper presented at Microscopy and Microanalysis 2004 in Savannah, Georgia, USA, August 1–5, 2004.

Extended abstract of a paper presented at Microscopy and Microanalysis 2011 in Nashville, Tennessee, USA, August 7–August 11, 2011.