Variable phase dark-field contrast has been developed as an illumination technique in light microscopy, which promises significant improvements and a higher variability in imaging of several transparent specimens. In this method, a phase contrast image is optically superimposed on a dark-field image so that a partial image based on the principal zeroth-order maximum (phase contrast) interferes with an image that is based on the secondary maxima (dark field). The background brightness and character of the resulting image can be continuously modulated from a phase-contrast-dominated to a dark-field-dominated character. The condenser aperture diaphragm can be used for modulations of the image's appearance. Specimens can either be illuminated concentrically or obliquely (eccentrically) when parts of the illuminating light beams are covered and blocked. Moreover, a bright-field-like partial image can be added. In this way, the illumination can be optimally adjusted to the specific properties of the specimen. The techniques described can lead to improved visual information especially in biological specimens consisting of phase structures and additional light-absorbing or -reflecting components. Moreover, the specimen's three-dimensionality can be accentuated with improved clarity because the illuminating light beams associated with phase contrast and dark field run to the specimen at different angles of incidence.

Hyperspectral cathodoluminescence mapping is used to examine a carbonado diamond. The hyperspectral dataset is examined using a data clustering algorithm to interpret the range of spectral shapes present within the dataset, which are related to defects within the structure of the diamond. The cathodoluminescence response from this particular carbonado diamond can be attributed to a small number of defect types: N-V0, N2V, N3V, a 3.188 eV line, which is attributed to radiation damage, and two broad luminescence bands. Both the N2V and 3.188 eV defects require high-temperature annealing, which has implications for interpreting the thermal history of the diamond. In addition, bright halos observed within the diamond cathodoluminescence, from alpha decay radiation damage, can be attributed to the decay of 238U.

Based on a scanning electron microscope operated at 30 kV with a homemade specimen holder and a multiangle solid-state detector behind the sample, low-kV scanning transmission electron microscopy (STEM) is presented with subsequent electron tomography for three-dimensional (3D) volume structure. Because of the low acceleration voltage, the stronger electron-atom scattering leads to a stronger contrast in the resulting image than standard TEM, especially for light elements. Furthermore, the low-kV STEM yields less radiation damage to the specimen, hence the structure can be preserved. In this work, two-dimensional STEM images of a 1-μm-thick cell section with projection angles between ±50° were collected, and the 3D volume structure was reconstructed using the simultaneous iterative reconstructive technique algorithm with the TomoJ plugin for ImageJ, which are both public domain software. Furthermore, the cross-sectional structure was obtained with the Volume Viewer plugin in ImageJ. Although the tilting angle is constrained and limits the resulting structural resolution, slicing the reconstructed volume generated the depth profile of the thick specimen with sufficient resolution to examine cellular uptake of Au nanoparticles, and the final position of these nanoparticles inside the cell was imaged.

The ability to see, or visualize, a phenomenon is an essential tool of modern biological research. Our ability to create static and dynamic images has grown exponentially in the 25 or so years since confocal microscopy became readily available. The ingenious and energetic application of insights from optical physics to biological imaging in recent years has brought us far-reaching extensions of simple imaging, including nonlinear (or multiphoton) excitation, total internal reflection imaging, and even single molecule counting techniques. The annual Omaha Imaging Symposium has since 2003 brought together experts in advanced biological imaging techniques for a one-day exposition of how these techniques help move biological science forward. The seventh iteration of the series was held Friday, April 8, 2011, at the Harper Student Life Center of Creighton University, in Omaha, Nebraska. This special section of Microscopy and Micronanalysis consists of papers from speakers at the symposium.

Alzheimer´s disease (AD) is characterized by extensive neuronal loss in brain areas related to memory and cognitive functions. Central to the neurodegenerative process is a peptide termed Abeta. The latter is the main component of senile plaques, one of the histopathological hallmarks of AD, and derives from proteolytic processing of the Alzheimer´s amyloid precursor protein (APP). Among the alterations induced by Abeta is increased cellular oxidative stress, imbalanced protein phosphorylation and cytoskeletal abnormalities, all factors that contribute to neuronal death.

Extended abstract of a paper presented at Microscopy and Microanalysis 2012 in Phoenix, Arizona, USA, July 29 – August 2, 2012.

For a few years, the biological effects on ecosystems and the public of the bioaccumulation of radionuclides in situations of chronic exposures have been studied. This work, in keeping with the ENVIRHOM French research program, presents the uranium microdistribution by secondary ion mass spectrometry (SIMS) technique in the renal cortex of rats following chronic exposure to this low level element in the drinking water (40 mg/L) as a function to exposure duration (6, 9, 12, and 18 months). The SIMS mass spectra and 238U+ ion images produced with a SIMS CAMECA 4F-E7 show the kinetic of uranium accumulation in the different structures of the kidney. For the rats contaminated up to 12 months, the radioelement is mainly fixed in the proximal tubules; then after 18 exposure months, uranium is detected in all the segments of the nephron. This work has also shown that ion microscopy is an analytical method to detect trace elements and give elemental cartography at the micrometer scale.

Conventional X-ray microcomputed tomography (micro-CT) is not usually sufficient to determine microscopic compositional distributions as it is limited to measuring the X-ray attenuation of the sample, which for a given dataset can be similar for materials of different composition. In contrast, the present work enables three-dimensional compositional analysis with a data-constrained microstructure (DCM) modeling methodology, which uses two or more CT datasets acquired with different X-ray spectra and incorporates them as model constraints. For providing input data for DCM, we have also developed a method of micro-CT data collection that enables two datasets with different X-ray spectra to be acquired in parallel. Such data are used together with the DCM methodology to predict the distributions of corrosion inhibitor and filler in a polymer matrix. The DCM-predicted compositional microstructures have a reasonable agreement with energy dispersive X-ray images taken on the sample surface.

We use a piezoelectric quartz tuning fork to calibrate the displacement of ceramic piezoelectric scanners that are widely employed in scanning probe microscopy. We measure the static piezoelectric response of a quartz tuning fork and find it to be highly linear, nonhysteretic and with negligible creep. These performance characteristics, close to those of an ideal transducer, make quartz transducers superior to ceramic piezoelectric actuators. Furthermore, quartz actuators in the form of a tuning fork have the advantage of yielding static displacements comparable to those of local probe microscope scanners. We use the static displacement of a quartz tuning fork as a reference to calibrate the three axis displacement of a ceramic piezoelectric scanner. Although this calibration technique is a nontraceable method, it can be more versatile than using calibration grids because it enables characterization of the linear and nonlinear response of a piezoelectric scanner in a broad range of displacements, spanning from a fraction of a nanometer to hundreds of nanometers. In addition, the creep and the speed dependent piezoelectric response of ceramic scanners can be studied in detail.

Atomic force microscopy (AFM) studies on observations of lead films deposited from the solutions containing an acetate buffer and different concentration of Pb(II) are presented. AFM images show considerable variability in morphology of the deposited lead layer depending on experimental conditions. To investigate effects of the Pb(II) concentration and pH of the supporting electrolyte on the accessible potential window of the lead film electrode (PbFE), voltammetric techniques were used. It was found that the useful potential window of PbFE is affected by the pH and Pb(II) concentration. Additionally, it was found that the distribution and large size of Pb particles on the electrode surface shown by AFM corresponded well to the mass of Pb expected on the glassy carbon support with respect to the voltammetric data. Results reveal that PbFE is an attractive nonmercury metallic electrode suitable for electrochemical detection of metal ions and a lot of organic compounds in a wide potential window. The accessible potential window of the PbFE in an acetate buffer (pH = 5.0) was compared to those obtained at the bismuth film electrode and antimony film electrode.

We investigated the effects of sample preparation and of the exposure to an electron beam on particles in cryo-electron tomographs. Various virus particles with icosahedral symmetry were examined, allowing a comparison of symmetrically related components that should be identical in structure but might be affected differently by these imaging artifacts. Comparison of tomographic reconstructions with previously determined structures established by an independent method showed that neither freezing nor electron beam exposure produced a significant amount of shrinkage along the z axis (thickness). However, we observed damage to regions of the particles located close to the surface of the vitreous ice.

In recent years great importance has been given to the adverse effects of particulate matter (PM) in health, and several epidemiological studies correlating the airborne particles with pulmonary injury have been carried out. The adverse effect of short- and long-term expositions to PM can, indeed, cause a variety of effects, from minor effects on the respiratory system to serious oncogenic effects that can lead to precocious death and are considered one of the top environmental public health concerns. Some cytogenetic techniques, as micronucleus assay, allow to detect chromosome breakage and loss by measuring the formation of micronuclei and proved to be a good tool in the evaluation of genotoxic damage induced by PM. Thus genotoxic effects can be evaluated by micronucleus test in order to identify a gradient of potential exposure and to assess environmental monitoring. However, cross investigations involving biomarkers of genotoxicity and electron microscopy PM-induced tracheal and lung damages in natural populations are not common.

Extended abstract of a paper presented at Microscopy and Microanalysis 2012 in Phoenix, Arizona, USA, July 29 – August 2, 2012.

The immune system is highly evolved and can respond to infection throughout the body. Pathogen-specific immune cells are usually generated in secondary lymphoid tissues (e.g., spleen, lymph nodes) and then migrate to sites of infection where their functionality is shaped by the local milieu. Because immune cells are so heavily influenced by the infected tissue in which they reside, it is important that their interactions and dynamics be studied in vivo. Two-photon microscopy is a powerful approach to study host-immune interactions in living tissues, and recent technical advances in the field have enabled researchers to capture movies of immune cells and infectious agents operating in real time. These studies have shed light on pathogen entry and spread through intact tissues as well as the mechanisms by which innate and adaptive immune cells participate in thwarting infections. This review focuses on how two-photon microscopy can be used to study tissue-specific immune responses in vivo, and how this approach has advanced our understanding of host-immune interactions following infection.

A comparative cathodoluminescence (CL) spectroscopic study of extraordinarily well-preserved versus diagenetically altered Turonian (∼92 Ma before present) calcitic and aragonitic microfossils was performed to document the cathodoluminescence characteristics of two common Cretaceous carbonate producers, foraminifera and calcareous dinoflagellates. Unaltered specimens reveal a conspicuous peak in the blue CL band at ∼400 nm that has rarely been previously reported for biogenic carbonates. We interpret this luminescence as an indicative feature of the primary bio-mineralized shells of calcareous dinoflagellates and foraminifera. Orange luminescence as the second important CL emission band (∼620 nm) in calcite generally increases with diagenetic cement overgrowth and recrystallization but can also be present in unaltered material. Thus, orange CL of biogenic calcite is not an unequivocal diagenetic indicator. Accordingly, spectroscopic investigation of both the ∼400 and ∼620 nm peaks represents a more objective criterion to evaluate the degree of diagenetic alteration. The ratio of relative intensities of the blue CL versus orange CL can provide a semiquantitative measure with relative intensity ratios blue:orange >2 occurring in the least diagenetically altered microfossils. Comparison of unaltered specimens of separate species reveals elemental differences that potentially indicate species-specific biomineralization or habitats.

Biologically and chemically modified nanoparticles are gaining much attention as a new tool in cancer detection and treatment. Herein, we demonstrate that an alizarin red S (ARS) dye coating on TiO2 nanoparticles enables visible light activation of the nanoparticles leading to degradation of neighboring biological structures through localized production of reactive oxygen species. Successful coating of nanoparticles with dye is demonstrated through sedimentation, spectrophotometry, and gel electrophoresis techniques. Using gel electrophoresis, we demonstrate that visible light activation of dye-TiO2 nanoparticles leads to degradation of plasmid DNA in vitro. Alterations in integrity and distribution of nuclear membrane associated proteins were detected via fluorescence confocal microscopy in HeLa cells exposed to perinuclear localized ARS-TiO2 nanoparticles that were photoactivated with visible light. This study expands upon previous studies that indicated dye coatings on TiO2 nanoparticles can serve to enhance imaging, by clearly showing that dye coatings on TiO2 nanoparticles can also enhance the photoreactivity of TiO2 nanoparticles by allowing visible light activation. The findings of our study suggest a therapeutic application of dye-coated TiO2 nanoparticles in cancer research; however, at the same time they may reveal limitations on the use of dye assisted visualization of TiO2 nanoparticles in live-cell imaging.

Advances in microscopy allow one to probe the structure of neurons and their interactions with astrocytes in brain slices and in vivo at ever increasing resolution. Moreover, the dynamic interactions between the cells can be examined in live preparation. In this paper we discuss how a variety of imaging approaches: confocal microscopy, electron microscopy, and multiphoton time-lapse microscopy are employed to probe neuron glia interactions in the developing cerebellum.

Extended abstract of a paper presented at Microscopy and Microanalysis 2012 in Phoenix, Arizona, USA, July 29 – August 2, 2012.