The present review is based on results of several years of investigation on the pathogenesis of plant wilt diseases using for one of the main aspects, several labeling techniques at the ultrastructural level: autoradiography with 3H-thymidine; gold-tagged lectins, enzymes, polyclonal, and monoclonal antibodies. Thus, opaque matter (OM), labeling for DNA with the appropriate markers, can be abundant in and associated with host tissue reactions and pronounced alterations (particularly in newly differentiated xylem). The presence of structures (called P-elements) in OM, paralleled by a similar occurrence in nuclei of both the host and pathogen cells, is considered as being also indicative of a cytoplasmic nature for this OM and most likely originating from fungal elements.

Fiber alignment plays a critical role in the structure and function of cells and tissues. While fiber alignment quantification is important to experimental analysis and several different methods for quantifying fiber alignment exist, many studies focus on qualitative rather than quantitative analysis perhaps due to the complexity of current fiber alignment methods. Speed and sensitivity were compared in edge detection and fast Fourier transform (FFT) for measuring actin fiber alignment in cells exposed to shear stress. While edge detection using matrix multiplication was consistently more sensitive than FFT, image processing time was significantly longer. However, when MATLAB functions were used to implement edge detection, MATLAB's efficient element-by-element calculations and fast filtering techniques reduced computation cost 100 times compared to the matrix multiplication edge detection method. The new computation time was comparable to the FFT method, and MATLAB edge detection produced well-distributed fiber angle distributions that statistically distinguished aligned and unaligned fibers in half as many sample images. When the FFT sensitivity was improved by dividing images into smaller subsections, processing time grew larger than the time required for MATLAB edge detection. Implementation of edge detection in MATLAB is simpler, faster, and more sensitive than FFT for fiber alignment quantification.

The biomechanical response of tissues serves as a valuable marker in the prediction of disease and in understanding the related behavior of the body under various disease and age states. Alterations in the macroscopic biomechanical response of diseased tissues are well documented; however, a thorough understanding of the microstructural events that lead to these changes is poorly understood. In this article we introduce a novel microbiaxial optomechanical device that allows two-photon imaging techniques to be coupled with macromechanical stimulation in hydrated planar tissue specimens. This allows that the mechanical response of the microstructure can be quantified and related to the macroscopic response of the same tissue sample. This occurs without the need to fix tissue in strain states that could introduce a change in the microstructural configuration. We demonstrate the passive realignment of fibrous proteins under various types of loading, which demonstrates the ability of tissue microstructure to reinforce itself in periods of high stress. In addition, the collagen and elastin response of tissue during viscoelastic behavior is reported showing interstitial fluid movement and fiber realignment potentially responsible for the temporal behavior. We also demonstrate that nonhomogeneities in fiber strain exist over biaxial regions of assumed homogeneity.

Contemporary research aims to understand biological processes not only by identifying participating proteins, but also by characterizing the dynamics of their interactions. Because Förster's Resonance Energy Transfer (FRET) is invaluable for the latter undertaking, its usage is steadily increasing. However, FRET measurements are notoriously error-prone, especially when its inherent efficiency is low, a not uncommon situation. Furthermore, many FRET methods are either difficult to implement, are not appropriate for observation of cellular dynamics, or report instrument-specific indices that hamper communication of results within the scientific community. We present here a novel comprehensive spectral methodology, SpRET, which substantially increases both the reliability and sensitivity of FRET microscopy, even under unfavorable conditions such as weak fluorescence or the presence of noise. While SpRET overcomes common pitfalls such as interchannel crosstalk and direct excitation of the acceptor, it also excels in removal of autofluorescence or background contaminations and in correcting chromatic aberrations, often overlooked factors that severely undermine FRET experiments. Finally, SpRET quantitatively reports absolute rather than relative FRET efficiency values, as well as the acceptor-to-donor molar ratio, which is critical for full and proper interpretation of FRET experiments. Thus, SpRET serves as an advanced, improved, and powerful tool in the cell biologist's toolbox.

Structured illumination fluorescence microscopy is a powerful super-resolution method that is capable of achieving a resolution below 100 nm. Each super-resolution image is computationally constructed from a set of differentially illuminated images. However, real-time application of structured illumination microscopy (SIM) has generally been limited due to the computational overhead needed to generate super-resolution images. Here, we have developed a real-time SIM system that incorporates graphic processing unit (GPU) based in-line parallel processing of raw/differentially illuminated images. By using GPU processing, the system has achieved a 90-fold increase in processing speed compared to performing equivalent operations on a multiprocessor computer—the total throughput of the system is limited by data acquisition speed, but not by image processing. Overall, more than 350 raw images (16-bit depth, 512 × 512 pixels) can be processed per second, resulting in a maximum frame rate of 39 super-resolution images per second. This ultrafast processing capability is used to provide immediate feedback of super-resolution images for real-time display. These developments are increasing the potential for sophisticated super-resolution imaging applications.

It is well known that c-Jun N-terminal kinase (JNK) plays pivotal roles in various mitotic events, but its function in mammalian oocyte meiosis remains unknown. In this study, we found that no specific JNK2 signal was detected in germinal vesicle stage. JNK2 was associated with the spindles especially the spindle poles and cytoplasmic microtubule organizing centers at prometaphase I, metaphase I, and metaphase II stages. JNK2 became diffusely distributed and associated with the midbody at telophase I stage. Injection of myc-tagged JNK2α1 mRNA into oocytes also revealed its localization on spindle poles. The association of JNK2 with spindle poles was further confirmed by colocalization with the centrosomal proteins, γ-tubulin and Plk1. Nocodazole treatment showed that JNK2 may interact with Plk1 to regulate the spindle assembly. Then we investigated the possible function of JNK2 by JNK2 antibody microinjection and JNK specific inhibitor SP600125 treatment. These two manipulations caused abnormal spindle formation and decreased the rate of first polar body (PB1) extrusion. In addition, inhibition of JNK2 resulted in impaired localization of Plk1. Taken together, our results suggest that JNK2 plays an important role in spindle assembly and PB1 extrusion during mouse oocyte meiotic maturation.

Quantification of very low density molecular coatings on large (60 nm) gold nanoparticles (AuNPs) is demonstrated via the use of antibody-mediated self-limiting self-assembly of small and large AuNPs into raspberry-like structures subsequently imaged by atomic force microscopy (AFM). AFM imaging is proposed as an automated, lower-cost, higher-throughput alternative to immunostaining and imaging by transmission electron microscopy. Synthesis of large AuNPs, containing one of three ligand molecules in one of three stoichiometries (1, 2, or 10 ligands per AuNP), and small probe AuNPs with one of three antibody molecules in a one antibody per AuNP ratio, enabled a range of predicted self-limiting self-assembled structures. A model predicting the probability of observing a given small to large AuNP ratio based on a topography measurement such as AFM is described, in which random orientational deposition is assumed and which accounts for the stochastic synthesis method of the library AuNPs with varied ligand ratios. Experimental data were found to agree very well with the predictive models when using an established AFM sample preparation method that avoids drying-induced aggregation.

A vascular corrosion cast of an entire mouse kidney was scanned with a modular multiresolution X-ray nanotomography system. Using an isotropic voxel pitch of 0.5 μm, capillary systems such as the vasa recta, peritubular capillaries and glomeruli were clearly resolved. This represents a considerable improvement over corrosion casts scanned with microcomputed tomography systems. The resolving power of this system was clearly demonstrated by the unique observation of a dense, subcapsular mat of capillaries enveloping the entire outer surface of the cortical region. Resolution of glomerular capillaries was comparable to similar models derived from laser scanning confocal microscopy. The high-resolution, large field of view and the three-dimensional nature of the resulting data opens new possibilities for the use of corrosion casting in research.

The teeth of the marine mollusk Acanthopleura hirtosa are an excellent example of a complex, organic, matrix-mediated biomineral, with the fully mineralized teeth comprising layers of iron oxide and iron oxyhydroxide minerals around a calcium apatite core. To investigate the relationship between the various mineral layers and the organic matrix fibers on which they grew, sections have been prepared from specific features in the teeth at controlled orientations using focused ion beam processing. Compositional and microstructural details of heterophase interfaces, and the fate of the organic matrix fibers within the mineral layers, can then be analyzed by a range of transmission electron microscopy (TEM) techniques. Energy-filtered TEM highlights the interlocking nature of the various mineral phases, while high-angle annular dark-field scanning TEM imaging demonstrates that the organic matrix continues to exist in the fully mineralized teeth. These new insights into the structure of this complex biomaterial are an important step in understanding the relationship between its structural and physical properties and may help explain its high strength and crack-resistance behavior.

Atom probe tomography (APT) represents a significant step toward atomic resolution microscopy, analytically imaging individual atoms with highly accurate, though imperfect, chemical identity and three-dimensional (3D) positional information. Here, a technique to retrieve crystallographic information from raw APT data and restore the lattice-specific atomic configuration of the original specimen is presented. This lattice rectification technique has been applied to a pure metal, W, and then to the analysis of a multicomponent Al alloy. Significantly, the atoms are located to their true lattice sites not by an averaging, but by triangulation of each particular atom detected in the 3D atom-by-atom reconstruction. Lattice rectification of raw APT reconstruction provides unprecedented detail as to the fundamental solute hierarchy of the solid solution. Atomic clustering has been recognized as important in affecting alloy behavior, such as for the Al-1.1Cu-1.7Mg (at. %) investigated here, which exhibits a remarkable rapid hardening reaction during the early stages of aging, linked to clustering of solutes. The technique has enabled lattice-site and species-specific radial distribution functions, nearest-neighbor analyses, and short-range order parameters, and we demonstrate a characterization of solute-clustering with unmatched sensitivity and precision.

In this study, we present a fully automated method to investigate and reconstruct the three-dimensional crack structure beneath an indent in a highly insulating material. This work concentrates on issues arising from a long automatic acquisition process, the insulating nature of the specimen, and the introduction of minimal damage to the original cracks resulting from indentation.

The elemental composition and microscopic-level shape of inclusions inside industrial materials are considered important factors in fracture analytical studies. In this work, a three-dimensional (3D) microscopic elemental analysis system based on a serial sectioning technique was developed to observe the internal structure of such materials. This 3D elemental mapping system included an X-ray fluorescence analyzer and a high-precision milling machine. Control signals for the X-ray observation process were automatically sent from a data I/O system synchronized with the precision positioning on the milling machine. Composite specimens were used to confirm the resolution and the accuracy of 3D models generated from this system. Each of the two specimens was composed of three metal wires of 0.5 mm diameter braided into a single twisted wire that was placed inside a metal pipe; the pipe was then filled with either epoxy resin or Sn. The milling machine was used to create a mirror-finish cross-sectional surface on these specimens, and elemental analyses were performed. The twisted wire structure was clearly observed in the resulting 3D models. This system enables automated investigation of the 3D internal structure of materials as well as the identification of their elemental components.

Three-dimensional (3D) analysis is an essential tool to obtain quantitative results from 3D datasets. Considerable progress has been made in 3D imaging techniques, resulting in a growing need for more flexible, complete analysis packages containing advanced algorithms. At the Centre for X-ray Tomography of the Ghent University (UGCT), research is being done on the improvement of both hardware and software for high-resolution X-ray computed tomography (CT). UGCT collaborates with research groups from different disciplines, each having specific needs. To meet these requirements the analysis software package, Morpho+, was developed in-house. Morpho+ contains an extensive set of high-performance 3D operations to obtain object segmentation, separation, and parameterization (orientation, maximum opening, equivalent diameter, sphericity, connectivity, etc.), or to extract a 3D geometrical representation (surface mesh or skeleton) for further modeling. These algorithms have a relatively short processing time when analyzing large datasets. Additionally, Morpho+ is equipped with an interactive and intuitive user interface in which the results are visualized. The package allows scientists from various fields to obtain the necessary quantitative results when applying high-resolution X-ray CT as a research tool to the nondestructive investigation of the microstructure of materials.

Various porous titania photocatalysts are analyzed three-dimensionally in real space by electron tomography. Shapes and three-dimensional (3D) distributions of fine pores and silver (Ag) particles (2 nm in diameter) within the pores are successfully reconstructed from the 3D data. Electron tomography is applied for measuring the specific surface area of the porous structures including open and closed porosity. Calculated specific surface areas of 22.8 m2/g for a conventional sol-gel TiO2 sample and 366 m2/g for a highly porous TiO2 sample prepared using the Pluronic P-123 self-assembly process are compared with those measured by the general BET method. The real-space surface measurement indicates that the highly porous TiO2 produced by the present method using block copolymers has a greater number of effective reaction sites for the degradation of methylene blue. Electron tomography shows a great potential to contribute considerably to the nanostructural analysis and design of such catalyst materials for photocatalysis.

We report the evolution of titanium dioxide nanostructures when Au nanoparticles, supported on single crystal TiO2 substrates, were heated under ∼260 Pa of flowing O2 in an environmental transmission electron microscope. Nanostructures with different morphologies were first observed around 500°C. Our measurements show that temperature, oxygen pressure, and the electron beam control the nanostructure growth. We propose a reaction-controlled growth mechanism where mobile Ti atoms generated by the electron- beam-induced reduction of TiO2 are preferentially reoxidized at the Au-TiO2 interface.

The transition from 10-fold to 5-fold symmetry was observed during the analysis of nanodiffraction patterns of a gold decahedral multiple twinned nanoparticle of 15 nm in diameter. The analysis shows that as the convergence of the beam is increased, the rotational symmetry of the diffraction pattern shifts from 10- to 5-fold. The 10-fold symmetry predicted by Friedel's law is lost by the asymmetric shift of the diffraction spots, an effect that becomes more noticeable as the electron beam convergence increases. Dynamical and kinematical diffraction calculations indicate this decrease in symmetry is the result of a double refraction effect coupled with the variation of the dynamical diffraction conditions arising from a varying electron beam convergence.

Herein, we characterized lead pipe scale removed from a drinking water distribution system using argon ion beam etching and a variety of solids analysis approaches. Specifically, pipe scale cross sections and solids were analyzed using scanning electron microscopy, energy dispersive X-ray analysis, X-ray diffraction, and acid digestion followed by inductively coupled plasma mass spectrometry analyses. The pipe scale consisted of at least five layers that contained Pb(II) and Pb(IV) minerals, and magnesium, aluminum, manganese, iron, and silicon solids. The outer layer was enriched with crystalline amorphous manganese and iron, giving it a dark orange to red color. The middle layers consisted of hydrocerussite and plattnerite, and the bottom layer consisted primarily of litharge. Over the litharge layer, hydrocerussite crystals were grown vertically away from the pipe wall, which included formations of plattnerite. Significant amounts of trace contaminant vanadium, likely in the form of vanadinite, and copper accumulated in the scale as well.

The degradation of Sydney sandstone used to build the heritage St Mary's Cathedral in Sydney, Australia, has been investigated using environmental scanning electron microscopy combined with energy dispersive X-ray spectroscopy. This technique provided the structural details of the cementing clay and an elemental characterization the sandstone. The observed differences in the elemental composition of the unweathered and weathered sandstones were associated with changes to the clay microstructure upon weathering. The results support the substitution theory that Fe3+ replaces Al3+ in the kaolinite clay component upon weathering. An examination of the impurities present prior to a nonstructural iron removal treatment revealed the presence of minerals that may provide a source of the elements responsible for the substitution process.

The M spectrum of the element uranium was reinvestigated by using both high-resolution wavelength dispersive (WD) spectrometry as well as energy dispersive (ED) spectrometry. Thereby we observed relative intensities that deviate from data in the literature. These discrepancies were not only observed for the weak U M lines but also for major lines. By measuring the Mα,β region of the spectrum with a PET crystal in second-order reflection, a sufficient energy resolution was achieved to separate Mα2 (M5N6) from Mα1 (M5N7). The intensity ratio I(M5N6)/I(M5N7) was determined to be approximately 5%, which is in strong contrast to the data tabulated by White and Johnson [White, E.W. & Johnson, G.G. (1970). X-Ray and Absorption Wavelengths and Two-Theta Tables. ASTM Data Series DS37A, 2nd ed. Philadelphia, PA: American Society for Testing and Materials]. Furthermore M5N7 was clearly observed as the strongest of the M lines that disagrees with data presented by Kleykamp [Kleykamp, H. (1981). Wavelengths of the M X-ray spectra of uranium, neptunium, plutonium, and americium. Z Naturforsch36a, 1388–1390], who reported Mβ (M4N6) as the strongest line. Also, after White and Johnson (1970), the line M2N4 should be more intense than M3O5 by a factor of 5. Both our WD and ED spectra show clearly that M3O5 is stronger than M2N4. Altogether, we observed in our WD spectra 26 M lines. In some cases untypical large differences between the line energies given by Bearden [Bearden, J.A. (1967). X-ray wavelengths. Rev Mod Phys39, 78–124] and measured by us were observed.

In this article, a new scanning electron microscopy (SEM) image composition technique is described, which can significantly reduce drift related image corruptions. Drift distortion commonly causes blur and distortions in the SEM images. Such corruption ordinarily appears when conventional image-acquisition methods, i.e., “slow scan” and “fast scan,” are applied. The damage is often very significant; it may render images unusable for metrology applications, especially where subnanometer accuracy is required. The described correction technique works with a large number of quickly taken frames, which are properly aligned and then composed into a single image. Such image contains much less noise than the individual frames, while the blur and deformation is minimized. This technique also provides useful information about changes of the sample position in time, which may be applied to investigate the drift properties of the instrument without a need of additional equipment.