Use of deep ultraviolet (DUV, below 350 nm) fluorescence opens up new possibilities in biology because it does not need external specific probes or labeling but instead allows use of the intrinsic fluorescence that exists for many biomolecules when excited in this wavelength range. Indeed, observation of label free biomolecules or active drugs ensures that the label will not modify the biolocalization or any of its properties. In the past, it has not been easy to accomplish DUV fluorescence imaging due to limited sources and to microscope optics. Two worlds were coexisting: the spectrofluorometric measurements with full spectrum information with DUV excitation, which lacked high-resolution localization, and the microscopic world with very good spatial resolution but poor spectral resolution for which the wavelength range was limited to 350 nm. To combine the advantages of both worlds, we have developed a DUV fluorescence microscope for cell biology coupled to a synchrotron beamline, providing fine tunable excitation from 180 to 600 nm and full spectrum acquired on each point of the image, to study DUV excited fluorescence emitted from nanovolumes directly inside live cells or tissue biopsies.

Computer vision tasks such as recognition and classification of objects and structures or image registration and retrieval can provide significant information when applied to microscopy images. Recently developed techniques for the detection and description of local features make the extraction and description of local image features that are invariant to various changes possible. The invariance and robustness of feature detection and description techniques play a key role in the design and implementation of object recognition, image registration, or image mosaicing applications. The scale-invariant feature transform (SIFT) technique is a widely used method for the detection, description, and matching of image features. In this article we present the results of our experiments regarding the repeatability of SIFT features, and to the precision of the SIFT feature matching, under image modifications specific to confocal scanning laser microscopy (CSLM). We have analyzed the behavior of SIFT while changing the pinhole aperture, photomultiplier gain, laser beam power, and electronic zoom. Our experiments, conducted on CSLM images, show that the SIFT technique is able to match detected key points between images acquired at different values of the acquisition parameters with good precision and represents a consistent tool for computer vision applications in CSLM.

The aim was to characterize the external structure, roughness, and absolute depth profile (ADP) of fluorotic enamel compared with healthy enamel. Eighty extracted human molars were classified into four groups [TFI: 0, control (C); 1–3, mild (MI); 4–5, moderate (MO); 6–9, severe fluorosis (S)] according to the Thylstrup-Fejerskov Index (TFI). All samples were analyzed by atomic force microscopy.

The mean values of enamel surface roughness (ESR) in nm were: Group C, 92.6; Group MI, 188.8; Group MO, 246.9; and Group S, 532.2. The mean values of absolute depth profile in nm were: C, 1,065.7; MI, 2,360.7; MO, 2,536.7; and S, 6,146.2. The differences between mean ESR and mean ADP among groups were statistically significant (p < 0.05). This structural study confirms at the nanometer level that there is a positive association between fluorosis severity, ESR, and ADP, and there is an association with the clinical findings of fluorosis measured by TFI as well.

Although bacterial biofilms have been studied in detail, adhesion of Candida albicans and non-albicans species to an intrauterine contraceptive device (IUD) is not clear. The objective of this study was to evaluate aspects of imaging of the ultrastructure and viability of vaginal yeasts adhered to different parts of an IUD, through scanning electron microscopy (SEM) and confocal scanning laser microscopy (CSLM). We studied yeasts isolated from different patients with vulvovaginal candidiasis: C. albicans, C. glabrata, C. guillermondii, C. parapsilosis, C. tropicalis, and Saccharomyces cerevisiae. A suspension of the each yeast was prepared and incubated with IUD parts (tail, without copper, and copper-covered). SEM and CSLM showed that all the vaginal yeasts adhered to all the parts of the IUD and demonstrated viability, including 30 days after contact for C. albicans. Possibly irregularities of IUD surface contribute to the adherence process. Although all of the IUD parts contribute to retention of yeasts in the genital tract, high concentration of yeast cells on the tail may indicate the importance of this segment in maintaining the colonization by yeast cells because the tail forms a bridge between the external environment, the vagina that is colonized by yeast cells, and the upper genital tract where there is no colonization.

We describe the architecture of nitrifying bacteria biofilms immobilized on a three-dimensional (3D) polyurethane foam that permits efficient water flow through a bioreactor. The 3D spatial organization of immobilized bacterial colonies is characterized on three resolution levels with X-ray tomography, light confocal microscopy, and scanning electron microscopy (SEM). Using these techniques we demonstrate biofilm distribution in the foam and the existence of several modes of binding of bacteria to the foam. Computed X-ray tomography permits observation of the distribution of the biofilm in the whole open cellular polyurethane material volume and estimation of biofilm volume. SEM and confocal laser scanning microscopy techniques permit 3D visualization of biofilm structure. Three distinct immobilization patterns could be observed in the open cellular polyurethane material: (1) large irregular aggregates of bacterial biofilm that exist as irregular biofilm fragments, rope-like structures, or biofilm layers on the foam surface; (2) spherical (pom-pom) aggregates of bacteria localized on the external surface of biofilm; and (3) biofilm threads adherent to the surface of polyurethane foam. Finally, we demonstrate that immobilized bacteria exhibit metabolic activity and growth.

The properties of activated sludge systems can be characterized using image analysis procedures. When these systems operate with high biomass content, accurate sludge characterization requires samples to be diluted. Selection of the best image acquisition magnification is directly related to the amount of biomass screened. The aim of the present study was to survey the effects of dilution and magnification on the assessment of aggregated and filamentous bacterial content and structure using image analysis procedures. Assessments of biomass content and structure were affected by dilutions. Therefore, the correct operating dilution requires careful consideration. Moreover, the acquisition methodology comprising a 100× magnification allowed data on aggregated and filamentous biomass to be determined and smaller aggregates to be identified and characterized, without affecting the accuracy of lower magnifications regarding biomass representativeness.

Plants have suberized cells that act as protective interfaces with the environment or between different plant tissues. A lamellar structure of alternating dark and light bands has been found upon transmission electron microscopy (TEM) observation of cork cells and considered a typical feature of the suberized secondary wall. We observed cork cells from periderms of Quercus suber, Quercus cerris, Solanum tuberosum, and Calotropis procera by TEM after uranyl acetate and lead citrate staining. A lamellated structure was observed in S. tuberosum and C. procera but not in Q. suber and Q. cerris where the suberized cell wall showed a predominantly hyaline aspect with only a dark dotted staining. Removal of suberin from Q. suber cells left a thinner secondary wall that lost the translucent aspect. We hypothesize that the species' specific chemical composition of suberin will result in different three-dimensional macromolecular development and in a different spatial location of lignin and other aromatics. A lamellated ultrastructure is therefore not a general feature of suberized cells.

Kalanchoe pinnata (Lamarck) Persoon (Crassulaceae) (air plant, miracle leaf) is popularly used to treat gastrointestinal disorders and wounds. Recently, the species was tested to treat cutaneous leishmaniasis with successful results. This medicinal activity was associated with the phenolic fraction of the plant. Blue light induces biosynthesis of phenolic compounds and many changes in anatomical characteristics. We studied the effects of supplementary blue light on the leaf morphology of in vitro K. pinnata. Plants cultured under white light (W plants) only and white light plus blue light (WB plants) show petioles with plain-convex section, amphistomatic leaf blades with simple epidermis, homogeneous mesophyll with densely packed cells, and a single collateral vascular bundle in the midrib. W plants have longer branches, a larger number of nodes per branch, and smaller leaves, whereas WB plant leaves have a thicker upper epidermis and mesophyll. Leaf fresh weight and leaf dry weight were similar in both treatments. Phenolic idioblasts were observed in the plants supplemented with blue light, suggesting that blue light plays an important role in the biosynthesis of phenolic compounds in K. pinnata.

Effects related with the attenuation and deflection suffered by an electron beam when it passes through a carbon conductive coating and an oxide film layer on the surface of bulk samples are studied by Monte Carlo simulations and energy dispersive spectroscopy with electron excitation. Analytical expressions are provided for the primary beam energy and intensity losses and for the deflection of the incident electrons in both layers, in terms of the incidence energy, the film mass thicknesses, and the atomic number of the oxidized element. From these analytical expressions, suitable corrections are proposed for the models used to describe the X-ray spectrum of the substrate, including also the contribution of the X-rays generated in the oxide and conductive films and the characteristic X-ray absorption occurring in those layers. The corrections are implemented in a software program for spectral analysis based on a routine of parameter refinement, and their influence is studied separately in experimental spectra of single-element standards measured at different excitation energies. Estimates for the layer thicknesses are also obtained from the spectral fitting procedure.

The discoloring interaction between the artist's pigments cadmium yellow and the copper-containing malachite, an interaction that is conjectured to cause black spotting in oil paintings of the 19th and early 20th centuries, was examined using X-ray mapping and scatter diagram analysis. The application of these coupled techniques confirmed that copper sulfide phases were produced during discoloration reaction. Scatter diagram analysis indicated that two copper sulfide stoichiometries (CuS and Cu3S2) were present as reaction products where previously only crystalline CuS (covellite) had been identified by X-ray diffraction. The results demonstrate the potential of X-ray mapping coupled with scatter diagram analysis for the identification of both crystalline and X-ray amorphous phases produced by such complex heterogeneous interactions and their applicability to the investigation of interactions of artists' pigments.

The scanning helium ion microscope has been used in transmission mode to investigate both the feasibility of this approach and the utility of the signal content and the image information available. Operating at 40 keV the penetration of the ion beam, and the imaging resolution achieved, in MgO crystals was found to be in good agreement with values predicted by Monte Carlo modeling. The bright-field and annular dark-field signals displayed the anticipated contrasts associated with beam absorption and scattering. In addition, the diffraction of the He ion beam within the sample gave rise to crystallographic contrast effects in the form of thickness fringes and dislocation images. Scanning transmission He ion microscopy thus achieves useful sample penetration and provides nanometer scale resolution, high contrast images of crystalline materials and crystal defects even at modest beam energies.

High-angle annular dark-field (HAADF) scanning transmission electron microscopy (STEM) images of electron-transparent samples show dominant atomic number (Z-) contrast with a high lateral resolution. HAADF STEM at low electron energies <30 keV is applied in this work for quantitative composition analyses of InGaAs quantum wells. To determine the local composition, normalized experimental image intensities are compared with results of Monte Carlo simulations. For verification of the technique, InGaAs/GaAs quantum-well structures with known In concentration are used. Transmission electron microscopy samples with known thickness are prepared by the focused-ion-beam technique. The method can be extended to other material systems and is particularly promising for the analysis of materials that are sensitive toward knock-on damage.

An electron diffraction technique called D-STEM has been developed in a transmission electron microscopy/scanning transmission electron microscopy (TEM/STEM) instrument to obtain spot electron diffraction patterns from nanostructures, as small as ∼3 nm. The electron ray path achieved by configuring the pre- and postspecimen illumination lenses enables the formation of a 1–2 nm near-parallel probe, which is used to obtain bright-field/dark-field STEM images. Under these conditions, the beam can be controlled and accurately positioned on the STEM image, at the nanostructure of interest, while sharp spot diffraction patterns can be simultaneously recorded on the charge-coupled device camera. When integrated with softwares such as GatanTM STEM diffraction imaging and Automated Crystallography for TEM or DigistarTM, NanoMEGAS, the D-STEM technique is very powerful for obtaining automated orientation and phase maps based on diffraction information acquired on a pixel by pixel basis. The versatility of the D-STEM technique is demonstrated by applying this technique to nanoparticles, nanowires, and nano interconnect structures.

We present a microfluidic system that maintains liquid flow in a specimen chamber for scanning transmission electron microscope (STEM) imaging. The specimen chamber consists of two ultrathin silicon nitride windows supported by silicon microchips. They are placed in a specimen holder that seals the sample from the vacuum in the electron microscope and incorporates tubing to and from the sample connected to a syringe pump outside the microscope. Using results obtained from fluorescence microscopy of microspheres flowing through the system, an equation to characterize the liquid flow through the system was calibrated. Gold nanoparticles of diameters of 30 and 100 nm moving in liquid were imaged with a 200 kV STEM. It was concluded that despite strong influences from Brownian motion, and sensitivity to small changes in the depth of the bypass channel, the electron microscopy flow data matched the calculated flow speed within an order of magnitude. The system allows for rapid (within a minute) liquid exchange, which can potentially be used, for example, to investigate the response of specimens, e.g., eukaryotic or bacterial cells, to certain stimuli.

The atomic force microscopes (AFM) images are obtained by keeping the bending of the cantilever unchanged in contact mode. However, it is found that changes in the tip-sample angle during parallel scan result in error in the topographic image. It is also discovered that measurement results obtained in the blind scan region contained large errors. In contrast, regions opposite the blind scan region gave more reliable result. To eliminate this topographic error caused by change in the tip-sample angle, a new operating method with lateral scan is utilized in AFM. Comparative experiments have been performed, and the results show that the error could be eliminated or decreased by using the operating method.

Three-dimensional atom probe tomography (APT) is successfully used to analyze the near-apex regions of an atomic force microscope (AFM) tip. Atom scale material structure and chemistry from APT analysis for standard silicon AFM tips and silicon AFM tips coated with a thin film of Cu is presented. Comparison of the thin film data with that observed using transmission electron microscopy indicates that APT can be reliably used to investigate the material structure and chemistry of the apex of an AFM tip at near atomic scales.

Local magnification effects and trajectory overlaps related to the presence of a second phase (clusters) are key problems and still open issues in the assessment of quantitative composition data in three-dimensional atom probe tomography (APT) particularly for tiny solute-enriched clusters. A model based on the distribution of distance of first nearest neighbor atoms has been developed to exhibit the variations in the apparent atomic density in reconstructed volumes and to correct compositions that are biased by local magnification effects. This model was applied to both simulated APT reconstructions and real experimental data and shows an excellent agreement with the expected composition of clusters.

Following is a list of microscopy-related meetings and courses. The editors would greatly appreciate input to this list via the electronic submission form found in the MSA World-Wide Web page at www.msa.microscopy.com. We will gladly add hypertext links to the notice on the web and insert a listing of the meeting in the next issue of the Journal. Send comments and questions to Nan Yao, [email protected].