We present an in vitro model of human skin that, together with nonlinear optical microscopy, provides a useful system for characterizing morphological and structural changes in a living skin tissue microenvironment due to changes in oxygen status and proteolytic balance. We describe for the first time the effects of chronic oxygen deprivation on a bioengineered model of human interfollicular epidermis. Histological analysis and multiphoton imaging revealed a progressively degenerating ballooning phenotype of the keratinocytes that manifested after 48 h of hypoxic exposure. Multiphoton images of the dermal compartment revealed a decrease in collagen structural order. Immunofluorescence analysis showed changes in matrix metalloproteinase (MMP)-2 protein spatial localization in the epidermis with a shift to the basal layer, and loss of Ki67 expression in proliferative basal cells after 192 h of hypoxic exposure. Upon reoxygenation MMP-2 mRNA levels showed a biphasic response, with restoration of MMP-2 levels and localization. These results indicate that chronic oxygen deprivation causes an overall degeneration in tissue architecture, combined with an imbalance in proteolytic expression and a decrease in proliferative capacity. We propose that these tissue changes are representative of the ischemic condition and that our experimental model system is appropriate for addressing mechanisms of susceptibility to chronic wounds.

The morphology of the osteocyte lacuno-canalicular system at the bone-biomaterial implant-interface has not been fully investigated. In this study, the resin-cast scanning electron microscopy technique was used, for the first time, to image the lacuno-canalicular network within neoformed bone around bioactive glass (BG) particles implanted in rat tibia bone marrow. The most salient finding was that the osteocyte canaliculi pass through the calcium-phosphorus layer formed at the bone-BG interface and reach the silica-rich layer of the reacted BG.

In this study the structure of collagen-chondroitin sulphate-hydroxyapatite porous composites is investigated by histochemical (Von Kossa staining), immunohistochemical, and transmission electron microscopy. Examination of composites on picrosirius red stained sections showed that polarization colors of collagen were generally in the range of orange-red. Immunofluorescence data indicate that chondroitin sulphate was either chemically incorporated into the bulk structure of collagen scaffolds or attached on surfaces of collagen bundles. Depending on the ratio between collagen:chondroitin sulphate:hydroxyapatite, von Kossa histochemical staining showed a progressive loading of collagen-chondroitin sulphate bundles with hydroxyapatite. Transmission electron microscopy studies have shown that composites contain mostly collagen fibrils aggregated with random orientation with very few collagen fibers showing the 67-nm banding pattern. Hydroxyapatite deposits of various sizes occurred among the collagen fibrils.

Bacteria flourish in nearly every environment on earth. Contributing to their ability to grow in many esoteric locations is their development into a biofilm structure. In an effort to more accurately model the growth environment of biofilms in nature, a Center for Disease Control and Prevention (CDC) biofilm reactor has been developed that mimics nature-like shear forces and renewable nutrient sources. To date, there has been no confirmation by scanning electron microscopy (SEM) that mature biofilms develop on a surface when grown using the CDC biofilm reactor. Three different SEM methods were used to collect images of Staphylococcus epidermidis ATCC 35984 that was to be grown using the CDC biofilm reactor. In addition, two different fixative techniques were used in each of the imaging methods. Results indicated that after 48 hours of growth in the reactor, S. epidermidis ATCC 35984 does produce a significant network of matrix components and 3D mushroom- or pillar-like structures with signs of water channel development. In conclusion, S. epidermidis ATCC 35984 grown using the CDC biofilm reactor does appear to display signs of mature biofilm development. These results could be important for studies wherein mature biofilms are needed for in vitro and/or in vivo applications.

Few simple labeling methods exist for simultaneous fluorescence and electron microscopy of bacteria and biofilms. Here we describe the synthesis, characterization, and application of fluorescent nanoparticle quantum dot (QD) conjugates to target microbial species, including difficult to label Gram-negative strains. These QD conjugates impart contrast for both environmental scanning electron microscopy (ESEM) and fluorescence microscopy, permitting observation of living and fixed bacteria and biofilms. We apply these probes for studying biofilms extracted from perennial cold springs in the Canadian High Arctic, which is a particularly challenging system. In these biofilms, sulfur-metabolizing bacteria live in close association with unusual sulfur mineral formations. Following simple labeling protocols with the QD conjugates, we are able to image these organisms in fully-hydrated samples and visualize their relationship to the sulfur minerals using both ESEM and fluorescence microscopy. We then use scanning transmission electron microscopy to observe precipitated sulfur around individual cells and within the biofilm lattice. All combined, this information sheds light on the possible mechanisms of biofilm and mineral structure formation. These new QD conjugates and techniques are highly transferable to many other microbiological applications, especially those involving Gram-negative bacteria, and can be used for correlated fluorescence and electron microscopy.

Different approaches using microscopy image analysis procedures were employed for characterization of activated sludge systems. The approaches varied mainly on the type of visualization and acquisition method used for collection of data. In this context, this study focused on the comparison of the two most common acquisition methods: bright field and phase-contrast microscopy. Images were acquired from seven different wastewater treatment plants for a combined period of two years. Advantages and disadvantages of each acquisition technique and the results are discussed. Bright field microscopy proved to be more simple and inexpensive and provided the best overall results.

In this article, it is shown that focused ion beam (FIB) systems can be used to study the inner structure of flax fibers, the use of which as a reinforcing material in polymer composites still draws much interest from multiple disciplines. This technique requires none of the specific preparations necessary for scanning electron microscopy or transmission electron microscopy studies. Irradiation experiments performed on FIB prepared cross sections with very low Ga+ ion beam currents revealed the softer material components of fibers. Thus, it confirmed the presence of pectin-rich layers at the interfaces between the fibers of a bundle, but also allowed the precise localization of such layers within the secondary cell wall. Furthermore, it suggested new insights on the transition modes between the sublayers of the secondary cell wall.

We evaluate the probe forming capability of a JEOL 2200FS transmission electron microscope equipped with a spherical aberration (Cs) probe corrector. The achievement of a real space sub-Angstrom (0.1 nm) probe for scanning transmission electron microscopy (STEM) imaging is demonstrated by acquisition and modeling of high-angle annular dark-field STEM images. We show that by optimizing the illumination system, large probe currents and large collection angles for electron energy loss spectroscopy (EELS) can be combined to yield EELS fine structure data spatially resolved to the atomic scale. We demonstrate the probe forming flexibility provided by the additional lenses in the probe corrector in several ways, including the formation of nanometer-sized parallel beams for nanoarea electron diffraction, and the formation of focused probes for convergent beam electron diffraction with a range of convergence angles. The different probes that can be formed using the probe corrected STEM opens up new applications for electron microscopy and diffraction.

Graphene is a single layer of carbon atoms arranged in a hexagonal lattice. The high carrier mobility and mechanical robustness of single layer graphene make it an attractive material for “beyond CMOS” devices. The current work investigates through high-resolution transmission electron microscopy (HRTEM) image simulation the sensitivity of aberration-corrected HRTEM to the different graphene stacking configurations AAA/ABA/ABC as well as bilayers with rotational misorientations between the individual layers. High-angle annular dark field–scanning transmission electron microscopy simulation is also explored. Images calculated using the multislice approximation show discernable differences between the stacking sequences when simulated with realistic operating parameters in the presence of low random noise.

Magnetic nanocomposite materials consisting of 5 and 10 wt% CoFe2O4 nanoparticles in a silica aerogel matrix have been synthesized by the sol-gel method. For the CoFe2O4-10wt% sample, bright-field scanning transmission electron microscopy (BF STEM) and high-resolution transmission electron microscopy (HREM) images showed distinct, rounded CoFe2O4 nanoparticles, with typical diameters of roughly 8 nm. For the CoFe2O4-5wt% sample, BF STEM images and energy dispersive X-ray (EDX) measurements showed CoFe2O4 nanoparticles with diameters of roughly 3 ± 1 nm. EDX measurements indicate that all nanoparticles consist of stoichiometric CoFe2O4, and electron energy-loss spectroscopy measurements from lines crossing nanoparticles in the CoFe2O4-10wt% sample show a uniform composition within nanoparticles, with a precision of at best than ±0.5 nm in analysis position. BF STEM images obtained for the CoFe2O4-10wt% sample showed many “needle-like” nanostructures that typically have a length of ∼10 nm and a width of ∼1 nm, and frequently appear to be attached to nanoparticles. These needle-like nanostructures are observed to contain layers with interlayer spacing 0.33 ± 0.1 nm, which could be consistent with Co silicate hydroxide, a known precursor phase in these nanocomposite materials.

The three-dimensional (3D) distribution of carbon nanotubes (CNTs) grown inside semiconductor contact holes is studied by electron tomography. The use of a specialized tomography holder results in an angular tilt range of ±90°, which means that the so-called “missing wedge” is absent. The transmission electron microscopy (TEM) sample for this purpose consists of a micropillar that is prepared by a dedicated procedure using the focused ion beam (FIB) but keeping the CNTs intact. The 3D results are combined with energy dispersive X-ray spectroscopy (EDS) to study the relation between the CNTs and the catalyst particles used during their growth. The reconstruction, based on the full range of tilt angles, is compared with a reconstruction where a missing wedge is present. This clearly illustates that the missing wedge will lead to an unreliable interpretation and will limit quantitative studies.

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.

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 http://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].