A variety of specimens including bacteria, ciliates, choanoflagellates (Salpingoeca rosetta), zebrafish (Danio rerio) embryos, nematode worms (Caenorhabditis elegans), and leaves of white clover (Trifolium repens) plants were high pressure frozen, freeze-substituted, infiltrated with either Epon, Epon-Araldite, or LR White resins, and polymerized. Total processing time from freezing to blocks ready to section was about 6 h. For epoxy embedding the specimens were freeze-substituted in 1% osmium tetroxide plus 0.1% uranyl acetate in acetone. For embedding in LR White the freeze-substitution medium was 0.2% uranyl acetate in acetone. Rapid infiltration was achieved by centrifugation through increasing concentrations of resin followed by polymerization at 100°C for 1.5–2 h. The preservation of ultrastructure was comparable to standard freeze substitution and resin embedding methods that take days to complete. On-section immunolabeling results for actin and tubulin molecules were positive with very low background labeling. The LR White methods offer a safer, quicker, and less-expensive alternative to Lowicryl embedding of specimens processed for on-section immunolabeling without traditional aldehyde fixatives.

To determine if matrix metalloproteinase (MMP)-28 mediates cardiac aging, wild-type (WT) and MMP-28−/− young (7 ± 1 months, n = 9 each) and old (20 ± 2 months, n = 7 each) female mice were evaluated. MMP-28 expression in the left ventricle (LV) increased 42% in old WT mice compared to young controls (p < 0.05). By Doppler echocardiography, LV function declined at 20 ± 2 months of age for both groups. However, dobutamine stress responses were similar, indicating that cardiac reserve was maintained. Plasma proteomic profiling revealed that macrophage inflammatory protein (MIP)-1 α, MIP-1β and MMP-9 plasma levels did not change in WT old mice but were significantly elevated in MMP-28−/− old mice (all p < 0.05), suggestive of a higher inflammatory status when MMP-28 is deleted. RT2-PCR gene array and immunoblotting analyses demonstrated that MIP-1α and MMP-9 gene and protein levels in the LV were also higher in MMP-28−/− old mice (all p < 0.05). Macrophage numbers in the LV increased similarly in WT and MMP-28−/− old mice, compared to respective young controls (both p < 0.05). Collagen content was not different among the WT and MMP-28−/− young and old mice. In conclusion, LV inflammation increases with age, and MMP-28 deletion further elevates inflammation and extracellular matrix responses, without altering macrophage numbers or collagen content.

A sample preparation method has been developed whereby sharp needle-shaped specimens for atom probe analysis are fabricated from multilayer thin films deposited onto silicon substrates. The specimens are fabricated in an orientation such that atom probe composition profiles across the layer interfaces can be determined with atomic-layer spatial resolution, i.e., the layer normals are parallel to the needle axis. The method uses standard silicon etching techniques and focused ion-beam milling. The feasibility and utility of this technique are shown through its application to a NiFe/CoFe/Cu/CoFe-based thin film structure.

Confocal microscopy allows for optical sectioning of tissues, thus obviating the need for physical sectioning and subsequent registration to obtain a three-dimensional representation of tissue architecture. However, practicalities such as tissue opacity, light penetration, and detector sensitivity have usually limited the available depth of imaging to 200 μm. With the emergence of newer, more powerful systems, we attempted to push these limits to those dictated by the working distance of the objective. We used whole-mount immunohistochemical staining followed by clearing with benzyl alcohol-benzyl benzoate (BABB) to visualize three-dimensional myocardial architecture. Confocal imaging of entire chick embryonic hearts up to a depth of 1.5 mm with voxel dimensions of 3 μm was achieved with a 10× dry objective. For the purpose of screening for congenital heart defects, we used endocardial painting with fluorescently labeled poly-L-lysine and imaged BABB-cleared hearts with a 5× objective up to a depth of 2 mm. Two-photon imaging of whole-mount specimens stained with Hoechst nuclear dye produced clear images all the way through stage 29 hearts without significant signal attenuation. Thus, currently available systems allow confocal imaging of fixed samples to previously unattainable depths, the current limiting factors being objective working distance, antibody penetration, specimen autofluorescence, and incomplete clearing.

Aberration correction leads to reduced focal depth of field in the electron microscope. This reduced depth of field can be exploited to probe specific depths within a sample, a process known as optical sectioning. An electron microscope fitted with aberration correctors for both the pre- and postspecimen optics can be used in a confocal mode that provides improved depth resolution and selectivity over optical sectioning in the scanning transmission electron microscope (STEM). In this article we survey the coherent and incoherent imaging modes that are likely to be used in scanning confocal electron microscopy (SCEM) and provide simple expressions to describe the images that result. Calculations compare the depth response of SCEM to optical sectioning in the STEM. The depth resolution in a crystalline matrix is also explored by performing a Bloch wave calculation for the SCEM geometry in which the pre- and postspecimen optics are defocused away from their confocal conditions.

Tannin-based rigid foams of different bulk densities and their carbonized counterparts were investigated for the first time by X-ray microtomography. This method allowed acquisition of three-dimensional pictures of such highly porous materials. Through mathematical treatment of the images, extremely useful physical characteristics such as porosity, fraction of open cells, connectivity, tortuosity, and pore-size distribution were determined as a function of the foam's density. The obtained information was compared with independent data derived from pycnometry measurements and scanning electron microscope image analysis. The agreement was shown to be acceptable in the limit of the accuracy of the laboratory microtomograph (4 μm). Moreover, recalculating properties like permeability were shown to be quite possible based on the results of standard microtomography data.

Nonlinear optical imaging techniques such as multiphoton and second harmonic generation (SHG) microscopy used in conjunction with novel signal analysis techniques such as spectroscopic and fluorescence excited state lifetime detection have begun to be used widely for biological studies. This is largely due to their promise to noninvasively monitor the intracellular processes of a cell together with the cell's interaction with its microenvironment. Compared to other optical methods these modalities provide superior depth penetration and viability and have the additional advantage in that they are compatible technologies that can be applied simultaneously. Therefore, application of these nonlinear optical approaches to the study of breast cancer holds particular promise as these techniques can be used to image exogeneous fluorophores such as green fluorescent protein as well as intrinsic signals such as SHG from collagen and endogenous fluorescence from nicotinamide adenine dinucleotide or flavin adenine dinucleotide. In this article the application of multiphoton excitation, SHG, and fluorescence lifetime imaging microscopy to relevant issues regarding the tumor-stromal interaction, cellular metabolism, and cell signaling in breast cancer is described. Furthermore, the ability to record and monitor the intrinsic fluorescence and SHG signals provides a unique tool for researchers to understand key events in cancer progression in its natural context.

Equations governing the elastic scattering of electrons are applied to the knock-on displacement of atoms along a substrate, yielding analytical expressions for the surface-translation energy, threshold incident energy, and displacement rate. For a surface perpendicular to the incident beam, scattering angles around 90° contribute most to the kinetic energy of surface atoms. Tilting the specimen lowers the threshold incident energy for displacement and leads to anisotropy in the atomic motion but has little effect on the directionally-averaged displacement rate. The rate of beam-induced adatom motion is predicted to exceed that of room-temperature thermal motion when the surface-diffusion energy is greater than about 0.5 eV.

Electron energy loss spectroscopy (EELS), X-ray photoelectron spectroscopy (XPS), and transmission electron microscopy have been used to study iron catalysts for Fischer–Tropsch synthesis. When silica-containing iron oxide precursors are activated in flowing CO, the iron phase segregates into iron carbide crystallites, leaving behind some unreduced iron oxide in an amorphous state coexisting with the silica binder. The iron carbide crystallites are found covered by characteristic amorphous carbonaceous surface layers. These amorphous species are difficult to analyze by traditional catalyst characterization techniques, which lack spatial resolution. Even a surface-sensitive technique such as XPS shows only broad carbon or iron peaks in these catalysts. As we show in this work, EELS allows us to distinguish three different carbonaceous species: reactive amorphous carbon, graphitic carbon, and carbidic carbon in the bulk of the iron carbide particles. The carbidic carbon K edge shows an intense “π*” peak with an edge shift of about 1 eV to higher energy loss compared to that of the π* of amorphous carbon film or graphitic carbon. EELS analysis of the oxygen K edge allows us to distinguish the amorphous unreduced iron phase from the silica binder, indicating these are two separate phases. These results shed light onto the complex phase transformations that accompany the activation of iron catalysts for Fischer–Tropsch synthesis.

Cantharidin, a type of terpenoid, is the blistering agent of blister beetles frequently used in traditional medicine. The isolation and anticancer activity of cantharidin from blister beetles, Mylabris cichorii has been recently reported by us. This study deals with changes in mitochondrial structure and function and understanding their significance in the underlying mechanism(s) in cantharidin-mediated antitumor effects in Dalton's lymphoma (DL) bearing mice. Cantharidin treatment caused the appearance of abnormal mitochondrial features which included roundish mitochondria with thickened membranes, irregularity in cristae, and appearance of small and large size vacuoles in mitochondria of DL cells. Cantharidin treatment resulted in a decrease in mitochondrial reduced glutathione, succinate dehydrogenase activity, mitochondrial membrane potential, and induced apoptosis and necrosis in DL cells. The decrease/release of mitochondrial cytochrome c were also observed after cantharidin treatment. Flow cytometry-based cell cycle analysis showed a time-dependent accumulation of the sub-G0 population of DL cells, thus, confirming the involvement of apoptosis in tumor cells in cantharidin-mediated antitumor activity. These finding signify that the apoptosis induced by cantharidin in DL cells should involve mitochondrial-dependent pathways. It is suggested that these cantharidin-mediated changes in mitochondria may play a crucial role in its antitumor activity.

Scanning transmission electron microscopy (STEM) of specimens in liquid, so-called Liquid STEM, is capable of imaging the individual subunits of macromolecular complexes in whole eukaryotic cells in liquid. This paper discusses this new microscopy modality within the context of state-of-the-art microscopy of cells. The principle of operation and equations for the resolution are described. The obtained images are different from those acquired with standard transmission electron microscopy showing the cellular ultrastructure. Instead, contrast is obtained on specific labels. Images can be recorded in two ways, either via STEM at 200 keV electron beam energy using a microfluidic chamber enclosing the cells, or via environmental scanning electron microscopy at 30 keV of cells in a wet environment. The first series of experiments involved the epidermal growth factor receptor labeled with gold nanoparticles. The labels were imaged in whole fixed cells with nanometer resolution. Since the cells can be kept alive in the microfluidic chamber, it is also feasible to detect the labels in unfixed, live cells. The rapid sample preparation and imaging allows studies of multiple whole cells.

Originally designed as position-sensitive detectors for particle tracking, silicon drift detectors (SDDs) are now used for high-count rate X-ray spectroscopy, operating close to room temperature. Their low-capacitance read-node concept places them among the fastest high-resolution detector systems. They have been used in a new spectrum of experiments in the wide field of X-ray spectroscopy: fluorescent analysis, diffrac-tometry, materials analysis, and synchrotron experiments such as X-ray holography and element imaging in scanning electron microscopes. The fact that the detector system can be used at room temperature with good spectroscopic performance and at −10°C with excellent energy resolution, avoiding liquid nitrogen for cooling and high-quality vacuum, guarantees a large variety of new applications, independent of the laboratory environment. A brief description of the device principles is followed by basics on low noise amplification. The performance results of a complete detector system are presented as well as some dedicated applications already realized, including use in a surface mapping instrument and use of a “mini-spectrometer” for the analysis of works of art. Fully depleted pn-charge-coupled devices (pn-CCDs) have been fabricated for the European X-ray Multi-Mirror mission (XMM) and the German X-ray satellite ABRIXAS, enabling high-speed, low-noise, position-resolving X-ray spectroscopy. The detector was designed and fabricated with a homogeneously sensitive area of 36 cm2. At −70°C it has a noise of 4 e- rms, with a readout time of the total focal plane array of 4 msec. The maximum count rate for single photon counting was 105 cps under flat field conditions. In the integration mode, more than 109 cps can be detected at 6 keV. Its position resolution is on the order of 100 μm. The quantum efficiency is higher than 90%, ranging from carbon K X-rays (277 eV) up to 10 keV.

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

The increased accessibility of soft-tissue data through diffusible iodine-based contrast-enhanced computed tomography (diceCT) enables comparative biologists to increase the taxonomic breadth of their studies with museum specimens. However, it is still unclear how soft-tissue measurements from preserved specimens reflect values from freshly collected specimens and whether diceCT preparation may affect these measurements. Here, we document and evaluate the accuracy of diceCT in museum specimens based on the soft-tissue reconstructions of brains and eyes of five bats. Based on proxies, both brains and eyes were roughly 60% of the estimated original sizes when first imaged. However, these structures did not further shrink significantly over a 4-week staining interval, and 1 week in 2.5% iodine-based solution yielded sufficient contrast for differentiating among soft-tissues. Compared to six “fresh” bat specimens imaged shortly after field collection (not fixed in ethanol), the museum specimens had significantly lower relative volumes of the eyes and brains. Variation in field preparation techniques and conditions, and long-term storage in ethanol may be the primary causes of shrinkage in museum specimens rather than diceCT staining methodology. Identifying reliable tissue-specific correction factors to adjust for the shrinkage now documented in museum specimens requires future work with larger samples.

Two issues that often impact the cryo-electron microscopy (cryoEM) specimen preparation process are agglomeration of particles near hole edges in holey carbon films and variations in vitreous ice thickness. In many cases, the source of these issues was identified to be the residues and topography often seen in commercially available films. To study and minimize their impact during specimen preparation, an improved holey carbon film has been developed. Rather than using a consumable template based on soft materials that must be removed prior to grid assembly, a method was developed that uses a hard template and a water-soluble release layer to replicate the template pattern into the carbon films. The advantages of this method are the improved purity and flatness of the carbon films, and these attributes are shown to have a dramatic improvement on the distribution of single particles embedded in vitreous ice suspended across the holes. Improving particle distribution is an enabling factor toward increasing the throughput of data collection for cryoEM.

As wood is prone to fungal degradation, fundamental research is necessary to increase our knowledge aiming at product improvement. Several imaging modalities are capable of visualizing fungi, but the X-ray equipment presented in this article can envisage fungal mycelium in wood nondestructively in three dimensions with submicron resolution. Four types of wood subjected to the action of the white rot fungus Coriolus versicolor (Linnaeus) Quélet (CTB 863 A) were scanned using an X-ray-based approach. Comparison of wood volumes before and after fungal exposure, segmented manually or semiautomatically, showed the presence of the fungal mass on and in the wood samples and therefore demonstrated the usefulness of computed X-ray tomography for mycological and wood research. Further improvements to the experimental setup are necessary to resolve individual hyphae and enhance segmentation.

The analytical performance of high-resolution scanning electron microscopy/energy dispersive X-ray spectroscopy (SEM/EDX) for accurate determination of the size, size distribution, qualitative elemental analysis of nanoparticles (NPs) was systematically investigated. It is demonstrated how powerful high-resolution SEM is by using both mono- and bi-modal distributions of SiO2 airborne NPs collected on appropriate substrates after their generation from colloidal suspension. The transmission mode of the SEM (TSEM) is systematically employed for NPs prepared on thin film substrates such as transmission electron microscopy grids. Measurements in the transmission mode were performed by using a “single-unit” TSEM transmission setup as manufactured and patented by Zeiss. This alternative to the “conventional” STEM detector consists of a special sample holder that is used in conjunction with the in-place Everhart–Thornley detector. In addition, the EDX capabilities for imaging NPs, highlighting the promising potential with respect to exploitation of the sensitivity of the new large area silicon drift detector energy dispersive X-ray spectrometers were also investigated. The work was carried out in the frame of a large prenormative VAMAS (Versailles Project on Advanced Materials and Standards) project, dedicated to finding appropriate methods and procedures for traceable characterization of NP size and size distribution.

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.

Alkali pretreatment is a promising pretreatment technology that can effectively deconstruct plant cell walls to enhance sugar release performance. In this study, multi-scale visualization of dynamic changes in poplar cell walls during sodium hydroxide pretreatment (2% w/v, 121°C) was carried out by light microscopy (LM), confocal Raman microscopy (CRM) and atomic force microscopy (AFM). LM observations indicated that swelling occurred primarily in the secondary wall (S) but alkali had little effect on the cell corner middle lamella (CCML). Correspondingly, there was a preferential delignification in the S at the beginning of pretreatment, while the level of delignification in CCML (~88%) was higher than that in the S (~83%) for the whole process revealed by Raman spectra. It also suggested that prolonging residence time to 180 min would not remove lignin completely but cause rapid loss of carbohydrates, which was further visualized by Raman spectroscopy images. Furthermore, AFM measurements illustrated that pretreatment with alkali exposed the embedded microfibrils from noncellulosic polymers clearly, enlarged the diameter of microfibrils, and decreased the surface porosity. These results suggested that there was a synergistic mechanism of lignocellulose deconstruction regarding cell wall swelling, main components dissolution, and microfibril morphological changes that occurred during alkali pretreatment.