OMNY-A tOMography Nano crYo stage.

For many scientific questions gaining three-dimensional insight into a specimen can provide valuable information. We here present an instrument called "tOMography Nano crYo (OMNY)," dedicated to high resolution 3D scanning x-ray microscopy at cryogenic conditions via hard X-ray ptychography. Ptychography is a lens-less imaging method requiring accurate sample positioning. In OMNY, this in achieved via dedicated laser interferometry and closed-loop position control reaching sub-10 nm positioning accuracy. Cryogenic sample conditions are maintained via conductive cooling. 90 K can be reached when using liquid nitrogen as coolant, and 10 K is possible with liquid helium. A cryogenic sample-change mechanism permits measurements of cryogenically fixed specimens. We compare images obtained with OMNY with older measurements performed using a nitrogen gas cryo-jet of stained, epoxy-embedded retina tissue and of frozen-hydrated Chlamydomonas cells.

OMNY PIN-A versatile sample holder for tomographic measurements at room and cryogenic temperatures.

Nowadays ptychographic tomography in the hard x-ray regime, i.e., at energies above about 2 keV, is a well-established measurement technique. At the Paul Scherrer Institut, currently two instruments are available: one is measuring at room temperature and atmospheric pressure, and the other, the so-called OMNY (tOMography Nano crYo) instrument, is operating at ultra-high vacuum and offering cryogenic sample temperatures down to 10 K. In this manuscript, we present the sample mounts that were developed for these instruments. Aside from excellent mechanical stability and thermal conductivity, they also offer highly reproducible mounting. Various types were developed for different kinds of samples and are presented in detail, including examples of how specimens can be mounted on these holders. We also show the first hard x-ray ptychographic tomography measurements of high-pressure frozen biological samples, in the present case Chlamydomonas cells, the related sample pins and preparation steps. For completeness, we present accessories such as transportation containers for both room temperature and cryogenic samples and a gripper mechanism for automatic sample changing. The sample mounts are not limited to x-ray tomography or hard x-ray energies, and we believe that they can be very useful for other instrumentation projects.

Direct observation of individual hydrogen atoms at trapping sites in a ferritic steel.

The design of atomic-scale microstructural traps to limit the diffusion of hydrogen is one key strategy in the development of hydrogen-embrittlement-resistant materials. In the case of bearing steels, an effective trapping mechanism may be the incorporation of finely dispersed V-Mo-Nb carbides in a ferrite matrix. First, we charged a ferritic steel with deuterium by means of electrolytic loading to achieve a high hydrogen concentration. We then immobilized it in the microstructure with a cryogenic transfer protocol before atom probe tomography (APT) analysis. Using APT, we show trapping of hydrogen within the core of these carbides with quantitative composition profiles. Furthermore, with this method the experiment can be feasibly replicated in any APT-equipped laboratory by using a simple cold chain.

Macromolecular 3D SEM reconstruction strategies: signal to noise ratio and resolution.

Three-dimensional scanning electron microscopy generates quantitative volumetric structural data from SEM images of macromolecules. This technique provides a quick and easy way to define the quaternary structure and handedness of protein complexes. Here, we apply a variety of preparation and imaging methods to filamentous actin in order to explore the relationship between resolution, signal-to-noise ratio, structural preservation and dataset size. This information can be used to define successful imaging strategies for different applications.

Mechanisms of haptoglobin protection against hemoglobin peroxidation triggered endothelial damage.

Extracellular hemoglobin (Hb) has been recognized as a disease trigger in hemolytic conditions such as sickle cell disease, malaria, and blood transfusion. In vivo, many of the adverse effects of free Hb can be attenuated by the Hb scavenger acute-phase protein haptoglobin (Hp). The primary physiologic disturbances that can be caused by free Hb are found within the cardiovascular system and Hb-triggered oxidative toxicity toward the endothelium has been promoted as a potential mechanism. The molecular mechanisms of this toxicity as well as of the protective activities of Hp are not yet clear. Within this study, we systematically investigated the structural, biochemical, and cell biologic nature of Hb toxicity in an endothelial cell system under peroxidative stress. We identified two principal mechanisms of oxidative Hb toxicity that are mediated by globin degradation products and by modified lipoprotein species, respectively. The two damage pathways trigger diverse and discriminative inflammatory and cytotoxic responses. Hp provides structural stabilization of Hb and shields Hb's oxidative reactions with lipoproteins, providing dramatic protection against both pathways of toxicity. By these mechanisms, Hp shifts Hb's destructive pseudo-peroxidative reaction to a potential anti-oxidative function during peroxidative stress.

Phase-contrast imaging in aberration-corrected scanning transmission electron microscopy.

Although the presence of phase-contrast information in bright field images recorded with a scanning transmission electron microscope (STEM) has been known for a long time, its systematic exploitation for the structural characterization of materials began only with the availability of aberration-corrected microscopes that allow sufficiently large illumination angles. Today, phase-contrast STEM (PC-STEM) imaging represents an increasingly important alternative to the well-established HRTEM method. In both methods, the image contrast is coherently generated and thus depends not only on illumination and collection angles but on defocus and specimen thickness as well. By PC-STEM, a projection of the crystal potential is obtained in thin areas, with the scattering sites being represented either with dark or bright contrast at two different defocus values which are both close to Gaussian defocus. This imaging behavior can be further investigated by image simulations performed with standard HRTEM simulation software based on the principle of reciprocity. As examples for the application of this method, PC-STEM results obtained on metal nanoparticles and dodecagonal quasicrystals dd-(Ta,V)1.6Te are discussed.

Minimization of amorphous layer in Ar+ ion milling for UHR-EM.

We present a comprehensive study on the influence of Ar(+) ion milling parameters in the range of low acceleration voltages (0.5-6 kV) and etching angles (3-10(°)) on the quality of standard high resolution Si TEM samples. The quality was assessed by the evaluation of HR-TEM images acquired from real TEM samples considering the thickness of the amorphous layer and the interlocking between crystalline and amorphous parts of the sample created by ion-beam induced amorphization, as well as topographical BSE-SEM investigation of the surface of those TEM samples. Increasing voltage clearly results in increased amorphous layer thickness as well as interlocking. The impact of the etching angle is less significant but still influences the amorphous layer thickness. It has, however, a strong effect on the preparation time, which is inversely correlated to the etching angle. Finally the experimental data were compared to model estimations by TRIM and the Schuhrke-Winterbon approximation, which fitted well to the experimental data for low voltage and angle, but were less accurate for higher voltage and angle. Despite their limitations, the models could reproduce trend and order of magnitude of the data, thus making them a useful tool for estimating the amorphous layer thickness after TEM sample preparation.

Focussed ion beam nanotomography reveals the 3D morphology of different solid phases in hardened cement pastes.

Due to the development of integrated low-keV back-scattered electron detectors, it has become possible in focussed ion beam nanotomography to segment not only solid matter and porosity of hardened cement paste, but also to distinguish different phases within the solid matter. This paper illustrates a method that combines two different approaches for improving the contrast between different phases in the solid matrix of a cement paste. The first approach is based on the application of a specially developed 3D diffusion filter. The second approach is based on a modified data-acquisition procedure during focussed ion beam nanotomography. A pair of electron images is acquired for each slice in the focussed ion beam nanotomography dataset. The first image is captured immediately after ion beam milling; the second image is taken after a prolonged exposure to electron beam scanning. The acquisition of complementary focussed ion beam nanotomography datasets and processing the images with a 3D anisotropic diffusion filter allows distinguishing different phases within the hydration products.

Three-dimensional reconstruction of biological macromolecular complexes from in-lens scanning electron micrographs.

Two helical samples: F-actin and the bacteriophage T4 tail sheath were reconstructed in three dimensions from contrast enhanced (rotational shadowing and negatively stained) in-lens cryo-field emission scanning electron micrographs, using the iterative real-space helical reconstruction method. The F-actin--and bacteriophage T4 reconstructions compare favourably to an atomic model refined against fibre diffraction data and a cryo-electron microscopy reconstruction, respectively. These results show that single-particle methods, developed for macromolecules imaged in the transmission electron microscope can be applied to cryo-field emission scanning electron micrographs data with appropriate symmetry.

Influence of pH on colloidal properties and surface activity of polyglycerol fatty acid ester vesicles.

Certain polyglycerol esters of fatty acids (PGE) form dispersions of uni- or multilamellar vesicles in dilute aqueous solution. These self-assembled aggregates reduce the surface-activity of PGE monomers such that interfacial films may take several hours to form. This is undesirable for processes, which rely on rapid surfactant adsorption, for example foaming. In the present work, we study the effect of pH on the colloidal (size distribution, morphology, surface charge) and interfacial (adsorption kinetics) properties of a commercial, non-purified PGE. Using dynamic light scattering, zeta-potential measurements and cryo-SEM, we show that changing the pH of the dispersion media can cause agglomeration and eventually osmotic rupture of PGE vesicles. The change in dispersion state also impacts the adsorption behavior at the water surface. Direct evidence that destabilized vesicle dispersion are more surface-active is provided by comparing the dynamic surface tension of solutions of different pH. The faster adsorption kinetics at low pH correlate with a remarkably increased foaming power. We suggest that an osmotic shock induced by changes in pH causes vesicles to deform and partially open, so that their hydrocarbon core is exposed to the dispersion media. This energetically unfavorable condition promotes the hydrophobically driven adsorption of surfactant monomers at surfaces and hence stimulates the foaming ability.

Visualizing nuclei in skin cryosections: viable options to 4'6-diamidino-2-phenylindol for confocal laser microscopy.

BACKGROUND: Visualization of nuclei in skin (cryo-) sections is essential for both, rapid overview and reliable orientation within skin samples. Therefore, nuclear staining is a very common counterstain for immunohistochemical studies of human skin as this nuclear staining precisely depicts the cellular distribution within the epidermis. Moreover, it clearly shows the epidermal-dermal border as well as the transition zone between the living and the cornified layers of the epidermis. For standard epifluorescence microscopy, 4'6-diamidino-2-phenylindol (DAPI) is commonly used. For confocal laser scanning microscopy (CLSM), however, DAPI is often not suitable because its excitation maximum is in the ultraviolet (UV) range (Ex(max) 359 nm) when bound to DNA, and UV lasers and the corresponding optics are not part of CLSM standard configuration. METHODS: In order to find an adequate DAPI substitute that is excitable with standard visible light lasers, the following nuclear stains were tested: LOLOt-1 iodide (Ex(max) 565 nm), TOTO s -3 iodide (Ex(max) 642 nm), LO-PROt-1 iodide (Ex(max) 567 nm), SYTO s 84 (Ex(max) 567 nm), SYTO s 85 (Ex(max) 567 nm), SYTOX s Green (Ex(max) 488 nm) and SYTOX s Orange (Ex(max) 547 nm), Propidium iodide (Ex(max) 535 nm). Besides optimal concentration and incubation time, following criteria were also evaluated: photobleaching, background, e.g. cytoplasmic staining of RNA, and sensitivity to different fixation conditions (unfixed, IEM fixation, PLP fixation and PFA fixation). RESULTS: According to these criteria Sytox s Green showed the best overall staining score and can be used for variously fixed skin samples and shows a distinct and stable green nuclear fluorescence.

Cryo-FIB-nanotomography for quantitative analysis of particle structures in cement suspensions.

Cryo-FIB-nanotomography is a novel high-resolution 3D-microscopy technique, which opens new possibilities for the quantitative microstructural analysis of complex suspensions. In this paper, we describe the microstructural changes associated with dissolution and precipitation processes occurring in a fresh cement paste, which has high alumina and sulphate contents. During the first 6 min, precipitation of ettringite leads to a general decrease of the particle size distribution. In the unhydrated cement paste almost no particles smaller than 500 nm are present, whereas after 6 min this size class already represents 9 vol%. The precipitation of ettringite also leads to a significant increase of the particle number density from 0.294*10(9)/mm(3) at t(0min) to 20.55*10(9)/mm(3) at t(6min). Correspondingly the surface area increases from 0.75 m(2)/g at t(0min) to 2.13 m(2)/g at t(6min). The small ettringite particles tend to form agglomerates, which strongly influence the rheological properties. The particular strength of cryo-FIB-nt is the potential to quantify particle structures in suspension and thereby also to describe higher-order topological features such as the particle-particle interfaces, which is important for the study of agglomeration processes.

Pros and cons: cryo-electron microscopic evaluation of block faces versus cryo-sections from frozen-hydrated skin specimens prepared by different techniques.

Over the last two decades, several different preparative techniques have been developed to investigate frozen-hydrated biological samples by electron microscopy. In this article, we describe an alternative approach that allows either ultrastructural investigations of frozen human skin at a resolution better than 15 nm or sample throughput that is sufficiently high enough for quantitative morphological analysis. The specimen preparation method we describe is fast, reproducible, does not require much user experience or elaborate equipment. We compare high-pressure freezing with plunge freezing, and block faces with frozen-hydrated slices (sections), to study variations in cell thickness upon hydration changes. Plunge freezing is optimal for morphological and stereological investigations of structures with low water content. By contrast, high-pressure freezing proved optimal for high-resolution studies and provided the best ultrastructural preservation. A combination of these fast-freezing techniques with cryo-ultramicrotomy yielded well-preserved block faces of the original biological material. Here we show that these block faces did not exhibit any of the artefacts normally associated with cryo-sections, and--after evaporating a heavy metal and carbon onto the surface--are stable enough in the electron beam to provide high-resolution images of large surface areas for statistical analysis in a cryo-SEM (scanning electron microscope). Because the individual preparation steps use only standard equipment and do not require much experience from the experimenter, they are generally more usable, making this approach an interesting alternative to other methods for the ultrastructural investigation of frozen-hydrated material.

Are sweat glands an alternate penetration pathway? Understanding the morphological complexity of the axillary sweat gland apparatus.

To build an effective barrier against the penetration of extrinsic agents is one of the skin's main functions. The barrier properties of the stratum corneum and the epidermis have been subject to extensive studies in the past while the role of skin appendages as possible pathways of penetration are only rarely described. In order to study the possible penetration barriers in these complex appendages, a careful investigation of their morphology and ultrastructure has to be done. Studying the morphology of axillary skin appendages requires clear-cut criteria for the differentiation between eccrine, apocrine and apoeccrine glands. Therefore we studied the distribution of proteins described to be specific for either eccrine or apocrine glands (CD15, CD44, S-100 and milk fat globulin) on axillary skin samples from healthy young adults by immunofluorescence. Additionally, we examined the distribution of cytoskeletal proteins such as cytokeratins (1/10/11, 14, 18) and F-actin. For a more detailed understanding of the possible versatile barrier elements of the axillary sweat glands, we studied the distribution of tight-junction-associated proteins (occludin, claudin 1, claudin 4). The coils and the dermal duct may provide an active barrier built of tight junctions as occludin and claudin 4 are co-localized. However, the intra-epidermal duct did not show any co-localization of the investigated proteins. By combining morphological features as revealed by F-actin staining and the distribution of the above-mentioned proteins, immunocytochemical typing of eccrine and apocrine glands becomes possible. With this tool, we could also confirm the existence of apoeccrine glands and locate them in their 'natural environment'.

Dead but highly dynamic--the stratum corneum is divided into three hydration zones.

Topically applied water exerts mechanical stress on individual corneocytes as well as on the whole stratum corneum (SC), resulting in an alteration of barrier function. In this study we used complete skin biopsies and showed that the SC reacts to water stress as a highly optimized and well-regulated structure against osmotic changes. Following a relatively new cryo-processing protocol for cryo-SEM, it is possible to reliably maintain and investigate the hydrated state of the SC and individual corneocytes after treatment with solutions of different ionic strength. Treatment with distilled water results in swelling of SC cells together with formation of massive water inclusions between adjacent cell layers. Treatment with 5-20% NaCl reveals three different hydration zones within the SC: Corneocytes near the live-dead transition zone can swell to nearly double their thickness. The second zone is the most compact, as the corneocytes here show the smallest thickness variation with all treatments. Within the outermost zone, again a massive swelling and loosening of intracellular filament packing can be observed. We therefore conclude that the SC itself is subdivided into three functional zones with individual water penetration and binding potentials. Since the second zone remains nearly unaffected by water stress, we propose that this zone hosts the functional SC barrier.

From tissue to cellular ultrastructure: closing the gap between micro- and nanostructural imaging.

Structural investigation of tissue biopsies requires the coupling of optimal structural sample preservation with detailed information collected at the light and electron microscopic level. Unfortunately, although cryo-immobilization by high-pressure freezing provides the best structural preservation, it is used routinely only for electron microscopic (EM) investigations, whereas for light microscopy chemical fixation protocols have been established. These chemically invasive fixation protocols have the drawback of introducing unpredictable fixation artefacts. Therefore, comparative histopathological (i.e. light microscopic) and ultrastructural (i.e. EM) results are usually obtained from parallel samples that have not been prepared identically and never by examining exactly the same features in exactly the same, optimally preserved sample. Finally, finding an area of interest for EM investigation within a complex tissue is like searching for a needle in a haystack. To overcome these handicaps, we modified the well-established freeze-substitution technique (FS) to allow us to investigate resin-embedded cryo-immobilized tissue by confocal laser scanning microscopy (CLSM) prior to EM examination. Thus (1) selected cells throughout the whole tissue block can be depicted by CLSM and subsequently (2) selectively prepared by targeted sectioning for follow-up investigation of the identical structure by transmission electron microscopy. This is facilitated by the addition of specific fluorescent dyes during the first FS exchange step. Selective binding properties of various dyes to different cellular structures allow a direct histological description of the tissue at the light microscope level. After embedding and preparation of a blockface, the specimen can first be examined by CLSM. For areas of interest, the depth in the resin block is determined followed by removal of the tissue lying above. Then, the cell layer can be cut into a series of ultrathin sections and examined by EM for determination of the subcellular and nanostructural organization.

Distribution of sunscreens on skin.

The effectiveness of sunscreens was originally achieved by incorporation of soluble organic UV absorbers such as cinnamates and others into cosmetic formulations. Determinations of the sun protection factor (SPF) of emulsions containing different organic UV absorbers clearly indicate that the efficacy depends on the absorption characteristics of each single UV filter substance. Nowadays, micronised pigments such as titanium dioxide or zinc oxide have also been found to be protective against harmful UV rays. Our investigations using optical and electron microscopy proved that neither surface characteristics, particle size nor shape of the micronised pigments result in any dermal absorption of this substance. Micronised titanium dioxide is solely deposited on the outermost surface of the stratum corneum and cannot be detected in deeper stratum corneum layers, the human epidermis and dermis.

Hydration dynamics of human fingernails: an ellipsometric study.

We use spectroscopic ellipsometry to obtain the complex refractive index, ñ=n+ik, of human fingernails. By studying the change of ñ upon hydration and dehydration, we reveal three different time domains with typical time constants of 4, 150, and 3200 min. A simple model that takes into account the presence of one fast and one slow process is fully consistent with the observed hydration and dehydration dynamics. We attribute these processes to "free" water incorporated between the keratin filaments and water more tightly "bound" in keratin complexes, respectively. From our model we determine the hydration profiles of "free" and "bound" water during, both, hydration and dehydration.

Localization of ceramide and glucosylceramide in human epidermis by immunogold electron microscopy.

Ceramides and glucosylceramides are pivotal molecules in multiple biologic processes such as apoptosis, signal transduction, and mitogenesis. In addition, ceramides are major structural components of the epidermal permeability barrier. The barrier ceramides derive mainly from the enzymatic hydrolysis of glucosylceramides. Recently, anti-ceramide and anti-glucosylceramide anti-sera have become available that react specifically with several epidermal ceramides and glucosylceramides, respectively. Here we demonstrate the detection of two epidermal covalently bound omega-hydroxy ceramides and one covalently bound omega-hydroxy glucosylceramide species by thin-layer chromatography immunostaining. Moreover, we show the ultrastructural distribution of ceramides and glucosylceramides in human epidermis by immunoelectron microscopy on cryoprocessed skin samples. In basal epidermal cells and dermal fibroblasts ceramide was found: (i) at the nuclear envelope; (ii) at the inner and outer mitochondrial membrane; (iii) at the Golgi apparatus and the endoplasmic reticulum; and (iv) at the plasma membrane. The labeling density was highest in mitochondria and at the inner nuclear membrane, suggesting an important role for ceramides at these sites. In the upper epidermis, ceramides were localized: (i) in lamellar bodies; (ii) in trans-Golgi network-like structures; (iii) at the cornified envelope; and (viii) within the intercellular space of the stratum corneum, which is in line with the known analytical data. Glucosylceramides were detected within lamellar bodies and in trans-Golgi network-like structures of the stratum granulosum. The localization of glucosylceramides at the cornified envelope of the first corneocyte layer provides further proof for the existence of covalently bound glucosylceramides in normal human epidermis.

Structure and assembly of intracellular mature vaccinia virus: isolated-particle analysis.

In a series of papers, we have provided evidence that during its assembly vaccinia virus is enveloped by a membrane cisterna that originates from a specialized, virally modified, smooth-membraned domain of the endoplasmic reticulum (ER). Recently, however, Hollinshead et al. (M. Hollinshead, A. Vanderplasschen, G. I. Smith, and D. J. Vaux, J. Virol. 73:1503-1517, 1999) argued against this hypothesis, based on their interpretations of thin-sectioned material. The present article is the first in a series of papers that describe a comprehensive electron microscopy (EM) analysis of the vaccinia Intracellular Mature Virus (IMV) and the process of its assembly in HeLa cells. In this first study, we analyzed the IMV by on-grid staining, cryo-scanning EM (SEM), and cryo-transmission EM. We focused on the structure of the IMV particle, both after isolation and in the context of viral entry. For the latter, we used high-resolution cryo-SEM combined with cryofixation, as well as a novel approach we developed for investigating vaccinia IMV bound to plasma membrane fragments adsorbed onto EM grids. Our analysis revealed that the IMV is made up of interconnected cisternal and tubular domains that fold upon themselves via a complex topology that includes an S-shaped fold. The viral tubules appear to be eviscerated from the particle during viral infection. Since the structure of the IMV is the result of a complex assembly process, we also provide a working model to explain how a specialized smooth-ER domain can be modulated to form the IMV. We also present theoretical arguments for why it is highly unlikely that the IMV is surrounded by only a single membrane.