X-ray microanalysis of any type of specimen in its natural state without the use of conventional SEM specimen preparation techniques has immense potential in a wide range of scientific and industrial applications. This capability would be particularly useful in microanalysis applications where it is highly desirable to preserve the integrity of the specimen, for example in semiconductor failure analysis and forensic investigations. in principle, this X-ray microanalysis goal can be achieved in an environmental or variable pressure scanning electron microscope (VPSEM) because specimen charging and vacuum stability problems are negated by the presence of a gas in the specimen chamber. However, the accuracy and spatial resolution of X-ray microanalysis in the VPSEM is significantly degraded by the chamber gas as it scatters primary beam electrons, generating spurious X-rays far from the analysis point. to date, two different X-ray measurement strategies have been developed to facilitate X-ray microanalysis at high chamber pressure in the VPSEM.

The demand for accurate mineralogical data is increasing rapidly as exploration methods, prospect evaluation procedures and metallurgical optimisation studies become more sophisticated. in response to these needs, semi-automated and automated image processing systems which detect minerals using optical microscopy, scanning electron microscopy or electron microprobe microanalysis (EPMA) are becoming increasingly important tools in the exploration, mining and mineral processing industries. CSIRO Minerals has developed an EPMA based imaging (or mapping) method for characterising ilmenite concentrates. The method uses a JEOL 8900R EPMA to collect elemental x-ray maps which are then processed using in-house developed software, Chimage. The mapping procedure differs from traditional automated identification systems in that no detailed a priori knowledge of the mineral phases is required. in addition, Chimage software enables complete processing and interpretation of the data set off-line. Elemental data can be displayed in either scatter or ternary diagrams showing clusters which allow mineral phases to be identified.

This work proposes a new consistency check total based on measurement of bremsstrahlung intensity which can be used to validate analyses even when concentrations have been normalised to 100%.

If elemental content of a material is unknown, spectral peaks have to be identified, either interactively, or by automatic procedures. When peak overlap is severe (e.g. Si K/ Ta M / W M, Al K/Br L), it is easy for an element to be misidentified or missed altogether and at low beam kV, some elements will not even produce a characteristic line suitable for analysis. When element identification or quantification requires deconvolution of closely spaced lines, minor miscalibration or inaccurate peak modelling produces inaccurate peak area estimates, Ai, instead of true peak areas, Ai*, and consequently inaccurate concentration estimates, Ci* . A common consistency check is to confirm that the analysis total is close to unity:

The mechanism for assembly of membrane lipid bilayers in vivo is generally considered to occur on existing membranes which act as templates for the assembly process. Not presently understood is the source of the membrane template. A thermodynamic theory describing a spontaneous process for assembly of unilamellar vesicles with properties of a critical state (1,2) has been tested in this study. The spontaneous formation of large unilamellar vesicles (LUV’s) from phospholipid components of cell membranes at a critical temperature T* has now been documented by cryo-electron microscope studies using aqueous dispersions of dimyrystoylphosphatidylcholine (T* = 29°C), and the total lipid extracts of E. coli membranes cultured at 32°C (T* = 32°C). The lipids were incubated at temperatures below, at and above the critical temperatures for LUV assembly. in conformity with thermodynamic theory, and other physical measurements that indicate unique bilayer properties exist at T* (3-5), LUV’s are seen to form only at T*.

Structured latex coatings find such applications as specialty paints, micro- and ultra-filtration membranes, templates for synthesizing inorganic complementary structures, and embedding media for whole-cell based bioreactors. to control well the microstructure and other properties of the final coatings requires understanding the film formation process that transforms a deposited layer of colloidal particles of polymer into a coherent solid coating as it dries, usually in air. This process cannot be visualized by light microscopy techniques when the latex particles are a few hundred down to a few tens of nanometers in size, as is normally the case. Conventional electron microscopy requires too high vacuum for liquid samples to survive. Environmental scanning electron microscopy tolerates modest partial pressure of solvent but is limited to a coating’s top surface and at low resolution. What is needed is to observe the interior of a drying, hydrated coating at nm-resolution. Cryo-SEM is a powerful means of studying microstructure evolution throughout a coating’s thickness in the wet and moist stages of film formation.

Low temperature scanning electron microscopy (LTSEM) avoids many of the artifacts associated with chemical fixation, dehydration and critical point drying. As a result, this technique has had numerous applications ranging from biology to hydrology. Unfortunately, experiments using LTSEM are frequently compromised by the limited sample manipulations that are available on most cryo-stages and by the cost of specimen holders. Whereas the conventional SEM stage enables 360° rotation and tilts as great as 90°, the braids or hoses required for cooling a cryo-stage generally limit rotation to about 30° and tilt to less than 40° from normal. Furthermore, the specimen holders for LTSEM may cost several hundred dollars each, thereby prohibiting experiments that require numerous holders. This study describes an inexpensive specimen holder that solves these problems.

Figure la illustrates a drawing of the specimen holder supplied with the standard transfer device from the Oxford CT 1500 Cryotrans System.

Cryo-techniques have been successfully used in scanning electron microscopy (SEM). They are especially promising for high-resolution SEM to improve specimen preservation and reduce radiation damage [1, 2]. A number of cryo-preparation systems are commercially available for SEM, however, our experience has shown that modifications are needed to perform highresolution imaging (>50,000x).

Emitech K1250 system consists of a sample preparation chamber, control unit, and cryo-stage. Magnetron sputter coating is standard and electron-beam evaporation is optional. A vacuum transfer device facilitates the sample transfer between the preparation chamber and the SEM to prevent contamination. The Emitech cryo-stage replaces the Hitachi S-4700 standard stage and the cryo-stage temperature is monitored and controlled by the Emitech control unit.

The specimen is mounted on a sample holder that mounts to the cryo-stage. Therefore, the distance from the specimen to cryo-stage, the thermal capacity of the sample holder, and the thermal contact between them will affect the actual temperature of the specimen.

Creating contrast of unstained frozen hydrated cells and isolated macromolecules in order to provide high fidelity recordings present several difficulties and therefor require attempts to maximize image information for characterization purposes. Adobe Photoshop 5.0 was employed to overlay (“layer“) topographic high resolution secondary electron (SE-I) images (HRSEM) onto STEM images with and without polarity reversal employed during digital image acquisition. Cells and molecular samples precipitated from an aqueous droplet or aerosol and supported on carbon and holey carbon coated grids were plunge frozen and Chromium (Cr) coated. Since Cr-coated cryosamples on carbon-grid supports have very low mass density and because signal to noise is low in HRSEM recordings, especially in regions which overhang holey carbon expanses, then the combined contrasts obtained in a composite image containing STEM and HRSEM contrasts result in greater detail recognition.

Tandem cryo-imaging, using an in-lens field emission (FE) SEM (DS-130F) operated at 25kV and fitted with above-lens secondary electron detector and below-lens scanning transmission detector, provided digital images of frozen cells and macromolecular complexes.

The recent increase in use of Field Emission Scanning Instruments in biology has shown the potential of these systems for sub-cellular imaging, as they essentially provide transmission EM levels of resolution for surface imaging. in structures lacking inherent contrast in thin sections, such as nuclear pore complexes,(NPC) high resolution surface imaging has provided significant complementary structural information to the macromolecular architecture of the NPC. (Goldberg and Allen, 1996). A series of intermediate structural forms of the NPC during its formation has also been described using FESEM, which would not have been resolvable in TEM.(Goldberg, Weise, Allen, Wilson, 1997). All this work was performed with chemical fixation, dehydration and critical point drying, followed by high resolution Chromium coating. We now are extending these studies to a cryo-preservation approach, mindful of the requirement of rapid freezing to preserve the more labile events of membrane dynamics, and also to circumvent the requirement to expose membranes to organic solvents during conventional dehydration.

Water makes up 60-80% of the wet weight of articular cartilage. The remaining parts of this tissue are made up of a variety of cells, generally referred to as chondrocytes, and an extracellular matrix of, collagen fibrils (types II, IX and XI), proteoglycans. The collagens and proteoglycans, with associated water, are assembled into a structure that is capable of withstanding considerable loading.

The micro-structure of adult rabbit articular cartilage has been described, based upon ultrastructural studies. The tissue is arranged into zones, mainly based on the general orientation of collagen fibrils (See Figure 1). The collagen framework in the radial zone forms a continuous area of radially oriented and tightly bound collagen fibrils, in which are embedded at regular interval, tubular inclusions of proteoglycans (Figure 2). in rabbit tibial plateau tissue these inclusions have diameter of 1-3 fim and can be continuous from the calcified to the tangential zone.

Imaging of fast frozen samples is the most direct approach for electron microscopy of biological specimen in a defined physiological state. It prevents chemical fixation and drying artifacts. High pressure freezing allows for ice-crystal-free cryo-fixation of tissue pieces up to a thickness of 200 urn and a diameter of 2 mm without prefixation. Such a frozen disc, however, is not directly amenable to electron microscopic observation: The structures of interest have to be made amenable to the electron beam, and the structures of interest must produce enough contrast to be recognized in the electron microscope. This can be achieved by freeze fracturing, cryo-sectioning or freeze substitution.

The figures show high pressure frozen bakers yeast saccharomyces cerevisiae in the cryo-SEM (Figures 1 and 2) and after freeze substitution in the TEM (Figure 3). For high pressure freezing either a Bal-Tec HPM 010 (Princ. of Liechtenstein; Figures 1 and 2), or a Wohlwend HPF (Wohlwend GmbH, Sennwald, Switzerland; Figure 3) were used.

It is well known that hemodynamic factors, such as blood pressure and flow, exert an important influence on native morphology of various animal organs. However, information about the significance of ultrastructures which are revealed by conventional preparation methods has been limited, because routine chemical fixation of cells and tissues takes considerable time, during which their morphology could easily change. Therefore, morphological studies with routine immersion or perfusion fixation have not revealed real ultrastructures of functioning animal organs with normal blood circulation.2 The ultimate goal of morphological study is that all features to be examined should reflect the physiological meaning under investigation. For that purpose, the preservation of cells and tissues in functioning organs is necessary for studies to define their ultrastructures. Most cryotechniques have been based on the use of prior excised tissues. Such small specimens are commonly frozen within several seconds following the excision of the tissues.

It is well known that flowing erythrocytes in blood capillaries were morphologically changing in vivo, as observed by light microscopy. Recently, dynamic morphological changes of flowing mouse erythrocytes in large blood vessels and hepatic sinusoids were demonstrated by scanning (SEM) or transmission (TEM) electron microscopy with our “in vivo cryotechnique”. Moreover, human erythrocyte deformability was already studied under artificially jetting conditions at different pressures by using “in vivo cryotechnique”, followed by freeze-substitution method for SEM. in this study, we have analyzed elemental changes of each human erythrocyte at different jetting pressures by “in vivo cryotechnique” combined with SEM for X-ray microanalysis.

Human blood was collected with heparin-coated syringes, divided into two groups and kept at 4°C and 36°C. They were directly jetted into isopentane-propane cryogen (-193°C) through tubes (21 gauge) at different pressures (0-220mmHg) (Fig.la). The frozen blood samples were freeze-dried (4-6×10-7torr,-95°C,24h) in Eiko FD-3AS apparatus (Eiko Engineering, Japan) (Fig. lb).

Cryo-scanning electron microscopy (cryo-SEM) and cryo-transmission electron microscopy (cryo- TEM), in conjunctions with rheological measurements, light and confocal microscopy, x-ray scattering, and solid state NMR, are used to characterize sodium stearate (NaSt) crystals dispersed in waterpropylene glycol (PG) mixtures at macroscopic, microscopic, molecular, and atomic levels. NaSt is a surface-active, structural agent in household and personal cleaning products, including deodorant sticks and soap bars. A better structural characterization of NaSt/PG/water systems has practical importance in personal care and cosmetic industries. NaSt crystals and other soap crystal morphologies have been studied by the TEM/replica technique. However, the replicas were made of the residue after the original sample or its aqueous dilution were dried, and the original structure may have been lost during drying. Cryo-SEM was not used to study NaSt crystals because of its lower resolution and because the crystals are highly susceptible to radiation damage by electron beam.

Conventional scanning and transmission electron microscopy (SEM and TEM) of the rhizosphere (soil around plant roots) has been used to describe the spatial organization of biotic (microorganisms including bacteria and fungi) and abiotic (organomineral complexes and clay platelets) constituents in soil.1 However, soil is deeper than plant rootS2 and the spatial distribution of microbes, the morphology of the microbial growth habit, and the spatial association of microbes with mineral substrates in the deeper vadose zone are little known. That subsurface terrestrial microbes catalyze most major nutrient-cycling reactions in the terrestrial biosphere3 punctuates the need to observe the true morphology of bacteria in association with mineral surfaces and microbemicrobe associations. Here, we present our use of environmental scanning electron microscopy (ESEM) to image bacteria on unsaturated sand, a simple surrogate for heterogeneous soil. ESEM is a choice tool for high resolution imaging of combined soft and hard matter, where delicate hydrated expolymer matrices bind microbes to each other and to soil particles as biofilm.4

The nucleocapsid of herpesviruses is assembled within the nucleus and transported to the perinuclear space by budding through the inner nuclear membrane. The route from the perinuclear space to the plasma membrane for exocytotic release is assumed to involve loss of the acquired envelope by fusion with the outer nuclear membrane followed by wrapping of the nucleocapsid by Golgi membranes. Alternatively, virions are thought to leave the perinuclear space via vacuoles originating from the outer nuclear membrane. Non of these processes has been shown so far. We thus examined the nuclear periphery of MDBK cells infected with BHV-1 by cryobased electron microscopy. Infected cells were high-pressure frozen at 4, 5 and 6 hours of incubation, and freezesubstituted employing a protocol yielding high resolution of membranes.

Thin sections stained with uranyl acetate and lead citrate showed 3 distinct stages of viral envelopment at any time of incubation.

The cytoplasm of the bacterium Thiobacillus neapolitanus contains 117 nm diameter polyhedral inclusions, “carboxysomes” (Fig. 1) that contain ribulose-1,5- bisphosphate carboxylase/oxygenase (RuBisCO). Surrounding the polyhedron are nonmembranous proteinaceous plates devoid of lipid. The carboxysomes are composed of at least 8 major peptides, all coded within the same operon. Six (CsoSIA, CsoSIB, CsoSIC, CsoS2A, CsoS2B, and CsoS3) make up the shell, and two are the large (CbbL) and small subunits (CbbS) of RuBisCO. Using immunogold labeling on ultrathin sections, peptides CsoS2A, CsoS2B, and CsoS3 have been localized to the shell. Since the original characterization of the csoSl gene, we have also immunolocalized the CsoSl peptide to the shell.

As part of our initial efforts to understand how these components are assembled into the symmetrical, functional entity, the carboxysome operon from T. neapolitanus was cloned into the pET-21a(+) plasmid, an expression vector that codes for resistance to ampicillin.

During a chromosome behavior study involving high-pressure freezing (HPF)/freeze substitution (FS) of Drosophila melanogaster testes, we discovered quasi-crystalline inclusions in the nuclei of adjacent gut epithelial cells (Fig. 1). The HPF and FS protocols were standard. The viscera of adult flies were packed in yeast paste for HPF. The tissue was fixed by FS with 1% osmium tetroxide in acetone for 72 hours at -90° C then 48 hours at -60° C. Afterwards, it was washed several times at room temperature in 100% acetone and embedded in Epon/Araldite. Thin sections were cut and stained with uranyl acetate and lead. As expected with HPF/FS, the material was well-preserved with straight microtubules, smooth membranes, dense mitochondria, and abundant ribosomes both on the rough endoplasmic reticulum and in the full cytoplasm (Fig. 1).

The inclusions consisted of virus-like particles packed loosely together in orderly arrays. Particles were usually hexagonally packed with spaces disrupting the periodicity (Figs. 2 and 3).

Caliciviruses (CacV) including Norwalk like virus and Sapporolike virus, papillomaviruses (PapV, and parvovirus B19 (B19V) are infectious viruses known to cause a variety of diseases. These viruses are distinctive because of their low titers in body fluids and fastidious nature to culture, making them difficult to detect by EM or serology. The genomes of several CacVs, PapVs, and B19Vs have been characterized facilitating expression of specific proteins via recombinant technologies. Recombinant proteins may be useful for obtaining the antibodies to be used in improved immunoassays and vaccines for preventing diseases associated with these viruses. CDC and Emory University investigators have designed techniques by which capsid proteins for a variety of viruses including CaCV, PapV, and B19V were expressed in Autographies califomicanuclear polyhedrosis virus (AcNPV) infected Spodoptera fruigiperda (Sf9) cells. Those proteins self-assembled into virus-like particles (VLPs)(Figs.l- 4). Culture-expressed capsids were “purified” and concentrated by traditional protein purification techniques including gel filtration, sucrose gradient centrifugation and cesium chloride gradient centrifugation.

Frog virus 3 (FV3), a member of the genus Ranavirus (family Iridoviridae), was isolated from the leopard frog Rana pipiens. Members of this genus are recognized pathogens of amphibians, reptiles and fish and have been associated with declines of amphibians in the United Kingdom and the USA. Neutron-scattering studies showed that FV3 virions are composed of four concentric layers: an outer lipid envelope containing protein VP58; an icosahedral capsid shell with major capsid protein VP48; an inner lipid membrane with VP63 and 44; and a central core of dsDNA (∼170 kbps) and associated proteins. The FV3 genome has a terminally-redundant and circularly-permuted sequence. Naked virions (i.e. without the envelope lipid) contain more than 20 different proteins and about 9% lipid, which is required for infectivity. to date, structural information about FV3 has primarily been obtained in electron microscopy studies by means of thin-sectioning and negative staining techniques.