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.

Phaeocystispouchetii virus (PpV0l), first isolated from Norwegian coastal water in 1995, infects and lyses the haptophyte Phaeocystis pouchetii Lagerheim. PpV0l has not yet been assigned to a family. Phaeocystis may play an important role in ocean climate control and PpV0l infection could alter the amount of this haptophyte. The virion contains a dsDNA genome (∼450 kbps) encapsulated within a large icosahedral shell. The most abundant protein has a mass of 59 kDa. More than five minor polypeptides with masses ranging from 30 to 50 kDa have also been identified. We have used cryo-electron microscopy (cryo-EM) and three-dimensional (3D) image reconstruction methods to examine the native morphology of PpV0l.

Purified PpV0l samples were prepared for Cryo-EM as described. Images were recorded on film at 33,000x nominal magnification in a Philips CM300 FEG microscope at 300kV, at 1.0μm to 2.0μm under focus, and with an electron dose of 2200e-/nm2 (Fig. 1A).

The vacuole of yeast, Saccharomyces cerevisae, is equivalent to the mammalian lysosome. Several genes and gene products have been identified in the vesicular transport of soluble hydrolases to the vacuole via an endosomal compartment. This postendosomal stage of vacuolar trafficking delivering biosynthetic, endocytosed, and autophagic/Cvt cargo specifically to the vacuole remains one of the least defined stages of vesicular transport. We have previously developed an immunological screen for mutants that internally accumulate a pre-vacuolar intermediate of the vacuolar protease, Carboxypeptidase Y (CPY), in a PEP4-independent manner while exhibiting normal secretion phenotypes. We hypothesize that such mutants would be defective at endosome to vacuole step of delivery, hence env mutants. in order to morphologically characterize our novel set of mutants we performed electron, fluorescent and Nomarski microscopy analysis.

Preliminary studies revealed pleiotrophic phenotypes with respect to vacuole structure by Nomarski and fluorescent microscopy. FM-464 uptake and staining assays shows punctate staining throughout the cell in mut79 and mut72.

Mule deer are infected with a herpesvirus associated with acute infectious keratoconjunctivitis (IKC) or “pink eye”. in December 2000, many young male mule deer in Wyoming had clinical signs of IKC including corneal edema, mucopuvulent conjuctivitis, blepharospasm, and blindness.

Conjunctival swab samples from 5 mule deer with IKC were processed for negative contrast electron microscopy (NCEM) at the Wyoming State Veterinary Laboratory (WSVL). Homogenized samples from each deer were centrifuged for 20 minutes at 3440g in a Beckman Avanti J-25 centrifuge (Beckman Instruments Inc., Fullerton, CA, USA). The supernatant from each sample was subsequently centrifuged for 60 minutes at 39,800g. The final pellet was resuspended in 210 μl of sterile deionized microfiltered water. 105 μ1 of the virus suspension was mixed with sodium phosphotungstic acid stain containing 84 μl of 4% aqueous phoshotungstic acid, pH 6.8 with NaOH, 420 μl sterile, deionized microfiltered water, and 21 μl 0.1% bovine serum albumin.

Cells of Pseudomonas aeruginosa were exposed to six different heavy metals, Al, Cd, Cu, Mn, Pb, Zn, exposure was done at the same concentrations of 20 ppm, except for the Cd which was done at 5 ppm. The cells were then analyzed using the STEM mode of a transmission electron microscope in conjunction with a PGT IMIX energy dispersive xray spectrometer. The data was then analyzed using a bulk sample analysis program (ZAF method) in standardless mode (w/w). We analyzed the cell wall the cytoplasm and polyphosphate bodies. Approximately 20 cells were analyzed per treatment and averages of the elements present were calculated. The cells of Pseudomonas aeruginosa were grown in nutrient broth and then embedded in Epon according to Luft's procedure. Quantitative analysis of the three different parts of the cell revealed that in cells exposed to Al, the wall contained on average 0(55%), C(20%) P( 8.0%) K(10%) Cl(2%) S(5.0%),Al(2.0%), the cytoplasm contained on average, 0(53%) C(20%) P(5.0%) Ca(4.0%) K(12%) Cl(4.0%) Al(2.0%).

Dissimilatory iron reducing bacteria have been extensively studied for their ability to reduce ferric iron Fe(III) to ferrous iron Fe(II), as well as several multivalent heavy metals and radionuclides as a mode of energy-yielding respiration. Shewanella putrefaciens strain CN32 was used to investigate the mechanism of biogenic metal reduction in systems simulating conditions of natural anaerobic iron reducing environments in the subsurface contaminated with U and Tc as a possible strategy for bioremediation of soils containing these contaminants. As previously reviewed, U(VI) is soluble in most groundwaters, while U(VI) generally precipitates as the insoluble mineral uraninite. Formation of bioreduced minerals can lead to immobilization of these contaminants in the subsurface, which might be a very useful strategy for in situ bioremediation.

To determine the metal reduction and the formation of biogenic Fe(II), U(IV) and Tc(IV) minerals, experiments with CN32 exposed to well-defined aqueous solutions were conducted. Metal reduction was measured with time, and the resulting solids were analyzed by X-ray diffraction, scanning electron microscopy (SEM) and energydispersive X-ray spectroscopy (EDS).

Magnetic bacteria are a group of motile microorganisms that orient to magnetic field lines because of membrane-bound organelles called magnetosomes. Magnetic bacteria precipitate magnetite (Fe3O4) or greigite (Fe3S4) in their magnetosomes. The ability of uncultured magnetic bacteria of accumulating minerals in different cell compartments is still poorly understood. We have been studying natural enrichments of magnetic bacteria from the Itaipu and Rodrigo de Freitas lagoons, in Rio de Janeiro, Brazil. Here, we have used energy-filtering transmission electron microscopy to study the element distribution in biocomposites found in magnetic bacteria from both lagoons. in Itaipu lagoon, electron microscopy and phylogenetic position determination by 16S rRNA genes sequencing identified at least four morphotypes of magnetic bacteria. Typical structures found in all gram-negative bacteria, in addition to some internal compartments were observed (Fig. la). Amorphous granules (Fig. la) and magnetosomes (Fig. lb) were the two well-defined compartments that accumulated significant amounts of minerals.

Multi-purpose microscopy laboratories can be found in a number of different places, including industry, hospitals, government research organizations and universities and the set up and operation of such a laboratory may well be quite different in each of these environments. in this tutorial the issues that are important in the running of a microscopy laboratory will be explored. The main topics include fundamental questions such as whether it is a good idea to have a centralized unit in the first place, how the operation of the laboratory should be funded and what the mix of staff and equipment should be.

Before we talk about how to run a multi-purpose laboratory we should consider the merits of having such a laboratory in a particular institution and whether that unit should be “centralized”. By centralized I mean that it is the preferred option for the institution to have most of the equipment in a central laboratory where it is essentially available to all the interested groups in the organization.

Low Voltage Scanning Electron Microscopy (LVSEM), defined as operation in the energy range below 5keV, has become perhaps the most important single operational mode of the SEM. This is because the LVSEM offers advantages in the imaging of surfaces, in the observation of poorly conducting and insulating materials, and for high spatial resolution X-ray microanalysis. These benefits all occur because a reduction in the energy E0 of the incident beam leads to a rapid fall in the range R of the electrons since R ∼ k.E01.66. The reduction in the penetration of the beam has important consequences. Firstly, volume of the specimen that is sampled by the beam shrinks dramatically (varying as about E05 ) and so the information generated by the beam is confined to the surface of the sample. Secondly, the yield 8 of secondary electrons is increased from a typical value of 0.1 at 20keV to a value that may be in excess of 1.0 at 1keV.

Introduction. Electron diffraction is sometimes considered to be a “difficult subject”. It is certainly one that can not be covered in the space available here. Rather this tutorial will present a few specific aspects of the topic. The topics have been chosen in the hope that they will provide illumination that spreads more widely than just onto the material presented. Several books treat electron diffraction with more generality.

Kikuchi lines Kikuchi lines are of great use in orienting a sample. Unfortunately, in modern microscopes, Kikuchi lines are not seen in selected-area diffraction (SAD). This is because immersion lenses send parallel electrons, from different parts of the sample (like the Kikuchi lines from a flat specimen), to different places in the diffraction pattern. Thus Kikuchi lines are blurred and generally not useful whenever, as in SAD patterns, a large area of the sample contributes to the diffraction pattern.