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.

Electron tomography has proven to be an invaluable tool for studying the 3-dimensional organization of a wide range of structures, from large cellular processes down to individual macromolecular complexes. An important requirement for electron tomography of thick specimens is the need for selective staining to delineate the structure of interest from other cellular constituents. of particular usefulness in this regard is the method of fluorescence photooxidation, whereby the reactive oxygen generated by a fluorescent compound is used to oxidize diaminobenzidine into a reaction product that can then be visualized with the electron microscope. The main advantages of this method are that not only does it allow for excellent correlated light and electron microscopy, but also the relatively small size of the oxidizing agent used allows for excellent 3-D labeling with high resolution.

This method has proven to be a particularly versatile technique.

A number of recent studies of grain boundaries and heterophase interfaces have demonstrated the power of combining Z-contrast STEM imaging, EELS and first-principles theoretical modeling to give an essentially complete atomic scale description of structure, bonding and energetics. Impurity sites and valence can be determined experimentally and configurations determined through calculations.

Here we present an investigation of the Si/SiO2 interface. The Z-contrast image in Fig. la, taken with the VG Microscopes HB603U STEM, shows that the atomic structure of Si is maintained up to the last layers visible. The decrease in intensity near the interface could originate from interfacial roughness of around one unit cell (∼0.5 nm), or may represent dechanneling in the slightly buckled columns induced by the oxide. Fig. lb, taken from a sample with ∼1 nm interface roughness, shows a band of bright contrast near the interface. This is not due to impurities or thickness variation since it disappears on increasing the detector angle from 25 mrad to 45 mrad (Fig. lc), and is therefore due to induced strain.

The tutorial presents 3D visualization as implemented at NCMI: beginning with a brief overview of the history and philosophy of scientific visualization, proceeding to a description of general methodologies used throughout the field of visualization, and concluding with specific applications in electron microscopy, confocal microscopy and x-ray diffraction. The majority of the tutorial uses biological examples of visualization to demonstrate concepts and tools.

History and Philosophy: Effective scientific visualization presents data in a simple, accurate conceptual formulation. Over the last one hundred years there has been a sea change in its economics caused by digital computers: beginning with calculated tables that were manually plotted, proceeding to two dimensional image graphics, and most recently multivariate 3D interactive graphics. The capability of scientific visualization has been greatly facilitated by the evolution of computers; particularly over the last ten years the 3D visualization of biological structures is quickly becoming a necessity for analysis, conceptualization, and presentations.

Variable pressure scanning electron microscopes (VPSEM) differ from conventional SEM by operating at pressures ranging from the ‘high vacuum’ SEM levels of 10-6 torr up to typically around 2 torr. The environmental SEM or ESEM is a commercial variant which employs an unique multistage pressure-limiting aperture (PLA) system to attain specimen chamber operating pressures of up to 50 torr. Early instruments used air or argon as the imaging gas but more commonly today water vapour is used. A wide range of gases have been employed, including potentially explosive hydrogen-methane mixtures. The choice of gas is operator-based and can be varied during the imaging session.

Early VPESM were restricted to backscattered electron imaging (BSE) until the development of the gaseous secondary electron detector in the ESEM. Gaseous secondary electron detectors are now available for all models of VPSEM and together with compatible cathodoluminescence and EDS XRay detectors, the full range of SEM-based imaging options is present.

The principal distinguishing feature of VPSEM is, of course, that samples can be examined uncoated. Gas-electron interaction generates a positive ion supply that can minimise conventional charging artefacts, in a simple imaging model.

Microscopy has always been concerned with the observation of samples in their natural states. The earliest instruments, optical microscopes, did not interfere with the samples which were under observation due to the pervasive presence of visible light in the normal evolution of these entities. Man and science persisted in this direction until Ruska in 1933 invented the electron microscope and the real world changed forever (see MSA Rudenberg for an enlightening description of the early days of man's understanding of the electron and the subsequent invention of the electron microscope). The earliest electron beam instruments were single vacuum chamber entities. As single chamber designs, every element in the optical path was under vacuum. These included the electron gun, the lens and coils, recording film, and of course, the specimen itself. The purpose of the vacuum was quite obvious...to eliminate the scattering of the electrons due to the presence of gas.

The environmental scanning electron microscope (ESEM™) and variable pressure electron microscope (VPSEM) have become accepted tools in the contemporary electron microscopy facility. Their flexibility and their ability to image almost any sample with little, and often no, specimen preparation has proved so attractive that each manufacturer of scanning electron microscopes now markets a low vacuum model.

The University of Michigan Electron Microbeam Analysis Laboratory (EMAL) operates two variable pressure instruments, an ElectroScan E3 ESEM and a Hitachi S3200N VPSEM. The E3 ESEM was acquired in the early 1990s with funding from the Amoco Foundation and it has been used to examine an extremely wide variety of different materials. Since EMAL serves the entire university community, and offers support to neighboring institutions and local industry, the types of materials examined span a wide range. There are users from Materials Science & Engineering, Chemical Engineering, Nuclear Engineering, Electrical Engineering, Physics, Chemistry, Geology, Biology, Biophysics, Pharmacy and Pharmacology.

When performing X-Ray microanalysis in the ESEM (Environmental Scanning Electron Microscope) or in the VP-SEM (Variable Pressure Scanning Electron Microscope), the operating conditions of the microscope must be optimized. This is to reduce the beam broadening of the incident electrons when they scatter with the gas molecules before entering into the specimen. As a result of this scattering, the incident beam is composed of two parts. The first part of the beam is the unscattered beam and the second part is the scattered beam, named the skirt. in high pressure and long working distances conditions, the diameter of the skirt may extend to several millimeters. in order to show the effect of the skirt on X-Ray generation, a copper strip was placed .5 mm away of the electron beam on a flat Al specimen. The peak to background ratio of the copper line was measured at different pressure (from 25 to 200 Pa) for Air as gas.

Charge contrast imaging (CCI) is a useful new method for imaging sub-micron features in crystalline materials using the unique gas/ion/electron imaging system of the environmental scanning electron microscope (Griffin, 1997; Doehne, 1998). Crystal growth zoning, microfractures, solution boundaries, and areas of chemical alteration or recrystallization can be imaged in a wide range of materials (Griffin, 2000; Watt, et al. 2000). While not fully understood, charge contrast images reflect differences in the ability of materials to accept, store and discharge deposited electrons from the primary electron beam. These differences are expressed, in turn, as contrasts in secondary electron emission from flat samples (e.g. these contrasts are not related to topography, as is usually the case). Charge contrast appears be related to differences in electronic properties which are often controlled by defect density. CCI is also affected by small-scale physical defects (such as microfractures) which appear to affect the distribution and timing of charge buildup and discharge in the sample (Johansen, et al. 1997).

Of all the commercially available scanning electron microscopes which operate at “low vacuum” the ESEM is the most suitable for examining biological samples. in order to maintain samples with liquid water present the specimen chamber must be capable of operating at a pressure of at least 4.6 Torr (611 pascals) of water vapour pressure (the vapour pressure of water at 0°C). Use of lower pressures or a chamber gas other than water vapour will result in evaporation of water from the sample at a rate dependant on the partial pressure difference between the sample and its surrounding environment. Tables of relative humidity as a function of water vapour pressure and temperature are readily available to calculate desired settings for the microscope.

One of the difficulties associated with examining fresh biological material is the need to have the microscope and sample available in the same location at the same time.If sample collection occurs at a site remote from the microscope inevitable necrotic changes will occur before examination can be carried out.

Environmental SEM has greatly extended the utility of SEM as a result of an ESEM's ability to image volatile and insulating samples. However, many institutions have older high-vacuum SEMs that they cannot afford to replace with an ESEM. For this reason, a simple and inexpensive pressure cell has been developed for performing low-vacuum SEM in a standard microscope. Rather than backfilling the entire analysis chamber with gas, a small volume of higher pressure is confined at the sample surface with the use of a pressure cell that has a small aperture for admitting the primary beam and a gas inlet. Figure 1 is a photograph of such a simple pressure cell.

The aperture for admitting the primary beam needs to be small enough to limit the escaping gas to an amount below what the standard SEM can pump and still maintain high vacuum. On the other hand, the aperture should be as large as possible to provide a useful field of view.

In a conventional scanning electron microscope, a thin, grounded conductive coating is applied to specimens that are poor electrical conductors to prevent retarding and deflection of the incident electron beam. in a variable pressure or environmental scanning electron microscope (ESEM), excess charge on the surface of uncoated poorly conducting specimens is balanced using ionized environmental gas. Ionized gas in environmental mode and grounded conductive coatings in conventional or high vacuum mode minimize charging at the specimen surface, however significant charge trapping may still occur in the implanted sub-surface regions of poorly conducting materials. A small fraction (<10-6) of the incident electrons are trapped at irradiation induced or pre-existing defects within the irradiated specimen. The trapped charge induces a highly localized electric field which can result in electro-migration and micro-segregation of charged mobile defect species within the irradiated micro-volume of specimen.