Single stained DNA molecules can be visualized by fluorescence microscopy (1). Therefore, perhaps single events of DNA metabolism can also be visualized by fluorescence microscopy. Problems to solve include (a) photobleaching of stained DNA, (b) photolytic damage to macromolecules necessary for metabolism, including DNA, (c) inhibition of metabolism by the process of preparation of the specimen, and (d) interference with image formation by macromolecules that either must be or by chance are present during metabolism. These problems have been solved, at least in part, during a study of the in vitro packaging of bacteriophage T7 (2). Packaging occurs in T7 capsids that had previously been assembled without assistance of DNA. The DNA substrate for packaging is a head-to-tail multimer (concatemer) of the terminally repetitious, nonpermuted T7 DNA. The metabolic event observed in ref. 2 was capsid-dependent specific cleavage of concatemers during packaging. The concatemers had formed a DNA network before cleavage.

The experimental approaches available to study the structure of most membrane proteins are limited. X-ray crystallography or electron crystallography rely on the availability or creation of 3D or 2D crystals to obtain high resolution structural information of membrane proteins. Single particleimaging of detergent solubilized protein is also possible, but removes the protein from its native environment. Torecreate suchan environment, we have developed a method which permits membrane proteins to be visualized in a lipid bilayer support.

A copper grid overlaid with a holey plastic film is vertically submerged into a lipid monolayer (Fig.l). This action creates planar lipid bilayers spanning 1 to 10 μm holes of the plastic film. These synthetic membranes have a thickness of approximately 40 Å, comparable to literature values for various cell membranes and are reasonably stable under a defocused beam in a transmission electron microscope (TEM)(Fig.2). Proteoliposomes with appropriate proteins in their membrane are added to the still-wet fresh lipid bilayers such that the liposomes fuse with the planar membrane (Fig.3).

It is increasingly suspected that negative staining should be capable of imaging protein structure at much higher resolution levels than are usually achieved. The heavy metal salts traditionally used as negative stains give structural information only to 20-25Å (2.0-2.5nm); this limit probably is caused by several defective properties of these compounds. If new reagents without these defects can be identified, then higher resolution details should be imaged. The finding that some light atom salts can be used successfully for negative staining greatly increases the range of candidate compounds available for evaluation.

A covalently modified monosaccharide, glucose-6-phosphate, recently was shown to function as an unconventional negative stain. This natural organic reagent directly combines the desirable structurepreserving actions of glucose with added scattering from phosphorus and potassium atoms. It is not known if sugar derivatives containing other light atoms in place of phosphorus can serve as a negative stain.

The Ehrlichia spp. are tick-borne pathogens that infect leucocytes and erythrocytes of human beings and animals (1-4). Three ehrlichial organisms, E. chaffeensis, E. equi, HGE (human granulocytic ehrlichiosis agent, equivalent to E. phagocytophila and closely related to E. equi), and Anaplasmamarginale were studied with respect to their morphology after using a combined fixative/potassium permanganate staining protocol (5). Cell cultures infected with the respective organisms were fixed and treated as follows: Tissues were initially fixed with Karnovsky's fixative [glutaraldehydeparaformaldehyde, 2.5/4.0% respectively], containing 0.1% tannic acid, in a Sorenson's phosphate buffer with 0.1M sucrose. Post fixation with OsO4 or without it occurred in some samples. En bloc staining with 1% aqueous uranyl acetate was always done. Dehydration was in a graded ethanol series followed by embedding in Spurr's™ resin. Sample sections were stained for 30-60 minutes at RT with 1% potassium permanganate. These were then washed for 5-15 seconds with 0.025-0.05% sodium citrate, jet washed with dH20, dried and examined (6).

Confocal microscopy is revealing associations between the internal organization of the nucleus and tissue architecture and function. Such associations may exist which are too subtle or complex for visual observation or quantitative analysis may be required, for example as input data to mathematical modeling of cellular processes. In these circumstances, it is necessary to perform the analysis using computer algorithms. We have developed 3D image analysis (IA) algorithms for segmenting nuclei from within intact tissue specimens, measuring the structure of the nuclei and for segmenting specifically- labeled punctate entities within nuclei. In this study we developed algorithms for measuring the spatial organization of the two copies of a specific DNA locus (labeled using fluorescence in situ hybridization (FISH) ) inside diploid nuclei and with respect to the nuclear organization of the tissue.

For each segmented nucleus, its center of mass (CoM) was determined, which informed us about its position in the tissue.

The high concentration of low-density lipoprotein in the plasma is the major risk factor of atherosclerosis. On the other hand, another plasma lipoprotein—high-density lipoprotein (HDL) — is inversely correlated with atherosclerosis. Recent studies have demonstrated that HDL mediates the transport of cholesterol from peripheral tissues to the liver. However there is considerable debate about the mechanisms by which high density lipoprotein removes excess cholesterol from cells. Two different pathway were suggested: (i) a docking receptor promoting cholesterol translocation, or (ii) a receptor mediated intracellular endosomal pathway termed “retroendocytosis”. In the present study, we performed immunofluorescence and electron microscopy to directly visualize the uptake of HDL using cholesterol-load rat aortic endothelial and smooth muscle cells.

Endothelial cells were obtained from rat aorta and cultured in medium under 5% CO2 / 95% air atmosphere. Then confluent monolayers of endothelial cells were incubated in cholesterol for 48 hr. The cells were precooled for 2 hr at 4°C with PBS containing HDL-Dil or HDL-gold (10 nm) at a concentration of 80 j± g protein per ml PBS. Internalization experiments were carried out by incubation at 37°C for 0, 5, 15, and 30 min, followed by three washes with PBS (pH 7.4). Then the cells were processed for fluorescence and transmission electron microscopy.

Chemically functionalized metal cluster compounds have demonstrated important advantages over colloidal gold as biological microscopy labels. They are covalently cross-linked to the targeting biomolecule, and therefore may be conjugated to a wide range of molecules which cannot be labeled with colloidal gold. The 1.4 nm Nanogold® cluster has been conjugated to peptides, lipids and oligonucleotides, some of which have been proposed as elements of novel molecular wires and novel materials. Dissociation of colloidal gold particles from the conjugate probe, a source of error in quantitative immunogold studies, is greatly reduced by covalent cross-linking. Nanogold® is an uncharged molecule, and because its surface is completely coordinated by organic ligands, nonspecific binding is greatly reduced. Nanogold® conjugates also show greatly enhanced penetration into cells and tissue sections. However, gold probes larger than Nanogold® are desirable for improved visualization in specimens with electron-dense regions or staining, or for applications such as double labeling studies with different sized gold labels, or visualizing wider antigen distributions.

In the fibroblast cell line, 3T3, lysophosphatidic acid (LPA) induces stress fiber formation. Stress fibers participate in physiological functions such as cell motility. LPA acts through a receptor coupled to a PTX-insensitive G-protein, G13. It was shown that a constitutively activated mutant of α13 (Q226L) induces stress fiber formation in Swiss3T3 cells through a second messenger cascade that involves a monomeric G-protein, Rho. The recently discovered guanine nucleotide exchange factor, p115 RhoGEF (p115) forms a link between a n and Rho A. In the presence of α13, p115 activates Rho. The N-terminus of p115 contains a regulator of G-protein signaling (RGS) box. RGS proteins act as negative regulators of G-protein dependent signaling by increasing GTPase activity and “locking” the G-protein in an inactive state. We have tested a role of p115 in the pathway coupling the LPA receptor to stress fiber formation by Rho in NTH-3T3 cells.

Taurine is transported from Purkinje cells into perineuronal glial cells during hypoosmotic swelling and is lost first from pyramidal cells during hypoxia/ ischemia, suggesting in situ cellular redistribution. Cultured isolated astrocytes or neurons and slices from rat hippocampus were treated first in 290 mOsm saline (ISO), then in 200 mOsm (HYPO) with or without taurine for 15 and 60 minutes.

Cells and slices were then fixed in 80 mM phosphate buffered (pH 7.4) fixative containing 1.25% paraformaldehyde and 2.5% glutaraldehyde followed by immuno-cytochemistry .Immunostaining was done either directly on the cell layers or on 1 micron araldite/eponate sections (Pelco). After incubation with antibody to taurine, GFAP, SNAP-25, or normal serum, immunoproduct was visualized using step-avidin-DAB (DAKO:LSAB+). Measurements of pyramidal cell diameters were made on toluidine blue-stained sections containing cells with prominent nucleoli.

The data suggest that taurine is initially lost from pyramidal cells within 15 minutes in HYPO to maintain normal cell volume, while taurine efflux from glia occurs over a 60 minute time peroid.

Tephritidae are the true fruit flies, which include the Mediterranean fruit fly, Ceratitis capitata (Wiedemann), the walnut husk fly, Rhagoletis completa Cresson, the Mexican fruit fly, Anastrephaludens (Loew), and the apple maggot fly, Rhagoletis pomonella (Walsh). Fruit fly infestation causes major damage to many economically-important food crops and is of major concern in agriculture worldwide.

Tephritidae are controlled primarily by repeated applications of organophosphate pesticides. Health concerns, environmental pollution, and costs associated with pesticide use press researchers to find new methods for control. One approach to finding new control methods involves investigating the associations that exist between tephritids and microorganisms. Currently, bacterial odors are being used to lure these pest tephritids into traps. These flies are specifically attracted to odors from certain bacteria found to inhabit their gut. Data suggest that these bacteria are likely involved in important aspects of fruit fly nutrition and reproduction.

Basic fibroblast growth factor (bFGF) is thought to play an important role in normal tissue repair and wound healing. It is a potent mitogenic and chemotactic factor for fibroblasts. To some degree bFGF regulates proliferation and extracellular matrix (ECM) production by these cells. In this study we investigate (i) the distribution of bFGF in fibroblasts and ECM of supporting tissues (ii) the subcellular location of this protein in vivo and, (iii) mechanisms for release of this growth factor from fibroblasts.

Spurr's resin embedded ultrathin sections from adult rat sciatic nerve, adrenal gland, kidney; and from normal human lung and skin were labeled with either polyclonal (F3393-Sigma) or monoclonal antibodies (F6162-Sigma, C3316-ZymoGenetics Inc.) specific for bFGF using a two-step immunogold labeling method. Controls were incubated with either an equivalent volume of blocking solution (omitting the primary antibody) or an irrelevant antibody (Factor VIII, anti-Histamine)(Fig. 4).

C57BL/6 mice, along with other mouse genotypes, have served as models of human presbycusis. C57BL/6 mice suffer from progressively severe hearing impairments, attributable primarily to losses of both hair cells (sensory receptors) and spiral ganglion cells (afferent neurons). Their age-related hair cell degeneration in the cochlea begins in the basal turn and progresses to the apex.

Auditory nerves also undergo age-related changes. The mammalian cochlear nerve is mixed, being composed largely of afferent (sensory) fibers and a small population of efferent fibers. Because efferent nerves are primarily cholinergic, we used acetylcholinesterase (AChE) as a marker enzyme for determining both age-related changes in AChE activity of the efferent fibers and also for tracing degenerative patterns in efferent pathways within the organ of Corti.

Twelve C57BL/6 mice of the following ages were used: four young (23 days), four middle-aged (13 months), and four old (22.5 months) mice.

Alport renal disease pathogenesis is characterized by a progressive irregular thickening, thinning, and splitting of the glomerular basement membrane (GBM), which culminates in a focal and segmental glomerulonephritis and progressive loss of glomerular filtration, leading to uremia and death. A mouse model for this disease was produced using a gene targeting approach (Cosgrove et al., 1996). The resulting model displays renal pathology that is very similar to that observed in humans. As matrix accumulation has long been associated with the thickened regions of the GBM, this model provided a means to study the molecular composition and ultrastructural localization of matrix in these rarefied regions of the GBM in the Alport mouse.

We examined three matrix molecules based on preliminary data; type IV collagen α l and α2 chains, laminin-1 and fibronectin. Immunohistochemical analysis showed that while all three of these molecules localize primarily to the mesangial matrix of normal mouse glomeruli, in the Alport glomeruli these molecules seem to be heavily deposited in the GBM.

The class B ,type I scavenger receptor (SR-BI) was the first molecularly well defined cell surface receptor which binds HDL and mediates the selective uptake of HDL cholesteryl ester. It is expressed primarily in liver and steroidogenic tissues. However, the recent studies also suggested a potentially important role of SR-BI in the initial steps of cholesterol efflux in the peripheral tissues. In this study, we have used immunoblotting and immunofluorescence microscopy to study the expression and subcellular localization of SR-BI in the cultured endothelial cells and the smooth muscle cells of rat aorta.

A peptide containing residues 495 to 509 from mSR-BI plus an NH2-terminal cysteine was coupled to hemocyanin. Then the peptide was used to generate mSR-BI495 antiserum in New Zealand white rabbits. The cells from the aortic ring of 1-month-old Spraque-Dawly rats were grown in the dishes containing complete medium (Dulbecco's modified Eagle's medium with 10% fetal bovine serum) and incubated at 37°C in 95% air/ 10 % CO2 atmosphere for 3-4 days.

Each astrocyte forms an extensive network of fine processes within the surrounding neural tissue, interacting extensively with neighboring neurons and blood vessels. Fine glial processes surround synapses and probably modulate synaptic transmission. Glial endfeet on capillaries are responsible for transport of ions and metabolites and possibly control blood flow. Alterations in these fine structures may be of significance in brain function and disease. Glial fibrillary acidic protein (GFAP) is an intermediate filament found in astrocytes of the central nervous system. GFAP is commonly found in the perikarya and processes of protoplasmic and fibrous type astrocytes. Immunohistochemical labeling of GFAP is extensively used as a means of determining the location and shape of astrocytes. However, its labeling pattern varies with brain region (e.g. cortex vs. hippocampus), with cell state (natural vs. reactive astrocytes), and with the specific α- GFAP antibody used. Furthermore, Golgi-stained or dye-filled astrocytes show numerous small appendages or vellate structures that conform to the surrounding tissue and do not stain for GFAP.

Some insects cover themselves with waxy particles including all parts of their body except the eyes. Two examples of insects that produce waxy particles from specialized glands on their abdominal surfaces are the adult stages of the silverleaf whitefly (Bemisia argentifolii) and one of its predators, the dusty wing (Semidalis flinti). In both species, the production of the waxy particles begins at adult eclosion. The silverleaf whitefly, a major plant pest, produces ribbons of waxy material that extrude from the numerous microtrichia that cover the surface of abdominal wax plates. Silverleaf whitefly males have 4 pair of wax plates and females have 2 pair. The adults use their tibia to break off the extruding ribbons, forming semicircular waxy particles about 1 micrometer in diameter. The semicircular waxy particles are composed of a mixture of long-chain aldehydes and alcohols, 34 carbons in length.

For the dustywing predator, the particles are formed by wax producing pores that cover most abdominal surfaces of the adults.

Variability among hairs of humans is dependent on body source and ethnicity. Variability in the hairs of non-human mammals is also dependent on somatic source, but may exhibit a wide range of characteristics among hairs from any given site. Humans do not have secondary guard hairs as defined below, but forensic scientists involved in trace analysis must recognize a different suite of variability that exists when examining animal hairs. Hairs other than primary guard hairs from the dorsum, which are often the only representative hairs illustrated in animal hair atlases, may be present as trace evidence in a forensic case in which the origin of the hair is unknown. An overview of the different grades of animal hair that could be present in a forensic case or natural history inquiry places the secondary, tertiary, etc. guard hairs in a continuum that is based on relative diameters.

Metallurgical failure analysis is generally straight forward in many cases. However, there are times when it is difficult to really discern the fracture characteristics due to surface degradation, improper handling, or subsequent mechanical damage from improper planning.

Dr. Alan Tettleman, a founder of Failure Analysis and Associates, in San Jose, CA, once told me you can not properly conduct any product failure analysis without metallography, and especially without scanning electron microscopy. In many ways, I agree with him. There are obvious times, however, when various physical properties and deformations are apparent. Those are generally when a consultant is not required. As in your field, there are many references that assist the usage and implementation of the scanning electron microscope and energy dispersive x-ray analysis system. Many of the references introduce the procedure and some of the cautionary steps that should be taken in preparation of samples and in the interpretation of the fracture.

Probably an example of every type of material ever manipulated and utilized by man through out history will eventually find its way to a conservator for treatment and preservation. The term “art conservator”, commonly used to describe those that work to protect and preserve the physical remains of mankind's invention and creativity, is a misnomer. We are better described as conservators of cultural materials, as our work involves the preservation of a huge range of materials and artifacts, many of which would not qualify as “art” under its most accepted definition. From the sublime (a Leonardo da Vinci cartoon) to the ridiculous (a 19th century saloon singer's dress made entirely from rattlesnake skins), from the ancient (paleolithic cave paintings) to the new (a pair of Nike® Air Jordans), if they are in a collection somewhere, they are candidates for examination by a conservator.

Despite the variety of materials and artifacts facing us, professional conservators use common approaches to the care of collections and we share the same arsenal of tools.

Recent celebrated criminal cases have raised the public's awareness of forensic science. The advent of DNA technology, in particular, has led to a perception of instrumental techniques that lead to a mathematical probability of guilt that approaches certainty. Mostly passed over in press accounts of forensic operations is the use of the microscope in the examination of physical evidence. By the use of an example from an adjudicated criminal case it is hoped that a greater appreciation is gained of the microscope, that in its various forms, is by far the most widely used instrument in a crime laboratory today.

A middle-aged woman was reported missing by her son after the Memorial Day weekend in 1997. An examination of her apartment by members of the Washington State Patrol Crime Scene Response Team revealed a small amount of blood on her bedding. This bedding, together with other items, was taken into evidence.