The formation of abnormal mitosis associated with cancer has been intriguing for many decades. While microtubules had been the focus of previous studies, recent research has focused on centrosomes, microtubule organizing centers which organize the mitotic apparatus during cell division. During normal mitosis centrosomes form two poles but in cancer, centrosomes can form three, four, or more poles, and organize tripolar, quadripolar, and multipolar mitoses, respectively. This has severe consequences for genomic stability because chromosomes are separated unequally to three, four, or more poles. This can result in aneuploidy and gene amplifications with multiple defects in cellular regulation. It can result in malignancy that is accompanied by cell cycle imbalances and abnormal cell proliferation. While radiation and chemical agents are known to damage DNA and can lead to cell cycle abnormalities, the damage of centrosome structure leading to abnormal mitosis deserves also consideration.

Spironolactone bodies (SB) were first described in 1963 by Janigan. These laminated, whorled structures are seen in cells of the adrenal zona glomerulosa in patients treated with the drug spironolactone. Spironolactone is an aldosterone antagonist. Hyperaldosteronism may result from excess production by the adrenal cortex. By both light microscopy and transmission electron microscopy (TEM), SB have a distinctive, laminated appearance. Kovacs, et al. observed that SB are composed of cellular constituents. To our knowledge, SB have not been analyzed using scanning electron microscopy (SEM) and electron probe microanalysis technology.

An adrenal gland with a 1 cm cortical mass was removed from a 39 year old female and received in 10% buffered formalin. Histologic examination of the mass showed a monotonous population of cells recapitulating zona glomerulosa cells. Intracytoplasmic structures compatible with SB were identified. Portions of the adrenal gland were processed for TEM and SEM analysis.

Our goal was to determine the cellular localization of left-handed Z-RNA, within preeguatorial zone (PZ) epithelium of the normal adult dog ocular lens (1.5 yr) (Fig. 1), employing anti-Z-RNA IgG polyclonal antibodies. B-DNA has the ability to adopt the Z-DNA configuration in vitro(1). A-RNA can be transformed into Z-RNA under certain conditions (2). Z-RNA has been localized in cultured cells (3). Strong evidence supports the presence of Z-DNA in vivo (1). Elimination of DNA binding proteins by certain fixatives can initiate DNA supercoiling which stabilizes Z-DNA sequences (1). Z-DNA may play a role in regulating in vivo transcriptional enhancement (1).

Anti-Z-RNA antibody probes were produced in 3 rabbits immunized with injections of Z-RNA: Br-poly[ribosomal(G-C)]. Concerning light microscopy [immunohistochemistry (ABC method)], lens tissues were fixed in Carnoy's, embedded in paraffin and sectioned (3 μm) (Fig. 2). Image analysis was performed using a Leitz DM-RB microscope and Leica Quantiment 500 + image analyzer.

The objective of this study was to evaluate the biological responses of three different mixtures of a bone substitute material placed in tooth extraction sites to determine which mixture best enhances bone formation. Under general anesthesia three adult mongrel canine had bilateral extractions of the second and fourth premolar bicuspids. Each site was modeled into 3-wall defect with box-like dimensions and having the window on the buccal (outer) side of the mandible. A synthetic hydroxyapatite bone cement (HAC) was prepared three different ways: 1) 2.5 gms HAC + 0.75 cc sterile water (W); 2) 2.5 gms HAC + 0.75 cc 2% Hydroxypropylmethylcellulose in sterile water (HPMC); 3) 2.5 gms HAC + 0.75 cc Sodium Phosphate [45mM/15mL Phosphate + 60 MEq/15mL Sodium] (SP). Each mixture was syringe injected into a different extraction site and the fourth site received no cement to serve as a control site, and after sites were filled, a periodontal flap closure was accomplished with a 4-0 VICRYL suture.

Aminoacyl-tRNA synthetases are the family of enzymes responsible for correctly linking tRNA molecules with amino acids so that the latter may be subsequently incorporated into growing polypeptide chains. A unique feature of aminoacyl-tRNA synthetases from higher eukaryotes is the existence of a high molecular mass multisynthetase complex. This approximately 1 × 106 Da particle measures approximately 27 nm in diameter and appears to be a cup or elongated “U“ shape as suggested by distinct orientations visible in negatively stained electron micrographs. A working model of the structure has been proposed which divides the particle into three domains. The composition of each domain as well as relative positions of individual polypeptides within each have been proposed, but to date there has been no precise localization of any specific component or site within the multisynthetase complex. This study describes the first localization of a particular polypeptide within the particle.

In principle, electron energy loss spectroscopy (EELS) in the scanning transmission electron microscope (STEM) should be able to detect single atoms of phosphorus in biological macromolecules. Such a capability would provide a direct method for measuring the degree of phosphorylation or nucleotide binding which are important in determining the functional states of protein assemblies. Although it has already been possible to map heavy elements, e.g., uranium, at near single atom sensitivity, this has not yet been achievable for lighter atoms like phosphorus. One difficulty in imaging light atoms in a biological specimen is that there is insufficient contrast to visualize the atoms directly so, in a stationary probe analysis, it is not known where to place the probe. Identifying single phosphorus atoms therefore necessarily involves compositional mapping in which EELS information is collected pixel by pixel, a technique known as spectrum-imaging. Recently, improvements have been made in the acquisition of such images, especially the ability to correct for specimen drift in a robust fashion.

Huntington’s Disease (HD) is a progressive neurodegenerative disease that is characterized by loss 19 of motor function and a decline in cognitive functions. HD is characterized morphologically by loss of neurons in the basal ganglia. During the progression of HD, striatal projection neurons die but cholinergic and somatostatin-containing intemeurons survive. The mutated gene for HD has been described and the protein transcribed by the disease (huntingtin or HT) described. Within the striatum, there is a lack of correlation between presence of HT and neuronal vulnerability. HT has been shown to be associated with intracellular vesicles in human tissues.

Female rats were transcardially perfused with 4% paraformaldehyde, 0.1 M lysine, 10 mM sodium periodate (PLP) in 0.1 M phosphate buffer. Following fixation, the brain was removed and the cortex and striatum removed from the brain as 5 mm3 pieces and transferred to fresh PLP containing 0.25% glutaraldehyde.

For the past ten years, research in our laboratory has focused on the synaptic circuitry of GABAergic and glutamatergic networks in the monkey basal ganglia, a group of subcortical brain structures involved in the control of motor and psychoaffective behaviors. Elucidating the distribution and synaptic localization of different subtypes of glutamate and GABA receptors and transporters is essential to understand the basic functioning of these brain structures and develop better therapies for neurodegenerative diseases such as Parkinson's and Huntington's diseases. Although immunoperoxidase labeling allows to localize receptors at the cellular level, this method is generally not suitable for quantification and exact synaptic location of receptors due to the diffuse nature of the peroxidase reaction product. Here, we describe a highly sensitive post-embedding immunogold technique on Lowicryl-embedded ultrathin sections to analyze the subsynaptic distribution of these receptors at the electron microscope level.

The cerebellar cortex of albino mice, hamsters, teleost fishes, primates and human have been examined by correlative microscopy to study the Golgi cell soma, dendritic processes, axonal plexus and synaptic connections in the granular and molecular layers. For light microscopy (LM) toluidinc blue stained-plastic embedded scmithin sections and Golgi light microscopy preparations were used. For confocal laser scanning microscopy (CLSM) of hamster cerebellum the FM4-64 fluorescent stain was used as intracellular tracer (1). Conventional and high resolution scanning electron microscopy (SEM) of teleost fishes, primates and human were coated with gold-palladium and chromium (2). I Transmission electron microscopy (TEM). either by ullrathin sections or frccze-clching replicas, were examined to characterize synaptic connections in the granular and molecular layers. The Golgi cells appeared in the granular layer as polygonal, stellate, round or fusiform microncurons. 10-25 μm in maximal dimension, surrounded by the granule cell groups. Golgi light microscopy.

Borrelia burgdorferi is the causative agent of Lyme disease. In addition to the characteristic effects of acute infection, Lyme disease can cause chronic effects including cardiac and neurological complications [1]. Several laboratories have shown the ability of B. burgdorferi to attach and invade several cell types such as lymphocytes [2] and fibroblasts [3]. Cultured normal human astrocytes were used to investigate the possible interaction between a cell type of the central nervous system and the Lyme disease spirochete.

Initially scanning electron microscopy was used to monitor and characterize the surface interaction between astrocytes and spirochetes. Astrocyte cell cultures were co-incubated with low passage B31 cultures over a time course from 1 hr to 18 hr. The co-cultures were washed extensively to discourage non-specific binding, glutaraldehyde fixed, critical point dried and mounted for field emission scanning electron microscopy. A time dependent association between the cultured astrocytes and B. burgdorferi were found to range from approximately 20% after 1 hr to almost 100% after 18 hr.

The signaling pathways which lead to the wave of photoreceptor cell death in the rd mouse during the second week after birth have yet to be defined. The photoreceptors of these mice develop normally until they begin to elaborate phototransducing outer segments at around postnatal day (P)8. At this point, their differentiation slows as intracellular levels of cGMP rise due to the inherited mutation in the phosphodiesterase-β subunit.

We have used an immunocytochemical approach to investigate potential signaling pathways leading to photoreceptor cell apoptosis in this mutant. The earliest change detected in the photoreceptor layer was an induction of CREB immunoreactivity in photoreceptor nuclei, commencing at P8. Although CREB is generally thought to be constitutively expressed and its function controlled primarily by its phosphorylation, we find that immature photoreceptors of normal mice are immunonegative for CREB until the time of eye opening and the initiation of phototransduction at around P14.

Copepods are an abundant and diverse group of crustaceans. One order of free living copepods, the calanoids, are usually < 3 mm in length, planktonic, and possess very rapid escape responses. These animals dominate planktonic communities and their escape reactions contribute to their success. Although all calanoids respond to hydrodynamic disturbances with an escape jump, minimum reaction times vary among species, ranging from 1.5 to 6 msec. To help us understand the physiological basis underlying the phenomenally short reaction times we initiated a comparative study of the internal structures by using transmission electron microscopy (TEM).

Traditional chemical fixation was adequate for several species of copepods, and sensory structures were clearly shown. In these calanoids, minimum reaction times ranged from 3 msec to 6 msec and can be partially explained by giant axons, the most common way invertebrates increase the rate of conduction of nerve impulses. However, the internal structures of two copepod species, Euchaetarimana and Undinula vulgaris, were distorted (Fig. 1).

Chronic wasting disease (CWD) is a transmissible spongiform encephalopathy (TSE) of mule deer and elk. Affected animals are characterized clinically by a combination of abnormal behavior and gradual loss of body condition. The disease is invariably fatal. Diagnosis of CWD is made by histologic examination of the central nervous system and microscopic lesions of CWD are typical of the TSEs. Brains from CWD-suspect elk and mule deer can be examined by transmission electron microscopy (TEM) for the presence of scrapie-associated fibrils (SAF). Western blot methods identify abnormal prion protein (PrPres) which accumulates in the brains of animals with TSE, including CWD.

Conventional SAF purification procedures for TEM examination of CWD-suspect brain tissue require both high speed and ultracentrifugation steps followed by Proteinase K enzyme treatment. Modifications used in this experiment include early Proteinase K, or Proteinase K and Collagenase treatment prior to high speed centrifugation and the elimination of the ultracentrifugation step.

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.