Brain inflammation is typically involved in the pathogenesis of acute conditions such as stroke and brain trauma and in chronic neurodegenerative diseases, for instance, Alzheimer’s disease. Accordingly, a well-characterized in vivo model of brain inflammation can be a useful tool to evaluate specific drug effects focused on various inflammation targets in the pharmaceutical drug discovery process. A model of brain inflammation was induced by the injection of β-amyloid peptide (αβ) or the bacterial endotoxin lipopolysaccharide (LPS) into the lateral ventricles of CD-I mice. The time course and dose response kinetics of cytokine and chemokine production were characterized. Levels of the pro-inflammatory cytokines IL-1α, IL-1β, IL-6 and the chemokine MCP-1 were significantly increased with respect to both time and dose in the cortex and hippocampus as determined by ELISA. Immunohistochemical assays were developed to detect IL-1β to determine cellular localization of this cytokine to microglia and astrocytes. Astrocytes were labeled with antibodies to glial fibrillary acidic protein (GFAP) and microglia were labeled with F4/80 antibodies. IL-1β was localized to cell type in frozen sections using double immunofluorescence (IMF) tags and were visualized with traditional fluorescence and confocal imaging.

The murine retinal vasculature develops in its entirety following birth through the process of angiogenesis. This developing system provides an extremely accessible model for investigation of angiogenic mechanisms in general and as they pertain to vascular diseases of the eye (e.g. diabetic retinopathy and macular degeneration). The model is particularly relevant clinically because the developmental events of retinal vascularization are nearly identical in mice and humans, although primate vessels develop in utero during the final trimester of pregnancy and are thus less accessible for experimental study. The model is also excellent for the testing of anti-angiogenic agents as potential treatments for vascular diseases. in this report we outline the use of confocal optical microscopy and multiphoton microscopy in the 3-dimensional characterization of retinal vascular development in the mouse.

We employed confocal microscopy to characterize the events of vascular development from postnatal days 0-56 (P0-P56). During this time the retinas are transformed from simple, thin bilaminar structures to more complex, functional, adult sensory organs. Retinal morphogenesis continues until P42. Retinas were dissected from eyes and vessels stained with collagen type-IV antibody (1) or Bandeiraea simplicifolia (Griffonia) lectin-dye conjugate (2) to outline the blood vessels. All patent vessels were stained by both techniques.

Embryonic development is a dynamic event and is best studied in live animals in real time. Much of our knowledge of the early events of embryogenesis, however, comes from immunofluourescent analysis of fixed embryos. While these studies provide an enormous amount of information about the organization of different structures during development, they can give only a static glimpse of a very dynamic event. More recently real-time fluorescent studies of living embryos have become much more routine and have given new insights to how different structures and organelles (chromosomes, centrosomes, cytoskeleton, etc.) are coordinately regulated. This is in large part due to the development of commercially available fluorescent probes, GFP technology, and newly developed sensitive fluorescent microscopes. For example, live confocal fluorescent analysis proved essential in determining the primary defect in mutations that disrupt early nuclear divisions in Drosophila melanogaster. For organisms in which GPF transgenics is not available, fluorescent probes that label DNA, microtubules, and actin are available for microinjection.

Tremendous advances have been made in the last twenty years in extending the usefulness of the light microscope in visualizing living biological specimens, and it is now possible to visualize most thin tissues in an unstained state at – or near – the limit of optical resolution. in general, however, these techniques have been used successfully on biological specimens in the transmitted light mode; unstained biological specimens generally do not have sufficient albedo or sufficient difference in refractive index from their surroundings to be efficiently visualized in epi-illumination. A number of applications, including studies of the growth of bacteria on metallic substrates, and studies of the studies of the formation of interfaces between living neurons and silicon chips, would benefit from being able to resolve living, unstained biological specimens growing on opaque substrates

We found that by imaging biological specimens on a reflective substrate in the reflection mode of the confocal microscope, we could get images that resemble transmission mode differential interference contrast images.

Angiotensin II (Ang II) plays an important role in heart development and has been shown to stimulate the formation of premyofibrils and differentiation of cardiac myocytes. Ang II signaling occurs through two types of receptors, the AT1 and AT2 receptors, both of which appear to play a role in the development of the myocardium. Through the use of specific Ang II receptor blockers it as been shown that blocking the AT1 receptor with Losartan (Merck) inhibits the formation of premyofibrils and sarcomere formation and that blocking the AT2 receptor with PD123,319 (Parke-Davis) inhibits the formation of trabeculae in the developing heart.

In a number of cases it has been shown that signal transduction mechanisms operate through different growth factor receptors and elicit specific responses within a cell type. Protein kinase C (PKC) is a family of closely related signal transduction enzymes that phosphorylate proteins on serine or threonine residues.

Conventional histological sectioning and staining techniques produce tissue sections, by large, independent from each other (e.g. the staining intensity depends on the total stainable substance within the section, but not influence by the section above or below it). Although the hardness of the structure in a given section may influence the quality of the following sections, never the less, each physical section can be generally regarded as an independent sampling event. However, the image intensity and relative contrast in an optical section obtained from confocal or non-linear optical microscopy varies depending on the position of the section where it was obtained. Therefore, in sampling terms, optical sections are dependent sampling events. Artifacts caused by the geometry and optical properties (e.g. absorption, scattering, refractive index) of objects within a specimen can interfere the interpretation of the result and hinder the usefulness of three-dimension reconstruction. Figure 1 demonstrates intensity variation in confocal and multi-photon micrographs due to the geometry of the specimen. Depends on the absorption coefficient and path length of the illumination and the fluorescecent light, a solid spherical object produced a 3D data set, when reconstructed, resemble an inverted Chinese rice bowl. Figure 2 shows a 3D reconstruction of an Arabidopsis thaliana mesophyll protoplast; note only half of the spherical cell facing the illumination is imaged as shown in the cut-off view in FIG 3.

The confocal laser-scanning microscope (CLSM) has enormous potential in many biological fields. It is remarkable that procedures to test the performance of these machines are not done routinely by most investigators and thus many of the machines in operation are working at levels of sub optimal performance. When these machines are checked, it is usually a subjective assessment of performance accomplished by primarily evaluating the system using a specific test slide provided by each user’s laboratory. We have devised test methods on the Leica TCS-SP and TCS-4D systems to ensure that these machines were working properly and delivering their correct performance. Tests were derived or perfected that measure field illumination, lens clarity, laser power, laser stability, dichroic functionality, spectral registration, axial resolution, overall machine stability, and system noise. It is anticipated this type of data will help to set performance standards for confocal microscopes and eliminate the current subjectivity in evaluating the CLSM. These tests will also serve as a guide for other investigators to insure that their machines are working correctly and providing data that is accurate and having the necessary resolution, sensitivity and precision.

Both surfaces of the butterfly wing are covered with thousands of colored scales that are arranged in precise linear arrays. Each scale contributes a single color to the overall pattern, which is usually different on the upper and lower surface of the wing. Moreover, the scales have a characteristic surface substructure that is visible using confocal autofluorescence (fig. 1). The ridges on the surface of all of the scales function to control the flow of air across the wing surface during gliding flight, and they contribute to the interference colors in a subset of the scales.

The fruit fly Drosophila melanogaster is the closest relative of butterflies for which detailed developmental, genetic and confocal imaging protocols have been established. Specimen preparation techniques for butterfly imaginal discs were largely developed from those for Drosophila tissues. The various stages of butterfly wing development have presented new challenges for confocal imaging. For example, butterfly fifth instar wing imaginal discs are much larger and thicker than those of the fruit fly.

Congenital heart disease is a leading cause of death in children with about 40,000 babies born in America each year with congenital heart disease. Common problems include atrial septal defects, patent ductus arteriosus and ventricular septal defects. However, very little is known about the underlying causes of congenital heart disease or the embryonic origin of cardiac malformation. The goal of our recent research has been to examine the role that specific growth factors and their receptors play in the development of the contractile apparatus of the heart and the experiments reported here further define the role of PDGF-AA and the PDGF alpha-receptor (PDGFR-α) during early heart growth and morphogenesis.

The PDGF ligand and receptor system consists of two well-characterized ligands, PDGF-A and B, and a third recently identified ligand, PDGF-C. The active forms of PDGFA and B exist as either AA or BB homodimers or an AB heterodimer. The PDGFR-α binds all of the dimers with high affinity while the PDGFR-β binds only the PDGF-BB homodimer with high affinity.

The non-linear nature of multi-photon fluorescence (FL) excitation, SHG and THG restricts the signal detecting volume to the vicinity of the focal point. As a result, the technology has intrinsic optical sectioning capability. The use of multi-photon fluorescence excitation also allows micro-fluorometry at high spatial resolution. Figure 1 shows a conventional optical micrograph of maize protoplasts, the time lapse fluorescence spectral change from a single chloroplast is shown in FIG 2. Under high intensity illumination, biological specimen not only emits fluorescence, but also generates harmonic emissions. in addition to the Ti-sapphire laser commonly used in multiphoton microscopy, the use of ultra-fast Cr-fosterite laser made simultaneous detecting two- and three-photon fluorescence, SHG and THG possible. in addition to the fluorescence signals generated by multi-photon excitation process, non-linear phenomena such as harmonic generation can also provide useful information about the structure and optical properties of a specimen (Kao et al., 2000). Simultaneous recording the spectral response in an image (x-y-λ) can provide insight about the nature of the signal.

We previously described two techniques to selectively label and/or stain myelinated and unmyelinated nerve fibers and simultaneously stain bone and cartilage. We used antiacetylated α-tubulin immunohistochemistry and anterograde and retrograde transport of selectively applied 3000 molecular weight (MW) biotin dextran amines (Molecular Probes, Inc. Eugene OR) to follow the course, peripheral branching, and origin of the ventral spinal nerve innervating the axial musculature. Those procedures were combined with an enzyme clearing and staining procedure for the simultaneous visualization of bone (alizarin red S) and cartilage (alcian blue) in whole-mount preparations. Use of the techniques enabled us to describe spatial and temporal changes of nerve fibers and the corresponding changes of the osseous elements during the transposition of the anal fin appendicular support of the Western Mosquitofish, Gambusia affinis affinis.

We have extended these techniques by selective multicolor anterograde and retrograde labeling of spinal motor neuron cell bodies, their dendritic arbors, and their ventral motor nerves. We used low (3000) molecular weight (MW) fluorescent lysine-fixable dextrans (Texas Red® and fluorescein) and biotinylated lysine-fixable dextrans (biotin and fluorescein conjugated to biotin), Nissl stains, and fluorescent conjugates of cholera toxin subunit B (Molecular Probes, Inc, Eugene OR).

There are three members of the caveolin (CAV) gene family that give rise to four polypeptides. These polypeptides are CAV-1α, CAV-1β, CAV-2, and CAV-3. The CAV-1β isoform is a truncated form of CAV-1α that lacks 31 amino acids at the N-terminus of the molecule. The CAV- 1β molecule arises through an alternative splicing mechanism.

Caveolae are specialized plasma membrane microdomains that are expressed at high levels in some cell types (e.g., endothelium, adipocytes, fibroblasts). These specialized regions of the plasma membrane have a characteristic omega-shaped appearance with diameters ranging from 40-90 run. They are distinct from clathrin-coated pits since they lack the characteristic coated appearance in electron microscopy. Caveolae were among the first structures to be discovered by biological electron microscopy. However, biochemical characterization of these structures did not begin in earnest until a marker protein was identified. The initial marker was the 22-kDa protein known as caveolin.

High resolution, quantitative cell labeling analysis for electron microscopy is typically accomplished using colloidal gold (cAu) particles which have a uniform diameter such that each particle conjugates no more than one ligand with monovalent specificity, such as an Fab antibody fragment. in this way, one particle labels exactly one target molecule. The corresponding particle size which satisfies this requirement, in the case of the Fab fragment, is 3-5nm. Under these constraints, multiple labeling cannot be performed using the standard cAu technology in which particles of various diameters are used such that each size corresponds to a unique label. Particles larger than 5nm may conjugate multiple Fab fragments, and thus a single particle may lable multiple target sites. Conversely, several particles smaller than 3nm may bind to a single Fab fragment so that several particles may label a single target molecule. Recent work in our lab has focused on the development of a labeling system which maintains the advantages of the standard cAu labeling technology, namely the use of electron-dense particles with excellent secondary and backscattered electron emission properties which make them readily detectable by both TEM and SEM, while at the same time offering greater versatility by permitting multiple labeling for high resolution, quantitative analysis. This novel labeling system makes use of colloidal particles which have unique and distinctive shapes, and composed of a variety of different metals including Au, Ag, Pt, Pd, Rh, and Ru. These elements all form stable aqueous colloidal suspensions, and the particles have surface charge properties similar to those of Au, permitting the adsorption of proteins and other ligands to them by the same conjugation techniques used with cAu (fig. 1).

Current computer chip technology is based on lithographic methods that limit components to ∼0.3 microns in size, due to the wavelength of light, and the photoresist/coating/etching processes. The size directly determines computer speed, complexity and cost, and advances in computers over the years have mostly been due to reduction in component size. It is here proposed to construct nanowires that are approximately 2 nm in diameter, or 150 times smaller than currently available. For 2 dimensions, this translates into a 1502 = 22,500-fold computational advantage. Additionally, 3 dimensional construction is proposed, bringing the potential improvement factor to 3,375,000. While it is probably unrealistic that this factor of packing density can be fully achieved, even several orders of magnitude improvement over current technology would be significant.

A wire width 2 nm may be achieved by placing gold quantum dots along a DNA template. Ends of the DNA-nanowire may be designed with sequences to attach by hybridization to complementary sequences on target connection pads, so that the two ends will seek and automatically wire correctly in solution. This strategy is easily adaptable to 3-dimensional wiring. Conduction between gold quantum dots may be studied as a function of spacing, size and coatings.

Molecular immunolabeling allows us to recognize location of molecules within complex biomolecular assemblies, cells, and organs. This technology helps us to facilitate correlations of the data available from biochemistry, molecular biology, pathology, and molecular imaging laboratories concerned with the gene expression as well as location and functions of its products. in this sense, molecular imaging serves as an integrative factor for life sciences endeavors (Albrecht et al. 1992, Bazett-Jones et al. 1996, Hainfeld and Powell 2000, Malecki et al. 1998, Robinson et al. 2000, Malecki 2000). Nevertheless, the essential requirement for pursuit of those projects is availability of molecular markers, which are detectable with various imaging instruments (Malecki 1996). Correlations between positron emission tomography (PET) providing metabolic information, magnetic resonance imaging (MRI) delineating precise anatomical location, and energy filtering transmission electron microscopy (EFTEM) expanding insights into the molecular level are particularly powerful. We have designed three main strategies for pursuit of this project: (a) expression of fusion proteins containing amplified metal binding sites (Malecki et al. 1998), (b) expression of recombinant single chain variable fragment (scFv) antibodies containing elemental recipient (Malecki et al. 2000), and (c) covalent coupling of IgG and Fab with organometallic clusters (Malecki 1996). Herewith, we report the results obtained through imaging of organometallic clusters inserted into the recombinant single chain variable fragment (scFv) antibodies.

A promising aspect of ESI is its application in the detection of elemental labels introduced into biomolecules for cell and molecular biological techniques. Even though colloidal gold labeling for electron microscopy (EM) is highly developed, availability of alternative labels, especially for double or triple labeling applications would be helpful because of difficulties with gold concerning i) detection (gold diameters ≤1nm), ii) discrimination due to gold particle size variations in one size class, and iii) different labeling efficiencies depending on gold granule size. An alternative labeling molecule should contain a high concentration of a specific chemical element which is not or in minor concentrations present in the system under surveillance, and has to have the potential to be discriminated from “biological” elements by ESI.

With respect to ESI, one candidate for elemental labeling is boron. It meets the criteria described above and substantial experience in the synthesis of labeling compounds exists. From the chemical point of view, the preferred labeling structure is a so called dendrimer, a highly branched regular three-dimensional monodisperse macromolecule. Dendritic structures offer a large variety of functionalities to incorporate an element detectable by energy filtering transmission electron microscopy (EFTEM).

Human CD46 (membrane cofactor protein) is expressed on all nucleated human cells and is a cellsurface regulator of activated complement as well as a receptor for both the measles and herpes 6 viruses. CD46 is also expressed in particularly high levels on some tumor cell lines . Complement leads to the recognition of foreign material, targeting it for removal and immune cytolysis. The primary role of CD46 is to bind to one of the complement components, C3b, thereby protecting host cells from autologous complement attack. in addition to its role as a complement regulatory factor, CD46 acts as a measles virus (MV) receptor. Despite the development and use of an effective MV vaccine, at least one million deaths occur each year. MV infection leads to downregulation of cell surface CD46 and immunosupression. MV (and probably also C3b) is internalized shortly after complex formation with CD46, yet, despite much work in this area, precisely how MV enters cells is largely unclear.

The purpose of this research project was to characterize the distribution of left-handed Z-RNA sequences within the epithelial cells of the adult noncataractous crystalline dog lens: the meridional rows (MR). This was achieved by using anti-Z-RNA IgG polyclonal antibody probes. Both light microscopy (LM) and electron microscopy (EM) were used to analyze the tissue binding of the anti- Z-RNA antibodies. Nucleic acids can adopt many different helical conformations (1), such as Z-DNA (Fig. 1) and Z-RNA (2,3). The lens is made up of a single cell type, which is a monolayer of undifferentiated epithelial cells covering its anterior surface (Fig. 2). At the equator of the lens, these cells elongate and form concentric layers of the secondary (nucleated) fiber cells, which undergo cell death-terminal differentiation (denucleation). The epithelial monolayer consists of several cell types, each of which has unique characteristics.

The production of anti-Z-RNA polyclonal antibody probes was achieved using four

The ultimate goal of the immunocytochemistry is to get the best signal with the lowest background while preserving ultrastructure. Unfortunaly too often the experimentator faces a compromise between density of the signal and ultrastructure preservation (1). with the advent of hight resolution confocal light microscopy, two photon imaging and other new technologies there is a need to provide the best immunocytochemical complementary results with the high resolution of the electron microscope. But often for exemple the high level of labelling observed by immunofluorescence microscopy is not matched by immunocytochemistry (2). One approach to obtain comparable levels of labelling is to use comparable protocols. We propose here such a new immunogold method. to illustrate our point we used a very convenient biological material, the adherent cells grown on 35 mm plastic culture dishes. The following procedure was applied : Confluent endothelial cells were fixed in situ for 3 hours with the following freshly prepared and filtered solution : 1 % 1-lysine, 4% paraformaldehyde, 0.04% glutaraldehyde, 0.25% sodium metaperiodate in 0.04M sodium cacodylate buffer, pH 7.4.

To obtain high resolution backscatter electron (BSE) images in field emission SEM (FESEM), one must consider selection of accelerating voltage, beam current, working distance between the specimen and the backscatter detector (in-lens or below-the-lens position for the specimen), the type of BSE detector, and the type of metal used to coat the specimen to improve conductivity and signal collection [1]. A new generation of below-the-lens FESEM have been tested for BSE imaging on biological samples, but no information exists on whether or not high resolution imaging is possible. Here we report the comparison of detection of a colloidal gold standard (6, 12, 18 nm) by high resolution BSE imaging using Autrata-modified YAG detectors in an in-lens FESEM and in a below-the-lens FESEM.

Standards were prepared by mixing colloidal gold particles of 6 nm, 12 nm, and 18 nm. The gold particles were attached via poly-l-lysine to glass chips and coated with <1 nm Pt by ion beam sputtering.