Previous studies have documented NADH oxidase as a source of the oxygen radicals, superoxide (O2) and hydrogen peroxide (H2O2), at sites of disruption of the blood retinal barrier (BRB) in a rat model of noninsulin dependent diabetes (NIDDM). Excess production of nitric oxide (NO) by inducible nitric oxide synthase (iNOS) has been implicated in the complications of diabetes. The effects of NO depend on its microenvironment and one mechanism for its role in complications of diabetes is interaction with oxygen radicals to form the toxic species, peroxynitrite (ONOO−). Since NO is a highly labile molecule, its potential localization is best achieved by immunocytochemical studies of NOS. The activity of iNOS can be detected by localization of nitrotyrosine, a marker indicative of the formation and activity of ONOO−.

Colloidal gold labeled immunocytochemical studies of iNOS and nitrotyrosine were done on sections of neural retina in eyes of obese, NIDDM BBZ/Wor rats in which NADH oxidase had been localized with cerium perhydroxide. Disruption of the BRB was documented by immunocytochemical localization of extravasated endogenous serum albumin. Age matched, nondiabetic control retinas were from eyes from BBDR/Wor rats.

An animal model for Cystic Fibrosis (CF) has been developed to further comprehend the pathogenesis of the disease as well as the development of therapeutic interventions. This is a mouse line from a phenotype of an inbred congenic strain of CFTR-knockout mouse that spontaneously develops lung disease. Pathology of the mice include severe impairment of the mucociliary transport, postbronchiolar over inflation of alveoli and parenchymal interstitial thickening with evidence of fibrosis and inflammatory cell invasion.

Although protein replacement therapy using liposomes containing purified reconstituted functional CFTR has been done on the nasal epithelium of CFTR-knockout mice the strain did not develop lung disease nor were the liposomes applied beyond the nasal cavity. Assessment of the treatments was done by measuring the transepithelial potential difference (PD), immunofluorescent labeling of the CFTR and immunogold labeling of the segments of nasal epithelium which were found to have a correction in their PD readings and CFTR present by immunofluorescence. It was clear after these studies that some form of correlative microscopy technique was needed to be devised in order to improve the efficiency of detection.

Gold labels such as Nanogold® and colloidal gold are enlarged and visualized in the electron microscope or optically by the selective deposition of silver onto their surfaces. This process, known as autometallography (AMG), silver amplification or silver enhancement, is initiated by exposing the particles to a solution containing silver (I) ions and a reducing agent such as hydroquinone or npropyl gallate. Particles may be enlarged to between 30 and 100 nm in diameter, giving a distinctive black, punctate staining in the light microscope. Nanogold® labeling with silver amplification is one of the most sensitive methods available for histopathology applications such as in situ hybridization. With Catalyzed Reporter Deposition (CARD; also called Tyramide Signal Amplification, or TSA® ; NEN Life Sciences, Boston MA), it has been used to detect as few as 1-2 copies of viral DNA or RNA per cell. However, its uses are restricted by reactions of silver (I) with halides and other elements in tissues. Also, after signal development, self-nucleation and non-specific background deposition begin quickly, which can make end-point selection difficult or prevent incorporation into automated procedures.

Multiple labeling for electron microscopy (EM) is typically accomplished by using colloidal gold (cAu) particles of different sizes. The size distribution of each cAu preparation, which may vary by up to 15%, is the principal factor that limits the number of labels which can be used simultaneously. Furthermore, there is no effective way to use multiple labeling for quantitative, high resolution EM studies. Such analysis requires the use of a single Fab antibody fragment conjugated to a single cAu particle in order to ensure that each particle corresponds to only one antigenic site. Whole antibody molecules cannot be used for quantitative analysis because they are at least divalent, and some, such as dimeric IgA or pentameric IgM, have even more antigen-binding sites. Consequently, it is impossible to deduce whether the presence of one whole antibody molecule corresponds to the presence of one, two, or more targets. Particle diameters ranging from 3 to 5nm are optimal for quantitation because one Fab fragment adsorbs to one particle, more than one Fab fragment may adsorb to a single particle larger than 5nm, and, when smaller than 3nm, several particles may bind to a single Fab fragment.

We are evaluating parameters apart from particle size variation to accomplish multiple labeling for both qualitative and quantitative analyses. One method relies on electron energy loss spectroscopy (EELS) to distinguish particles of several metallic compositions, including Au, Ag, Pt, Pd, Rh, and Ru. EELS is performed using a LEO 912 energy filtering transmission electron microscope (EFTEM) with an in-column Omega spectrometer.

Non-imaging microscopy has developed somewhat independently of both traditional light microscopy and laser confocal microscopy. Flow cytometry is the chief commercial and research technology among these microscopies, and, like other nonimaging detection systems, developed around the theme of automation in clinical laboratory medicine. It is an important correlative or parallel microscopy to several image forming microscopical methods. Cell sorting is an important option as well.

The basic structure of the flow cytometer certainly parallels light, laser and electron microscopes. The flow cytometer has a light source, a set of adjustable optics to focus the beam on the specimen, objective optics to collect the light and direct it to appropriate sensors, and the sensors themselves. A real image is not formed because the sensors are not in an even plane with the projection, such as provided by the retina in light microscopy or an image plane or film plate in electron microscopy, and the objective optics may not focus in the image plane.

While early flow cytometers were developed primarily for the automatic counting of cells and particles, modern instruments offer particular advantages for the analysis of fluorescence, fluorescent chemicals and probes and cellular auto fluorescence.

Hard metal is an alloy of tungsten carbide and cobalt along with other components such as chromium carbide, molybdenum carbide, tantalum carbide, and nickel. Chronic exposure to hard metal particles by inhalation causes alveolitis leading to interstitial fibrosis, the pathogenesis of which is still undefined. The initial inflammatory response includes a change in epithelial cell permeability barrier function (1) which has been shown to be regulated by the state of assembly and organization of the actin cytoskeletal network (2, 3). Therefore, the objective of this study was to evaluate the effect hard metal particles have on F-actin organization of rat lung epithelial cells in an in vitro culture system.

Rat lung epithelial cells (L2: ATCC, CCL-149) were grown to confluence on glass coverslips and exposed to various concentrations of hard metal particles for 24 hours. The effect on F-actin organization was visualized by confocal microscopy following Bodipy-Phallacidin staining, while changes in cell morphology were assessed by phase contrast microscopy. Hard metal particles of cobalt, tungsten carbide, and tungsten carbide/cobalt (6 % cobalt) were tested at concentrations of 1, 3, and 5 μg/ml. There was a dose-dependent change in the F-actin organization in the cells. The actin microfilaments lost their uniform distribution and aggregated into homogeneous masses of F-actin staining. Significant change in F-actin state was observed even at a 1 μg/ml concentration of tungsten carbide/cobalt particles. This is consistent with previous observations that pathological effects of tungsten carbide/cobalt particles are more pronounced compared to either metal alone. Phase contrast microscopy revealed no significant change in the cell morphology at this short incubation time.

The Western Mosquitofish, Gambusia affinis affnis, a sexually dimorphic internal fertilizing species of the family Poeciliidae, shows distinct sexual dimorphism in the anal fin and its axial and appendicular support.

The development of the regional vertebral differences (i.e. precaudal [vertebra bearing ribs] and caudal [vertebra bearing hemal spines]) in female and male G. a. affinis is generated through a development program of first posterionzing more anterior vertebra (formation of hemal spines more anteriorly) then antenonzing more posterior vertebra (loss of hemal spines and formation of parapophysis with pleural ribs) . This developmental program is reminiscent of effects obtained by disturbing developmental programs by exogenous treatment with retinoic acid.

Recently we generated data suggesting that all-trans retinoic acid (tRA), a non-steroidal lipophilic molecule may play a role in the regulation and patterning of sexual dimorphic phenotype(s), an area considered the domain of steroid hormones.

We wanted to identify the specific neural cell type(s) synthesizing endogenous tRA within the developing spinal cord which might coordinate sexually dimorphic patterning of spinal cord levels in G. a. affinis. To achieve this, we employed retrograde transport studies using 3,000 molecular weight (MW) dextran amine and biocytin combined with immunogold and/or lmmunohistochemistry to identify the specific neural type(s) which express tRA synthesizing enzymefs] (i.e. retinaldehyde dehydrogenase [RALDH]).

This study was conducted to compare the immunohistochemical and morphometric characteristics of blood vessels in brain metastasis with blood vessels of the adjacent uninvolved brain tissue. This is based on the hypothesis that new vessels formed in the tumor express antigens that are not expressed in blood vessels of the adult brain parenchyma. Thus, a panel of antibodies known to react with mouse endothelial cells at various stages of differentiation or stimulation was tested on frozen sections of experimental brain metastasis. Intratumoral microvessel density which has been reported to reflect the intensity of tumor angiogenesis was inapplicable to experimental brain metastasis therefore other parameters were measured.

Brain metastasis was produced in nude mice following intracarotid injection of tumor cells. Three human tumors and three murine tumors were injected into the carotid artery of nude mice. The animals were sacrificed when they were moribund, the brains were embedded in OCT and frozen in liquid nitrogen. Some animals were injected with bromodeoxyuridine (BrdU) 1 h before they were necropsied. Immunohistochemistry was performed using antibodies directed to the following antigens: CD31, CD34, MECA-32, endoglin, ICAM-1, ICAM-2, and BrdU. Antibodies to leukocyte antigens, F4/80 and CD3 were also tested to determine the amount of host cells infiltrating the tumors. SEM of immunogold labeled preparations determined the cellular localization of some antigens.

Determination of the fine organization of actin networks is important for understanding different functional aspects of the central nervous system (CNS) such as plastic events. Early studies investigating the localization of actin filaments at the ultrastructural level relied on structural methods or antibodies raised against actin isoforms. More recently, many investigators have employed the actin-binding peptide phalloidin conjugated to various fluorescent molecules. By conjugating phalloidin to the fluorophore eosin, we have been able to localize f-actin at the electron microscopic level using photooxidation of diaminobenzidine (DAB) by eosin. The goal of this study is to provide a better description of the cellular and subcellular localization of actin filaments at the light and electron microscopy level based on the high binding affinity of phalloidin for actin and utilizing this novel method.

Tissues were obtained from male Sprague Dawley rats that were perfused transcardially under deep anesthesia with normal rat Ringer's solution at 35°C followed by fixative. The fixative contained 4 % paraformaldehyde and different concentrations of glutaraldehyde, ranging from 0.1- 2.5%. Sections of cerebellum, striatum and hippocampus which were cut at a thickness of 50-80 μm with a Vibratome were incubated with phalloidin-eosin in a solution of 0.05% in 0.5% cold water fish gelatin/50mM glycine-PBS (“working buffer”) for 2 hrs. As a control, phalloidin-eosin was omitted for one set of tissues.

A dye, Phloxine B, a common food coloring and one of the active components of a photoreactive insecticide was recently approved by Food and Drug Administration as D&C Red #28 for use in drugs and cosmetics. Phloxine B is also one of the most widely use stain in fluorescence microscopy. However, in spite of the widespread interest in multi-photon spectroscopy and imaging, no information is available on the electronic transitions properties of Phloxine B with red edge fluorescence excitation and with multi-photon excitation.

In the present report we described the steady state and time-resolved fluorescent properties of Phloxine B with up-convert photon excitation and with two-photon excitation. We examined the electronic transitions properties of Phloxine B when was excited by femtosecond pulses from a mode-locked titanium sapphire laser or a mode-locked optical parametric oscillator laser. Phloxine B under excitation wavelengths above 775 nm was found to display two-photon excitation fluorescence with a spectrum maximum at 580 nm, which emission is consistent with one-photon excitation fluorescence spectrum. At the red edge excitation of Phloxine B, from 590 nm to 650 nm, we observed one-photon excitation fluorescence indicating that Phloxine B behaves like upconverting dye with one-photon excitation process.

Dental plaque is the biofilm which forms on the surface of teeth, comprising a multitude of bacterial species embedded in microbe- and host-derived polymers. Confocal microscopy (CLSM) studies revealed an open architecture in which palisades of biofilm biomass were shown to be interspersed with water channels of lower density. However, the penetration depth of CLSM in these samples is limited to around 20-40 μm, depending on the density of the samples. Therefore, thick biofilms associated with, for example, fissures in the tooth surface, can not be investigated using this technique. The limited depth penetration of CLSM can be overcome by employing two-photon excitation microscopy (TPEM). Due to the use of the longer wavelength excitation light in TPEM, a two to four fold higher penetration into the sample is obtained than in CLSM, see fig. 1. This, together with the reduced photobleaching of the fluorescent probes, makes TPEM well suited for in-depth imaging studies of strongly scattering samples such as biofilm.

The biofilm nature of plaque allows the development of gradients of key biochemical parameters such as ion concentrations. These gradients may result in extreme conditions at certain sites within the plaque that are often associated with the development of the diseased state. Conditions in the oral cavity are overtly aerobic, and the pH of saliva is near neutral.

Historically, high-resolution optical microscopy has played a vital role in biomedical research. The resolution of conventional optical microscopy is limited by the diffraction effect to about 200 nm and 500-900 nm in the radial and axial directions, respectively. This modest resolution is a major limitation in using optical microscopy for demanding applications. New techniques such as near field optical microscopy, scanning tunneling microscopy, and atomic force microscopy have been developed to circumvent this problem and have achieved atomic resolution in solid state specimens. Unfortunately, the applications of these new techniques have been difficult in biological systems. First, scanning probe techniques require mechanical interaction with the specimens. The mechanical forces exerted by the probe tip may cause sample deformation. This interaction further degrades the achievable image resolution. Resolutions in the range of 50 to 100 nm are often reported for near field optical systems. Second, scanning probe technique has a relatively low frame rate since the required high precision position control imposes severe bandwidth limitations. Scanning probe techniques are more suitable for studying static sample but often have difficulties in addressing many inherently dynamical phenomena in biology. Third, these are surface imaging techniques and are inherently limited in their ability to investigate the 3-D structures of biological specimens.

Chemical sensing based on fluorescence is presently undergoing rapid development. As one example, fluorescence lifetime measurements are now being used for measuring cation and anion concentrations in clinical chemistry, cellular imaging and flow cytometry. Additionally, there is an increasing use of proteins as sensors because of their high affinity and specificity for ligands.

In this report we describe some recently developed approaches to fluorescence sensing. These new methods are based on the use of a fluorescence reference either within the sample or external to the sample, but within the optical path. Two types of references are used; microsecond lifetime metalligand complexes (MLCs) or oriented films of ns decay time fluorophores. A protein sensor for glucose was developed using the glucose-galactose binding protein (GGBP) from E.coli (Figure 1).

The cAMP dependent-protein kinase (PKA) signaling pathway plays a key role in the sympathetic regulation of muscle contraction in adult cardiac myocytes. This pathway may be regulated via compartmentalization of its components, whereby A-kinase anchoring proteins (AKAPs) tether PKA to specific subcellular areas to give each receptor binding event specificity. The purpose of this study is to investigate the binding kinetics between Ht31, a peptide containing the PKA binding portion of an AKAP from human thyroid, and the regulatory subunit of PKA (R).

Fluorescence resonance energy transfer (FRET) was used to monitor binding events between the type II regulatory subunit of PKA (RE) and Ht31 or Ht31P, a mutated form of Ht31 which does not bind RII. Each protein was fused to a derivative of the green fluorescent protein (GFP) so that the excitation-emission spectra of the two fluorescent proteins overlap.

The widespread availability of automated fluorescence microscope systems has led to an explosion in the acquisition of digital images by biologists. This has created a need for computer applications that automate the analysis of these images and an opportunity to develop new approaches to classical problems. An example is the determination of the subcellular location of a protein from immunofluorescence images (or, more recently, images of GFP fluorescence). Current practice is to compare such images to mental images that a cell biologist has developed over time, and to reach a tentative conclusion about the structure (i.e., organelle) that a protein is found in. Since this determination is subjective, it often must be followed up by double labeling with a marker protein from the suspected structure.

As an initial exploration of the feasibility of automating the determination of subcellular location, we developed a system that is able to classify the localization patterns characteristic of five cellular molecules (proteins and DNA) in Chinese Hamster Ovary (CHO) cells. Images were acquired on an epifluorescence microscope after the cells had been fixed, permeabilized, and labeled with appropriate fluorescent reagents (usually antibodies conjugated to fluorescent dyes). The labels used were directed against a Golgi protein, a lysosomal protein, a nuclear protein, a cytoskeletal protein, and DNA.

A partnership between California State University, Hayward and Ohlone College, Fremont, California resulted in a program to introduce undergraduate students to scanning electron microscopy (SEM) sample preparation procedures. Ten students enrolled in a biology major's course at the Ohlone Community College are selected each semester to participate in the program. In addition to these students, ten others from a Basic Skills Enhancement program for underrepresented students participate through a short course offered during the summer. The program, in effect for two years, resulted in the compilation of a laboratory manual describing basic sample preparation techniques that are appropriate for an outreach program. We select samples that are simple to prepare yet yield results that are germane to a biology student's curriculum. A selection of plants, pollen grains, bacteria, fungi, yeast, Drosophilia melanogaster, and various other invertebrates are typical samples that lend themselves to an outreach program that emphasizes biological concepts. Initially our program required the Ohlone College students to come to the Hayward campus to prepare their samples. External funding has allowed us to purchase sample preparation equipment for the community college.

Bugscope is a new educational project in the World Wide Laboratory. The World Wide Laboratory provides Web browser based control of scientific imaging instrumentation using the Internet [1]. Providing K-12 classrooms with web based remote access to sophisticated scientific imaging systems was initially demonstrated by us in 1996 in the Chickscope project [2,3]. Chickscope allowed students to study chicken embryo development using a remotely controlled magnetic resonance imaging (MRI) system from their classrooms. While the Chickscope project was highly successful, the resources required to provide operational support for the remote imaging aspects of the project for a small number of classrooms were enormous and the project was not sustainable. The Bugscope project builds on the methods developed and the lessons learned from the Chickscope project. The primary goal is to demonstrate that relatively low cost, sustainable access to an environmental scanning electron microscope (ESEM) can be made available to K-12 classrooms to examine arthropods.

Methods: Classrooms use a standard web browser over the Internet to control and acquire images from a Philips XL-30FEG ESEM. The architecture to support remote acquisition is shown in fig. 1. The client/server control architecture for the ESEM remote control server is based on the emScope control library [4].

The focused ion beam (FIB) instrument has been developed and exploited by the microelectronics arena for specimen preparation for both scanning and transmission electron microscopy (TEM). The inception [1] and subsequent development [2] of the FIB TEM lift-out (LO) technique has enabled electron transparent membranes of generally uniform thickness to be produced for TEM analysis. The primary advantage of the FIB technique is that site specific cross sections (or plan view sections [3]) may be fabricated quickly and reproducibly. The FIB LO technique has been used extensively in our laboratory for a wide range of materials [4] and biological applications [5] which are summarized in figure 1.

The FIB LO method consists of milling a series of trenches around an area of interest. Then the bulk sample is tilted up to ∼60 degrees to allow the beam to impinge on the lower portion of the specimen surface so that cuts can be made along the bottom edge and the lower 2/3 of the distance up one side of the specimen.

X-ray mapping is one of the most popular modes for displaying information obtained with x-ray spectrometry performed in the scanning electron microscope. This popularity arises from the ready accessibility and apparent simplicity of information presented in a pictorial fashion, especially when used in conjunction with other SEM imaging modes, such as backscattered, secondary, and specimen current electron images. Further, the rise of powerful, inexpensive computer systems capable of image processing and display has given the analyst a dedicated, on-line tool with the capacity and flexibility needed for problem solving. Figure 1 shows a typical example of mapping. Although the interpretation of x-ray images obtained with a modern digital control and recording system would seem to be straightforward and relatively trivial, there are significant pitfalls and limitations that can easily fool the unwary. In Figure 1, within an individual x-ray map, the observer can reasonably judge where the concentration is lower or higher, at least for a group of contiguous pixels. Can such judgments be made among a set of maps of the same region for different elements, or even for the same element from different regions of the same specimen? With current x-ray processing and display systems, the answers are generally no. In fact, problems that can influence interpretation can arise at each stage of x-ray generation/emission, x-ray spectral collection, processing, and display.

Since the introduction of personal computers in the early 1980s, most documents and manuscripts have been prepared from within a word processing program on such a computer. Indeed, most documents on personal computers are created on a limited number of word processing packages that have come to dominate the market. Most of the data acquisition that attendees of Microscopy and Microanalysis currently perform is digital, i.e. directly into a personal computer or desktop workstation. It is frequently the case that images, spectra and diffraction patterns are only committed to paper when they are dispatched for publication. With the advent of the World Wide Web in 1994, and its subsequent explosive growth, entirely electronic publishing has become possible. It is possible to envisage the day when most publications will be available solely electronically and material will be only printed out occasionally by the reader.

While there are a growing number of journals that are only available electronically, entirely paperless publication is not yet the norm and there are no definite plans, as yet, to publish the proceedings of the Microscopy and Microanalysis meetings solely in electronic format. However, there are a number of advantages to applying some of these emerging technologies in the production of the future proceedings. Electronic submission of abstracts is one advance that is being considered by the organizers of the meetings scheduled for the early 2000s. The National Science Foundation now receives almost all proposals electronically. There are several reasons for moving to electronic submission. The first is that proofing and correcting the abstracts would then be straightforward.