Reconstruction of phase images from off-axis electron holograms recorded in a field-emission TEM was for many years a tedious and time-consuming process. The advent of digital imaging systems and ever-faster desktop computers has recently resulted in processing times of a few seconds for a useful phase reconstruction. However, a goal of the “holographer” has been to view the phase images essentially in “live-time”, that is, at least at a few frames per second. This enables effective observation of phase change, through “a window to the phase world,” while the sample is being searched. Chen et al. used a mixture of digital and analog techniques to obtain phase images at nearly TV scan rates. Because optical lens systems provide the fast Fourier processing required for phase reconstructions, their geometry allows phase images to be displayed while the sample is being moved and/or timedependent specimen changes are occurring. Voelkl et al. have described a purely digital system for phase reconstructions from electron holograms that provides several phase images per second, and has several advantages over the mixed system. The refresh rate is presently limited more by the readout time of the slow-scan digital camera than by the computational time of the computer.

Dental enamel is the most durable bioceramics produced by a vertebrate as it is designed to perform masticatory functions throughout its lifetime. The understanding of the mechanism of enamel formation and effects of proteins during the biomineralization process are fundamental issues, essential for both potential enamel regeneration and as a base for synthesis, via self-assembly, of biomimetic composites.

The biomineralization process of enamel is carried out by ameloblast cells that line the inner enamel epithelium and secrete an extracellular protein matrix onto a mineralized dentin surface at the dentin-enamel junction (DEJ). A major matrix protein, amelogenin, is believed to regulate the mineralization of hydroxyapatite (HAP) in the enamel tissue. It has been shown to undergo self-assembly in vitro and in vivo to form nanospheres of ∼20nm in diameter. Previous TEM studies have shown that the nanospheres align along the length (c-axis) of hydroxyapatite (HA) crystals. There are two domains, namely A (residues 1-42) and B (residues 157-173), that control the self-assembly behavior of the nanospheres.

Electron probe microanalysis has been a useful method for biological research for some years. Its advantages include the low background of x-ray spectra and range to include characteristic peaks of most biological elements. The traditional method of analyzing biological X-ray spectra filtering both the experimental and reference spectra with a top-hat filter, before performing a linear least squares fit, to remove the slowly varying background. We show that, as expected, this procedure introduces an additional error in the quantitation results, and reduces the sensitivity and precision of this method. Our experience with quantitating trace elements with EELS shows that direct fit of unfiltered spectra yields the highest sensitivity, when the data contain only statistical errors. Biological specimens usually contain high K and low Ca, and in X-ray spectra the K Kβ peak is overlapped with the Ca Kα peak. A small error in extracting the K signal will dramatically affect the quantitation of Ca. We demonstrate here the application of a direct fitting procedure, which focuses on the Ca and K region of the X-ray spectra (2800 ∼ 4900 eV), and yields smaller fitting uncertainties compared to the traditional filtered fit method in trace Ca quantitation.

The low survival of sugar maple (Acer saccharum) seedlings growing in unglaciated acidic forest soils (pH<4.0) in north-central Pennsylvania may be due in part to aluminum (Al) or manganese (Mn) phytotoxicity. Although foliar analysis has shown high concentrations (>2,000 ppm) of Mn, the location of potentially toxic elements in leaves or absorptive fine roots has not been studied in field-grown seedlings. This study of vascular (xylem and phloem) cells was conducted to determine by energy-dispersive X-ray microanalysis (EDX) whether Mn and/or Al could be detected in fine root or leaf midvein vascular cells. A secondary objective was to determine if vascular cells prepared by chemical fixation (CF) for conventional TEM or by freeze-substitution (FS) for EDX showed similar ultrastructural characteristics.

Four sugar maple seedlings from an unglaciated field site (Potter County PA) were excavated, and roots were washed carefully. Two mm-long segments of fine roots and leaf midveins from each seedling were immersed in 3% glutaraldehyde for 6 hours, transferred to 0.1 M phosphate buffer, and transported to the Delaware Research Lab where tissues were post-fixed in OsO4 (in buffer), dehydrated in ethanol, and embedded in PolyBed-Araldite epoxy resin (CF method).

The airways are lined with a thin film consisting of two layers. A watery layer, the airway surface liquid (ASL) surrounds the cilia and its major function is to serve as a medium for ciliary boating of mucus out of the respiratory system. The other layer is a viscous mucus layer consisting mainly of airway cellular secretions and cellular debris.

The composition and effects of altered concentrations of the constitutive elements in the ASL and mucus layer remain a mystery. in human patients, attempts have been made to determine the composition of these layers by sampling with a filter paper by touching it to the mucosa and wetting it by capillary action. This material was then removed from the filter paper and quantitative analyses using energy dispersive x-ray spectrometry (EDS) and a Hall’s thin film correction routine performed on the residue. Although differences were detected between normal and pathological material, these results did not reflect the composition of the individual layers but an average of both.

Attempts have been made to determine the elemental composition of these layers in intact and in cultured rabbit trachea, bovine trachea and hamster trachea.

To make specimens for the biological materials, especially the retina and palpebral conjunctiva, and to detect the elements and organic substances.

Twelve-week-old male Wistar Kyoto rats were used. Immediately after the eyes were enucleated under pentobarbital anesthesia, the eyeballs and eyelids were put into liquid nitrogen without any fixation and covered by O.C.T. compound (TISSUE-TEK, Miles Inc.). Semi-thin sections (5 μm) were cut with a cryomicrotome at −30 °C (Zeiss, Germany). Sections of the retina and palpebral conjunctiva were placed on siliconwafer plates 10 × 7 × 1 mm and air-dried for 30 min or freeze-dried for 8 hrs. A metal microscope (Leica- DMRBE) was used to confirm the morphological features of the unstained conjunctiva and retina on the siliconwafer plate prior to analysis with a TOF-SIMS microscope. Without coating, the specimens were examined with a TOF-SIMS microscope (TRIFID) (Physical Electronics, USA). Positive and negative ion images were examined with a Ga+ source at an acceleration voltage of 15 kV. Secondary ion acceleration voltage of 4.5 keV was used. The amount of trace elements was analyzed by Wincadence (Physical Electronics, USA) software.

Digital fluorescence microscopy is now a standard tool for determining the localization of cellular components in fixed and living cells. Two fundamentally different imaging technologies are available for imaging fluorescently labelled cells and tissues, in either the fixed or living state. The laser scanning microscope uses a diffraction-limited focused beam to scan the sample and develop an image point by point. in addition, a pinhole placed in a plane confocal to the specimen prevents emitted out-of focus fluorescence from reaching the photomultiplier tube (PMT) detector. By combining spot illumination and selection of infocus fluorescence signal, the laser scanning confocal microscope (LSCM) creates an image of the specimen largely free of out-of-focus blur. By contrast, a wide-field microscope (WFM) illuminates the whole specimen simultaneously and detects the signal with a spatial array of point detectors, usually a charge-coupled device camera (CCD). This approach collects an image of all points of the specimen simultaneously and includes all the out-of-focus blurred light. Subsequent restoration by iterative deconvolution generates an estimate of the specimen, largely free of out-of-focus blur. While many other fluorescence imaging modalities exist, these two methods represent the majority of the fluorescence imaging systems currently in use in biomedical research.

Here we show a confocal microscope system where a vertical-cavity surface-emitting laser (VCSEL) array is introduced for the parallel probing beams.

A VCSEL array is a semiconductor laser device that emits circular beams vertically to the surface of the chip, which can directly be used as confocal laser microscope applications. The VCSEL device itself can be the parallel beam source of the confocal laser system, whereas in conventional Nipkow disc-based system, several lenses are required to form probing beams. Figure 1 shows the basic structure of VCSEL-based confocal microscope systems. Since there is typically no correlation between individual channels of VCSEL, there are no interferences among dense parallel beam arrays. Additionally, in the case of Nipkow disc system, the signal to noise ratio of the detected light is another problem: some of the light from the source laser reflects at the surface of the disc, which eventually could be detected by the sensor as noise. in the proposed system no such problem occurs as shown in Fig. 2(a). in addition, “all” optical beam from VCSEL is used as probing beam whereas most of the power is lost at the disc as compared in Fig. 2(b). in addition, pixel parallel illumination control is also achievable by controlling each channel of the VCSEL so that the dynamic range of the detected signal is maximized.

Continuing improvements on laser scanning microscopy now allow easy acquisition of multi-gigabyte-sized data sets from single living specimens. These multi-dimensional data comprise stacks of serial optical sections (3D) collected repeatedly over time (4D), often recording separate signal channels simultaneously (5D). in our work imaging developmental and cell biological processes in living embryos, we try to take advantage of the full scope of spatial and temporal detail that microscopes can now accumulate. Aside from the obvious difficulties of data management and archival, we still struggle to visually appreciate both the holistic view of the specimen and the smaller dynamic details that we know are captured within these recordings. Ideally, the multi-dimensional microscopist should view and analyze the specimen as the full set of tens of thousands of images. Although strides have been made, visualization software and computer hardware for browsing and analyzing multi-dimensional data are not yet up to the task of giving the microscopist full kinesthetic access to the thriving subject that the microscope hardware and software have captured. Several pieces of software allow the viewer to fluidly browse the original optical sections through space and over time. Moving beyond the constraints of the raw image plane by rendering, re-slicing, digitally dissecting, and animating the time-varying volumes is the task that is currently so demanding that all approaches are only partially satisfactory.

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).