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PubMedDate not supplied

Quantification of dental microwear by tandem scanning confocal microscopy and scale-sensitive fractal analyses.

Dental microwear analysis is among the most commonly used approaches to reconstructing the diets of extinct animal species and past peoples. The usual procedure involves imaging tooth wear surfaces by scanning electron microscopy (SEM). Surfaces are characterized quantitatively by measurement of individual wear features (pits and scratches) on photomicrographs. Recent studies of living animals have shown associations between diets on one hand and patterns of dental microwear on the other. Furthermore, patterns on fossil teeth have been used to reconstruct diets in extinct forms. However, conventional methods for microwear analysis are limited. Scanning electron microscopy does not provide a true representation of these surfaces in three dimensions, and identification and measurement of individual features is time consuming, subjective, and subject to high interobserver error. This paper describes a new approach to the analysis of dental microwear using tandem scanning confocal microscopy and scale-sensitive fractal analyses. The instrument used in this study provides three-dimensional coordinates representing surfaces at a resolution equivalent to that employed by most SEM microwear studies. Fractal analyses offer objective, repeatable, quantitative characterization of surfaces. This approach eliminates major sources of error and increases power to resolve differences between species. Moreover, rapid surface characterization will allow examination of large samples to assess within species variation and to make finer distinctions between species.

Animals↗
PubMedDate not supplied

The structure of proteoglycan aggregate determined by atomic force microscopy.

Proteoglycan aggregate is the major extracellular matrix component in cartilage, comprising about 18% of the dry weight of hyaline cartilage. The proteoglycan aggregate is the major substance in cartilage which resists compression in the joint. The purpose of this study was to utilize the newly developed imaging technique, Atomic force Microscopy (AFM), to visualize the ultrastructure of proteoglycan aggregates. The proteoglycan aggregate molecules were imaged in air using the tapping mode of the AFM. The images illustrated the ultrastructure of the aggregates, especially the individual proteoglycan and the core hyaluronic acid. In addition to the length and width of each molecule, the height of the proteoglycan aggregates and the individual proteoglycans could be directly measured. The images of the ultrastructures of proteoglycan aggregates visualized from the AFM are comparable with those using conventional electron microscopy approaches. Nevertheless, the sample preparation for AFM imaging does not involve fixation, staining, coating, and other routine procedures required for traditional electron microscopy imaging. Thus, this technique could be a simple alternative approach for future analysis of proteoglycan aggregate and its assembly.

Animals↗
PubMed1995

Utility of fluorescence microscopy in embryonic/fetal topographical analysis.

For topographical analysis of developing embryos, investigators typically rely on scanning electron microscopy (SEM) to provide the surface detail not attainable with light microscopy. SEM is an expensive and time-consuming technique, however, and the preparation procedure may alter morphology and leave the specimen friable. We report that by using a high-resolution compound epifluorescence microscope with inexpensive low-power objectives and the fluorochrome acridine orange, we were able to obtain surface images of fixed or fresh whole rat embryos and fetal palates of considerably greater topographical detail than those obtained using routine light microscopy. Indeed the resulting high-resolution images afford not only superior qualitative documentation of morphological observations, but the capability for detailed morphometry via digitization and computer-assisted image analysis.

Animals↗
PubMed2000

Determination of the chelatable iron pool of single intact cells by laser scanning microscopy.

We have previously established a method of detecting intracellular chelatable iron in viable cells based on digital fluorescence microscopy. To quantify cellular chelatable iron, it was crucial to determine the intracellular indicator concentration. In the present study, we therefore adapted the method to confocal laser scanning microscopy, which should allow the determination of the indicator concentration on the single-cell level. The fluorescent heavy-metal indicator phen green SK (PG SK), the fluorescence of which is quenched by iron, was loaded into cultured rat hepatocytes. The hepatocellular fluorescence increased when cellular chelatable iron available to PG SK was removed from the probe by an excess of the membrane-permeable transition metal chelator 2,2'-dipyridyl (2, 2'-DPD, 5 mM). We optimized the scanning parameters for quantitatively recording changes in fluorescence and determined individual intracellular PG SK concentrations from the unquenched cellular fluorescence (after 2,2'-DPD) compared with PG SK standards in a "cytosolic" medium. An ex situ calibration method based on laser scanning microscopy was set up to determine the concentration of cellular chelatable iron from the increase of PG SK fluorescence after addition of 2,2'-DPD (5 mM). As the stoichiometry of the PG SK:Fe(2+) complex was 3:1 as long as PG SK was not limiting, cellular chelatable iron was calculated directly from absolute changes in cellular fluorescence. Using this method, we found 2.5 +/- 2.2 microM chelatable iron in hepatocytes. This method makes it possible to determine the pool of chelatable iron in single vital cells independently of cellular differences (e.g., dye loading, cell volume) in heterogeneous cell populations.

Animals↗
PubMed1997

Tertiary structure of the hepatic cell protein fibrinogen in fluid revealed by atomic force microscopy.

Fibrinogen participates in important cellular physiological processes, such as cell adhesion and blood clotting. Although the primary and secondary structures of fibrinogen are known, its tertiary structure is yet to be determined. In attempts to understand the tertiary structure of this important hydrated cellular and plasma membrane protein, the present study using atomic force microscopy was carried out. The techniques presented in this manuscript may also be applicable to enhance the imaging of live cells as well as their subcellular components. The authors have imaged fibrinogen by Tapping Mode atomic force microscopy in fluid. Purified human fibrinogen, together with 15-nm colloidal gold particles serving as an internal calibration standard, were adhered to a poly-L-lysine substrate on freshly cleaved mica. Atomic force microscopy images were obtained using oxide-sharpened silicon nitride probes, either unaltered or with an electron beam deposited extended tip. Although various structures were observed, the predominant forms consisted of a bi- or trinodular slightly curved linear shape. Approximately 300 of these structures were observed with six different tips (1 unaltered and 5 electron beam deposited) and their lengths and heights were analyzed. The mean length of the fibrinogen molecules was 65.8 nm and the mean height was 3.4 nm. The quantitative measurements were little influenced by the shape of the tip, whereas the sharper electron beam deposited tips seemed to produce qualitatively superior images.

Fibrinogen↗
PubMed1994

The structure of F-actin. Results of global searches using data from electron microscopy and X-ray crystallography.

The structure of F-actin was investigated by fitting the crystallographically determined actin monomer structure to F-actin electron microscopy data sets obtained by a variety of methods. A reciprocal-space global search procedure was applied to non-equatorial reciprocal-space amplitudes and phases of the microscopy data to locate minima. Fits were performed over a range of cross-sectional radii-of-gyration encompassing values obtained from X-ray solution scattering measurements. Five data sets from four laboratories were investigated: one from frozen-hydrated single filaments, three from negatively stained single filaments, and one from negatively stained single-layer paracrystals. In this last case the paracrystal data were straightened to improve resolution. The best fits nearly unanimously favored a monomer orientation having long-pitch connectivity that was close to that obtained by fitting X-ray fiber diffraction patterns. In certain cases where the resolving power was low, competitive fits were obtained with a quite different orientation, one having only protomer connectivity along the genetic helix. Using a running 10-residue deletion from the monomer in the Holmes-type orientation, subtle differences between the monomer structure and the protomer structure in F-actin could be detected. In particular, differences in the "DNase loop" (residues 41 to 50) and the "hydrophobic loop" (residues 264 to 273) were seen in single-filament data. In addition, a perturbation of the structure was seen in a region near residues 81 to 90. A rearrangement within the protomer structure reported in the literature did not produce fits as good as those obtained when using the undistorted monomer crystallographic structure without 10-residue deletions. These results, taken as a whole, provide strong support for a structure of Mg(2+)-ADP F-actin similar to that originally suggested by Holmes et al. but with alterations of the hydrophobic loop, the DNase loop and in the region near residues 81 to 90. These latter two regions have been proposed as secondary binding sites for myosin heads. The available evidence from electron microscopy and from other sources suggests that residues 40 to 49 are disordered in Mg(2+)-ADP F-actin.

Actins↗
PubMed1996

Freeze fracture electron microscopy of lyotropic lipid systems: quantitative analysis of the inverse micellar cubic phase of space group Fd3m (Q227).

An inverse micellar cubic phase of cubic aspect 15 formed by dioleoylglycerol/dioleoylphosphatidylcholine mixtures has been studied by freeze fracture electron microscopy. The structure was well preserved after freezing samples which had been hydrated either in pure water or in 30 vol% aqueous glycerol solutions. Electron microscopy images of high quality and resolution have been obtained. Four types of fracture planes, perpendicular to the [111], [110], [311] and [100] crystallographic axes, were identified by optical diffraction of the images from selected areas of the replicas. This is the largest number of different fracture planes yet observed in any lipid mesophase by electron microscopy. These planes are also perpendicular to the directions of the lowest order, and most intense reflections in the X-ray patterns from this cubic phase. The images were filtered using correlation averaging techniques, and they revealed the presence of mirror planes, which establishes that the space group is Fd3m (Q227) rather than Fd3. The interpretation of the images was aided by the novel use of standard deviation (s.d.) information obtained from the averaging procedures. The results are easily interpreted with the structure model deduced from X-ray diffraction and consisting of a complex packing of two different sizes of quasi-spherical inverse micelles located at positions (a) and (d) of the Fd3m unit cell. The results also show clearly that the fracture pathways always coincide with the regions of high CH3 concentration, located between the crystallographic planes containing the larger inverse micelles located at positions (a).

Diglycerides↗
PubMedDate not supplied

The Electron Microscopy Outreach Program: A Web-based resource for research and education.

We have developed a centralized World Wide Web (WWW)-based environment that serves as a resource of software tools and expertise for biological electron microscopy. A major focus is molecular electron microscopy, but the site also includes information and links on structural biology at all levels of resolution. This site serves to help integrate or link structural biology techniques in accordance with user needs. The WWW site, called the Electron Microscopy (EM) Outreach Program (URL: http://emoutreach.sdsc.edu), provides scientists with computational and educational tools for their research and edification. In particular, we have set up a centralized resource containing course notes, references, and links to image analysis and three-dimensional reconstruction software for investigators wanting to learn about EM techniques either within or outside of their fields of expertise.

Academies and Institutes↗
PubMed2000

Atomic force microscopy of the cell nucleus.

In mammals and plants, the cell nucleus is organized in dynamic macromolecular domains involved in DNA and RNA metabolism. These domains can be visualized by light and electron microscopy and their composition analyzed by using several cytochemical approaches. They are composed of chromatin or ribonucleoprotein structures as interchromatin and perichromatin fibers and granules, coiled bodies, and nuclear bodies. In plants, DNA arrangement defines chromocentric and reticulated nuclei. We used atomic force microscopy to study the in situ structure of the plant cell nucleus. Samples of the plants Lacandonia schismatica and Ginkgo biloba were prepared as for electron microscopy and unstained semithin sections were mounted on glass slides. For comparison, we also examined entire normal rat kidney cells using the same approach. Samples were scanned with an atomic force microscope working in contact mode. Recognizable images of the nuclear envelope, pores, chromatin, and nucleolus were observed. Reticulated chromatin was observed in L. schismatica. Different textures in the nucleolus of G. biloba were also observed, suggesting the presence of nucleolar subcompartments. The observation of nuclear structure in situ with the atomic force microscope offers a new approach for the analysis of this organelle at high resolution.

Animals↗
PubMed2001

Multi-photon microscopy in the evaluation of human saphenous vein.

BACKGROUND: The use of conventional fluorescence microscopy to image biological systems at the cellular level is limited by its inability to spatially resolve thick tissues. We have applied the technique of multi-photon fluorescence microscopy to study the structure and function of endothelial cells in living human saphenous vein taken from patients undergoing coronary artery bypass surgery. MATERIALS AND METHODS: Vein segments were preserved for 1-4 h to determine the temporal effects of storage. The effect of pH on endothelial and smooth muscle cell viability was examined by storing segments at pH 6.0, 7.4, and 8.0. Calcein-mediated green fluorescence and ethidium homodimer-mediated red fluorescence were used to differentiate cell viability. Increases in diaminofluorescein fluorescence were used to measure bradykinin activation of endothelial nitric oxide synthase (eNOS) with or without N-nitro-l-arginine (L-NNA). Multi-photon imaging was performed with the BioRad MRC1024ES system. RESULTS: Successful imaging of endothelial and smooth muscle cells of vein segments was achieved. Cell viability was well preserved up to 3 h of storage but dramatically decreased after 4 h. Cell viability was maintained at pH 7.4, diminished at pH 8.0, and was completely lost at pH 6.0. A two- to threefold increase in eNOS activity was observed upon activation by bradykinin which was completely inhibited in L-NNA-treated samples. CONCLUSIONS: We have demonstrated the successful application of multi-photon microscopy in imaging and quantifying nitric oxide production and cell viability under various storage conditions in human saphenous veins. This imaging technique allows for the functional imaging of cellular processes and may have diagnostic potential in cardiovascular surgery for patients undergoing bypass operations.

Cell Survival↗
PubMed2001

Imaging protein-protein interactions using fluorescence resonance energy transfer microscopy.

Fluorescence resonance energy transfer (FRET) detects the proximity of fluorescently labeled molecules over distances >100 A. When performed in a fluorescence microscope, FRET can be used to map protein-protein interactions in vivo. We here describe a FRET microscopy method that can be used to determine whether proteins that are colocalized at the level of light microscopy interact with one another. This method can be implemented using digital microscopy systems such as a confocal microscope or a wide-field fluorescence microscope coupled to a charge-coupled device (CCD) camera. It is readily applied to samples prepared with standard immunofluorescence techniques using antibodies labeled with fluorescent dyes that act as a donor and acceptor pair for FRET. Energy transfer efficiencies are quantified based on the release of quenching of donor fluorescence due to FRET, measured by comparing the intensity of donor fluorescence before and after complete photobleaching of the acceptor. As described, this method uses Cy3 and Cy5 as the donor and acceptor fluorophores, but can be adapted for other FRET pairs including cyan fluorescent protein and yellow fluorescent protein.

Animals↗
PubMed1999

Red and green laser Doppler compared with capillary microscopy to assess skin microcirculation in the feet of healthy subjects.

Skin microvasculature consists of nutritive capillaries and subpapillary arteriolar and venular plexus connected by arteriolovenular anastomoses. Capillary perfusion is of paramount importance for skin viability. Recently a new combined laser Doppler instrument has become available, featuring a combination of near-infrared (RL; 780 nm) and green (GL; 543 nm) laser light sources. Theoretically, the red laser will penetrate deeper, whereas the green laser will read fairly superficially. This may enable differentiation between the more superficial, i.e., capillary, and the deeper skin layers. To test this hypothesis, the combined laser Doppler technique was compared with nail fold capillary microscopy in the feet of 10 healthy subjects. Seven males and 3 females with a median age of 26 (range 20-42) years and without arterial pathology were investigated. The laser Doppler (Periflux 4001, Perimed) was equipped with a special dual probe conducting both GL and RL. The probe was attached to the pulp of the big toe (with many AV-shunts) and to the nail fold, at the site where capillary microscopy was performed too. Laser Doppler and capillary perfusion was assessed at rest and during postocclusive reactive hyperemia. These measurements were performed both in the sitting and the supine positions to test the postural vasoconstriction response. Median resting and hyperemic skin perfusion with GL were lower (P < 0.01) than with the RL in both areas and positions, except for the resting value in the sitting position on the dorsum of the toe. Plantar perfusion was found significantly higher than dorsal perfusion only with the RL in the supine position (P < 0.01). GL and RL on the plantar, but not the dorsal, side showed a significantly decreased perfusion upon dependency (P < 0.05), both at rest and during hyperemia. In contrast, resting and peak capillary velocity did show a decrease on dependency (P < 0.05). Although the green laser measures a lower perfusion than does the red laser, which is likely to be derived from more superficial skin layers, it does not show a reactivity similar to that measured with capillary microscopy. Thus, it is questionable whether the green laser exclusively measures capillary perfusion.

Adult↗
PubMed2006

Multiphoton laser scanning microscopy as a tool for Xenopus oocyte research.

Multiphoton laser scanning microscopy (MPLSM) has become an increasingly invaluable tool in fluorescent optical imaging. There are several distinct advantages to implementing MPLSM as a Xenopus oocyte research tool. MPLSM increases signal-to-noise ratio and therefore increases image quality because there is no out-of-focus fluorescence as would be created in conventional or confocal microscopy. All the light that is generated can be collected and used to generate an image because point detection of descanned fluorescence is not required. This is particularly useful when imaging deep into tissue sections, as is necessary for Xenopus oocytes, which are notoriously large (approximately 1-mm diameter). Because multiphoton lasers use pulsed energy in the infrared wavelengths, the energy can also travel further into tissues with much less light scattering. Because there is no out-of-focus excitation, phototoxicity, photodamage, and photobleaching are significantly reduced, which is particularly important for long-term experiments that require the same region to be scanned repeatedly. Finally, multiple fluorophores can be simultaneously excited because of the broader absorption spectra of multiphoton dyes. In this chapter, we describe the advantages and disadvantages of using MPLSM to image Xenopus oocytes as compared to conventional and confocal microscopy. The practical application of imaging oocytes is demonstrated with specific examples.

Animals↗
PubMed2005

Higher harmonic generation microscopy.

Higher harmonic-generation, including second harmonic generation and third harmonic generation, leaves no energy deposition to the interacted matters due to its virtual-level transition characteristic, providing a truly non-invasive modality and is ideal for in vivo imaging of live specimens without any preparation. Second harmonic generation microscopy provides images on stacked membranes and arranged proteins with organized nano-structures due to the bio-photonic crystalline effect. Third harmonic generation microscopy provides general cellular or subcellular interface imaging due to optical inhomogeneity. Due to their virtual-transition nature, no saturation or bleaching in the generated signal is expected. With no energy release, continuous viewing without compromising sample viability can thus be achieved. Combined with its nonlinearity, higher harmonic generation microscopy provides sub-micron three-dimensional sectioning capability and millimeter penetration in live samples without using fluorescence and exogenous markers, offering morphological, structural, functional, and cellular information of biomedical specimens without modifying their natural biological and optical environments.

Equipment Design↗
PubMed1991

Some contributions of electron microscopy to the study of the rickettsiae.

Electron microscopy has provided valuable insights into the study of rickettsiae as intracellular parasites from several important perspectives. This tool has allowed researchers to delineate the fine structural features of these organisms and to show that they truly resemble free-living bacteria. Furthermore, it has been shown that there are subtle, but distinct differences in the outer envelope structure of some members of the genus Rickettsia that may explain reported differences in tinctorial properties and in their sensitivity to certain antibiotics. With Coxiella burnetii, electron microscopy has helped significantly in the characterization of the pleomorphic nature of the organism including formation of terminal bodies that resemble endospores of gram-positive bacteria. Electron microsxopy has also helped to define the relationship of the rickettsiae to their host cells. For example, ultrastructural analysis can reveal whether organisms exist free within the cytoplasm or nucleus (members of the genus Rickettsia), or whether they are bound by a phagosomal or phagolysosomal membrane (Ehrlichia and Coxiella). Finally, although all rickettsiae eventually destroy their host cell, it has been shown through transmission electron microscopy that this destruction might be mediated by different mechanisms that are specific for different rickettsial species.

Animals↗
PubMed1978

The salivary glands of the cockroach Nauphoeta cinerea (Olivier). A study of its innervation by light and scanning electron microscopy.

The innervation of the salivary gland of the cockroach Nauphoeta cinerea (Olivier) has been investigated with the use of light and scanning electron microscopy. Light microscopy of methylene blue stained glands reveals the presence of a dual innervation arising from the ventral nerve cord and the stomodeal nervous system; the principal innervation is that from the ventral nerve cord which passes to the gland via the reservoir ducts. Branches of these nerves form a plexus on the acinar surface, the axons of which exhibit swelling at irregular intervals. The presence of this surface plexus and the axonal swellings was confirmed by scanning electron microscopy both in normal glands and in those in which the basal lamina had been removed by means of an HCl-collagenase digestion method. No acinar plexus was seen to be formed by branches of the stomatogastric nerve that were associated with the gland. However, other branches of this nerve were clearly connected with a complex network of multipolar neurones on the surfaces of the anterior regions of both salivary reservoirs.

Animals↗
PubMed1976

The primate median eminence. II. Correlative high-voltage transmission electron microscopy.

The ultrastructure of the normal median eminence of the male rhesus monkey (Macaca mulatta) is described using high-voltage electron microscopy. Surface specializations of ependymal cells lining the infundibular recess included cilia, apical extrusions, and microvilli. Supraependymal cells were predominantly macrophage-like, but examples of lymphocytic types were also seen. Tanycytes had long, branching, basal processes filled with numerous microtubules, some lipid droplets, and granules. The zona interna was composed of large unmyelinated neurosecretory fibers. A few myelinated fibers were also seen, but their character as neurosecretory fibers could not be established. The zona externa was composed of densely-packed profiles of neuro secretory fibers of small diameter, was well-vascularized and contained the terminations of tanycytes. Perivascular glial cells, vesiculated elements, pituicytes, and cellular elements common to connective tissue were observed. The intricate relationships between both the cellular and fibrous elements of the median eminence can be appreciated with the capability of high-voltage electron microscopy to discern ultrastructure in sections 10 times thicker than those used for low-voltage electron microscopy. The median eminence of this primate species has an ultrastructural organization similar to that described for most other species.

Animals↗
PubMed1994

High resolution detection of uncoated metaphase chromosomes by means of field emission scanning electron microscopy.

HeLa metaphase chromosomes were examined by means of "in lens" field emission scanning electron microscopy, which permits high resolution detection of uncoated biological samples. By using uncoated chromosomes as a model for comparison we report evidence of how traditional scanning electron microscopy techniques such as metal coating and conductive methods can generate errors in chromosome structure evaluation, since both give rise to morphological artifacts. By comparing the morphology of uncoated chromosomes obtained by two different isolation procedures, such as that utilized in standard cytogenetics and the polyamine method, we have drawn the following conclusions: (a) the standard cytogenetic method gives rise to a chromosome structure consisting of a flattened network of 10 nm fibers, in which higher order chromatin organization is absent. (b) Chromosomes obtained by the polyamine method show both three-dimensional profile and higher level folding of chromatin fibers, supporting the loop chromosome organization previously suggested by scanning electron microscopy observation of hexylene glycol isolated chromosomes.

Chromosomes, Human↗