Electron microscopic visualization of proteoglycans with Alcian Blue.

The intercellular matrices of bovine nasal cartilage, chick embryo perichordal cartilage, and chick embryo mesenchymal cells cultured in vitro have been examined by electron microscopy after staining them with Alcian Blue in salt solutions according to Scott & Dorling (1965). Matrix granules, which are typical components of cartilage at the ultrastructural level, are not visible after Alcian Blue staining and are replaced by alcianophilic rod-like particles, varying in length and width. With tissue cultures, Alcian Blue stains 40-120 A thick filaments which display an orthogonal and longitudinal relationship to collagen fibrils. We assume that cartilage matrix granules represent linear proteoglycans that are coiled as a consequence of the usual glutaraldehyde-osmium fixation. It is thought that Alcian Blue, on the other hand, contributes to the stabilization of the proteoglycans in their original structural arrangement. This stabilizing property presumably also results in the sharp visualization of fine filaments in the tissue culture matrix.     

Fluorescence microscopic visualization of a DNA-cationic fullerene complex.

Cationic fullerene derivative 1, which is soluble in aqueous medium, bound to double stranded DNA. Observation of Coliphage T4 DNA in the absence and presence of 1 by fluorescence microscopy suggested that 1 bound to DNA along its groove. Upon irradiation of visible light on the DNA complex of 1, DNA was cleaved dramatically. The process of DNA photo-cleavage could be monitored continuously by fluorescence microscopy.     

Electron microscopic visualization of restriction sites on DNA molecules.

DNA molecules were adsorbed to a polylysine-treated carbon film and digested directly on the film by restriction enzymes. After washing the film with 1 M NaCl, 0.4% Kodak Photo-Flo and 9% formamide, each cleavage site introduced was visualized as a gap under the electron microscope. By measuring the gapped positions on linear DNA molecules induced by other enzymes, a single EcoRI site on a lambda dv1 molecule and three HinHI sites on an fd1RF molecule were mapped at the positions expected from the cleavage maps, respectively. This electron-microscopic procedure may be useful for the construction of a cleavage map.     

Scanning electron microscopic visualization of collagen fibers in embryonic chick skin.

The disposition of collagen fibers in embryonic chick skin can be visualized by use of scanning microscopy (SEM). Chick back skin was removed from 8-day embryos, the epithelial and mesenchymal components were separated, and the mesenchyme was subjected to 10% trypsin treatment (Stuart, E. S., and Moscona, A. A. (1967) Science 157, 947–948), after which it was prepared for SEM by critical point drying and coating. Such preparations were largely free of cellular material. Cavities which presumably had contained the cells were present in a network of fibers. Skin of the scaleless mutant was also studied. In this mutant the collagen network was more irregular and collagen fiber diameter was more variable. These findings are discussed in connection with the formation of feather germ pattern.     

3D parallel coordinate systems--a new data visualization method in the context of microscopy-based multicolor tissue cytometry.

BACKGROUND: Presentation of multiple interactions is of vital importance in the new field of cytomics. Quantitative analysis of multi- and polychromatic stained cells in tissue will serve as a basis for medical diagnosis and prediction of disease in forthcoming years. A major problem associated with huge interdependent data sets is visualization. Therefore, alternative and easy-to-handle strategies for data visualization as well as data meta-evaluation (population analysis, cross-correlation, co-expression analysis) were developed. METHODS: To facilitate human comprehension of complex data, 3D parallel coordinate systems have been developed and used in automated microscopy-based multicolor tissue cytometry (MMTC). Frozen sections of human skin were stained using the combination anti-CD45-PE, anti-CD14-APC, and SytoxGreen as well as the appropriate single and double negative controls. Stained sections were analyzed using automated confocal laser microscopy and semiquantitative MMTC-analysis with TissueQuest 2.0. The 3D parallel coordinate plots are generated from semiquantitative immunofluorescent data of single cells. The 2D and 3D parallel coordinate plots were produced by further processing using the Matlab environment (Mathworks, USA). RESULTS: Current techniques in data visualization primarily utilize scattergrams, where two parameters are plotted against each other on linear or logarithmic scales. However, data evaluation on cartesian x/y-scattergrams is, in general, only of limited value in multiparameter analysis. Dot plots suffer from serious problems, and in particular, do not meet the requirements of polychromatic high-context tissue cytometry of millions of cells. The 3D parallel coordinate plot replaces the vast amount of scattergrams that are usually needed for the cross-correlation analysis. As a result, the scientist is able to perform the data meta-evaluation by using one single plot. On the basis of 2D parallel coordinate systems, a density isosurface is created for representing the event population in an intuitive way. CONCLUSIONS: The proposed method opens new possibilities to represent and explore multidimensional data in the perspective of cytomics and other life sciences, e.g., DNA chip array technology. Current protocols in immunofluorescence permit simultaneous staining of up to 17 markers. Showing the cross-correlation between these markers requires 136 scattergrams, which is a prohibitively high number. The improved data visualization method allows the observation of such complex patterns in only one 3D plot and could take advantage of the latest developments in 3D imaging.     

Electron microscopic visualization of DNA single strand breaks

DNA single strand breaks (ssb) have been induced in FLC/C cells in culture. They have been visualized in the electron microscope after decoration with biotin-avidin-ferritin complexes and spreading as monomolecular mixed films. This allowed one to determine the average number of decorated ssbs per unit of DNA length applying straight-forward and simple evaluation methods. This method has been used to investigate the DNA alterations by benzo[a]pyrene (B[a]P) on FLC/C culture cells. Thus a B[a]P-DNA damage curve can be constructed as a regression with a correlation coefficient of r = 0.97, while its isomer benzo[e]pyrene (B[e]P) known to have only low mutagenicity under the same experimental conditions is virtually without effect. The method has further informational potential regarding damage distribution and repair of DNA.     

Simultaneous visualization of multiple protein interactions in living cells using multicolor fluorescence complementation analysis

The specificity of biological regulatory mechanisms relies on selective interactions between different proteins in different cell types and in response to different extracellular signals. We describe a bimolecular fluorescence complementation (BiFC) approach for the simultaneous visualization of multiple protein interactions in the same cell. This approach is based on complementation between fragments of fluorescent proteins with different spectral characteristics. We have identified 12 bimolecular fluorescent complexes that correspond to 7 different spectral classes. Bimolecular complex formation between fragments of different fluorescent proteins did not differentially affect the dimerization efficiency of the bZIP domains of Fos and Jun or the subcellular sites of interactions between these domains. Multicolor BiFC enables visualization of interactions between different proteins in the same cell and comparison of the efficiencies of complex formation with alternative interaction partners.     

Immunoelectron microscopic visualization of neurohypophyseal hormones: evaluation of some tissue preparations and staining procedures.

To obtain optimal electron microscopical localization of vasopressin in the rat neurohypophyses two immunocytochemical staining procedures and several tissue treatments were evaluated. The peroxidase-antiperoxidase technique allowed greater dilution of the first antibody than the indirect method using a commercial peroxidase conjugate. This appeared crucial for the dilution-dependent specificity in the localization of vasopressin. Immersion fixation with formalin gave better results than those obtained with perfusion fixation with glutaraldehyde-paraformaldehyde (resulting in similar preservation of immunoreactivity) and freeze substitution (showing the best preservation of immunoreactivity). However, these latter two tissue fixation methods resulted in less than optimal preservation of general ultrastructure than immersion fixation in formalin alone. Immersion fixation with glutaraldehyde-paraformaldehyde followed by OsO4 improved ultrastructural detail, but immunoreactivity decreased considerably. Fixation with paraformaldehyde-picric acid resulted in poorest preservation of morphologic detail. Immunoreactivity was similar in both Epon 812 and Araldite 6005 embedded tissue.     

Poly(ADP-ribose) polymerase auto-modification and interaction with DNA: electron microscopic visualization.

The interaction between purified calf thymus poly(ADP-ribose) polymerase and its activating co-purified DNA (sDNA) was investigated by electron microscopy. We have shown that the enzyme-DNA complex possesses a nucleosome-like structure. The enzyme-bound DNA (sDNA) was found to be enriched in single-stranded regions and branched structures, presumed to be replication forks. The auto-ribosylated polymerase as well as the branched poly(ADP-ribose) formed were visualized by dark field electron microscopy during the auto-ADP-ribosylation reaction and the possible mechanism of this phenomenon is discussed.     

Simultaneous visualization of two protein complexes in a single plant cell using multicolor fluorescence complementation analysis

Bimolecular fluorescence complementation (BiFC) is an approach used to analyze protein–protein interaction in vivo, in which non-fluorescent N-terminal and C-terminal fragments of a fluorescent protein are reconstituted to emit fluorescence only when they are brought together by interaction of two proteins to fuse both fragments. A method for simultaneous visualization of two protein complexes by multicolor BiFC with fragments from green fluorescent protein (GFP) and its variants such as cyan and yellow fluorescent proteins (CFP and YFP) was recently reported in animal cells. In this paper we describe a new strategy for simultaneous visualization of two protein complexes in plant cells using the multicolor BiFC with fragments from CFP, GFP, YFP and a red fluorescent protein variant (DsRed-Monomer). We identified nine different BiFC complexes using fragments of CFP, GFP and YFP, and one BiFC complex using fragments of DsRed-Monomer. Fluorescence complementation did not occur by combinations between fragments of GFP variants and DsRed-Monomer. Based on these findings, we achieved simultaneous visualization of two protein complexes in a single plant cell using two colored fluorescent complementation pairs (cyan/red, green/red or yellow/red).     

Electron microscopic visualization of the filament binding mode of actin-binding proteins

A large number of actin-binding proteins (ABPs) regulate various kinds of cellular events in which the superstructure of the actin cytoskeleton is dynamically changed. Thus, to understand the actin dynamics in the cell, the mechanisms of actin regulation by ABPs must be elucidated. Moreover, it is particularly important to identify the side, barbed-end or pointed-end ABP binding sites on the actin filament. However, a simple, reliable method to determine the ABP binding sites on the actin filament is missing. Here, a novel electron microscopic method for determining the ABP binding sites is presented. This approach uses a gold nanoparticle that recognizes a histidine tag on an ABP and an image analysis procedure that can determine the polarity of the actin filament. This method will facilitate future study of ABPs.     

Electron microscopic visualization of the outer membrane permeability of Bacteroides fragilis

Abstract Electron microscopy of negatively stained Bacteroides fragilis cells treated with hypertonic solutions of di- or trisaccharides showed the shrinkage of whole cells, whereas the pentose- or hexose-treated cells shrank less significantly. Electron microscopy of thin sections of the monosaccharide-treated cells showed plasmolysis. Determination of the diffusion rate by the liposome-swelling method suggested that the apparent exclusion limit of the outer membrane pore is close to the size of uncharged saccharides of approx. M _r 250. These results suggested that the outer membrane of B. fragilis contains small diffusion pores.