Real time imaging of rotating molecular machines.
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Observation of true rotation has been relatively rare in living systems, but there may be many molecular machines that rotate. Molecular rotations accompanying function can be imaged in real time under an optical microscope by attaching to the protein machine either a small tag such as a single fluorophore or a tag that is huge compared with the size of the protein. As an example of the former approach, axial rotation of an actin filament sliding over myosin has been measured quantitatively by attaching a fluorophore rigidly to the filament and imaging the orientation of the fluorophore continuously by polarization microscopy. As a huge tag in the latter approach, an actin filament turned out to be quite useful. Using this tag, the enzyme F1-ATPase has been shown to be a rotary stepper motor made of a single molecule. Further, the efficiency of this ATP-fueled motor has been shown to reach almost 100%. The two examples above demonstrate that one can now image conformational changes, which necessarily involve reorientation, in a single protein molecule during function. Single-molecule physiology is no longer a dream. (+info)
Microscopic investigation in fossil hominoidea: a clue to taxonomy, functional anatomy, and the history of diseases.
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Ten selected samples of fossilized bones (including Australopithecus, Homo erectus, Homo neandertalensis, and Homo sapiens sapiens) were examined by light microscopy using plane and polarized light. The histomorphological findings show that microscopic research adds much to what can be ascertained by marcoscopic examination or by X-ray techniques. In particular, emphasis was placed on taxonomy, functional anatomy of bones, and causes of some of the diseases of early hominids. Anat Rec (New Anat): 257:225-232, 1999. (+info)
Assessment of bronchial inflammation using an automated cell recognition system based on colour analysis.
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The aim of this study was to develop an automated system of cell recognition based upon colour analysis suitable for microscopic examination of bronchial inflammation. Human bronchi obtained from 17 patients undergoing thoracotomy were embedded in glycolmethacrylate to perform immunohistochemistry with antibodies against: neutrophil elastase, tryptase, chymase, eosinophil cationic protein, CD68, CD3 and immunoglobulin E. The image analysis system calculates three independent criteria (optic density, hue density, hue) combined with morphological parameters to specifically recognize a positive staining. This automated analysis was applied to the study of bronchial inflammation in smokers and nonsmokers in terms of the absolute number of cells and the expression of different markers by a single cell. The use of these criteria enabled the characterization of a positive stain on single (intraclass correlation coefficient (ICC) = 0.88 or serial (ICC = 0.84) sections. Cell counts obtained by the automated system were highly reproducible. Regarding bronchial inflammation, it was found that the number of inflammatory cells was significantly higher in smokers than in nonsmokers, the majority of these cells bearing immunoglobulin E. These results demonstrate that such computerized analysis of colours is a valuable method for quantifying inflammatory cells in bronchial tissue and for analysing the expression of different markers by a single cell. (+info)
The inhibitory effects of recombinant plasminogen kringle 1-3 on the neovascularization of rabbit cornea induced by angiogenin, bFGF, and VEGF.
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Angiostatin is a potent angiogenesis inhibitor that is composed of the first four kringles of plasminogen fragment. Angiostatin with one less kringle molecule (kringle 1 to 3) was recently demonstrated to be an effective angiogenic inhibitor. To determine whether recombinant plasminogen kringle 1-3 (rPK1-3) can inhibit the corneal neovascularization induced by potent angiogenic factors; angiogenin, bFGF, or VEGF, hydron polymer discs each containing 2.0 microg of angiogenin, 500 ng of bFGF, or 500 ng of VEGF respectively were implanted into the corneal stroma of 138 rabbit eyes, and then discs each containing 10 microg, 12.5 microg, 20 microg or 30 microg of rPK1-3 were implanted randomly. Discs containing phosphate buffered saline were also implanted as a control. The angiogenesis score on number and length of newly formed vessels on the each of the rabbit's cornea were recorded daily by two observers (blinded). The treated corneas were also examined histologically. Recombinant PK1-3 treated corneas showed less neovascularization induced by all angiogenic factors (p < 0.05). and the extent of inhibition of neovascularization was proportional to the concentration of rPK1-3 (p < 0.05). Histologic examination showed leukocyte infiltration into the corneal stroma on the PBS treated eyes whereas rPK1-3 treated eyes showed only traces of leukocytes. These results of the effective rPK1-3 inhibition of corneal neovascularization induced by angiogenin, bFGF, or VEGF suggest that this angiostatin related fragment, rPK1-3, may be useful in the treatment of various neovascular diseases. (+info)
Identification of and spatio-temporal differences between microbial assemblages from two neighboring sulfurous lakes: comparison by microscopy and denaturing gradient gel electrophoresis.
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The microbial assemblages of Lake Ciso and Lake Vilar (Banyoles, northeast Spain) were analyzed in space and time by microscopy and by performing PCR-denaturing gradient gel electrophoresis (DGGE) and sequence analysis of 16S rRNA gene fragments. Samples obtained from different water depths and at two different times of the year (in the winter during holomixis and in the early spring during a phytoplankton bloom) were analyzed. Although the lakes have the same climatic conditions and the same water source, the limnological parameters were different, as were most of the morphologically distinguishable photosynthetic bacteria enumerated by microscopy. The phylogenetic affiliations of the predominant DGGE bands were inferred by performing a comparative 16S rRNA sequence analysis. Sequences obtained from Lake Ciso samples were related to gram-positive bacteria and to members of the division Proteobacteria. Sequences obtained from Lake Vilar samples were related to members of the Cytophaga-Flavobacterium-Bacteroides phylum and to cyanobacteria. Thus, we found that like the previously reported differences between morphologically distinct inhabitants of the two lakes, there were also differences among the community members whose morphologies did not differ conspicuously. The changes in the species composition from winter to spring were also marked. The two lakes both contained sequences belonging to phototrophic green sulfur bacteria, which is consistent with microscopic observations, but these sequences were different from the sequences of cultured strains previously isolated from the lakes. Euryarchaeal sequences (i.e., methanogen- and thermoplasma-related sequences) also were present in both lakes. These euryarchaeal group sequences dominated the archaeal sequences in Lake Ciso but not in Lake Vilar. In Lake Vilar, a new planktonic population related to the crenarchaeota produced the dominant archaeal band. The phylogenetic analysis indicated that new bacterial and archaeal lineages were present and that the microbial diversity of these assemblages was greater than previously known. We evaluated the correspondence between the abundances of several morphotypes and DGGE bands by comparing microscopy and sequencing results. Our data provide evidence that the sequences obtained from the DGGE fingerprints correspond to the microorganisms that are actually present at higher concentrations in the natural system. (+info)
Single-target molecule detection with nonbleaching multicolor optical immunolabels.
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We introduce and demonstrate the use of colloidal silver plasmon-resonant particles (PRPs) as optical reporters in typical biological assays. PRPs are ultrabright, nanosized optical scatterers, which scatter light elastically and can be prepared with a scattering peak at any color in the visible spectrum. PRPs are readily observed individually with a microscope configured for dark-field microscopy, with white-light illumination of typical power. Here we illustrate the use of PRPs, surface coated with standard ligands, as target-specific labels in an in situ hybridization and an immunocytology assay. We propose that PRPs can replace or complement established labels, such as those based on radioactivity, fluorescence, chemiluminescence, or enzymatic/colorimetric detection that are used routinely in biochemistry, cell biology, and medical diagnostic applications. Moreover, because PRP labels are nonbleaching and bright enough to be rapidly identified and counted, an ultrasensitive assay format based on single-target molecule detection is now practical. We also present the results of a model sandwich immunoassay for goat anti-biotin antibody, in which the number of PRP labels counted in an image constitutes the measured signal. (+info)
Virtual neuropathology: three-dimensional visualization of lesions due to toxic insult.
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A first-pass approach incorporating high-field magnetic resonance imaging (MRI) was used for rapid detection of neuropathologic lesions in fixed rat brains. This inherently 3-dimensional and nondestructive technique provides high-resolution, high-contrast images of fixed neuronal tissue in the absence of sectioning or staining. This technique, magnetic resonance microscopy (MRM), was used to identify diverse lesions in 2 well-established rat neurotoxicity models. The intrinsic contrast in the images delineated lesions that were identified using a battery of histologic stains, some of which would not be used in routine screening. Furthermore, the MRM images provided the locations of lesions, which were verified upon subsequent sectioning and staining of the same samples. The inherent contrast generated by water properties is exploited in MRM by choosing suitable pulse sequences, or proton stains. This approach provides the potential for a comprehensive initial MRM screen for neurotoxicity in preclinical models with the capability for extrapolation to clinical analyses using classical MRI. (+info)
Noncontact specular microscopy of human lens epithelium.
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PURPOSE: To obtain in vivo specular images of human lens epithelial cells (LECs) from persons with or without age-related cataract (ARC); to identify features that describe individual aspects of these complex images; to develop feature scales to quantify the severity of each feature; and to study the association of these features with LEC count, age, Lens Opacity Classification System III (LOCS III) classifications and microscopic features of lens epithelium in ARC. METHODS: One hundred fifty-two individuals underwent ophthalmic examinations and LOCS III cataract classifications. Specular images of lenses were captured using a modified noncontact corneal specular microscope (SML-2; Konan, Hyogo, Japan). Enhanced images were graded in a masked fashion, and the presence or absence and severity of each of four features in the specular image ("columnar organization," "linear furrows," "puffy clouds," and "black holes") was graded on a four-step scale. The generalized linear model with intraclass correlation was used to ascertain the statistical significance of associations between age, sex, LOCS III grade, cell count, and feature grade. Capsulorrhexis specimens from 29 patients were studied with correlative light and electron microscopy. RESULTS: LEC density declined with age and was inversely correlated with the scalar grade for puffy clouds and for the size and number of black holes. The scalar grade for columnar organization was inversely associated with the severity of posterior subcapsular and nuclear cataracts, which was the only feature associated with the LOCS III grade of ARC. No statistically significant associations were found between average cell count and LOCS III grade. CONCLUSIONS: With the use of the corneal specular microscope excellent in vivo specular images of the LECs were obtained, the features in these images that correlated well with microscopic findings were classified, and cell density in vivo was estimated. (+info)