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At least 1,603 records · Page 89Linked to original sources
PubMed1991

Laser scanning and confocal microscopy of daunorubicin, doxorubicin, and rhodamine 123 in multidrug-resistant cells.

The multidrug-resistant gene (MDR1) encodes an energy-dependent drug efflux pump (P-glycoprotein) for many anti-cancer drugs. We have studied the intracellular distribution of rhodamine 123 (R123), daunorubicin (DN), and doxorubicin (DOX) in cells expressing a human MDR1 gene. The distribution of these fluorescent drugs was measured by laser scanning microscopy and confocal microscopy. We devised a new method for analysis of fluorescence line scan data to determine the intracellular distribution of fluorescent probes. This method and confocal microscopy showed that R123, DN, and DOX are localized to both plasma membrane and intracellular compartments in multidrug-resistant cells. When the cells are treated with verapamil, an inhibitor of the multidrug transporter, the amount of DOX, DN, and R123 associated with the cell rises. After inhibition, the relative distribution of DOX and DN between the cell surface and intracellular structures does not change dramatically. However, R123 tends to relocalize to intracellular sites from predominantly plasma membrane sites, indicating that this dye behaves differently than the anti-cancer drugs. These results show the subcellular distributions of R123, DN, and DOX in plasma membrane, cytoplasm, and intracellular membrane systems, but do not allow definitive distinctions among existing models of how P-glycoprotein affects the distribution of drugs.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1989

Verifying copper toxicosis in Bedlington terriers by analytical electron microscopy of needle biopsies of liver.

Diagnosis of copper toxicosis in Bedlington terriers was performed by analytical electron microscopy of needle biopsy of the liver. The animals were four Bedlington terriers clinically suspected of hereditary copper toxicosis, and four healthy control animals of the same breed. Light microscopy of the livers of the diseased animals revealed dense bodies in the hepatocytes which stained histochemically for copper. Analytical electron microscopy showed that the intrahepatocytic bodies consisted almost entirely of copper.

Animals↗
PubMed1991

Macrophage phagocytosis: use of fluorescence microscopy to distinguish between extracellular and intracellular bacteria.

One of the challenges of phagocytosis research is to differentiate bacteria adherent to a host cell from bacteria which the cell has internalized. To address this question, various techniques such as fluorescence microscopy, electron microscopy, and flow cytometry have been used. We have adapted a flow cytometric method (Fattorossi et al., 1989) to use fluorescence microscopy for studying phagocytosis of fluorescein-labeled Listeria by inflammatory mouse peritoneal macrophages. In this assay, ethidium bromide is used as a quenching agent and is added to cells after they have phagocytosed labeled bacteria. Ethidium bromide causes extracellular FITC-labeled Listeria to fluoresce red-orange, whereas intracellular bacteria are not exposed to the dye and remain green. This process allows distinction between intracellular and extracellular bacteria by simultaneous visualization of both populations.

Animals↗
PubMed1988

Direct localization of the tRNA--anticodon interaction site on the Escherichia coli 30 S ribosomal subunit by electron microscopy and computerized image averaging.

Previous immunoelectron microscopy studies have shown that the anticodon of valyl-tRNA, photocrosslinked to the ribosomal P site at the C1400 residue of the 16 S RNA, is located in the vicinity of the cleft of the small ribosomal subunit of Escherichia coli. In this study we used single-particle image-averaging techniques to demonstrate that the 30 S-bound tRNA molecule can be localized directly, without the need for specific antibody markers. In agreement with the immunoelectron microscopy results, we find that the tRNA molecule appears to be located deep in the cleft of the 30 S subunit. We believe that the use of computer image averaging to localize ligands bound to ribosomes and other macromolecular complexes will become widespread because of the superior sensitivity, precision and objectivity of this technique compared with conventional immunoelectron microscopy.

Anticodon↗
PubMed1991

Secondary structure of coliphage Q beta RNA. Analysis by electron microscopy.

The secondary structure of genomic RNA from the coliphage Q beta has been examined by electron microscopy in the presence of varying concentrations of spermidine using the Kleinschmidt spreading technique. The size and position of structural features that cover 70% of the viral genome have been mapped. The structural features that are visualized by electron microscopy in Q beta RNA are large. They range in size from 170 to 1600 nucleotides. A loop containing approximately 450 nucleotides is located at the 5' end of the RNA. It includes the initiation region for the viral maturation protein. A large hairpin containing approximately 1600 nucleotides is located in the center of the molecule. It is multibranched and includes most of the viral coat gene, the readthrough region of the A1 gene, and approximately one third of the viral replicase gene. Within the central hairpin, the initiation region for the viral replicase gene pairs with a region within the distal third of the viral coat gene. This structure may participate in the regulation of translational initiation of the viral replicase gene. Two structural variants of the central hairpin were observed. One of them brings the internal S and M viral replicase binding regions into juxtaposition. These observations suggest that the central hairpin may also participate in the regulation of translation of the viral coat gene. The secondary structures that are observed in Q beta RNA differ significantly from structures that we described previously in the genomic RNA of coliphage MS2 but are similar to structures we observed by electron microscopy in the related group B coliphage SP.

Coliphages↗
PubMed1992

Insect crossbridges, relaxed by spin-labeled nucleotide, show well-ordered 90 degrees state by X-ray diffraction and electron microscopy, but spectra of electron paramagnetic resonance probes report disorder.

The structure of glycerinated Lethocerus insect flight muscle fibers, relaxed by spin-labeled ATP and vanadate (Vi), was examined using X-ray diffraction, electron microscopy and electron paramagnetic resonance (e.p.r.) spectra. We obtained excellent relaxation of MgATP quality as determined by mechanical criteria, using vanadate trapping of 2' spin-labeled 3' deoxyATP at 3 degree C. In rigor fibers, when the diphosphate analog is bound in the absence of Vi, the probes on myosin heads are well-ordered, in agreement with electron microscopic and X-ray patterns showing that myosin heads are ordered when attached strongly to actin. In relaxed muscle, however, e.p.r. spectra report orientational disorder of bound (Vi-trapped) spin-labeled nucleotide, while electron microscopic and X-ray patterns both show well-ordered bridges at a uniform 90 degrees angle to the filament axis. The spin-labeled nucleotide orientation is highly disordered, but not completely isotropic; the slight anisotropy observed in probe spectra is consistent with a shift of approximately 10% of probes from angles close to 0 degrees to angles close to 90 degrees. Measurements of probe mobility suggest that the interaction between probe and protein remains as tight in relaxed fibers as in rigor, and thus that the disorder in relaxed fibers arises from disorders of (or within) the protein and not from disorder of the probe relative to the protein. Fixation of the relaxed fibers with glutaraldehyde did not alter any aspect of the spectrum of the Vi-trapped analog, including the slight order observed, showing that the extensive inter- and intra-molecular cross-linking of the first step of sample preparation for electron microscopy had not altered relaxed crossbridge orientations. Two models that may reconcile the apparently disparate results obtained on relaxed fibers are presented: (1) a rigid myosin head could possess considerable disorder in the regular array about the thick filament; or (2) the nucleotide site could be on a disordered, probably distal, domain of myosin, while a more proximal region is well ordered on the thick filament backbone. Our findings suggest that when e.p.r. probes signal disorder of a local site or domain, this is complementary, not contradictory, to signals of general order. The e.p.r. spectra show that a portion of the myosin molecule can be disordered at the same time as the X-ray diffraction and electron microscopy show the bulk of myosin head mass to be uniformly oriented and regularly arrayed.

Animals↗
PubMed1992

Quantitation of molecular densities by cryo-electron microscopy. Determination of the radial density distribution of tobacco mosaic virus.

We have determined the absolute mass and radial scattering density distribution of tobacco mosaic virus in the frozen-hydrated state by energy-filtered low-dose bright-field transmission electron microscopy. The absolute magnitude of electron scattering from tobacco mosaic virus in 150 nm of ice was within 3.0% of that predicted, with inelastic scattering accounting for approximately 80% of the scattering contrast. In order to test the accuracy of the radial reconstruction, a computer model of tobacco mosaic virus was built from the atomic co-ordinates assuming uniform solvent density. The validity of the model was confirmed by comparison of X-ray scattering and predictions of the model (R factor = 0.05). First-order corrections for the microscope contrast transfer function were necessary and sufficient for conversion of the cryo-electron microscopy images into accurate representations of the mass density. At 1.9 nm resolution the compensated reconstruction and model had density peaks of similar magnitude at 2.4, 4.2, 6.0 and 7.8 nm radius and a central hole of 2 nm radius. Equatorial Fourier transforms of the corrected electron images were in excellent agreement with predictions of the model (R factor = 0.12). Thus, the uniform solvent approximation was adequate at 1.9 nm resolution to describe quantitatively X-ray scattering in liquid water and electron imaging in vitreous ice. This is the first demonstration that cryo-electron microscopy images can be used to quantitate the absolute mass, mass per unit length and internal density distributions of proteins and nucleic acids.

Computer Simulation↗
PubMed1990

Analysis of glass ionomer cement with use of scanning electron microscopy.

In the study, scanning electronmicroscopy and x-ray microanalysis techniques were used to examine a silver-reinforced glass ionomer cement. Scanning electron microscopy was used to evaluate surface topography and microstructure. Microcracking and a bimodal pore distribution were observed. The cement consisted of unreacted glass particles surrounded by a silica gel "halo" and embeded in an organic salt matrix. Back-scattered electron microscopy and energy-dispersive spectroscopy detected discrete clusters of silver atoms within the material. Other elements, such as calcium and aluminum, were dispersed homogeneously. Wave-length dispersive spectroscopy was used to detect fluoride, which also appeared to be homogeneously distributed. Electron microscopy and x-ray microanalysis techniques could prove valuable in clarifying the microstructure and fluoride release mechanisms of glass ionomer cements. Refinement of some aspects, including sample preparation, will be necessary to obtain consistently reliable results.

Aluminum↗
PubMed1984

Distribution of capillary blood flow in the microcirculation of the hamster: an in vivo study using epifluorescent microscopy.

In vivo epifluorescent microscopy (EPI) was used to study capillary perfusion in superfused hamster cheek pouch, and cremaster and sartorius muscle preparations. In cheek pouches and cremaster muscles, in vivo epifluorescence microscopy (EPI) was compared to in vivo transillumination microscopy (TRANS) and the former was found to allow detection of a larger number of capillaries--34% more capillaries in cheek pouch and 21% more in cremasters were observed with EPI. The fraction of capillaries containing erythrocytes alone, plasma alone or plasma plus red cells was determined in all three tissues. Also, the fraction of capillaries unperfused was noted. Less than 2% of the capillaries contained plasma alone. The number of capillaries which contained stationary erythrocytes varied with vasomotor tone. In control cremaster and sartorius muscles 17 and 13% of observed capillaries were unperfused but contained erythrocytes. Ninety-eight percent of capillaries contained stationary erythrocytes in cremaster muscles vasoconstricted with 21% oxygen. From these observations we conclude (1) functional capillary counts obtained with TRANS may represent underestimates of the true number; (2) plasma shunts do not appear to be a significant factor in normal microcirculatory function in the hamster cheek pouch, or in cremaster and sartorius muscles; (3) since unperfused capillaries contain red cells, red cell counts in histological sections will overestimate functional capillarity. The magnitude of the overestimation will be a function of vascular tone and capillary hematocrit.

Animals↗
PubMed1992

Effect of calcium on the vasoactive response of arterioles to light during fluorescent intravital microscopy of the tibialis anterior muscle of the hamster.

Arterioles may undergo a transient vasoactive response when exposed to light during fluorescent intravital microscopy. We hypothesized that the type and frequency of the vasoactive responses by arterioles to light is calcium dependent. In order to test this hypothesis we quantitated the type and frequency of vasoactive responses by arterioles to light in the presence and absence of calcium within the suffusate bathing the muscle. In addition, we determined whether the presence or absence of calcium also influenced the reactivity of these arterioles to adenosine and norepinephrine. In separate experiments, we determined the effect of increases in suffusate calcium concentration on the resting diameter of arterioles. The concentration of calcium in the suffusate significantly influenced the frequency of light-induced vasoactivity of the arterioles of the tibialis anterior muscle: a normal suffusate calcium concentration of 2 x 10(-3) M was associated with an incidence of light-induced vasoreactivity of 77% but decreased significantly to 58% when calcium was removed from the suffusate. Although the frequency of the vasoactive response to light was different for the two experimental conditions, the type of vasoactive response, predominately vasomotion, was similar. The vasoactive responses of arterioles to adenosine and norepinephrine were similar in the presence and absence of calcium in the suffusate. However, the concentration of calcium in the suffusate did significantly influence resting arteriolar diameters: 2 x 10(-3) M calcium CaCl2 caused a mean decrease in the arteriolar diameter of 11.6 (+/- 3.0)%, 4 x 10(-3) M CaCl2 caused a mean decrease of 41.2 (+/- 12)%, and 8 x 10(-3) M CaCl2 caused a mean decrease of 55.4 (+/- 14.4)%. These results show that the concentration of calcium in the suffusate bathing the tibialis anterior muscle during fluorescent intravital microscopy significantly influences the vasoactive response of arterioles to light. Further investigation of the mechanisms of light-induced effects on microcirculation during fluorescent intravital microscopy will become important as this technique is used more widely in the study of the microcirculation of solid tissues and organs.

Adenosine↗
PubMed1990

Detection of rubella virus in amniotic fluid by electron microscopy.

We report in this paper the observation of rubella virus by electron microscopy in an amniotic fluid sample, collected from a pregnant woman with rubella infection. Virological investigations by inoculation of cell cultures with amniotic fluid and fetal blood remained negative, due probably to the presence of neutralizing antibodies in the samples. Electron microscopy is a rapid but weakly sensitive method to detect viruses in clinical specimens. However, this unusual observation would indicate that in some cases electron microscopy could be a useful technique to evidence a fetal rubella infection.

Adult↗
PubMedDate not supplied

Specular microscopy in the identification of deep corneal opacities.

Specular microscopy provides a level of magnification and differentiation of detail which approaches that of a histological examination, and has the advantage of being a noninvasive procedure. This makes specular microscopy useful not only in examining the corneal endothelium, but also in identifying deep corneal opacities, which fall into two broad groups: 1) exogenous materials deposited in the cornea; and 2) endogenous pathologic corneal changes. Exogenous materials in the cornea have a wide variety of appearances, including the typical appearance of caterpillar hairs, the multifaceted appearance of glass foreign bodies, the regular geometric appearance of cholesterol crystals and cigar-shaped myeloma crystals or the more irregular deposits of chlorpromazine. Endogenous changes include ghost vessels, larger striate corneal nerves, fasciculi of fine parallel lines in keratoconus, criss-cross irregularly running lines in resolved keratitis, and a shagreen or cellophane-like appearance suggesting thickening of Descemet's membrane. While some deep corneal opacities are easily diagnosed clinically, the nature of others may be difficult to determine and specular microscopy is a valuable aid in such cases.

Corneal Diseases↗
PubMed1987

The distinction by light and electron microscopy of two types of cone containing colorless oil droplets in the retina of the turtle.

In turtle retina the spectral types of cones can be distinguished by the characteristic colors of their oil droplets. Blue cones until recently were thought to be identified by their colorless oil droplets. Now it has been shown that the cones containing colorless oil droplets can be subclassified into two types: one type in which the oil droplet fluoresces under ultraviolet stimulation and the other type in which it does not. We have attempted to recognize these two subclasses of cones with colorless oil droplets by the following anatomical techniques. (1) Light microscopy of fresh retina, (2) light microscopy of isolated cells in tissue culture and (3) light and electron microscopy of photoreceptors stained with horseradish peroxidase in whole-mount. The cone type with the colorless fluorescent oil droplet (FC cone) has an oil droplet size intermediate between those contained in the green single cones and the cone with the clear colorless oil droplet (CC cone); it typically has a short (15 microns long) oblique axon and it forms approximately 13% of the cones in the retina. The CC cone, on the other hand, is recognized by having the smallest oil droplet of all cone types which is nonfluorescent; it has a very long (30 microns long) oblique axon and forms 5% of the cones in the retina. We discuss the similarity of the FC cone morphology and distribution to blue cones in other vertebrate retinas.

Animals↗
PubMed1993

Use of remote video microscopy (telepathology) as an adjunct to neurosurgical frozen section consultation.

We investigated the use of remote video microscopy (telepathology) to assist in the diagnosis of 52 neurosurgical frozen section cases. The TelMed system (Discovery Medical Systems, Overland Park, KS), in which the referring pathologist selects appropriate fields for transmission to the consultant, was used for the study. There was a high degree of concordance between the diagnosis rendered on the basis of transmitted video images and that rendered on the basis of direct evaluation of frozen sections; however, in seven cases there was substantial disagreement. Remote evaluation was associated with a more rapid consultation from the standpoint of the consultant, who spent approximately 2 minutes less per case when using remote microscopy; this was achieved at the expense of considerably greater effort on the part of the referring pathologist, who spent approximately 16 minutes per case selecting an average of 4.5 images for transmission to the consultant. The use of remote video microscopy for pathology consultation is associated with a complex series of tradeoffs involving cost, information loss, and timeliness of consultation.

Frozen Sections↗
PubMed1996

Histomorphometrical analysis of the bone-implant interface: comparison of microradiography and brightfield microscopy.

Section thickness has been shown to affect the histomorphometrical measurement of bone-implant contact when analysed under brightfield microscopy. This study investigated whether microradiography of the bone-implant interface eliminated the errors associated with thick section analysis. Seven implant containing sections were utilized. Microadiographs of the thick (approximately 100 microns) sections were taken and the sections were subsequently ground to thicknesses of 50 microns and 25 microns. Photomicrographs were taken of the microradiographs and of the sections at each thickness (100, 50 and 25 microns) under brightfield microscopy. The photomicrographs were analysed for direct bone-implant contact in the cortical passage region and along the total length of the implant. The effect of section thickness on multiple fluorochrome labelling in 10 rabbit femur specimens was also examined. Centre-to-centre interlabel distance was measured for each label pair at a thickness of 100 microns and then again after the sections were ground to 50 microns and 25 microns. The thick (100 microns) sections showed a significantly greater amount of bone-implant contact than either the thin sections or the microradiographs. There was no difference in direct bone-implant contact measured by microradiography or thin sections. However, the microradiographic analysis showed a much lower variability of the bone-implant contact than the sections evaluated under brightfield microscopy. In addition, they have the added benefit of providing information on bone mineral density. Centre-to-centre interlabel distance was not significantly different for any label pair owing to section thickness. Data from this study provides evidence that the use of microradiographs for histomorphometrical analysis of the bone-implant interface is superior to brightfield analysis of thin sections owing to the lower variability of microradiographical data and the ability to obtain bone mineral density measures. Additionally, given that interlabel distance was not significantly affected by section thickness, the use of 100 microns thick sections for analysis of fluorochrome labels in cortical bone is supported.

Analysis of Variance↗
PubMed1992

Subcellular distribution of cytosolic Ca2+ in isolated rat hepatocyte couplets: evaluation using confocal microscopy.

Ca2+ agonists induce Ca2+ waves and other non-uniform Ca2+ patterns in the cytosol of epithelial cells. To define subcellular Ca2+ transients in the cytosol of hepatocytes we examined Fluo-3-loaded isolated rat hepatocyte couplets using confocal microscopy. Optical sections of less than 1 micron in thickness were observed in couplets, and fluorescence from cytosolic Ca2+ signals was readily distinguished from nuclear, mitochondrial, and lysosomal fluorescence. The nature of the noncytosolic components of the fluorescent images was verified by double labelling with the mitochondrial dye DiOC6(3) and with the lysosomal marker acridine orange. Using the line scanning mode of confocal microscopy, measurements of cytosolic Ca2+ were made with a frequency of up to 250 Hz and without significant bleaching. It was found that phenylephrine-induced Ca2+ signals generally began at the basal pole of the hepatocytes, then spread to the canaliculus at average speeds of 80 micron/s. These findings demonstrate the utility of confocal line scanning microscopy for detecting rapid changes in the subcellular distribution of cytosolic Ca2+ in hepatocyte couplets, and suggest that phenylephrine-induced Ca2+ waves radiate in a basal-to-apical direction in this cell type.

Animals↗
PubMed1995

Stereoscopic and biplanar microphotography of Golgi-impregnated neurons: a correlative study using conventional and real-time, direct-image confocal microscopies.

A correlative study of neuronal reconstruction methods was made using both conventional (non-confocal) and real-time confocal microscopies. Simple and sophisticated (totally automated) methods are described for making both biplanar microphotographs using conventional transmitted light, and stereoscopic microphotographs using real-time confocal microscopy of Golgi-impregnated neurons. Confocal microscopy discriminates against out-of-focus layers to produce optical sections which can be summed on photographic film to obtain neuronal reconstructions. Biplanar images are obtained by fusion, using a stereo-viewer, of two adjacent optical sections obtained with a conventional transmitted light microscope. Stereoscopic and biplanar microphotographs of 3-day-old chick neurons are presented.

Animals↗
PubMed1990

Detection by electron microscopy of a small subband 13q14.11 deletion in an hereditary retinoblastoma.

High-resolution banding, specific for electron microscopy, was applied to chromosomes of synchronized blood lymphocytes obtained from a child with bilateral retinoblastoma. Ultrastructural analysis of the subbands in region q14.1, after synchronization and immunochemical banding, showed that the deletion in the abnormal chromosome 13 corresponds to subband 14.11, thus evidencing that the retinoblastoma gene is located within subband q14.11. This first application to a diagnostic problem of immunochemical banding suggests that, coupled with electron microscopy, this banding provides a higher resolution than that obtained with light microscopy and should be useful to pinpoint important localizations.

Child, Preschool↗