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At least 1,135 records · Page 63Linked to original sources
PubMed2000

Attachment of Escherichia coli O157:H7 to the surfaces and internal structures of apples as detected by confocal scanning laser microscopy.

Confocal scanning laser microscopy (CSLM) was used to demonstrate the attachment of Escherichia coli O157:H7 transformed with a plasmid encoding for green fluorescent protein (GFP) to the surface and within the internal structures of nonwaxed Red Delicious cv. apples. Apples at 2 or 25 degrees C were inoculated with an E. coli O157:H7 cell suspension at 2 or 25 degrees C. The effect of a negative temperature differential (cold inoculum, warm apple), a positive differential (warm inoculum, cold apple), and no differential (warm inoculum, warm apple), in combination with a pressure differential (atmospheric versus 10,130 Pa), on the attachment and infiltration of cells was determined. CSLM stereo images of external surfaces of apples subjected to all combinations of test parameters showed preferential cellular attachment to discontinuities in the waxy cuticle on the surface and to damaged tissue surrounding puncture wounds, where the pathogen was observed at depths up to 70 microm below the skin surface. Attachment to lenticels was sporadic but was occasionally observed at depths of up to 40 microm. Infiltration through the floral tube and attachment to seeds, cartilaginous pericarp, and internal trichomes were observed in all apples examined, regardless of temperature differential during inoculation. The pressure differential had no effect on infiltration or attachment of E. coli O157:H7. Image analysis to count cells at various depths within tissues was used to quantitatively compare the extent of infiltration into various apple structures as well as the effects of the temperature differential. Puncture wounds harbored greater numbers of the pathogen at greater depths than did other sites examined. Attachment or infiltration of cells was greater on the intact skin and in lenticels, russet areas, and the floral tube of apples inoculated under a negative temperature differential compared to those inoculated under no temperature differential. The results suggest that E. coli O157:H7 attached to internal core structures or within tissues of apples may evade decontamination treatments. Interventions designed to deliver disinfectants to these locations or to remove viable cells of E. coli O157:H7 and other pathogens from apples by other means need to be developed and validated.

Animals↗
PubMed1999

Attachment of Escherichia coli O157:H7 to lettuce leaf surface and bacterial viability in response to chlorine treatment as demonstrated by using confocal scanning laser microscopy.

Confocal scanning laser microscopy was used to observe the location of Escherichia coli O157:H7 on and within lettuce leaves. Sections of leaves (ca. 0.5 by 0.5 cm) were inoculated by submersion in a suspension of E. coli O157:H7 (ca. 10(7) to 10(8) CFU/ml) overnight at 7 degrees C. Fluorescein isothiocyanate-labeled antibody was used to visualize the attached bacteria. E. coli O157:H7 was found attached to the surface, trichomes, stomata, and cut edges. Three-dimensional volume reconstruction of interior portions of leaves showed that E. coli O157:H7 was entrapped 20 to 100 microm below the surface in stomata and cut edges. Agar plate culturing and microscopic observation indicated that E. coli O157:H7 preferentially attached to cut edges, as opposed to the intact leaf surface. Dual staining with fluorescein isothiocyanate-labeled antibody and propidium iodide was used to determine viability of cells on artificially contaminated lettuce leaves after treatment with 20 mg/liter chlorine solution for 5 min. Many live cells were found in stomata and on cut edges following chlorine treatment. E. coli O157:H7 did not preferentially adhere to biofilm produced by Pseudomonas fluorescens on the leaf surface. In contrast to E. coli O157:H7, Pseudomonas adhered to and grew mainly on the intact leaf surface rather than on the cut edges.

Bacterial Adhesion↗
PubMed2003

Three-dimensional distribution of GFP-labeled Pseudomonas putida during biofilm formation on solid PAHs assessed by confocal laser scanning microscopy.

Confocal laser scanning microscopy was used to monitor the colonization pattern of the gfp-labeled derivative strain of Pseudomonas putida ATCC 17514 on fluorene and phenanthrene crystals. The in situ experiments showed that P. putida tends to grow directly on phenanthrene, forming a biofilm on accessible crystalline surfaces. On the other hand, no significant biofilm formation was observed in the presence of fluorene. The results obtained showed that substrate properties affected bacterial strategy regarding uptake.

Biofilms↗
PubMed2001

Non destructive analysis of the wax layer of apple (Malus domestica Borkh.) by means of confocal laser scanning microscopy.

Confocal laser scanning microscopy (CLSM) was used to non-destructively analyse the changes in the structure and thickness of the cuticle during storage of apples (Malus domestica Borkh.). Interpretation of the confocal images was performed by comparison with scanning electron microscopy and environmental scanning electron microscopy images. The natural reflectance of the wax and the auto-fluorescence of the underlying cells made it possible with CLSM to distinguish the wax from the underlying layers without any pretreatment of the fruit. The thickness of the consecutive layers (wax, cutin, cells) could be estimated from measurements of the reflection and fluorescence intensities as a function of the number of pixels. The mean wax-layer thickness measured in this way amounted to 2.58 microm, 3.41 microm or 4.14 microm for the cultivars Jonagold, Jonagored and Elstar, respectively. Changes in the wax structure and cells of the same important Belgian apple cultivars as mentioned above were monitored during nine months of storage in ultra low oxygen and after exposure to ambient conditions. The changes in the wax ultrastructure and cell morphology are likely related to water losses and specific protection of the apple cultivars against water losses during storage and shelf life.

Fruit↗
PubMed2001

delta- and gamma-Sarcoglycan localization in the sarcoplasmic reticulum of skeletal muscle.

Sarcoglycans are transmembrane proteins that are members of the dystrophin complex. Sarcoglycans cluster together to form a complex, which is localized in the cell membrane of skeletal, cardiac, and smooth muscle fibers. However, it is still unclear whether or not sarcoglycans are restricted to the sarcolemma. To address this issue, we examined alpha-, beta-, delta-, and gamma-sarcoglycan expression in femoral skeletal muscle from control and dystrophin-deficient mice and rats using confocal microscopy and immunoelectron microscopy. Confocal microscopy of the tissues in cross-section showed that all sarcoglycans were detected under the sarcolemma in rats and control mice. delta- and gamma-sarcoglycan labeling demonstrated striations in the longitudinal section, suggesting that the proteins were expressed in the sarcoplasmic reticulum (SR) or transverse tubules (T-tubules). Moreover, such striations of both sarcoglycans were recognized in the dystrophin-deficient mouse skeletal muscle. Double labeling with phalloidin or alpha-actinin and delta- or gamma-sarcoglycan showed different labeling patterns, indicating that delta-sarcoglycan localization was distinct from that of gamma-sarcoglycan. Immunoelectron microscopy clarified that delta-sarcoglycan was localized in the terminal cisternae of the SR, while gamma-sarcoglycan was found in the terminal cisternae and longitudinal SR over I-bands but not over A-bands. These data demonstrate that delta- and gamma-sarcoglycans are components of the SR in skeletal muscle, suggesting that both sarcoglycans function independent of the dystrophin complex in the SR.

Actinin↗
PubMed1994

Morphological and quantitative analyses of normal epidermal Langerhans cells using confocal scanning laser microscopy.

Confocal scanning laser microscopy (CSLM), when used in conjunction with computerized image processing systems, provides a powerful tool for morphological and quantitative analyses of biological tissues. In this study, normal human epidermal sheets were stained by an indirect immunofluorescence method using anti-CD1a monoclonal antibody. Positively stained epidermal Langerhans cells (LCs) were visualized using the Bio-Rad MRC-600 Confocal Imaging System. Images obtained from the confocal microscope were volumetrically rendered and quantitatively analysed using ANALYZE (Version 4.0) running on a Sun SPARC 2 Workstation. Normal epidermal LCs were shown to be large disc-like structures with five to nine long dendritic processes per cell, orientated with their flat surfaces parallel to the skin surface. LCs form a monolayer network of cells distributed evenly throughout the suprabasal layers of the epidermis, with no direct physical contact between dendritic processes. Mean LC density was estimated to be 582 per mm2 (95% confidence intervals, CI = 233-940), and mean cell volume was 612 microns3 (95% CI = 257-1020). LCs in sun-exposed sites were significantly lower in mean cell density, but larger in mean cell volume, than in covered sites. Mean surface area projected by LCs was estimated to be 26.8% (95% CI = 18.9-34.2), and this value did not show significant regional or individual variation. Our data support the notion that epidermal LCs are organized in such a way as to maximize their surface area for efficient trapping of antigens, and a reduction in LC density per unit area in sun-exposed sites is compensated for by an increase in the mean cell volume.

Cell Count↗
PubMed2005

Atomic force microscopy and confocal laser scanning microscopy on the cytoskeleton of permeabilised and embedded cells.

We describe a technical method of cell permeabilisation and embedding to study the organisation and distribution of intracellular proteins with aid of atomic force microscopy and confocal laser scanning microscopy in identical areas. While confocal laser scanning microscopy is useful for the identification of certain proteins subsequent labelling with markers or antibodies, atomic force microscopy allows the observation of macromolecular structures in fixed and living cells. To demonstrate the field of application of this preparatory technique, cells were permeabilised, fixed, and the actin cytoskeleton was stained with phalloidin-rhodamine. Confocal laser scanning microscopy was used to show the organisation of these microfilaments, e.g. geodesic dome structures. Thereafter, cells were embedded in Durcupan water-soluble resin, followed by UV-polymerisation of resin at 4 degrees C. This procedure allowed intracellular visualisation of the cell nucleus or cytoskeletal elements by atomic force microscopy, for instance to analyse the globular organisation of actin filaments. Therefore, this method offers a great potential to combine both microscopy techniques in order to understand and interpret intracellular protein relations, for example, the biochemical and morphological interaction of the cytoskeleton.

Actins↗
PubMed1994

Development of glomerular structure in rabbit olfactory bulb: three-dimensional reconstruction under the confocal laser scanning microscopy.

Confocal laser scanning imaging was used to reconstruct the three-dimensional distribution of the aggregates of olfactory receptor axons terminating within individual glomeruli in the rabbit main olfactory bulb. Two monoclonal antibodies, R2D5 and R4B12, were used to mark selectively the olfactory receptor axons. Thick coronal sections (100-200 microns in thickness) through the bulb were labeled with either of the antibodies, and then serial optical sectioning was performed to reconstruct three-dimensional optic images of the labeled axons in the glomeruli. The results revealed an intricate internal structure of the glomeruli with a mesh-like arrangement of bundles of terminal olfactory axons. Developmental study showed that primitive glomeruli lacking the internal structure of the adult form first appear in the 22-day embryo and that the characteristic internal structure of the glomeruli develops mainly during postnatal days. The confocal laser scanning image method together with specific molecular markers provides a simple tool for the three-dimensional analysis of the glomerular structure.

Age Factors↗
PubMed2005

Monopolar radiofrequency treatment of partial-thickness cartilage defects in the sheep knee joint leads to extended cartilage injury.

BACKGROUND: The application of radiofrequency energy to smooth and stabilize the cartilage surface has become increasingly controversial. There is little knowledge on extended-term effects, such as cartilage viability. PURPOSE: To analyze the effect of radiofrequency treatment on artificially created partial-thickness defects in the femoral cartilage of sheep knee joints 24 weeks after surgery. STUDY DESIGN: Controlled laboratory study. METHODS: Grade II cartilage surface defects on the medial and lateral femoral condyles were artificially created in sheep for in vivo analysis. The cartilage lesions were treated alternately on the lateral or the medial condyle using a monopolar radiofrequency probe. Radiofrequency treatment was performed in a freehand technique until surface smoothing without change of cartilage color was seen. At 24 weeks after surgery, cartilage samples were harvested and were processed for macroscopic and histological evaluation. To analyze the effect of radiofrequency at time zero, samples of sheep femoral condyle cartilage with and without artificially created clefts were treated in vitro with radiofrequency. Evaluation was performed by scanning electron and confocal microscopy. RESULTS: At 24 weeks after surgery, grade IV cartilage defects were detected in all radiofrequency-treated samples. The histological findings showed a central ulcer and dead chondrocytes in the radiofrequency-treated regions. The radiofrequency-treated cartilage revealed partial surface irregularities with partial-defect repair. After radiofrequency treatment in vitro, samples at time zero showed smoothing of the artificially created clefts, as seen by scanning electron microscopy. Confocal microscopy showed necrosis of chondrocytes over approximately one fourth of the upper cartilage thickness. CONCLUSION: Even if chondrocyte death is seen only in approximately one fourth of the upper cartilage layers in the sheep femur after in vitro application, radiofrequency treatment can cause damage to cartilage 24 weeks after application. CLINICAL RELEVANCE: Caution is recommended in the application of monopolar radiofrequency energy by visual control to partial-thickness cartilage defects. Irregular fronds of chondromalacia may be unattractive but represent viable articular cartilage. Using radiofrequency to obtain a more visually pleasing smooth surface may be counterproductive.

Animals↗
PubMed2004

Analysis of passive mixing behavior in a poly(dimethylsiloxane) microfluidic channel using confocal fluorescence and Raman microscopy.

Confocal fluorescence microscopy (CFM) and confocal Raman microscopy (CRM) have been applied to monitor the laminar flow mixing behavior in a poly(dimethylsiloxane) (PDMS) microfluidic channel. Two passive PDMS micromixing devices were fabricated for this purpose: a two-dimensional round-wave channel and a three-dimensional serpentine channel. The microscale laminar flow mixing of ethanol and isopropanol was evaluated using the CFM and CRM at various flow rates. The mixing behavior of confluent streams in the microchannel was assessed by determining the degree of color change in Rhodamine 6G dye on mixing using the CFM. However, it was also possible to quantitatively evaluate the mixing process without employing a fluorescence label using the CRM. The results show a strong potential for CRM as a highly sensitive detection tool to measure fundamental fluid mixing processes and to provide detailed information on chemical changes of non-fluorescent reaction mixtures in a PDMS microfluidic channel.

2-Propanol↗
PubMedDate not supplied

Validation of three-dimensional surface characterising methods: scanning electron microscopy and confocal laser scanning microscopy.

Surface characteristics of enosseous titanium implants have been known to influence the quality of osseointegration. Parameters recommended for metrical analysis should be supplemented by a topographical description. In this study, Ra values obtained by established tactile and optical profilometric methods are correlated with those obtained by stereo scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). As test specimens, standardised CVD diamond-coated titanium alloys with different microwave coating power ranging from 2,000 to 3,000 W were used. A non-CVD-coated corund grit-blasted specimen (NC) was used as reference. After coating with a CVD, power of 2,000 W Ra ranges from 4.33 to 5.69 microm depending on the method used. With increasing power of the coating process, the amplitude of the surface roughness is significantly increased to 4.53 to 6.89 microm. Ra values of the same sample obtained by different methods are also significantly different (p = 0.001). Compared with the established methods of tactile and optical profilometry, SEM and CLSM offer valid data on the surface roughness accompanied by a topographical imaging. In future studies, the underlying method should be specified to interpret roughness values correctly, as not every method is suitable for each specimen and values obtained by different methods vary extremely.

Diamond↗
PubMed2000

Measurement of ligand distribution in individual adsorbent particles using confocal scanning laser microscopy and confocal micro-Raman spectroscopy.

Two methods, confocal scanning laser microscopy and confocal micro-Raman spectroscopy were used to analyse the distribution of IgG antibodies immobilized on CNBr-activated agarose beads. In the first method the internal distribution profile of fluorescent labelled Protein A was used as an indirect measure of the distribution of IgG, while the second method detects vibrations originating from aromatic amino acids present in the immobilized antibodies. Both these methods indicate an homogeneous ligand distribution within IgG Sepharose 4 Fast Flow and IgG Sepharose 6 Fast Flow.

Adsorption↗
PubMed1991

The development of exo-erythrocytic schizonts of Plasmodium berghei in vitro from gamma-irradiated and non-irradiated sporozoites: a study using confocal laser scanning microscopy.

Confocal scanning laser microscopy has been used to study the distribution of antigens expressed by the liver stages of Plasmodium berghei in cultured hepatoma cells. The 3-dimensional images obtained of intact parasites clearly show complex patterns of antigen expression not apparent when using conventional IFAT or immunoelectron microscopy. A liver-stage specific antigen (Pbl 1) was shown to be confined to the parasitophorous vacuole; the vacuole has extensive diverticulae extending into the host cell. Small parasites were detected for the first time in 'mature' cultures. These did not represent a distinct population, but the 'tail' of a broad continuum of parasite sizes. Irradiated sporozoites produce a transient population of slow-growing parasites which express a very limited range of antigens de novo in the invaded hepatoma cell. A comparison of the reactivity of normal EE parasites with anti-circumsporozoite antibody and with anti-Pbl 1 suggests that the former reagent may reliably be used to identify sporozoites invading host cells, but should not be used to determine the number of parasites that successfully undergo intrahepatic development. Anti-Pbl-1 indicates on 33% of invaded sporozoites identified by anti-CSP subsequently differentiate.

Animals↗
PubMed2005

Radon track imaging in CR-39 plastic detectors using confocal scanning laser microscopy.

Confocal scanning laser microscopy (CSLM) has been used to provide the first images of radon track populations in two external CR-39 plastic detectors. Measurements of variables including track area distribution and estimates of the angle of track inclination (dip) derived from surface CSLM sections are presented. CSLM depth slices, combined with three-dimensional (3D) visualization techniques, provide a new, non-destructive way of examining the 2D and 3D geometry of the etched tracks within solid-state nuclear track detectors that may prove useful in complementing existing optical microscopy methods.

Journal Article↗
PubMed1992

Quantitative analysis of Langerhans' cells in epidermis at irritant contact reactions using confocal laser scanning microscopy.

Confocal laser scanning microscopy (CLSM) was used for quantitative analysis of CD1a+ cells in epidermis at irritant reactions. Sodium lauryl sulphate (2% and 4%) or non-anoic acid (20% and 80%) were applied to the skin of healthy volunteers under occlusion for 24 h. Skin biopsy specimens were taken after additional 24 h and were snap frozen. Freeze-sections, 25 microns thick, were stained with anti-CD1a antibodies (Leu-6) followed by FITC-labelled rabbit anti-mouse IgG. The sections were viewed and optically sectioned in the CLSM at four depth levels. The data was analysed using a threshold value for the fluorescence. The obtained result is presented as the proportion of specimen area having a fluorescence intensity above the threshold. The result demonstrates that the CLSM is a useful tool for obtaining not only structural information but also quantitative information from a defined tissue volume. In the present investigation it was possible to demonstrate variations in CD1a+ reactivity in epidermis at detergent-induced irritant reactions with a marked decrease in CD1a+ after 80% non-anoic acid exposure and only minor differences in the CD1a+ after 2% and 4% sodium lauryl sulphate exposure.

Cell Count↗
PubMed2006

Combined phase-sensitive acoustic microscopy and confocal laser scanning microscopy.

Combined phase-sensitive acoustic microscopy (PSAM) at 1.2 GHz and confocal laser scanning microscopy (CLSM) in reflection and fluorescence has been implemented and applied to polymer blend films and fluorescently labeled fibroblasts and neuronal cells in order to explore the prospects and the various contrast mechanisms of this powerful technique. Topographic contrast is available for appropriate samples from CLSM in reflection and, with significantly higher precision, from the acoustic phase images. Material contrast can be gained from acoustic amplitude V(z) graphs. In the case of the biological cells investigated, the optical and acoustic images are very different and exhibit different features of the samples.

3T3 Cells↗