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PubMed1999

Response to high-pressure, low-temperature treatment in vegetables: determination of survival rates of microbial populations using flow cytometry and detection of peroxidase activity using confocal microscopy.

Application of high hydrostatic pressure (200, 300, 350 and 400 MPa) at 5 degrees C for 30 min to different micro-organisms, including Gram-positive and Gram-negative bacteria, moulds and yeasts, proved to be more effective in inactivating these organisms than treatments at 20 degrees C for 10 min and at 10 degrees C for 20 min. Moulds, yeasts, Gram-negative bacteria and Listeria monocytogenes were most sensitive, and their populations were completely inactivated at pressures between 300 and 350 MPa. The same conditions of pressure, temperature, and time were applied to different vegetables (lettuce, tomato, asparagus, spinach, cauliflower and onion), achieving reductions of from 2-4 log units in both viable mesophiles and moulds and yeasts at pressures of between 300 and 400 MPa. Sensory characteristics were unaltered, especially in asparagus, onion, tomato and cauliflower, though slight browning was observed in cauliflower at 350 MPa. Flow cytometry was applied to certain of the microbial populations used in the above experiment before and after the pressurization treatment. The results were indicative of differing percentage survival rates depending on micro-organism type, with higher survival rates for Gram-positive bacteria, except L. monocytogenes, than in the other test micro-organisms. Growth of survivors was undetectable using the plate count method, suggesting that micro-organisms suffering from pressure stress were metabolically inactive though alive. The pressurization treatments did not inactivate the peroxidase responsible for browning in vegetables. Confocal microscopic examination of epidermal tissue from onion showed that the enzyme had been displaced to the cell interior. Use of low temperatures and moderately long pressurization times yielded improved inactivation of micro-organisms and better sensorial characteristics of the vegetables, and should lower industrial costs.

Aerobiosis↗

PubMed2001

Microtensile bond strength and confocal microscopy of dental adhesives bonded to root canal dentin.

PURPOSE: To evaluate the bond strength of two dentin-bonding systems to root canal dentin. MATERIALS AND METHODS: Six central incisors, extracted for periodontal reasons, were endodontically treated, no obturation of the root canal space was performed. The teeth were cut parallel to their long axis. One half of each tooth was randomly assigned to one of two experimental groups. The teeth of the first group were treated with All-Bond 2 dental adhesive and those of the second group with Panavia F. The root canal dentin was conditioned as suggested by the manufacturers and, after the application of the dental adhesives, a layer of resin-based composite was polymerized over the adhesive layer. After the composite had completely set, the sections were prepared for microtensile test and kept in a wet environment at a relative humidity of 90 +/- 2% measured with a digital relative humidity meter and at a temperature of 22 degrees C for 24 hrs. The specimens were sectioned perpendicularly to the bonded interface, into slabs 1.5 mm thick (10 sections for each experimental group). The slabs were then trimmed by super fine diamond burs for the microtensile bond test with the narrowest region located at the respective bonded interface. The thickness of the slab was then exactly established by three measurements performed in three points of each slab by a digital micrometer. The specimens were then subjected to a tensile force at a crosshead speed of 0.2 mm/min. The test was observed using a confocal microscope and the failure modes were noted. The load at failure was registered and the bonded area was measured using a confocal microscope at a magnification of x25 in conjunction with an image-processing program. The failure data were analyzed using the Wilcoxon-Gehan test with exact non-parametric inference. RESULTS: The mean value at failure was the same for the two experimental groups (17.1 MPa). There was no statistically significant difference between Panavia F and All-Bond 2 (P> 0.8671). Most of the specimens showed a debonding of the adhesive from the hybrid layer. The results showed that the bond strength of the two bonding systems tested to the dentin of the root canal are slightly lower than to those obtained with the latest adhesive systems, in the coronal dentin.

Composite Resins↗

PubMed1993

Three-dimensional organization of the ribosomal genes and Ag-NOR proteins during interphase and mitosis in PtK1 cells studied by confocal microscopy.

The three-dimensional (3-D) organization of rDNA-containing chromatin and the set of protein markers of active ribosomal genes, the Ag-NOR proteins, were investigated by confocal laser scanning microscopy (CLSM). The rDNA genes of marsupial cells (PtK1) were mapped using biotinylated DNA probes for 45S rDNA sequences and the Ag-NOR protein distribution was revealed by specific Ag-NOR staining. We used PtK1 cells because each nucleolus possesses only one nucleolar organizer region (NOR). In metaphase chromosomes, nonisotopic in situ hybridization demonstrated the presence of rDNA in the secondary constriction of the X chromosomes with an axial distribution and also lateral expansions. 3-D reconstruction of the Ag-NOR protein signals revealed the presence of these proteins in the secondary constriction where they formed a crescent-shaped structure around the axial chromatin pedicule. The organization of the secondary constriction in PtK1 chromosomes is discussed. During interphase, nonisotopic in situ hybridization in intact cell monolayers and isolated nuclei showed the rDNA genes distributed as intense fluorescent spots linked by weak signals in the inner regions of the nucleoli. We conclude that the rDNA is not homogeneously distributed in the internal regions of the nucleoli. In the same nucleolar regions, the Ag-NOR proteins were revealed as granules linked by thin filaments. These images indicate similar 3-D distributions for rDNA probes and Ag-NOR proteins. The beaded organization of the transcriptional regions in the nucleoli is discussed.

Animals↗

PubMed2006

Measurement and restoration of the point spread function of fluorescence confocal microscopy.

The point spread function of an objective lens of a fluorescence confocal microscope was directly measured by imaging fluorescent beads. We analysed how the measurement of the point spread function was influenced by the diameter of the fluorescent beads and how the restoration technique with a deconvolution algorithm improved the measuring performance. Numerical and experimental results are presented for a typical point spread function and a zero-centred point spread function.

Journal Article↗

PubMedDate not supplied

The microanatomy of the alveolar duct of the human lung imaged by confocal microscopy and visualised with computer-based 3D reconstruction.

The most widely accepted model of human lung alveolar duct systems is that they are constructed from central helical fibres between the turns of which lie alveolar opening. Experimental difficulties of handling and sectioning lung tissue have made it difficult to confirm this. Confocal laser scanning microscopy (CLSM) was therefore used to generate optical serial sections of the lung that were reconstructed in three dimensions and displayed using volume rendering techniques. From images of the reconstructions, a new structure is proposed in which alveolar ducts consist of collections of connected oval, twisted loop structures with eccentric openings.

Adult↗

PubMed2002

Internal features of graphite in cast irons. Confocal microscopy: useful tool for graphite growth imaging.

Spherulitic crystallisation is a mode of growth of crystals from the melt. Considerable attention has been given to spheroidal graphite formation, providing detailed information about the internal microstructure of the spherulites in spheroidal (SG irons) and compacted graphite irons (CG irons) (Stefanescu, D., 1990. Cast Irons. ASM Handbook, 10th ed., vol. 1). Nevertheless, the mechanisms responsible for this mode of crystallisation are not fully understood. This study deals with the inoculation mechanisms, with particular emphasis on the study of the inclusions for the heterogeneous nucleation of graphite. It is shown that the graphite nuclei are sulfide products of the nodularizing treatment. It has been observed that when rare-earth treatment is applied, the central nucleus consists of a core and an envelope from which the graphite grows. Confocal Scanning Laser Microscopy (CSLM), in reflection mode, was used to study the internal features of the spheroidal graphite growth. Confocal reflection imaging, which has a capacity for optical sectioning of the sample, provides high-resolution images of surface and subsurface regions of interest contained within a semi-transparent sample. Furthermore, three-dimensional reconstruction of these optical sections can provide insight into the mechanism of graphite growth mechanism interpretation. With CSLM the radial growth of graphite was seen. Other techniques, such as TEM, SEM-EDS, WDS, AES and SAM were also used to corroborate the results.

Journal Article↗

PubMed1994

Fluorescent in situ hybridization of maize meiotic chromosomes as visualized by confocal microscopy.

Knob heterochromatin served as the model for the development of fluorescent chromosome in situ hybridization on maize meiotic chromosomes. The meiotic chromosomes were hybridized with a digoxigenin-labeled RNA probe of the knob repeat sequence that is a component of the morphologically determined knob heterochromatin. The fluorescein-labeled knob probe and propidium iodide counter-stained chromosomes were imaged using confocal laser scanning microscopy, which allowed for the individual analysis of each fluorescent probe emission intensity, the ability to utilize image processing techniques and the generation of high-resolution images. A composite, in register, merged image of the knob probe signal and meiotic chromosomes demonstrated exact co-localization of the knob probe and the morphologically identified knobs. The establishment of the fluorescent in situ hybridization technique in maize allows for the expanded study of the biological role of knob heterochromatin and the possibility of locating other repeat sequences on maize chromosomes.

Biotechnology↗

PubMed2002

Behavior of the point-spread function in photon-limited confocal microscopy.

We study the behavior of the point-spread function (PSF) of the confocal scanning optical microscope (CSOM) when the available optical energy density from the sample plane is low (<7.5 microJoule/micrometers2). The PSF profile is analyzed under three photon-limited imaging conditions: (1) reflection-type CSOM with a weak source and a perfectly reflecting sample, (2) reflection-type CSOM with a strong illumination source and a weak sample, and (3) fluorescence CSOM with a weak fluorescent sample. Linfoot's image quality criteria of fidelity, structural content, and correlation quality are used to assess the reproducibility of the PSF profile as a function of the photon number. Low photon numbers yield a PSF profile that is difficult to maintain from one location in the sample plane to another. The optical sectioning capability of the CSOM was found to deteriorate more quickly against light power reduction than its transverse resolving power. The signal-to-noise ratio of the scanned CSOM image improves exponentially with the photon number from the sample plane. The noise that is generated by an unstable PSF has an average amplitude that decreases exponentially with the photon number and is significant only at low photon numbers. The CSOM image quality deteriorates because of spurious high-frequency components, degradation in the PSF dynamic range, and varying resolving power.

Journal Article↗

PubMed1991

Confocal microscopy and three-dimensional reconstruction of electrophysiologically identified neurons in thick brain slices.

Three-dimensional morphology and electrophysiology were correlated from individual neurons in a thick brain slice preparation. The hippocampal formation from immature and adult rats was cut transverse to the longitudinal axis into 500 microns-thick slices which were maintained under physiologic conditions. Individual neurons were impaled and physiologically characterized using microelectrodes. Recordings were made from the soma and in some cases from a dendrite. The impaled neurons were filled through the microelectrode with the fluorescent dye lucifer yellow and imaged by confocal scanning laser microscopy using an analog preprocessor. As many as 180 optical sections were recorded as a function of depth through the slices. Images are presented as a series of optical sections, stereo pairs, or three-dimensional reconstructions. Both stereo contouring and volume rendering methods were employed, and the reconstructions were viewed from any arbitrary perspective. Dendritic and axonal fields were separated from each other and displayed separately or as different pseudocolors. The three-dimensional reconstructions provided perspectives that were difficult or impossible to appreciate by viewing the optical sections or conventionally formed stereo pairs.

Animals↗

PubMed1990

Confocal microscopy as a tool for the study of the intranuclear topography of chromosomes.

A scanning confocal microscope was used to investigate the spatial positions of specific regions within blood cell nuclei. These centromeric regions were fluorescently labelled by in-situ hybridization to suspended nuclei with a centromere-1-specific DNA probe. The 3-D image data sets, obtained by optical sectioning of the cells, were used to determine the spatial position of the centromeric regions in the nuclei by means of specially developed software. The centromeres were found to be localized near the nuclear boundary. This spatial pattern was tested against a random distribution model by means of the Kolmogorov-Smirnov test. The difference between the two patterns was at a P less than 0.01 significance level.

Blood Cells↗

PubMed2004

Quantitative analyses of Streptococcus mutans biofilms with quartz crystal microbalance, microjet impingement and confocal microscopy.

Microbial biofilm formation can be influenced by many physiological and genetic factors. The conventional microtiter plate assay provides useful but limited information about biofilm formation. With the fast expansion of the biofilm research field, there are urgent needs for more informative techniques to quantify the major parameters of a biofilm, such as adhesive strength and total biomass. It would be even more ideal if these measurements could be conducted in a real-time, non-invasive manner. In this study, we used quartz crystal microbalance (QCM) and microjet impingement (MJI) to measure total biomass and adhesive strength, respectively, of S. mutans biofilms formed under different sucrose concentrations. In conjunction with confocal laser scanning microscopy (CLSM) and the COMSTAT software, we show that sucrose concentration affects the biofilm strength, total biomass, and architecture in both qualitative and quantitative manners. Our data correlate well with previous observations about the effect of sucrose on the adherence of S. mutans to the tooth surface, and demonstrate that QCM is a useful tool for studying the kinetics of biofilm formation in real time and that MJI is a sensitive, easy-to-use device to measure the adhesive strength of a biofilm.

Journal Article↗

PubMed1992

The fate of medial edge epithelial cells during palatal fusion in vitro: an analysis by DiI labelling and confocal microscopy.

Fusion of bilateral shelves, to form the definitive mammalian secondary palate, is critically dependent on removal of the medial edge cells that constitute the midline epithelial seam. Conflicting views suggest that programmed apoptotic death or epithelial-mesenchymal transformation of these cells is predominantly involved. Due in part to the potentially ambiguous interpretation of static images and the notable absence of fate mapping studies, the process by which this is achieved has, however, remained mechanistically equivocal. Using an in vitro mouse model, we have selectively labelled palatal epithelia with DiI and examined the fate of medial edge epithelial (MEE) cells during palatal fusion by localisation using a combination of conventional histology and confocal laser scanning microscopy (CLSM). In dynamic studies using CLSM, we have made repetitive observations of the same palatal cultures in time-course investigations. Our results concurred with the established morphological criteria of seam degeneration; however, they provided no evidence of MEE cell death or transformation. Instead we report that MEE cells migrate nasally and orally out of the seam and are recruited into, and constitute, epithelial triangles on both the oral and nasal aspects of the palate. Subsequently these cells become incorporated into the oral and nasal epithelia on the surface of the palate. We hypothesize an alternative method of seam degeneration in vivo which largely conserves the MEE population by recruiting it into the nasal and oral epithelia.

Animals↗

PubMed1990

Histochemical and immunocytochemical localization of prolactin receptors on Nb2 lymphoma cells: applications of confocal microscopy.

We studied prolactin (PRL) binding sites on Nb2 lymphoma cells using two different light microscopic methods. First, histochemical detection was accomplished by using an aminomethyl coumarin-acetic acid-conjugated ovine prolactin molecule (AMCA-oPRL) on both glutaraldehyde-fixed and unfixed Nb2 lymphoma cells. Binding of AMCA-oPRL was studied after UV illumination and appeared as punctate fluorescence associated with many but not all cells. Binding was abolished when tissue sections were treated with excess unlabeled lactogenic hormones and was unchanged when a non-lactogenic hormone was used for displacement. Counting revealed significant differences between the number of labeled cells in populations known to exhibit up- or down-regulated PRL receptors. Second, indirect immunocytochemistry of Nb2 PRL receptors was accomplished by immunological detection of exogenously added ovine PRL using two antisera directed against ovine PRL. Visualization of the ligand-antibody complexes was accomplished by confocal laser scanning microscopy. Staining was restricted to a subpopulation of cells. The morphological results presented here add to the previous physiological and biochemical data on the presence of lactogenic hormone receptors on Nb2 lymphoma cells.

Animals↗

PubMed1991

Spatial and temporal distribution of DNA replication sites localized by immunofluorescence and confocal microscopy in mouse fibroblasts.

The temporal course of replication monitored by 2- or 5-min pulses of bromodeoxyuridine (BrdUrd) incorporation in synchronized 3T3 cells was mapped by high-resolution light microscopy employing a charge-coupled device (CCD) camera and a confocal laser scanning microscope (CLSM). The cells were labeled simultaneously with monoclonal antibodies directed against BrdUrd and nuclear lamin, and stained with the A+T-specific dye 4',6-diamidino-2-phenylindole (DAPI). Stereoscopic reconstructions of cells showing both the lamin and BrdUrd distributions demonstrate that DNA replication occurs at discrete sites in the nucleus, the locations of which progress through a programmed sequence during S phase. Replication begins in a small number of sites in the interior of the nucleus exclusive of the nuclear membrane and proceeds rapidly in early S phase to encompass a relatively large number of small, discrete sites located throughout the nucleus, with the exception of the condensed heterochromatic regions. Replication is primarily confined to the condensed heterochromatic regions in mid-to-late S phase, and to the nuclear periphery at the end of S phase. These distinctive patterns demonstrate a programmed control of replication sites in the spatial domain in differentiated cell nuclei.

Animals↗

PubMed1991

The use of confocal microscopy and STERECON reconstructions in the analysis of sea urchin embryonic cell division.

A laser scanning confocal microscope has been used to investigate the development of the sea urchin embryo. The samples were fixed in Carnoy's solution at various developmental stages, stained for DNA with the Feulgen reaction, and optically sectioned with a BioRad MRC-500 confocal microscope. Computer-generated stereographic projection images and a three-dimensional contour tracing and reconstruction system were employed to investigate the cleavage pattern during the 6th cleavage division. Cell division is found to be asynchronous during the 6th cleavage, with macromere derivatives completing division first, followed by mesomeres, and finally by the outer quartet of micromeres (which begins division only after macromeres and mesomeres have completed their respective divisions). Sixth cleavage produces an embryo comprising 60 cells. Asynchronous division was also observed within individual tiers of blastomeres. Variations in the orientations of cell division axes within individual tiers of cells were also observed. The utility of computer-graphics reconstruction techniques for both quantitative and qualitative developmental analysis are discussed.

Animals↗

PubMed1992

Characterization and minimization of cellular autofluorescence in the study of oligonucleotide uptake using confocal microscopy.

Intrinsic autofluorescent signals can interfere with extrinsic fluorophore signals when living cells are viewed under a confocal laser scanning microscope. The general pattern of this endogenous fluorescence is initially diffuse and cytoplasmic, but it can redistribute and intensify to become punctate and perinuclear as cells age. To reduce the contribution of autofluorescence when tracking the location of an extrinsic fluorophore, such as a fluorescently-labeled oligonucleotide, laser power settings, aperture settings, laser scanning rates, pH buffering environments, and excitatory wavelengths can be modulated. Decreasing laser power settings and aperture sizes, increasing laser scanning rates and excitatory wavelengths, and surrounding cells in a pH buffer all act to delay the signal transformation. In addition, the presence of an exogenous fluorophore can hasten the autofluorescent redistribution and intensification when compared to similar untreated cells.

Cells, Cultured↗

PubMed1993

Three-color immunofluorescence imaging of Drosophila embryos by laser scanning confocal microscopy.

We present a simple means for triple-labeling biological specimens by immunofluorescence using a laser scanning confocal microscope for imaging with a krypton/argon laser as a light source. Three separate images of fluorescein-, lissamine rhodamine- and cyanine-5-labeled antibodies are collected and subsequently merged to form the triple-labeled image, which is displayed at full-image resolution (24 bit) on a second image processing system. The technique is illustrated using immunofluorescence localization of three segmentation proteins in Drosophila embryos.

Animals↗

PubMed1994

A method to stain nuclei of Drosophila for confocal microscopy.

We report a method of staining nurse cell and follicle cell nuclei in Drosophila ovaries and nuclei in Drosophila embryos with the fluorescent dye propidium iodide. This technique was used to replace more commonly used 4', 6-diamidino-2-phenylindole (DAPI) and Hoechst staining as a method of visualizing nuclear material in Drosophila. Propidium iodide has its maximum excitation at about 530 nm and maximum fluorescence at 615 nm, and therefore it can be used as a fluorescent marker with confocal microscopes that do not have a UV excitation source. Another advantage of the described method is the convenience of simultaneous use of fluorescein as a second fluorophore in multicolor fluorescence. We show that the nuclear material in Drosophila ovaries and early embryos can be visualized with propidium iodide using both confocal and conventional fluorescence microscopy. We also test the combination of two fluorophores-propidium iodide for nuclear staining and fluorescein-labeled phalloidin for membrane-bound actin--in the same tissue.

Animals↗