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G J Brakenhoff

Publications and source records attributed to G J Brakenhoff.

15 recordsLinked to original sources
PubMed1992

Dynamics of three-dimensional replication patterns during the S-phase, analysed by double labelling of DNA and confocal microscopy.

The temporal and spatial progression of DNA replication in interphase nuclei of eukaryotic cells has been investigated. Application of a recently developed technique for the immunofluorescence double staining of cell nuclei labelled first with iododeoxyuridine (IdUrd) and subsequently with chlorodeoxyuridine (CldUrd) allows the visualization of two replication patterns in the same nucleus originating from two different periods of the S-phase. We have analysed changes in the three-dimensional replication patterns during the S-phase. To record dual colour three-dimensional images of doubly stained nuclei, a confocal microscope is used. This CSLM is equipped with a specific laser/filter combination to collect both fluorescence signals (FITC and Texas Red) in a single scan, thus precluding pixel shift between the images. A method for the quantitative evaluation of the degree of overlap between DNA regions replicated in two different periods of the S-phase is applied. The results confirm the generally accepted theory that DNA is replicated coordinately in a specific temporal order during the S-phase. The replication time of a DNA domain (i.e. the time between initiation and termination of DNA replication within a domain) at the very beginning of the S-phase was known to be one hour (Nakamura et al., 1986). Our observations show that in the rest of the S-phase, the replication time of a DNA region is also about one hour. We conclude that replicon clusters located in the same region are replicated in the same relatively short period of time. After this period there is no unreplicated DNA left in this region.

Animals↗
PubMed1991

Single beam optical trapping integrated in a confocal microscope for biological applications.

Confocal microscopy is very useful in biology because of its three dimensional imaging capacities and has proven to be an excellent tool to study the 3D organization of, for instance, cell structures. This property of confocal microscopy makes it also very suitable for observation during guidance of the three dimensional manipulation of single cells or cell elements. Therefore we decided to integrate a confocal microscope and a single beam optical manipulator into a single instrument. The advantage of optical manipulation over mechanical techniques is that it is non-invasive and therefore may be applied on living (micro-) organisms and cells. The creation of an effective single beam optical trap requires the use of a high numerical aperture (N.A.) objective to focus the laser beam. In this paper we briefly discuss the vertical or axial force exerted on a sphere in a single beam trap. The axial force on a sphere placed on the optical axis, caused by reflection and refraction, is calculated applying a electromagnetic vector diffraction theory to determine the field distribution in the focal region. One of the results is that the particle also experiences a vertical trapping force towards the focusing lens when it is in the strongly convergent part of the field in addition to the known negative signed trapping force in the divergent part of the field. Further we describe an instrumental approach to realize optical trapping in which the optical trap position is controlled by moving the focusing objective only.(ABSTRACT TRUNCATED AT 250 WORDS)

Algorithms↗
PubMed1990

Spatial topography of a pericentromeric region (1q12) in hemopoietic cells studied by in situ hybridization and confocal microscopy.

A fluorescent in situ hybridization procedure with a chromosome 1-specific (1q12) repetitive satellite DNA probe was used to label the 1q12 regions of the chromosomes 1 in spherical and polymorphic hemopoietic cell nuclei. The entire procedure was performed in suspension to preserve nuclear morphology. The result was studied by three-dimensional analysis, as provided by a scanning laser confocal microscope. The 1q12 regions of chromosome 1 were measured to be closely associated with the nuclear envelope in isolated nuclei of unstimulated diploid human lymphocytes. The relative positions to each other in the periphery of these spherical nuclei could not be distinguished from a random distribution pattern. In the diploid and tetraploid polymorphic nuclei of cells of the promyelocytic leukemia cell line HL60 these pericentromeric sequences were also associated with the nuclear surface.

Cell Nucleus↗
PubMed1990

Three-dimensional reconstruction of pericentromeric (1q12) DNA and ribosomal RNA sequences in HL60 cells after double-target in situ hybridization and confocal microscopy.

A fluorescent in situ hybridization procedure was applied to simultaneously label intranuclear pericentromeric (1q12) sequences of the chromosomes 1 and cytoplasmic ribosomal RNA sequences in whole cells of the promyelocytic HL60 cell line. For this purpose biotinated chromosome 1-specific (1q12) repetitive satellite DNA and 28S ribosomal ssRNA probes were used. The entire procedure was performed in suspension to preserve nuclear morphology. The result was studied by three-dimensional analysis, as provided by a scanning laser confocal microscope. The intracellular positions of both cytoplasmic rRNA and intranuclear centromere 1 DNA could easily be distinguished. This approach could be useful as a framework for the study of the 3-D localization of genes and gene transcripts.

Cell Line↗
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↗
PubMed1990

Three-dimensional distribution of DNase I-sensitive chromatin regions in interphase nuclei of embryonal carcinoma cells.

In situ nick-translation allows the visualization of nuclease-sensitive chromatin regions in interphase nuclei. We have analyzed the three-dimensional (3-D) distribution of DNase I-sensitive regions of chromatin in nuclei from mouse P19 embryonal carcinoma cells by making optical sections using confocal scanning laser microscopy. In undifferentiated as well as embryonal carcinoma cells differentiated in vitro, DNase I-sensitive regions of chromatin are observed as discrete spots in the nucleus. These spots represent clusters of DNase I-sensitive sites. By optical sectioning, we show that these spots are preferentially, but not exclusively, localized at the nuclear periphery. No differences were observed in the spatial distribution of DNase I-sensitive sites in P19 EC cells or the differentiated P19 END-2 cells. Furthermore, we did not observe differences in the distribution of DNase I-sensitive chromatin regions during the cell cycle. These findings indicate, at least for P19 mouse embryonal carcinoma cells and their differentiated derivative END-2, that the compartmentalization of DNase I-sensitive chromatin regions is a general characteristic of the nucleus, independent of cell cycle stage or differentiation state. Since evidence has been presented that DNase I-sensitive sites are associated with actively transcribed chromatin, our results indicate that active transcribing chromatin is compartmentalized, preferentially in the periphery of the nucleus.

Animals↗
PubMed1989

Three-dimensional visualization methods for confocal microscopy.

Three-dimensional images of microscopic objects can be obtained by confocal scanning laser microscopy (CSLM). The imaging process in a CSLM consists of sampling a specific volume in the object and storing the result in a three-dimensional memory array of a digital computer. Methods are needed to visualize these images. In this paper three methods are discussed, each suitable in a specific area of application. For purposes where realistic rendering of solid or semi-transparent objects is required, an algorithm based on simulation of a fluorescence process is most suitable. When speed is essential, as for interactive purposes, a simple procedure to generate anaglyphs can be used. Both methods have in common that they require no previous interpretation or analysis of the image. When the study of an object imaged by CSLM involves analysis in terms of a geometrical model, sophisticated graphics techniques can be used to display the results of the analysis.

Algorithms↗
PubMed1989

Three-dimensional imaging in fluorescence by confocal scanning microscopy.

The improved resolution and sectioning capability of a confocal microscope make it an ideal instrument for extracting three-dimensional information especially from extended biological specimens. The imaging properties, also with finite detection pinholes are considered and a number of biological applications demonstrated.

Animals↗
PubMed1989

Ki-67 detects a nuclear matrix-associated proliferation-related antigen. I. Intracellular localization during interphase.

Ki-67 is a commercially available mouse monoclonal antibody, which reacts with a nuclear antigen in proliferating cells. The antibody can be used to determine the growth fraction of human tumours in situ and has been shown to be of prognostic importance. In this study it is shown that in interphase cells Ki-67 reacts with an antigen, mainly present in the nucleoli. Confocal scanning laser microscopy and immunoelectron microscopy on human MR65 monolayer cells revealed that this nucleolar antigen is predominantly localized in the nucleolar cortex and in the dense fibrillar components. The Ki-67 antigen appeared to be preserved in nuclear matrix preparations obtained after in situ fractionation of MR65 cells. Despite many efforts, we could not identify the antigen in immunoblotting or immunoprecipitation assays. Testing of cell cultures of different species by means of indirect immunofluorescence revealed that the antibody reacted with human cells and with the Rhesus monkey kidney-derived cell line LLC-MK2.

Antibodies, Monoclonal↗
PubMed1989

Three-dimensional chromosome arrangement of Crepis capillaris in mitotic prophase and anaphase as studied by confocal scanning laser microscopy.

To estimate the extent of ordering of chromosomes, confocal scanning laser microscopy was used to make three-dimensional images from optical sections. For Crepis capillaris, which has 2n = 6 easily recognizable chromosomes, a statistically significant sample of 75 Feulgen-stained root tip anaphases was analysed. A comparison of the observed chromosome ordering and the expected random distribution showed a significant surplus of one of the arrangements with a juxtaposition of the two chromosomes with a nucleolus organizer region. Two of the arrangements with these chromosomes in opposite positions were never observed in our material. Another analysis of 30 mithramycin A-stained prophases and 30 meta- and anaphases showed partly different patterns of non-random chromosome distribution in the two stages of mitosis. A preference for an association of the homologues was observed for all pairs of chromosomes in prophase cells, whereas in meta- and anaphase the association only persisted for the nucleolus organizer chromosomes. This indicates that there may be some relocation of the chromosome positions during the transition from prophase to metaphase. In meta- and anaphase one of the arrangements with juxtaposed NOR chromosomes was preferred, i.e. the ordering in which chromosomes 1 and 3 occupied alternate positions. Probably, the nucleolus is an important factor in producing a non-random distribution, but there could be other factors that influence chromosome ordering as well. A comparison of the anaphase chromosome ordering in C. capillaris plants from very different localities, indicated that the observed non-random distribution was independent of the origin of the material. Existing models of chromosome disposition are not sufficient to explain the observed non-random chromosome ordering in C. capillaris.

Cell Division↗
PubMed1989

Ki-67 detects a nuclear matrix-associated proliferation-related antigen. II. Localization in mitotic cells and association with chromosomes.

In interphase cells the proliferation-associated antigen recognized by monoclonal antibody Ki-67 is almost exclusively located in the nucleoli. When cells at several stages of mitosis were examined for the localization of the Ki-67 antigen, a striking redistribution could be observed. During prophase the distinct nucleolar Ki-67 fluorescence changed to a bright irregular meshwork throughout the nucleoplasm. At metaphase the antigen appeared to be distributed in a reticulate structure surrounding the condensed chromosomes, while at late telophase a punctated staining of the entire nucleoplasm was observed, which preceded the typical nucleolar localization pattern in each of the two daughter cells. Immunolabelling with Ki-67 of metaphase chromosome spreads revealed a circumferential staining of the individual chromosomes. The Ki-67 antigen is preserved in nuclear matrix preparations obtained after in situ fractionation of interphase cells. When mitotic cells were exposed to such treatments, the obtained fluorescence data suggested that the antigen may be part of the chromosome scaffold. Quantification of the Ki-67 fluorescence signal using flow cytometry revealed the highest staining intensities in mitotic cells. Furthermore, it was shown that nutritionally deprived cells became negative for Ki-67.

Antibodies, Monoclonal↗
PubMed1988

Visualization and analysis techniques for three dimensional information acquired by confocal microscopy.

Confocal Scanning Laser Microscopy (CSLM) is particularly well suited for the acquisition of 3-dimensional data of microscopic objects. In the CSLM a specific volume in the object is sampled during the imaging process and the result is stored in a digital computer as a three-dimensional memory array. Optimal use of these data requires both the development of effective visual representations as well as analysis methods. In addition to the well known stereoscopic representation method a number of alternatives for various purposes are presented. When rendering in terms of solid-looking or semitransparent objects is required, an algorithm based on a simulated process of excitation and fluorescence is very suitable. Graphic techniques can be used to examine the 3-dimensional shape of surfaces. For (near-)real time applications a representation method should not require extensive previous data-processing or analysis. From the very extensive field of 3-D image analysis two examples are given.

Cell Nucleus↗
PubMed1988

3-dimensional imaging of biological structures by high resolution confocal scanning laser microscopy.

Imaging in confocal microscopy is characterized by the ability to make a selective image of just one plane inside a specimen, virtually unaffected -within certain limits- by the out-of-focus regions above and below it. This property, called optical sectioning, is accompanied by improved imaging transverse to the optical axis. We have coupled a confocal microscope to a computer system, making the combination of both an excellent instrument for mapping the 3-dimensional structure of extended specimens into a computer memory/data array. We measured that the volume element contributing to each data point has, under typical fluorescence conditions, a size of 0.2 X 0.2 X 0.72 micron. The data can be analysed and represented in various ways, i.e., stereoscopical views from any desired angle. After a description of the experimental arrangement, we show various examples of biological and food-structural studies. The microscope can be operated either in reflection or in fluorescence. In the latter mode a spectral element allows selection of the wavelength band of fluorescence light contributing to the image. In this way, we can distinguish various structures inside the cell and study their 3-dimensional relationships. Various applications in biology and the study of food structure are presented.

Animals↗