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Phototoxicity and photobleaching are major limitations in live-cell fluorescence microscopy. They are caused by fluorophores in an excited singlet or triplet state that generate singlet oxygen and other reactive oxygen species. The principle of controlled light exposure microscopy (CLEM) is based on non-uniform illumination of the field of view to reduce the number of excited fluorophore molecules. This approach reduces phototoxicity and photobleaching 2- to 10-fold without deteriorating image quality. Reduction of phototoxicity and photobleaching depends on the fluorophore distribution in the studied object, the optical properties of the microscope and settings of CLEM electronics. Here, we introduce the CLEM factor as a quantitative measure of reduction in phototoxicity and photobleaching. Finally, we give a guideline to optimize the effect of CLEM without compromising image quality.

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The detrimental effects of a refractive-index mismatch on the image formation in a two-photon microscope were investigated. Point-spread functions (PSF's) were recorded with an oil-immersion objective numerical aperture (NA) of 1.3 and a water-immersion objective NA of 1.2 in an aqueous sample at different depths. For the oil-immersion objective the enlargement of the PSF volume with increasing depth yields an axial and a lateral loss in resolution of approximately 380% and 160%, respectively, at a 90-microm depth in the sample. For the water-immersion objective no resolution decrease was found. Measurements on a thick aqueous biofilm sample shows the importance of matching the refractive index between immersion fluid and sample. With a good match, no loss in image resolution is observed.

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We report three-dimensional (3D) microscopy with nearly isotropic resolution in the lambda/5-lambda/10 range. Our approach combines 4PI-confocal two-photon fluorescence microscopy with image restoration. The 3D resolution is demonstrated with densely clustered beads as well as with F-actin fibers in mouse fibroblast cells. A comparison with unrestored two-photon confocal images reveals a total reduction of the uncertainty volume up to a factor of 15.

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A study on the chromatin organisation of synchronised G1 and G2 populations of maize root cell nuclei is reported using 3-D images acquired with a confocal fluorescence microscope. The analysis is based on the concept of accessibility. Accessibility of a position x is the effort to arrive at x, when choosing the minimum effort path to arrive at x from the nuclear border. The effort is then taken to be proportional to the amount of all mass encountered on the path, and computed by a technique called the grey valued distance transform. The approach relies heavily on quantitative analysis of the intensity information. Hence, considerable attention was paid to the quantitative modification of the confocal intensity values by diffraction, absorption and scatter corrections. Three texture features are extracted from the accessibility maps: the global object inaccessibility, the relative object accessibility, and the object homogeneity. On the basis of individual texture features, no distinction between the G1 and G2 populations could be established. However, the three features combined did show a clear difference with a high significance.

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The PML protein is a human growth suppressor concentrated in 10 to 20 nuclear bodies per nucleus (PML bodies). Disruption of the PML gene has been shown to be related to acute promyelocytic leukaemia (APL). To obtain information about the function of PML bodies we have investigated the 3D-distribution of PML bodies in the nucleus of T24 cells and compared it with the spatial distribution of a variety of other nuclear components, using fluorescence dual-labeling immunocytochemistry and confocal microscopy. Results show that PML bodies are not enriched in nascent RNA, the splicing component U2-snRNP, or transcription factors (glucocorticoid receptor, TFIIH, and E2F). These results show that PML bodies are not prominent sites of RNA synthesis or RNA splicing. We found that a large fraction of PML bodies (50 to 80%) is closely associated with DNA replication domains during exclusively middle-late S-phase. Furthermore, in most cells that we analysed we found at least one PML body was tightly associated with a coiled body. In the APL cell line NB4, the PML gene is fused with the RAR alpha gene due to a chromosomal rearrangement. PML bodies have disappeared and the PML antigen, i.e., PML and the PML-RAR fusion protein, is dispersed in a punctated pattern throughout the nucleoplasm. We showed that in NB4 cells the sites that are rich in PML antigen significantly colocalize with sites at which nascent RNA accumulates. This suggests that, in contrast to non-APL cells, in NB4 cells the PML antigen is associated with sites of transcription. The implications of these findings for the function of PML bodies are consistent with the idea that PML bodies are associated with specific genomic loci.

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The glucocorticoid receptor and the mineralocorticoid receptor are hormone-dependent transcription factors. They regulate the excitability of rat hippocampus CA1 neurons in a coordinated fashion. We studied the spatial distribution of these transcription factors in nuclei of CA1 neurons by dual labeling immunocytochemistry and confocal microscopy, combined with novel image restoration and image analysis techniques. We found that both receptors are concentrated in about one thousand clusters within the nucleus. Some clusters contain either mineralocorticoid receptors or glucocorticoid receptors, but a significant number of clusters contains both receptors. These results indicate that the two receptor types are targeted to specific compartments in the nucleus. The coordinated action of the glucocorticoid and mineralocorticoid receptor on gene expression may be established in a specific set of nuclear domains that contain both receptors.

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In this paper we present a three-dimensional visualization technique for multi-channel volume data. The technique simulates the physical process of fluorescence, hence its name: achromatic multi-channel simulated fluorescent process (amSFP). The data set is simulated as 3D distribution of different fluorescent dyes, where each channel is represented by a particular type of dye. Apart from the spatial density map, no additional characteristics about the data set have to be defined; no image segmentation is needed prior to visualization. The degree of interaction among the channels in the fluorescence process can be adapted to optimally render specific structures in the image. 3D multi-channel data can be obtained by a three-dimensional imaging device that is able to measure a number of physical quantities at a given location within a specimen. The fluorescence principle, the algorithm, and its implementation are presented. We have used the technique to investigate the relative spatial arrangement of blood vessels and astrocytes in the cat retina. The two components have been stained with different fluorescence dyes and recorded in a confocal light microscope to form a two-channel 3D data set.

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When studying the three-dimensional shape of prophase chromosomes (or any other tubular structure), it is useful to represent these structures as a string of three-dimensional Cartesian coordinates along the medial axis. This procedure was automated in order to limit the number of human interactions and to improve reproducibility. In this paper the design, implementation, and validation of the automated method is presented. From the data presented it can be concluded that the cursor algorithm provides an objective and therefore reproducible method to trace the medial axes of prophase chromosomes automatically. This method could allow a more extensive understanding of the (changes in) chromosome organisation throughout the cell cycle, its relation to cell function, and the complex process of chromosome condensation.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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The relationship between cell shape and function has long been of interest. However, although the behaviour of the cytoskeleton during the cell cycle has been studied extensively variations in the shape and three-dimensional substructure of the nucleus are less well documented. The spatial distribution of chromatin has previously been studied by a mathematical analysis of the optical densities of stained nuclei, allowing an indirect derivation of the three-dimensional distribution of chromatin. More direct information on chromatin organization can be obtained from electron-microscopic serial sections, although this is very laborious. Using an iterative deconvolution algorithm, Agard and Sedat achieved a degree of optical sectioning in conventional fluorescence microscopy and reconstructed the three-dimensional arrangement of polytene chromosomes. We report here on the three-dimensional structure of cultured mammalian cells as visualized by confocal scanning laser microscopy (CSLM). The exceptionally short depth of field of this imaging technique provides direct optical sectioning which, together with its higher resolution, makes CSLM extremely useful for studying the three-dimensional morphology of biological structures.

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The nucleoid of living and OsO4- or glutaraldehyde-fixed cells of Escherichia coli strains was studied with a phase-contrast microscope, a confocal scanning light microscope, and an electron microscope. The trustworthiness of the images obtained with the confocal scanning light microscope was investigated by comparison with phase-contrast micrographs and reconstructions based on serially sectioned material of DNA-containing and DNA-less cells. This comparison showed higher resolution of the confocal scanning light microscope as compared with the phase-contrast microscope, and agreement with results obtained with the electron microscope. The effects of fixation on the structure of the nucleoid were studied in E. coli B/r H266. Confocal scanning light micrographs and electron microscopic reconstructions showed that the shape of the nucleoid remained similar after OsO4 or glutaraldehyde fixation; however, the OsO4 nucleoid appeared to be somewhat smaller and more centralized within the cell.

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