Light microscope based analysis of three‐dimensional structure: Applications to the study of Drosophila salivary gland nuclei. I. Data collection and analysis

Mathog, David R.; Hochstrasser, Mark; Sedat, John W.

doi:10.1111/j.1365-2818.1985.tb02582.x

Cited by 22 publications

(17 citation statements)

References 26 publications

(27 reference statements)

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“…The specimen is stained with a DNA-specific fluorescent dye and prepared for microscopy. Fluorescence images from serial sections of nuclei are collected, and from such image stacks, the paths of the five major chromosome arms are traced using an interactive modeling program (27)(28)(29). Subsequently, quantitative properties of the resulting stick figure models are measured and displayed.…”

Section: Resultsmentioning

confidence: 99%

“…The models and quantitation plots are used to derive a detailed structural description of chromosome folding. Details of methodology are published elsewhere (27)(28)(29), and any modifications are noted under Materials and Methods.…”

Section: Resultsmentioning

confidence: 99%

“…The model building and analysis program IMP has also been delineated (27)(28)(29), although its capabilities have been expanded. None of the images from the aqueous samples were processed computationally.…”

Section: Microscopy and Model Buildingmentioning

confidence: 99%

“…The computer-controlled Zeiss Axiomat microscope for serial optical sectioning of polytene nuclei was as described (27,29). Some hardware changes have been made.…”

Section: Microscopy and Model Buildingmentioning

confidence: 99%

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Spatial organization of chromosomes in the salivary gland nuclei of Drosophila melanogaster.

Hochstrasser¹,

Mathog²,

Gruenbaum³

et al. 1986

The Journal of Cell Biology

251

221

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Abstract. Using a computer-based system for model building and analysis, three-dimensional models of 24Drosophila melanogaster salivary gland nuclei have been constructed from optically or physically sectioned glands, allowing several generalizations about chromosome folding and packaging in these nuclei. First and most surprising, the prominent coiling of the chromosomes is strongly chiral, with fight-handed gyres predominating. Second, high frequency appositions between certain loci and the nuclear envelope appear almost exclusively at positions of intercalary heterochromatin; in addition, the chromocenter is always apposed to the envelope. Third, chromosomes are invariably separated into mutually exclusive spatial domains while usually extending across the nucleus in a polarized (Rabl) orientation. Fourth, the arms of each autosome are almost always juxtaposed, but no other relative arm positions are strongly favored. Finally, despite these nonrandom structural features, each chromosome is found to fold into a wide variety of different configurations. In addition, a set of nuclei has been analyzed in which the normally aggregrated centromeric regions of the chromosomes are located far apart from one another. These nuclei have the same architectural motifs seen in normal nuclei. This implies that such characteristics as separate chromosome domains and specific chromosomenuclear envelope contacts are largely independent of the relative placement of the different chromosomes within the nucleus.E UKARYOTIC cells contain enormous lengths of DNA that must undergo considerable packing to fit inside the interphase nucleus. The packing is accomplished through multiple foldings of the DNA molecule within each chromosome and the folding of the chromosomes themselves inside the nucleus. This hierarchy of foldings is under several general constraints. First, the DNA must be available to regulatory factors and transcriptional machinery in such a way that readout of the genome can be precisely controlled. Second, the resident copies of DNA must be faithfully duplicated and repackaged with histones and other proteins; in dividing cells, the daughter helices must also be topologically resolved. Finally, nuclear division depends on a number of tightly orchestrated events: condensation of chromatin into compact chromosomes, dissolution of the nuclear envelope, movement of chromosomes to a central plate with subsequent splitting of daughter chromatids toward opposite poles, decondensation of daughter chromosomes, and reassembly of the nuclear envelope around them.Despite the interest in elucidating the means by which cells accommodate these constraints and package their genomes, only a meager sketch of higher order structures can currently be claimed. Beyond the level of the 10-nm nucleosomal fiber, little.consensus has been reached. This is particularly true of the highest level of chromosome organization, the threedimensional arrangement of chromosomes in the interphase nucleus. For reviews, see references 13 and 21.The giant ...

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Microscopy and Model Buildingmentioning

confidence: 99%

“…The computer-controlled Zeiss Axiomat microscope for serial optical sectioning of polytene nuclei was as described (27,29). Some hardware changes have been made.…”

Section: Microscopy and Model Buildingmentioning

confidence: 99%

See 2 more Smart Citations

Spatial organization of chromosomes in the salivary gland nuclei of Drosophila melanogaster.

Hochstrasser¹,

Mathog²,

Gruenbaum³

et al. 1986

The Journal of Cell Biology

251

221

View full text Add to dashboard Cite

show abstract

“…Our OM system is built around a Zeiss inverted Axiomat microscope that was modified so that stage, focus, and data collection are fully computer controlled (4,9). A CCD camera made by Photometrics (Tucson, Arizona) that consists of a dewar for liquid nitrogen cooling and associated electronics was attached to the straight-through port of the Axiomat.…”

Section: System Descriptionmentioning

confidence: 99%

The Use of a Charge-Coupled Device for Quantitative Optical Microscopy of Biological Structures

Hiraoka

Sedat

Agard

1987

Science

Self Cite

291

132

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The properties of a charge-coupled device (CCD) and its application to the high-resolution analysis of biological structures by optical microscopy are described. The CCD, with its high resolution, high sensitivity, wide dynamic range, photometric accuracy, and geometric stability, can provide data of such high quality that quantitative analysis on two- and three-dimensional microscopic images is possible. For example, the three-dimensional imaging properties of an epifluorescence microscope have been quantitatively determined with the CCD. This description of the imaging properties of the microscope, and the high-quality image data provided by the CCD, allow sophisticated computational image processing methods to be used that greatly improve the effective resolution obtainable for biological structures. Image processing techniques revealed fine substructures in Drosophila embryonic diploid chromosomes in two and three dimensions. The same approach can be extended to structures as small as yeast chromosomes or to other problems in structural cell biology.

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Three‐dimensional light microscopy of diploid Drosophila chromosomes

Agard

Hiraoka

Sedat

1988

Cell Motil. Cytoskeleton

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Fluorescence microscopy, uniquely, provides the ability to examine specific components within intact, even living, cells. Unfortunately, high-resolution conventional fluorescence microscopy is intrinsically a two-dimensional technique and performs poorly with specimens thicker than about 0.5 micron. Probing the spatial organization of components within cells has required the development of new methods optimized for three-dimensional data collection, processing, display, and interpretation. Our interest in understanding the relationship between chromosome structure and function has led us to develop the necessary methodology for exploring cell structures in three dimensions. It is now possible to determine directly the three-dimensional spatial organization of diploid chromosomes within intact nuclei throughout most of the mitotic the cell cycle.

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Light microscope based analysis of three‐dimensional structure: Applications to the study of Drosophila salivary gland nuclei. I. Data collection and analysis

Cited by 22 publications

References 26 publications

Spatial organization of chromosomes in the salivary gland nuclei of Drosophila melanogaster.

Spatial organization of chromosomes in the salivary gland nuclei of Drosophila melanogaster.

The Use of a Charge-Coupled Device for Quantitative Optical Microscopy of Biological Structures

Three‐dimensional light microscopy of diploid Drosophila chromosomes

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