Three‐dimensional light microscopy of diploid <i>Drosophila</i> chromosomes

Agard, David A.; Hiraoka, Yasushi; Sedat, John W.

doi:10.1002/cm.970100106

Cited by 11 publications

(7 citation statements)

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“…Since image quality was visibly better towards the center of the maximum image frame of 1024 × 1024 pixels, we decided to use an image frame of 768 × 768 pixels. Three-dimensional deconvolution microscopy [7] requires acquisition of a stack of images at narrowly spaced parallel focal planes. Microscope objectives also collect light from out-offocus objects, which normally blurs each image.…”

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Analysis of the Trypanosoma brucei cell cycle by quantitative DAPI imaging

Siegel

Hekstra

Cross

2008

Molecular and Biochemical Parasitology

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Trypanosoma brucei has two DNA compartments: the nucleus and the kinetoplast. DNA replication of these two compartments only partially coincides. Woodward and Gull [J Cell Sci 1990;95:49-57] comprehensively studied the relative timing of the replication and segregation of nuclear and kinetoplast DNA. Others have since assumed the consistency of morphological indicators of cellcycle stage among strains and conditions. We report the use of quantitative DAPI imaging to determine the cell-cycle stage of individual procyclic cells. Using this approach, we found that kinetoplast elongation occurs mainly during nuclear S phase and not during G2, as previously assumed. We confirmed this finding by sorting cells by DNA content, followed by fluorescence microscopy. In addition, simultaneous quantitative imaging at two wavelengths can be used to determine the abundance of cell-cycle-regulated proteins during the cell cycle. We demonstrate this technique by co-staining for the non-acetylated state of lysine 4 of histone H4 (H4K4), which is enriched during nuclear S phase. KeywordsCell cycle; Deconvolution microscopy; DNA quantification; Fluorescence-activated cell sorting; Histone modification; Trypanosoma brucei Members of the order Kinetoplastidae are characterized by the presence of the kinetoplast, the uniquely structured mitochondrial DNA that consists of an interlocked network of several thousand minicircles and more than twenty maxicircles. Understanding the mechanism of kinetoplast DNA (kDNA) replication has been of great interest [1,2]. Initiation of kDNA and nuclear DNA (nDNA) replication probably coincide, but kDNA segregation is completed before the onset of mitosis [3]. Any cell can hence be assigned to a specific cell-cycle stage by comparing nuclear and kinetoplast morphology. For example, cells with an elongated kinetoplast would be in the G2. However, our recent studies suggested that elongated kinetoplasts exist in nuclear S phase as well as [4].The goal of this study was to determine the cell-cycle stage of individual cells solely by their nDNA content. Using modern fluorescence microscopy and deconvolution algorithms, we * Corresponding author. Tel.: +1 212 327 7571; fax: +1 212 327 7845. E-mail address: george.cross@rockefeller.edu (G.A.M. Cross). Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Quantification of images requires images of high quality. Spherical aberration, nonuniform illumination, and differential response of sensor pixels, need to be avoided or corrected for. Spherical aberration occurs when light waves passing through the periphery of...

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“…Three-dimensional deconvolution microscopy [7] requires acquisition of a stack of images at narrowly spaced parallel focal planes. Microscope objectives also collect light from out-offocus objects, which normally blurs each image.…”

mentioning

confidence: 99%

Analysis of the Trypanosoma brucei cell cycle by quantitative DAPI imaging

Siegel

Hekstra

Cross

2008

Molecular and Biochemical Parasitology

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“…In dramatic contrast, the diameter of a polytene nucleus is 30/zm and each polytene chromosome has a width of 3 #m. Thus, an increase of nearly an order of magnitude in resolution over that obtained for the studies on polytene chromosomes was required to examine diploid chromosomes. Recent technological developments in light microscopy, using computational image processing (reviewed in Agard et al, 1989;Fay et al, 1989) or confocal microscopes (reviewed in Brakenhoff et al, 1989) to remove out-of-focus image information, have made it possible to examine the three-dimensional organization of diploid chrom'osomes directly in intact cells (Agard et al, 1988;Rawlins and Shaw, 1988;Oud et al, 1989). Unfortunately, currently available confocal microscopes have proved to be too insensitive to record useful three-dimensional data for the very small Drosophila embryonic chromosomes.…”

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Temporal and spatial coordination of chromosome movement, spindle formation, and nuclear envelope breakdown during prometaphase in Drosophila melanogaster embryos.

Hiraoka

Agard

Sedat

1990

The Journal of Cell Biology

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Abstract. The spatial and temporal dynamics of diploid chromosome organization, microtubule arrangement, and the state of the nuclear envelope have been analyzed in syncytial blastoderm embryos of Drosophila melanogaster during the transition from prophase to metaphase, by three-dimensional optical sectioning microscopy. Time-lapse, three-dimensional data recorded in living embryos revealed that congression of chromosomes (the process whereby chromosomes move to form the metaphase plate) at prometaphase occurs as a wave, starting at the top of the nucleus near the embryo surface and proceeding through the nucleus to the bottom. The time-lapse analysis was augmented by a high-resolution analysis of fixed embryos where it was possible to unambiguously trace the three-dimensional paths of individual chromosomes. In prophase, the centromeres were found to be clustered at the top of the nucleus while the telomeres were situated at the bottom of the nucleus or towards the embryo interior. This polarized centromere-telomere orientation, perpendicular to the embryo surface, was preserved during the process of prometaphase chromosome congression. Correspondingly, breakdown of the nuclear envelope started at the top of the nucleus with the mitotic spindle being formed at the positions of the partial breakdown of the nuclear envelope. Our observations provide an example in which nuclear structures are spatially organized and their functions are locally and coordinately controlled in three dimensions.

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“…But biological samples are three-dimensional, and microscopy has increasingly been used over the past three-decades to develop three-dimensional fluorescence images by moving the focal plane of the microscope objective through the depth of the sample to create a stack of two-dimensional views. [1][2][3] In three-dimensional microscopy, as the imaging plane moves deeper into the sample, the image quality becomes degraded due to optical aberrations and scattering. Both scattering and optical aberrations are caused by refractive index variations, but scattering is the result of sub-micron inhomogeneities in the tissue and can best be treated as multiple scattering events in the sample which lead to a randomized phase and amplitude distribution of the light.…”

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Adaptive optics in wide-field microscopy

et al. 2011

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Three-dimensional live imaging in cell biology is hindered by optical aberrations which degrade the resolution and signal-to-noise ratio as the focal plane is moved deeper into the sample. The solution to this problem is to use adaptive optics to correct the aberrations. In this paper, we discuss our work on applying adaptive optics to wide-field fluorescence microscopy. We demonstrate correction of depth-aberrations and focusing using a deformable mirror in open-loop operation. We then discuss the use of phase retrieval and phase diversity in adaptive optics.

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Three‐dimensional light microscopy of diploid Drosophila chromosomes

Cited by 11 publications

References 14 publications

Analysis of the Trypanosoma brucei cell cycle by quantitative DAPI imaging

Analysis of the Trypanosoma brucei cell cycle by quantitative DAPI imaging

Temporal and spatial coordination of chromosome movement, spindle formation, and nuclear envelope breakdown during prometaphase in Drosophila melanogaster embryos.

Adaptive optics in wide-field microscopy

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