Three-dimensional intranuclear DNA organization in situ: three states of condensation and their redistribution as a function of nuclear size near the G1-S border in HeLa S-3 cells

1984 ◽  
Vol 65 (1) ◽  
pp. 123-138
Author(s):  
A. Belmont ◽  
F.M. Kendall ◽  
C. Nicolini
Keyword(s):  
Optical Density ◽  
Chromatin Condensation ◽  
Physiological State ◽  
Three Dimensional ◽  
Nuclear Size ◽  
Nuclear Morphology ◽  
Discrete States ◽  
Dna Organization ◽  
Density Values

Characteristic variations in nuclear morphology occurring with variations in the physiological state of the cell have been observed in a number of systems to date. In this paper, we have critically examined the relationship between nuclear morphology and intranuclear DNA organization near the G1-S transition in HeLa S-3 cells, by the study of both the spatial distribution of optical density values and the optical density histograms for individual Feulgen-stained nuclei. Our results demonstrate that the majority of the DNA is located in a narrow shell surrounding the nuclear and nucleolar borders, and present evidence for at least three discrete states of chromatin condensation. Greater than 90% of the genome appears distributed among the two classes with larger density, and a redistribution between these two classes occurs as a function of changing nuclear size. Numerical simulations indicate that the observed distribution does not arise as an artifact related to overlapping but, in fact, actually represents discrete states of condensation. Interestingly, the extrapolated nuclear area at which the fraction of DNA in the state of highest density is reduced to zero, corresponds closely to the nuclear size shown elsewhere as representing the critical size that HeLa S-3 nuclei must exceed in order to initiate S phase.

Detection and mapping of interactions between chromatin and the nuclear envelope in drosophila embryos

1995 ◽  
Vol 53 ◽  
pp. 764-765
Author(s):  
W.F. Marshall ◽  
A.F. Dernburg ◽  
B. Harmon ◽  
J.W. Sedat
Keyword(s):  
Nuclear Envelope ◽  
Chromatin Condensation ◽  
Three Dimensional ◽  
Physiological Role ◽  
Nuclear Surface ◽  
Intact Cells ◽  
Molecular Determinants ◽  
Surface Harmonic ◽  
Drosophila Embryos

Interactions between chromatin and nuclear envelope (NE) have been implicated in chromatin condensation, gene regulation, nuclear reassembly, and organization of chromosomes within the nucleus. To further investigate the physiological role played by such interactions, it will be necessary to determine which loci specifically interact with the nuclear envelope. This will not only facilitate identification of the molecular determinants of this interaction, but will also allow manipulation of the pattern of chromatin-NE interactions to probe possible functions. We have developed a microscopic approach to detect and map chromatin-NE interactions inside intact cells.Fluorescence in situ hybridization (FISH) is used to localize specific chromosomal regions within the nucleus of Drosophila embryos and anti-lamin immunofluorescence is used to detect the nuclear envelope. Widefield deconvolution microscopy is then used to obtain a three-dimensional image of the sample (Fig. 1). The nuclear surface is represented by a surface-harmonic expansion (Fig 2). A statistical test for association of the FISH spot with the surface is then performed.

Three-dimensional imaging of the living eye

1991 ◽  
Vol 49 ◽  
pp. 170-171 ◽  
Author(s):  
Barry R. Masters
Keyword(s):  
Laser Scanning ◽  
Water Immersion ◽  
Physiological State ◽  
Three Dimensional ◽  
Confocal Microscope ◽  
Image Stack ◽  
Rabbit Eye ◽  
Reflected Light

The structure of the in situ rabbit cornea can be observed at high resolution and contrast with reflected light confocal microscopy. In vivo confocal images of the living cornea have been made at lower resolution and lower contrast using a SIT video camera together with a real-time Nipkow disk confocal microscope adapted for in vivo observations. This paper describes the three dimensional reconstruction of the in situ cornea from an enucleated rabbit eye with confocal reflected light microscopy and volume rendering computer techniques.A laser scanning confocal microscope (BioRad MRC 600) was used in the reflected light mode to obtain the two-dimensional image stack from the cornea of a freshly enucleated rabbit eye. The eye was maintained in a physiological state with aerated Ringer's solution. The light source was an argon ion laser with a 488 nm wavelength. The microscope objective was a Leitz X25, NA 0.6 water immersion lens. The 400 micron thick cornea was optically sectioned into 133, three micron sections. The optical sectioning was performed perpendicular to the optical axis of the eye globe.

Correlated Studies of Cellular Interactions Using TEM, Freeze Etching, and SEM

1974 ◽  
Vol 32 ◽  
pp. 320-321
Author(s):  
J. P. Revel
Keyword(s):  
Developmental Stages ◽  
Three Dimensional ◽  
Cell Movement ◽  
Cellular Interactions ◽  
Serial Sections ◽  
Cell Sheets ◽  
Freeze Etching ◽  
Early Developmental

Movement of individual cells or of cell sheets and complex patterns of folding play a prominent role in the early developmental stages of the embryo. Our understanding of these processes is based on three- dimensional reconstructions laboriously prepared from serial sections, and from autoradiographic and other studies. Many concepts have also evolved from extrapolation of investigations of cell movement carried out in vitro. The scanning electron microscope now allows us to examine some of these events in situ. It is possible to prepare dissections of embryos and even of tissues of adult animals which reveal existing relationships between various structures more readily than used to be possible vithout an SEM.

In vitro and in vivo polysaccharides assembly: ultrastructural and cytochemical study of growing plant cell wall components.

1978 ◽  
Vol 36 (2) ◽  
pp. 434-435
Author(s):  
D. Reis ◽  
B. Vian ◽  
J. C. Roland
Keyword(s):  
Cell Wall ◽  
Self Assembly ◽  
Plant Cell Wall ◽  
Higher Plants ◽  
Three Dimensional ◽  
Cytochemical Study ◽  
Wall Components

Wall morphogenesis in higher plants is a problem still open to controversy. Until now the possibility of a transmembrane control and the involvement of microtubules were mostly envisaged. Self-assembly processes have been observed in the case of walls of Chlamydomonas and bacteria. Spontaneous gelling interactions between xanthan and galactomannan from Ceratonia have been analyzed very recently. The present work provides indications that some processes of spontaneous aggregation could occur in higher plants during the formation and expansion of cell wall.Observations were performed on hypocotyl of mung bean (Phaseolus aureus) for which growth characteristics and wall composition have been previously defined.In situ, the walls of actively growing cells (primary walls) show an ordered three-dimensional organization (fig. 1). The wall is typically polylamellate with multifibrillar layers alternately transverse and longitudinal. Between these layers intermediate strata exist in which the orientation of microfibrils progressively rotates. Thus a progressive change in the morphogenetic activity occurs.

In situ investigation of the primary recrystallization of alpha titanium in HVEM

1978 ◽  
Vol 36 (1) ◽  
pp. 634-635
Author(s):  
S. Naka ◽  
R. Penelle ◽  
R. Valle
Keyword(s):  
Dimensional Analysis ◽  
Texture Evolution ◽  
Cold Work ◽  
Three Dimensional ◽  
Primary Recrystallization ◽  
Thin Foils ◽  
In Situ Investigation ◽  
Grain Growth Model ◽  
Alpha Titanium

The in situ experimentation technique in HVEM seems to be particularly suitable to clarify the processes involved in recrystallization. The material under investigation was unidirectionally cold-rolled titanium of commercial purity. The problem was approached in two different ways. The three-dimensional analysis of textures was used to describe the texture evolution during the primary recrystallization. Observations of bulk-annealed specimens or thin foils annealed in the microscope were also made in order to provide information concerning the mechanisms involved in the formation of new grains. In contrast to the already published work on titanium, this investigation takes into consideration different values of the cold-work ratio, the temperature and the annealing time.Two different models are commonly used to explain the recrystallization textures i.e. the selective grain growth model (Beck) or the oriented nucleation model (Burgers). The three-dimensional analysis of both the rolling and recrystallization textures was performed to identify the mechanismsl involved in the recrystallization of titanium.

Three-Dimensional Subcellular Organization of Hepatocytes in Intact Liver: Simultaneous Visualization of Cytoskeletal and Membrane Markers by Confocal Microscopy

1996 ◽  
Vol 54 ◽  
pp. 288-289
Author(s):  
Greg V. Martin ◽  
Ann L. Hubbard
Keyword(s):  
Three Dimensional ◽  
Integral Membrane Proteins ◽  
Polarized Secretion ◽  
Microscope Images ◽  
Computer Aided ◽  
Intracellular Organelles ◽  
Cytoskeletal Elements ◽  
Simultaneous Visualization

The microtubule (MT) cytoskeleton is necessary for many of the polarized functions of hepatocytes. Among the functions dependent on the MT-based cytoskeleton are polarized secretion of proteins, delivery of endocytosed material to lysosomes, and transcytosis of integral plasma membrane (PM) proteins. Although microtubules have been shown to be crucial to the establishment and maintenance of functional and structural polarization in the hepatocyte, little is known about the architecture of the hepatocyte MT cytoskeleton in vivo, particularly with regard to its relationship to PM domains and membranous organelles. Using an in situ extraction technique that preserves both microtubules and cellular membranes, we have developed a protocol for immunofluorescent co-localization of cytoskeletal elements and integral membrane proteins within 20 µm cryosections of fixed rat liver. Computer-aided 3D reconstruction of multi-spectral confocal microscope images was used to visualize the spatial relationships among the MT cytoskeleton, PM domains and intracellular organelles.

Conformational characterization of nucleosome structure by Electron Microscopy

1993 ◽  
Vol 51 ◽  
pp. 194-195
Author(s):  
Gregory J. Czarnota
Keyword(s):  
Electron Microscopy ◽  
Structural Changes ◽  
Physiological State ◽  
Gene Activation ◽  
Three Dimensional ◽  
Macromolecular Complex ◽  
Ultrastructural Studies ◽  
Ionic Environment ◽  
Nucleosome Structure ◽  
Dimensional Reconstruction

Chromatin structure at the fundamental level of the nucleosome is important in vital cellular processes. Recent biochemical and genetic analyses show that nucleosome structure and structural changes are very active participants in gene expression, facilitating or inhibiting transcription and reflecting the physiological state of the cell. Structural states and transitions for this macromolecular complex, composed of DNA wound about a heterotypic octamer of variously modified histone proteins, have been measured by physico-chemical techniques and by enzyme-accessibility and are recognized to occur with various post-translational modifications, gene activation, transformation and with ionic-environment. In spite of studies which indicate various forms of nucleosome structure, all current x-ray and neutron diffraction studies have consistently resulted in only one structure, suggestive of a static conformation. In contrast, two-dimensional electron microscopy studies and three-dimensional reconstruction techniques have yielded different structures. These fundamental differences between EM and other ultrastructural studies have created a long standing quandary, which I have addressed and resolved using spectroscopic electron microscopy and statistical analyses of nucleosome images in a study of nucleosome structure with ionic environment.

Mathematical Model Investigation of Far-Field Transport of Ocean-Dumped Sewage Sludge Related to Remote Sensing

1982 ◽  
Vol 14 (3) ◽  
pp. 33-39
Author(s):  
C Y Kuo
Keyword(s):  
New York ◽  
Sewage Sludge ◽  
Laboratory Data ◽  
Three Dimensional ◽  
Approximate Model ◽  
Transport Processes ◽  
Far Field ◽  
New York Bight ◽  
Mean Current

An existing, three-dimensional, Eulerian-Lagrangian finite-difference model was modified and used to examine the far-field transport processes of dumped sewage sludge in the New York Bight. Both in situ and laboratory data were utilized in an attempt to approximate model inputs such as mean current speed, vertical and horizontal diffusion coefficients, particle size distributions, and specific gravities. Concentrations of the sludge near the sea surface predicted from the computer model were compared qualitatively with those remotely sensed.

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