scholarly journals Spindle microtubules and their mechanical associations after micromanipulation in anaphase.

1982 ◽  
Vol 95 (1) ◽  
pp. 91-104 ◽  
Author(s):  
R B Nicklas ◽  
D F Kubai ◽  
T S Hays
Keyword(s):  
Electron Microscopy ◽  
Three Dimensional ◽  
Chromosome Movement ◽  
Serial Sections ◽  
Microtubule Bundle ◽  
Uncertain Significance ◽  
Glutaraldehyde Solution ◽  
Spindle Microtubules

Micromanipulation of living grasshopper spermatocytes in anaphase has been combined with electron microscopy to reveal otherwise obscure features of spindle organization. A chromosome is pushed laterally outside the spindle and stretched, and the cell is fixed with a novel, agar-treated glutaraldehyde solution. Two- and three-dimensional reconstructions from serial sections of seven cells show that kinetochore microtubules of the manipulated chromosome are shifted outside the confusing thicket of spindle microtubules and mechanical associations among microtubules are revealed by bent or shifted microtubules. These are the chief results: (a) The disposition of microtubules invariably is consistent with a skeletal role for spindle microtubules. (b) The kinetochore microtubule bundle is composed of short and long microtubules, with weak but recognizable mechanical associations among them. Some kinetochore microtubules are more tightly linked to one other microtubule within the bundle. (c) Microtubules of the kinetochore microtubule bundle are firmly connected to other spindle microtubules only near the pole, although some nonkinetochore microtubules of uncertain significance enter the bundle nearer to the kinetochore. (d) The kinetochore microtubules of adjacent chromosomes are mechanically linked, which provides an explanation for interdependent chromosome movement in "hinge anaphases." In the region of the spindle open to analysis after chromosome micromanipulation, microtubules may be linked mechanically by embedment in a gel, rather than by dynein or other specific, cross-bridging molecules.

scholarly journals Anatomical basis for cell transplantation into mouse seminiferous tubules

Reproduction ◽  
2012 ◽  
Vol 144 (3) ◽  
pp. 385-392 ◽  
Author(s):  
Unai Silván ◽  
Juan Aréchaga
Keyword(s):  
Electron Microscopy ◽  
Cell Transplantation ◽  
Soft Tissues ◽  
Partial Information ◽  
Three Dimensional ◽  
Seminiferous Tubules ◽  
Serial Sections ◽  
Pathological Conditions ◽  
Anatomical Basis ◽  
Scanning Electron

Cell transplantation into the seminiferous tubules is a useful technique for the study of physiological and pathological conditions affecting the testis. However, the precise three-dimensional organization and, particularly, the complex connectivity of the seminiferous network have not yet been thoroughly characterized. To date, the technical approaches to address these issues have included manual dissection under the stereomicroscope, reconstruction of histological serial sections, and injection of contrast dyes, but all of them have yielded only partial information. Here, using an approach based on the microinjection of a self-polymerizing resin followed by chemical digestion of the surrounding soft tissues, we reveal fine details of the seminiferous tubule scaffold and its connections. These replicas of the testis seminiferous network were studied by scanning electron microscopy. The present results not only establish a morphological basis for more precise microinjection into the mouse seminiferous tubules but also enable a more profound investigation of physiological and embryological features of the testis.

Electron microscopy of spermatocytes previously studied in life: methods and some observations on micromanipulated chromosomes

1979 ◽  
Vol 35 (1) ◽  
pp. 87-104
Author(s):  
R.B. Nicklas ◽  
B.R. Brinkley ◽  
D.A. Pepper ◽  
D.F. Kubai ◽  
G.K. Rickards
Keyword(s):  
Electron Microscopy ◽  
Electron Microscope ◽  
Target Cell ◽  
Living Cell ◽  
Chromosome Movement ◽  
Electron Microscopic ◽  
Microscopic Studies ◽  
Direction Of Movement ◽  
Spindle Microtubules ◽  
Future Work

A new method is offered for combined living cell and electron-microscopic studies of spermatocytes (or other cells) which normally do not adhere to glass. The key step is micro-injection of glutaraldehyde near the target cell whenever desired during observation in life. Fixation begins and simultaneously the cell is stuck very firmly to the underlying coverslip. The method is easy and reliable: cells are almost never lost and are well preserved, except for membranes. The application of the method is illustrated by studies of micromanipulated grasshopper spermatocytes. A chromosome was detached from the spindle and placed in the cytoplasm. Before or after the beginning of chromosome movement back toward the spindle, the cell was fixed, sectioned and the manipulated chromosome observed in the electron microscope. If the detached chromosome had not moved by the time of fixation, no or only one or two microtubules were seen at its kinetochore, but if movement had occurred, a few microtubules were always present. The arrangement of these microtubules corresponded to the direction of movement, but they commonly were at an unusual angle relative to the kinetochore. The origin and role in chromosome movement of the microtubules seen near moving chromosomes far from the spindle is not yet established, but a speculation is offered. A goal for future work is the detailed analysis of the microtubules associated with individual moving chromosomes. Such an analysis is feasible because the moving chromosome is far removed from the confusing mass of spindle microtubules, and its value is enhanced because the direction of movement at the time of fixation is known.

scholarly journals Meiosis in Sciara coprophila: structure of the spindle and chromosome behavior during the first meiotic division.

1982 ◽  
Vol 93 (3) ◽  
pp. 655-669 ◽  
Author(s):  
D F Kubai
Keyword(s):  
Chromosome Segregation ◽  
Chromosome Movement ◽  
Serial Sections ◽  
Chromosome Behavior ◽  
Monopolar Spindle ◽  
Prophase Nucleus ◽  
Fungus Gnat ◽  
Spindle Microtubules ◽  
Conical Radiation ◽  
Meiosis I

Light microscope descriptions of meiosis I in males of the fungus gnat Sciara coprophila suggested the presence of a monopolar spindle in which maternal and limited chromosomes move poleward while paternal chromosomes "back away" from the pole. The ultrastructural analysis reported here, based upon serial sections of cells in different stages of meiosis I, shows that the spindle is indeed monopolar with a distinctive differentiation, the polar complex, at one pole. This complex is the focus of a conical radiation of spindle microtubules. Kinetochores of paternal chromosomes face the complex and microtubules associated with these kinetochores run toward the complex. No kinetochore microtubules were discovered on maternal or limited chromosomes. When the position of paternal, maternal, and limited chromosomes is compared at various stages, it is found that limited chromosomes always remain near the polar complex, paternal chromosomes remain far from it and only maternal chromosomes move closer to the pole. Apparently, chromosome segregation does not depend on paternal chromosomes "backing away" from the pole, and the required movement of maternal chromosomes take place in the absence of kinetochore microtubules. In the prophase nucleus, limited and maternal chromosomes are already spatially separate from paternal chromosomes before the spindle forms. Thus, the monopolar spindle functions only to increase the distance between already segregated sets of chromosomes. An extensive system of microtubule-associated membranes outlines the spindle; the possibility that maternal chromosome movement is somehow related to the presence of this membrane is discussed.

Unorthodox Mitosis in Trichonympha Agilis: Kinetochore Differentiation and Chromosome Movement

1973 ◽  
Vol 13 (2) ◽  
pp. 511-552
Author(s):  
DONNA F. KUBAI
Keyword(s):  
Nuclear Envelope ◽  
Direct Contact ◽  
Direct Interaction ◽  
Stage Iv ◽  
Spindle Pole ◽  
Stage Ii ◽  
Nuclear Surface ◽  
Chromosome Movement ◽  
Serial Sections ◽  
Spindle Microtubules

Changes in rostral structures and the nuclear events which occur in dividing cells of Trichonympha agilis (obtained from experimentally refaunated termites) were studied by means of electron microscopy of serial sections. It is possible to characterize 5 stages of division: Stage I. During this earliest recognizable division stage, the bilaterally symmetrical hemirostra have begun to separate and spindle microtubules appear in the intervening space. As in interphase, the kinetochore regions of chromosomes are distinguishable as fibrillar masses underlying the intact nuclear envelope; and, in individual sections, they are often seen to occur in pairs. These pairs are taken to be sister kinetochores. Stage II. The extranuclear spindle has become established between the posterior ends of well separated hemirostral tubes. Elaboration of daughter rostral structures begins and will continue through the subsequent stages of division. Kinetochores differentiate, becoming bipartite structures consisting of a fibrillar element underlain by a dense disk. The fibrillar kinetochore element is associated with the still-intact nuclear envelope which lies between kinetochores and cytoplasmic spindle microtubules. Reconstruction from serial sections shows all kinetochores to be disposed in pairs which are distributed randomly over the nuclear surface. Stage III. The fibrillar elements of kinetochores are enclosed in evaginations of the nuclear envelope, while the disk elements have come to lie in the plane of the nuclear surface. Kinetochores remain separated from the extranuclear spindle microtubules by the intact nuclear envelope. The distribution of kinetochores has changed relative to that seen in stage II: kinetochores no longer appear to be paired, and they are confined to that hemisphere of the nuclear surface closest to the spindle. Stage IV. The nuclear envelope opens at the sites of kinetochores, leaving the dense disk kinetochore element inserted in pore-like discontinuities of the nuclear envelope and the fibrillar element in the cytoplasm. Direct interaction between fibrillar kinetochore element and extranuclear spindle microtubules is, however, not yet established. Stage V. The cytoplasmically situated fibrillar elements of ‘inserted’ kinetochores are now in direct contact with spindle microtubules. As seen in reconstructions of the nucleus from serial sections, kinetochores have become segregated in 2 groups on the nuclear surface, one near each spindle pole. It is during this stage that final elaboration of rostral structures takes place. On the basis of the observed changes in kinetochore distribution which occur between stages II and III while the intact nuclear envelope prevents any direct interaction between intra-nuclear kinetochores and extranuclear spindle microtubules, it is suggested that kinetochore-membrane interaction is involved in early chromosome movement in Trichonympha agilis. Only during stage V, when direct contact between kinetochores and spindle microtubules is established, may the microtubules assume their usual role in chromosome movement.

Staining Serial Thick Sections For High-Voltage Electron Microscopy

1985 ◽  
Vol 43 ◽  
pp. 708-709
Author(s):  
John C. Kinnamon ◽  
Terri A. Sherman
Keyword(s):  
Electron Microscopy ◽  
High Voltage ◽  
Carbon Coating ◽  
Three Dimensional ◽  
Taste Bud ◽  
Serial Sections ◽  
High Voltage Electron Microscopy ◽  
Ultrastructural Studies ◽  
Voltage Electron ◽  
High Voltage Electron

Ultrastructural studies often require three-dimensional reconstructions from serial electron micrographs. The use of thick sections and high voltage electron microscopy can reduce the numbers of serial sections needed, but we have found that over 300 serial sections (0.5 μm thick) are still needed for our studies of taste bud ultrastructure. Serial reconstructions are almost always done with sections mounted onto formvar-coated slot grids, because they provide an unobscured view of the specimen. Staining and carbon coating several hundred formvar-coated slot grids often results in the loss of serial sections because the formvar films break due to handling during the staining and carbon-coating procedures. In addition, the staining process itself can be tedious and time-consuming when staining thick sections because staining times of hours are often required. Such lengthy staining times may result in staining artifacts due to exposure of the stain to the atmosphere, especially with lead stains.

Structure of Drosophila polytene chromosomes. Evidence for a toroidal organization of the bands

1982 ◽  
Vol 57 (1) ◽  
pp. 73-113
Author(s):  
L.I. Mortin ◽  
J.W. Sedat
Keyword(s):  
Electron Microscopy ◽  
Fluorescent Microscopy ◽  
Polytene Chromosomes ◽  
Three Dimensional ◽  
Structural Features ◽  
Serial Sections ◽  
High Voltage Electron Microscopy ◽  
Voltage Electron ◽  
Chromosome Bands ◽  
Whole Mounts

Avoiding acid fixation or squashing, the structure of Drosophila salivary gland polytene chromosomes has been examined in detail in nuclei, with special emphasis on the organization of the DNA in the chromosome bands. Cut serial sections, optical serial sections, scanning electron microscopy (SEM) on whole mounts, high-voltage electron microscopy (HVEM) on whole mounts, and pancreatic DNase I digestion monitored by fluorescent microscopy have been used to complement one another in this analysis. With all five of these techniques, stereo pairs were used to aid in the three-dimensional reconstruction of chromosomal structures. Evidence is presented that most, if not all, of the polytene chromosome bands are torus-shaped. The DNA of these bands is largely confined to the rim, with the interior essentially DNA-free. The chromatin in each polytene band is also seen to have an extremely regular and highly ordered substructure. This substructural organization is largely radially symmetric in the bands and generally parallel to the chromosome axis. In addition, each band appears to be a distinct architectural entity with regard to its exact structural features and dimensions. A model is presented that follows these organizational boundary conditions.

scholarly journals INTERPRETATIONS OF ELECTRON MICROGRAPHS OF SINGLE AND SERIAL SECTIONS

1955 ◽  
Vol 1 (4) ◽  
pp. 301-314 ◽  
Author(s):  
Robley C. Williams ◽  
Frances Kallman
Keyword(s):  
Electron Microscopy ◽  
Three Dimensional ◽  
Electron Microscope Observation ◽  
Serial Sections ◽  
Total Thickness ◽  
High Angle ◽  
Dimensional Reconstruction ◽  
Fine Structures ◽  
Sectioned Tissue ◽  
Made In

A method of securing serial sections for electron microscopy is described. Serial sections present certain anomalies of interpretation of a nature such that a complete and detailed three-dimensional reconstruction of the sectioned tissue cannot be made. These anomalies are discussed, as well as those which have been encountered in the interpretation of single sections. Observations of the following kinds have been made in an attempt to elucidate the interpretation of single and serial sections: differing methods of mounting adjacent sections, observation of the same section by high-angle stereoscopy, and examination of sections which have been shadowed prior to and subsequent to electron microscopy. It is found that the appearance of sections is independent of the choice of side to be placed against the formvar films. Stereoscopy shows that the appearance of fine structures is strongly dependent upon the direction of the penetrating electron beam with respect to the plane of the structures. Stereoscopy, combined with shadowing, shows quantitatively that extensive sublimation of polymer occurs upon normal exposure in the electron microscope. Observation of sections shadowed prior to electron microscopy indicates that varying amounts of material are removed between sections by the action of microtomy; i.e., it is probable that the sum of the thicknesses of several serial sections is considerably less than the total thickness of material removed from the block. It is believed that this effect, combined with the effect of sublimation, aids in explaining the failure of adjacent sections to exhibit continuity in their detailed structures.

scholarly journals Interpolar spindle microtubules in PTK cells.

1993 ◽  
Vol 123 (6) ◽  
pp. 1475-1489 ◽  
Author(s):  
D N Mastronarde ◽  
K L McDonald ◽  
R Ding ◽  
J R McIntosh
Keyword(s):  
Electron Microscopy ◽  
Cross Section ◽  
Specific Interaction ◽  
Spindle Pole ◽  
Serial Sections ◽  
Spindle Poles ◽  
Late Anaphase ◽  
Computer Image Processing ◽  
Spindle Microtubules ◽  
Anaphase B

Spindle microtubules (MTs) in PtK1 cells, fixed at stages from metaphase to telophase, have been reconstructed using serial sections, electron microscopy, and computer image processing. We have studied the class of MTs that form an interdigitating system connecting the two spindle poles (interpolar MTs or ipMTs) and their relationship to the spindle MTs that attach to kinetochores (kMTs). Viewed in cross section, the ipMTs cluster with antiparallel near neighbors throughout mitosis; this bundling becomes much more pronounced as anaphase proceeds. While the minus ends of most kMTs are near the poles, those of the ipMTs are spread over half of the spindle length, with at least 50% lying > 1.5 microns from the poles. Longitudinal views of the ipMT bundles demonstrate a major rearrangement of their plus ends between mid- and late anaphase B. However, the minus ends of these MTs do not move appreciably farther from the spindle midplane, suggesting that sliding of these MTs contributes little to anaphase B. The minus ends of ipMTs are markedly clustered in the bundles of kMTs throughout anaphase A. These ends lie close to kMTs much more frequently than would be expected by chance, suggesting a specific interaction. As sister kinetochores separate and kMTs shorten, the minus ends of the kMTs remain associated with the spindle poles, but the minus ends of many ipMTs are released from the kMT bundles, allowing the spindle pole and the kMTs to move away from the ipMTs as the spindle elongates.

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