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  • 1
    ISSN: 0886-1544
    Keywords: marginal band ; spectrin ; vimentin ; surface-associated cytoskeleton ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Platinum-carbon (Pt-C) replicas of freeze-dried erythrocyte cytoskeletons of the toad, Bufo marinus, were prepared using a modified Balzers 300 system. Examination in stereo of replicas of the microtubule-containing marginal band revealed filaments projecting from the microtubule walls to form links between adjacent microtubules. These cross-bridging proteins may bundle the microtubules into the configuration of the marginal band (MB) and may also serve to stabilize the structure. The MB appears to have linkages to components of the surface-associated cytoskeleton (SAC). The SAC forms a continuous matrix that spreads across the upper and lower surfaces of the cell adjacent to the plasma membrane and extends around the outer perimeter of the MB. Thus, the SAC encapsulates the MB and the central nucleus. After lysis, the elements of the cytoskeleton remain in a configuration similar to that found in the whole cell. Spectrin (fodrin) and actin were identified by immunofluorescence in the region of the SAC. When labeled with antibodies specific for vimentin and synemin, a network of intermediate filaments can be detected in the region between the nucleus and the MB. These vimentin filaments are also enclosed within the SAC and appear in Pt-C replicas to emerge from the area of the nuclear envelope. As the filaments extend toward the periphery of the cell, they form attachments to the SAC. Attachments of intermediate filaments to both the nucleus and the SAC thus appear to anchor the nucleus in its central position within the cytoskeleton.
    Additional Material: 11 Ill.
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  • 2
    ISSN: 0741-0581
    Keywords: TEM ; Parallax equation ; Freeze-etch ; Pt-C replication ; Hydrated spermidine-condensed DNA toruses ; Stereoheight measurements ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: Stereoimaging of hydrated single complex macromolecules requires thin freeze-etch platinum-carbon replicas (≤200 Å) and that the transmission electron microscope (TEM) be equipped with a tilt-rotation eucentric goniometer stage. The original parallax equation is an accurate approximation for high-magnification work, micrographs (105 ×) being less than 0.3% in error. In addition, we have derived formulas for high-magnification work to measure heights, lateral distances, and the object tilt angle for an object not lying flat on the film surface. The accuracy of the height measurements is evaluated on spermidine-condensed DNA toruses. By using the maximum error equation derived from the original parallax equation, we discuss methods to improve the height measurement precision (95% fractile) to the 5-10 Å range.
    Additional Material: 6 Ill.
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  • 3
    ISSN: 0741-0581
    Keywords: Balzer's freeze-etch machine cold stage modification ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: The cold stage specimen surface in our Balzer's 300 Freeze-etch instrument, averages 29-43°C warmer than the reading on the GA-1 stage temperature control unit. By introduction of Indium foil seals above and below the stainless steel plug in the stage tower, the specimen cold stage surface temperatures were still 9-10°C warmer than the GA-1 reading. After replacing the stainless steel plug with a copper plug and using Indium seals, the cold stage specimen surface was brought within 2.6°C of the GA-1 temperature reading. In addition, the copper plug, Indium seal modified stage cools about two times faster in going from -100°C to -150°C than the normal stainless steel stage modified with Indium seals.
    Additional Material: 4 Ill.
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  • 4
    ISSN: 1059-910X
    Keywords: Lactose repressor ; Mitochondrial ATPase ; F1 ; Freeze-etching method ; 45° unidirectional Pt-C replication of globular proteins ; 45° and vertical replication of gold ball standards on indirect carbon films ; Transmission electron microscopy ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: The real size of platinum-carbon (Pt-C) replicated particles is not directly equivalent to either its metal-coated diameter or its shadow width. This paper describes two indirect methods, shadow widths and coated particle diameters, for determining a particle's actual size beneath a Pt-C replication film. Both produce equivalent measurements using the same standardized conditions: 2.3 nm Pt-C films deposited at a 45° angle on an ≈ -100°C surface in a 10 -6 torr vacuum. For the first method, gold balls nucleated in a partial pressure of helium and deposited on flat indirect carbon films (root mean square roughness of 0.8 nm) on 400 mesh grids were used as test particles for calibrating shadow widths as a function of particle size. The gold ball test specimens were replicated, and a distribution of Pt-C shadow widths orthogonal to the Pt-C deposition direction was measured and averaged for gold balls 1.5 ± 0.25 nm, 2.0 ± 0.25 nm, etc. The diameter of each gold ball was measured within the Pt-C film along with its shadow width because the Pt-C did not obscure or adhere well to the gold. The shadow width distributions for each gold size do not differ significantly from log normal. Two proteins, the lactose repressor and the mitochondrial ATPase, F1, were also used as replication test objects. Negative staining of both proteins was conducted to measure their average diameters. In the second method, a distribution of Pt-C-coated lac repressor diameters perpendicular to the shadow direction was measured. The Pt-C film thickness measured on the quartz crystal monitor was subtracted from the average metal-coated protein diameter to obtain the lac repressor's diameter. The Pt-C-coated particle diameter distributions also did not differ significantly from log normal. While doing this work it was discovered that outgassing the Pt-C electron gun greatly affected Pt-C film granularity: 19 sec produced a high contrast, granular Pt-C film, whereas 120 sec yielded a low contrast, less granular Pt-C film. Both gold balls and protein particles were subjected in separate experiments to either 19 or 120 sec of outgassing of the Pt-C gun prior to Pt-C replication. Outgassing had a profound effect on the average size of the Pt-C shadow widths on both gold and protein particles. The Pt-C gun outgassing procedure also determined the smallest replicated particle that could be resolved. The frequency of some smaller gold ball sizes detected after replication was reduced disproportionately with 19 sec vs. 120 sec outgassing. However, Pt-C gun outgassing did not affect the average measured diameter of the Pt-C-coated protein particles. The “geometric assumption” that each metal-coated particle creates a shadow width the same size as the metal-coated particle diameter was tested using a globular protein. Pt-C replication of protein particles at a 45° and 20° angle could not confirm the geometric assumption because an average shadow width was always significantly larger than its average Pt-C-coated particle diameter. A model for how the large shadow widths are formed is presented. Gold balls were also replicated at a 45° angle with current high resolution conditions at a substrate temperature of -185°C, and the results of these replicas were compared to the results reported here at ˜-100°C. © 1995 Wiley-Liss, Inc.
    Additional Material: 18 Ill.
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  • 5
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Microscopy Research and Technique 28 (1994), S. 13-28 
    ISSN: 1059-910X
    Keywords: Collagen IV ; Laminin ; Human amnion ; Mouse EHS tumor ; Bovine lens capsule ; stroma ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: High angle platinum/carbon (Pt/C) replication has proved to be a valuable tool in analyzing basement membrane structure in human amnion, bovine lens capsule, and the Engelbreth-Holm-Swarm (EHS) tumor. High resolution replicas for transmission electron microscopy (TEM) have been achieved by depositing 1.0 ± 0.1 nm thick Pt/C films backed with rotary deposited 12.5 ± 2.5 nm thick carbon films. The basement membrane collagen IV network was observed to consist of fine branching filaments containing globular domains intrinsic to the filaments. A second quasi-regular network is formed by laminin. Unidirectional 45° angle Pt/C replication was used for most of this work. The merits and deficiencies of unidirectional vertical replication (80° angle), unidirectional 45° angle, and 20° low angle rotary replication are discussed. Vertical replication produces the highest resolution replicas and has the potential for revealing the overall pattern of basement membrane structural assembly if basement membrane preparations freeze-dried in low salt can faithfully maintain their in vivo structure. © 1994 Wiley-Liss, Inc.
    Additional Material: 14 Ill.
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  • 6
    ISSN: 0741-0581
    Keywords: Sol gels ; DNA helix ; Pectin ; Immobilon ; Polyvinylidene fluoride ; Polymethacrylate ; Stainless steel ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: Single molecule resolution in beam-sensitive, uncoated, noncrystalline materials has heretofore not been possible except in thin (≤150 Å) platinum-carbon (Pt-C) replicas, which are resistant to electron beam destruction. Previously, the granularity of metal film replicas limited their resolution to ≥20 Å. This paper demonstrates that Pt-C film granularity and resolution are a function of the method of replication and other controllable factors. Low-angle 20° rotary, 45° unidirectional, and vertical 9.7 ± 1 Å Pt-C films deposited on mica under the same conditions were compared. Vertical replication had a 5 Å granularity, the highest resolution, and evenly coated the whole surface. A 45° replication had a 9.5 Å granularity, a slightly poorer resolution, and a discontinuous surface coating. The use of 20° rotary replication proved to be unsuitable for high-resolution imaging, with 20-25 Å granularity and resolution two to three times poorer. Vertical and 45° Pt-C replicas can visualize the deep-etched DNA helix and the 13.3 Å 32 helix of pectin in a gel. The DNA double helix, the complex structures of sol-gel glasses, Immobilon filters (polyvinylidene fluoride), a polymethacrylate plastic, the metal oxide surfaces of 440c stainless steel, and aluminum are illustrated. This high-resolution vertical Pt-C replica technique can image in the context of solutions, gels, or solids, single molecular chains 3-7 Å wide, their associations, and their conformation. Included in the present article are first time descriptions for removing replicas from metals and plastics and for making high-magnification photographic prints of normal contrast using a reversal rephotographic process.
    Additional Material: 16 Ill.
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  • 7
    ISSN: 0006-3525
    Keywords: Chemistry ; Polymer and Materials Science
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: The solid state secondary structure of myoglobin, RNase A, concanavalin A (Con A), poly(L-lysine), and two linear heterooligomeric peptides were examined by both far-uv CD spectroscopy1 and by ir spectroscopy. The proteins associated from water solution on glass and mica surfaces into noncrystalline, amorphous films, as judged by transmission electron microscopy of carbon-platinum replicas of surface and cross-fractured layer. The association into the solid state induced insignificant changes in the amide CD spectra of all α-helical myoglobin, decreased the molar ellipticity of the α/β RNase A, and increased the molar ellipticity of all-β Con A with no change in the positions of the bands' maxima. High-temperature exposure of the films induced permanent changes in the conformation of all proteins, resulting in less α-helix and more β-sheet structure. The results suggest that the protein α-helices are less stable in films and that the secondary structure may rearrange into β-sheets at high temperature. Two heterooligomeric peptides and poly (L-lysine), all in solution at neutral pH with “random coil” conformation, formed films with variable degrees of their secondary structure in β-sheets or β-turns. The result corresponded to the protein-derived Chou-Fasman amino acid propensities, and depended on both temperature and solvent used. The ir and CD spectra correlations of the peptides in the solid state indicate that the CD spectrum of a “random” structure in films differs from random coil in solution. Formic acid treatment transformed the secondary structure of the protein and peptide films into a stable α-helix or β-sheet conformations. The results indicate that the proteins aggregate into a noncrystalline, glass-like state with preserved secondary structure. The solid state secondary structure may undergo further irreversible transformations induced by heat or solvent. © 1993 John Wiley & Sons, Inc.
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