TEM‐, SEM‐ and STEM‐studies of sputter‐coated cytoskeletons

Lindroth, Margaretha; Bell, Paul B.; Fredriksson, Bengt-Arne

doi:10.1002/sca.4950090202

Cited by 8 publications

(17 citation statements)

References 11 publications

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“…Preservation of all the major cytoskeletal components is in line with previous studies in which 0.1-0.5% Triton X-100, together with buffered or stabilizing buffers (Pudney & Singer, 1979;Henderson & Weber, 1979;Heuser & Kirschner, 1980;Schliwa & Blerkom, 1981;Bell et al, 1987Bell et al, , 1989Lindroth et al, 1987), or replica technique combined with detergentextraction (Heuser & Kirschner, 1980), were used.…”

Section: Composition Of the Detergent-extracted Whole-mount Cytoskelesupporting

confidence: 51%

“…Whole-mount electron microscopy is a widely used technique to study three-dimensional relationships of various cell components, such as nuclei, the plasma membrane and cytoskeletal structures (Heuser & Kirschner, 1980;Schliwa & Blerkom, 1981;Lindroth et al, 1987;Bell et al, 1987Bell et al, , 1989. In order to make wholemount preparations, cultured cells are usually treated with a detergent in an appropriate buffer to extract the soluble cytoplasmic components (Osbom & Weber, 1977).…”

Section: Introductionmentioning

confidence: 99%

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?-Spectrin in detergent-extracted whole-mount cytoskeletons of chicken embryo heart fibroblasts

Sormunen

1993

Histochem J

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The distribution of alpha-spectrin, and its relation to other cytoskeletal structures and to the plasma membrane, was studied in detergent-extracted whole-mount cytoskeletons of chicken embryo heart fibroblasts by using immunogold labelling and electron microscopy (IEM). The cell surface was labelled with gold-conjugated wheat germ agglutinin (WGA-gold), microtubules with anti-tubulin antibodies, and spectrin by using antibodies raised to chicken erythrocyte alpha-spectrin. Additionally, the effect of fixation and drying on the labelling pattern was evaluated. In electron microscopy, a three-dimensional filamentous network was observed in detergent-extracted whole-mount preparations. Filaments of diameter 7-10 nm and 15 nm, microtubules of diameter 30 nm, and filament bundles (40-50 nm in diameter) were seen. In IEM, alpha-spectrin was seen on the surface of the cytoskeletal network, especially along the thick filament bundles. In some cells, a distinct membrane skeleton which was labelled with alpha-spectrin antibodies, was seen in close association with the cytoskeletal network. The cells which were labelled first with WGA-gold, and then permeabilized, fixed and labelled with alpha-spectrin, showed a co-localization of the WGA binding sites and alpha-spectrin along the surface of the filament bundles. Reversing the order of the staining, such that fixation was done before WGA labelling and permeabilization, led to a greatly diminished labelling for alpha-spectrin and less pronounced co-localization of spectrin and WGA. Comparison of the conventional critical point drying method with Peldri II, a novel drying agent, indicated a better stability of the cellular structures under the electron beam when Peldri II was used.(ABSTRACT TRUNCATED AT 250 WORDS)

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Section: Composition Of the Detergent-extracted Whole-mount Cytoskelesupporting

confidence: 51%

Section: Introductionmentioning

confidence: 99%

?-Spectrin in detergent-extracted whole-mount cytoskeletons of chicken embryo heart fibroblasts

Sormunen

1993

Histochem J

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“…Thus the platinum grain structure can be resolved in the microscope in both SEM and STEM modes, but it is not sharply focused. Tungsten coats of similar thickness do not show this grainy substructure in either scanning mode (Lindroth et al, 1987).…”

Section: Resultsmentioning

confidence: 97%

“…PBS-A: phosphate buffered saline without calcium or magnesium (137 mM NaC1, 3 mM KC1, 0.5 mM Na2HP04, 1.5 mM KH2P04, pH 7.4); PFA: paraformaldehyde, 20% aqueous stock solution diluted to 4% just prior to use. SB: microtubule-stabilizing buffer (1mM EGTA, 4% polyethylene glycol 6000,100 mM PIPES, pH 6.9) (Bell et al, 1987;Henderson and Weber, 1979). Thsb: 0.5% Triton in HSB.…”

Section: Reagentsmentioning

confidence: 99%

“…The monitor was calibrated by optical interferometry of platinum deposited onto a glass microscope slide. With the Microvac coater, sputtering of platinum or tungsten was carried out in pure argon at 5 x Tom, 1 kV, and 50 mA, as further described by Lindroth et al (1987). Some samples for TEM were subsequently coated with carbon by thermal evaporation in a JEOL vacuum evaporator.…”

Section: Sputter Coatingmentioning

confidence: 99%

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Use of sputter coating to prepare whole mounts of cytoskeletons for transmission and high‐resolution scanning and scanning transmission electron microscopy

Bell

Lindroth

Fredriksson

1987

J. Elec. Microsc. Tech.

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This paper describes the use of sputter coating to prepare detergent-extracted cytoskeletons for observation by scanning (SEM), scanning transmission (STEM), inverted contrast STEM, and transmission (TEM) electron microscopy. Sputtered coats of 1-2 nm of platinum or tungsten provide both an adequate secondary electron signal for SEM and good contrast for STEM and TEM. At the same time, the grain size of the coating is sufficiently fine to be just at (platinum) or below (tungsten) the limit of resolution for SEM and STEM. In TEM, the granular structure of platinum coats is resolved, and platinum decoration artifacts are observed on the surface of structures. The platinum is deposited as small islands with a periodic distribution that may reveal information about the underlying molecular structure. This method produces samples that are similar in appearance to replicas prepared by low-angle rotary shadowing with platinum and carbon. However, the sputter-coating method is easier to use; more widely available to investigators; and compatible with SEM, STEM, and TEM. It may also be combined with immunogold and other labeling methods. While TEM provides the highest resolution images of sputter-coated cytoskeletons, it also damages the specimens owing to heating in the beam. In SEM and STEM cytoskeletons are stable and the resolution is adequate to resolve individual microfilaments. The best single method for visualizing cytoskeletons is inverted contrast STEM, which images both the metal-coated cytoskeletal structures and electron-dense material within the nucleus and cytoplasm as white against a dark background. STEM and TEM were both suitable for visualizing colloidal gold particles in immunolabeled samples.

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Preservation and visualization of molecular structure in detergent‐extracted whole mounts of cultured cells

Lindroth

Bell

Fredriksson

et al. 1992

Microscopy Res & Technique

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Today's electron microscopes have a resolution sufficient to resolve supramolecular structures. However, the methods used to prepare biological samples for electron microscopy often limit our ability to achieve the resolution that is theoretically possible. We use whole mounts of detergent-extracted cells grown on Formvar-coated gold grids as a model system to evaluate various steps in the preparation of biological samples for high resolution scanning electron microscopy (SEM). Factors that are important in determining the structure and composition of detergent-extracted cells include the nature of the detergent and the composition of the extraction vehicle. Chelation of calcium is extremely important to stabilize and preserve the cytoskeletal filaments. We have also demonstrated both morphologically and by gel electrophoresis that treatment of cells with bifunctional protein crosslinkers before or during extraction with detergent can significantly enhance the preservation of both proteins and supramolecular structures. The methods used to dry samples are a major determinant of the quality of structural preservation. For cytoskeletons freeze-drying (FD) is superior to critical point-drying (CPD), one reason being that CPD samples have to be dehydrated, thereby causing more shrinkage as compared to FD samples. The high pressures to which samples are exposed during CPD may also cause increased shrinkage, and water contamination during CPD causes severe structural damage. We have obtained the best structural preservation of detergent-extracted and fixed cells by manually plunging them into liquid propane and drying over night in a freeze-dryer. The factor that most limits achievement of high resolution in SEM is the metal coat, which has to be very thin, uniform, and free of grain in order not to hide structures or to create artifactual ones. We have found that sputter-coating with 1-3 nm of tungsten (W) or niobium (Nb) gives extremely fine-grained films as well as satisfactory emission of secondary electrons. These samples can also be examined at high resolution by transmission electron microscopy (TEM) and scanning transmission electron microscopy (STEM). The best preservation and visualization of supramolecular structures have been obtained using cryosputtering, in which the samples are freeze-dried and then sputter-coated within the freeze-dryer while still frozen.

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TEM‐, SEM‐ and STEM‐studies of sputter‐coated cytoskeletons

Cited by 8 publications

References 11 publications

?-Spectrin in detergent-extracted whole-mount cytoskeletons of chicken embryo heart fibroblasts

?-Spectrin in detergent-extracted whole-mount cytoskeletons of chicken embryo heart fibroblasts

Use of sputter coating to prepare whole mounts of cytoskeletons for transmission and high‐resolution scanning and scanning transmission electron microscopy

Preservation and visualization of molecular structure in detergent‐extracted whole mounts of cultured cells

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