A new inexpensive specimen carrier for freeze‐fracturing

Müller, Werner; Pscheid, Peter

doi:10.1111/j.1365-2818.1979.tb00159.x

Cited by 8 publications

(5 citation statements)

References 2 publications

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“…The stamping device produces hat-shaped sample-holders as proposed by Gulik-Krzywicki & Costello (1978). For an equivalent thickness, this shape presents a higher mechanical rigidity than the U shape proposed by Muller & Pscheid (1978). In the stamping device described here the mechanical tolerances are studied in order to provoke a cold-hammering of the cylindrical walls during the forming which increases their mechanical rigidity.…”

Section: Discussionmentioning

confidence: 96%

New instruments which facilitate rapid freezing at 83 K and 6 K

Escaig¹

1982

Journal of Microscopy

216

140

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SUMMARY We have developed three instruments to freeze different biological specimens for various purposes in the easiest and most economical way: a punching device, a rapid immersion device and a device to freeze the specimens against a copper block cooled by liquid helium. Cooling rate measurements are made by a thin flat thermocouple. Freezing against a copper block cooled down to 4 K gives cooling rates 3–5 times higher than freezing by immersion into propane at 85 K.

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Section: Discussionmentioning

confidence: 96%

New instruments which facilitate rapid freezing at 83 K and 6 K

Escaig¹

1982

Journal of Microscopy

216

140

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“…The samples were examined with a Philips EM 300 electron microscope, operating at 80 kV. For freeze-fracture experiments the vesicles were deposited on phosphor bronze specimen carriers, prepared by the method of Mueller and Pscheid [18] and immediately quenched in a slush of solid and liquid nitrogen. The samples were fractured at -160~ in a freeze-fracture device as described elsewhere [31].…”

Section: Methodsmentioning

confidence: 99%

Calcium-induced fusion of didodecylphosphate vesicles: The lamellar to hexagonal II (HII) phase transition

et al. 1987

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Electron microscopic techniques have been employed to investigate the ability of didodecylphosphate vesicles (diameter approx. 900 A) to fuse in the presence of Ca2+. As revealed by negative staining, Ca2+ induces extensive fusion and large vesicles with diameters up to 7000 A are formed. In a process secondary to fusion, the fused vesicles display a tendency to flatten and are subsequently transformed into extended tubular structures. Freeze-fracture electron microscopy, in conjunction with 31P NMR and selected area electron diffraction measurements indicate that the tubes are packed in a hexagonal (HII) array and that the amphiphiles are converted from the lamellar to the hexagonal HII phase. The relationship between membrane fusion and the lamellar-to-hexagonal phase transition is discussed in terms of formation and abundance of transiently stable inverted micellar intermediates at contact regions between two interacting membranes. A model for the conversion of the (vesicular) lamellar into the (tubular) hexagonal HII phase is presented, taking into account the molecular shape of the amphiphile. The relevance of using simple synthetic amphiphiles as models for phospholipid bilayers and complex biomembrane behavior is briefly discussed.

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“…The cells appear to be kept by microvilli at a distance from the substratum in all figures. x 18,000. advantage of using very thin free-fracture holders, mainly from copper (Pfenninger & Rinderer, 1975;Moor et al, 1976;Gulik-Krzywicki & Costello, 1978;Muller & Pscheid, 1978). (4) Furthermore, a cryogen jet was already used by Riehle (1968), Moor et al (1976, Burstein & Maurice (1978).…”

Section: Comments On New Methodsmentioning

confidence: 99%

“…Umrath, 1977). In a separate communication (Muller & Pscheid, 1978) it was already shown that object holders should be of Table 1. Cooling rates [K s-l]* (determined with thermocouples in the range between 293 K and 193 Kt).…”

Section: Preparation Of Cell Cultures F O R Fast Freezingmentioning

confidence: 99%

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Cryofixation of monolayer cell cultures for freeze‐fracturing without chemical pre‐treatments

Pscheid

Schudt

Plattner

1981

Journal of Microscopy

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A cryofixation method is presented which gives excellent ultrastructural preservation of monolayer cell cultures without any chemical pretreatments. Rat hepatocytes in primary culture were used in this study. The equipment needed is inexpensive and easy to manufacture. Cells are grown on a usual tissue culture support material (Thermanox plastic sheets). For cryofixation, samples are prepared essentially by a combined sandwich-cryogen-jet technique. 3 mm large discs are punched out and sandwiched with Cu-or Au-object holders of little mass; a 15 pm spacer is put in between. The viability of the cells is not impaired by the manipulations before freezing. The sandwich sample is quickly frozen by shooting a propane jet from a simple pressure chamber on to the metal object holder. The relevant parameters were optimized by parallel freeze-fracture analyses of 5" , , glycerol as a model system and by thermocouple measurements. Sandwich samples are then mounted in an appropriate double replication specimen table for further analysis by freeze-fracturing. It is possible to obtain a certain selectivity of the fracture plane with regard to apical, lateral or basal aspects of the cell layer. Alternatively, disc samples can be processed by chemical fixation methods (including freeze substitution to determine the freeze-fracture plane), since the support material Thermanox is insensitive to organic solvents and easy to cut. In each case the cells remain attached to their substratum throughout the whole procedure. Thus, the ultrastructural data can be directly correlated with parallel functional analyses obtained from the same cell cultures.

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A new inexpensive specimen carrier for freeze‐fracturing

Cited by 8 publications

References 2 publications

New instruments which facilitate rapid freezing at 83 K and 6 K

New instruments which facilitate rapid freezing at 83 K and 6 K

Calcium-induced fusion of didodecylphosphate vesicles: The lamellar to hexagonal II (HII) phase transition

Cryofixation of monolayer cell cultures for freeze‐fracturing without chemical pre‐treatments

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