Simultaneous Freeze-Etching of Eight Specimens

1969 ◽  
Vol 27 ◽  
pp. 398-399
Author(s):  
Russell L. Steere ◽  
Michael Moseley
Keyword(s):  
Freezing Point ◽  
Specimen Holder ◽  
Cold Stage ◽  
Freeze Etching

A specimen cap for the Denton Freeze-Etching Module has been modified (Fig. 1) to permit the easy manipulation and simultaneous freeze-etching of eight specimens. A slot has been cut in one side of the cap to permit the insertion of a brass washer and a thin copper retainer plate above the threads, but below the supporting top of the cap. The inside rims of the washer are elevated, and the end of each specimen holder flange is beveled down and out to the base to form a wedge which can be forced under the washer. Small caps are placed over the specimen in each holder. Specimens are frozen by rapid immersion in Freon 22. The assembled cap is then mounted on a stage, standing in liquid Freon near its freezing point and slightly loosened. The eight frozen specimens, with caps in place, are picked up individually with tweezers and wedged under the washer, lifting it in the process. These operations can be accomplished with cold stage and cap under a dissecting microscope, if desired.

New Dimensions in Freeze-Etching

1969 ◽  
Vol 27 ◽  
pp. 202-203 ◽  
Author(s):  
Russell L. Steere ◽  
Michael Moseley
Keyword(s):  
Fracture Surfaces ◽  
Aluminum Specimen ◽  
Specimen Holder ◽  
Freeze Etching ◽  
The Right

A redesigned specimen holder and cap have made possible the freeze-etching of both fracture surfaces of a frozen fractured specimen. In principal, the procedure involves freezing a specimen between two specimen holders (as shown in A, Fig. 1, and the left side of Fig. 2). The aluminum specimen holders and brass cap are constructed so that the upper specimen holder can be forced loose, turned over, and pressed down firmly against the specimen stage to a position represented by B, Fig. 1, and the right side of Fig. 2.

Electron Microscopic Observation of Biological Specimens in Their Native State by Employing Cryogenic Techniques

1972 ◽  
Vol 30 ◽  
pp. 410-411 ◽  
Author(s):  
Tokio Nei ◽  
Haruo Yotsumoto ◽  
Yoichi Hasegawa ◽  
Yuji Nagasawa
Keyword(s):  
Electron Microscopic Observation ◽  
Surface Structures ◽  
Specimen Preparation ◽  
Native State ◽  
Electron Microscopic ◽  
Biological Specimen ◽  
Specimen Holder ◽  
Cold Stage ◽  
Preparation Techniques ◽  
Scanning Electron

In order to observe biological specimens in their native state, that is, still containing their water content, various methods of specimen preparation have been used, the principal two of which are the chamber method and the freeze method.Using its recently developed cold stage for installation in the pre-evacuation chamber of a scanning electron microscope, we have succeeded in directly observing a biological specimen in its frozen state without the need for such conventional specimen preparation techniques as drying and metallic vacuum evaporation. (Echlin, too, has reported on the observation of surface structures using the same freeze method.)In the experiment referred to herein, a small sliced specimen was place in the specimen holder. After it was rapidly frozen by freon cooled with liquid nitrogen, it was inserted into the cold stage of the specimen chamber.

HVEM Cryomicroscopy of Frozen Hydrated PTK1 Cells

1990 ◽  
Vol 48 (1) ◽  
pp. 500-501
Author(s):  
E.T. O’Toole ◽  
G.P. Wray ◽  
J.R. Kremer ◽  
J.R. Mcintosh
Keyword(s):  
Liquid Nitrogen ◽  
Phase Contrast ◽  
Culture Medium ◽  
Low Dose ◽  
Ammonium Acetate ◽  
Freezing Point ◽  
Voltage Electron ◽  
Cold Stage ◽  
Humidity Chamber ◽  
Coated Carbon

Ultrarapid freezing and cryomicroscopy of frozen hydrated material makes it possible to visualize samples that have never been exposed to chemical fixatives, dehydration, or stains. In principle, freezing and cryoimaging methods avoid artifacts associated with chemical fixation and processing and allow one to visualize the specimen in a condition that is close to its native state. Here we describe a way to use a high voltage electron microscope (HVEM) for the cryoimaging of frozen hydrated PTK1 cells.PTK1 cells were cultured on formvar-coated, carbon stabilized gold grids. After three days in culture, the grids were removed from the culture medium and blotted in a humidity chamber at 35° C. In some instances, the grids were rinsed briefly in 0.16 M ammonium acetate buffer (pH 7.2) prior to blotting. After blotting, the grids were transferred to a plunging apparatus and plunged into liquid ethane held directly above its freezing point. The plunging apparatus consists of a vertical slide rail that guides the fall of a mounted pair of forceps that clamp the specimen. The forceps are surrounded by a plexiglass humidity chamber mounted over a dewar of liquid nitrogen containing an ethane chamber. After freezing, the samples were transferred to liquid nitrogen and viewed in a JEOL JEM 1000 equipped with a top entry cold stage designed and built by Mr. George Wray (Univ. Colorado). The samples were routinely exposed to electron doses of 1 e/Å2/sec, and viewed at a temperature of −150° C. A GATAN video system was used to enhance contrast and to estimate the correct amount of underfocus needed to obtain phase contrast at various magnifications. Low dose micrographs were taken using two second exposures of Kodak 4463 film. The state of the solid water in the specimen was determined by diffraction using a 30/μm field limiting aperture and a camera length of 1 meter.

The plasma membrane of pityrosporum has natural markers to show how the cell grows

1978 ◽  
Vol 36 (2) ◽  
pp. 404-405
Author(s):  
Kanji Takeo ◽  
Ei-Ichi Nakai
Keyword(s):  
Plasma Membrane ◽  
Spiral Groove ◽  
Left Handedness ◽  
Left Handed ◽  
Specimen Holder ◽  
Inner Surface ◽  
Freeze Etching ◽  
Pityrosporum Orbiculare ◽  
Tinea Versicolor ◽  
Spiral Grooves

Pityrosporum is a lipophilic yeast containing the causative agent of tinea versicolor, and has spiral grooves on the inner surface of the cell wall and the plasma membrane. Detailed studies on the plasma membrane of this organism revealed the existence of asymmetry around the plasma membrane, peculiar mode of the growth of this organism, mode of spiral groove formation, and a possible mechanism of groove formation. One strain each of Pityrosporum orbiculare and P. pachydermatis and ten strains of P. ovale were grown on a potato yeast extract agar, supplemented with 1% olive oil at 27-37°C for 1-20 days. Cultures were directly transferred to the specimen holder of the freeze-etching apparatus without no pretreatment. 40% glycerol was added before cooling.All the strains tested of the genus Pityrosporum had only left-handed spiral grooves of the plasma membrane. Left-handedness of the spiral grooves of Pityrosporum was confirmed by scanning electron microscopy which occasionally revealed left-handed spiral grooves on the outer surface of the wall.

scholarly journals Hexagonal and Cubic Ice at Low Temperatures

1968 ◽  
Vol 7 (49) ◽  
pp. 95-108 ◽  
Author(s):  
Motoi Kumai
Keyword(s):  
Coefficient Of Thermal Expansion ◽  
Gold Foil ◽  
Ice Crystal ◽  
Structural Form ◽  
Lattice Constants ◽  
Specimen Holder ◽  
Hexagonal Form ◽  
Cold Stage ◽  
Cubic Forms ◽  
Collodion Film

AbstractThe formation of hexagonal and cubic forms of ice was studied by the use of a cold stage in an electron microscope within the temperature range −90° to −180° C. Ice crystal specimens were made on cold substrates, i.e. a collodion film, gold foil, or copper grid on the specimen holder of the cold stage. The hexagonal form of ice formed on the cold substrates at temperatures from−90° to−100° C. At −100° to −130° C, both hexagonal and cubic forms of ice were detected. From −130° to −160° C only cubic ice was found. At temperatures below −160° C, minute crystals of cubic ice were detected. No transformation of the structural form of ice from hexagonal to cubic or from cubic to hexagonal occurred when the temperature of the specimens was varied in the range −90° to −160° C. The lattice constants of hexagonal and cubic ice, and the coefficient of thermal expansion of ice were calculated from the experimental results.

scholarly journals The Location of Impurities in Antarctic Ice

1988 ◽  
Vol 11 ◽  
pp. 194-197 ◽  
Author(s):  
E.W. Wolff ◽  
R. Mulvaney ◽  
K. Oates
Keyword(s):  
Freezing Point ◽  
Ice Sheet ◽  
Chemical Substance ◽  
Triple Junctions ◽  
Sea Salts ◽  
X Ray ◽  
Cold Stage ◽  
Micro Analysis ◽  
The Antarctic ◽  
Scanning Electron

Analysis of an ice sample with an estimated age of 125 years, from the Antarctic Peninsula, using a scanning electron microscope with a cold stage and an X-ray micro-analysis facility, shows that H2SO4 occurs mainly at triple junctions. Sea salts show no such localization. The different behaviour may be due to the freezing-point behaviour of each chemical substance, and to the effect this has both in the atmosphere and during recrystallization in the ice sheet. If this finding applies generally to other parts of the Antarctic ice sheet, it has major implications for many of the physical properties of Antarctic ice. In particular, it leads to a better understanding of the d.c. electrical conductivity of such ice.

Cryoultramicrotomy of plant protoplasts

1978 ◽  
Vol 36 (2) ◽  
pp. 36-37
Author(s):  
Francis A. Williamson
Keyword(s):  
Cell Wall ◽  
Freezing Point ◽  
Lectin Binding ◽  
Enzymatic Digestion ◽  
Allium Porrum ◽  
General Description ◽  
Plant Protoplasts ◽  
Sodium Cacodylate ◽  
Concentrated Suspension ◽  
Specimen Holder

IntroductionNaked plant cells (protoplasts) have been used for the study of lectin binding to the plasma membrane, and of the initial stages of cell wall formation (2,3 and refs, therein). In this paper, I describe methods for the cryoultramicrotomy of plant protoplasts and give a general description of their ultrastructure. In addition, labelled lectins have been applied to cryo-sections to locate intracellular binding sites, and the superior resolution of the negatively stained cryo-sections has been applied to a preliminary study of cell wall fibril formation.Materials and MethodsProtoplasts were prepared from meristematic leaves of leek (Allium porrum) by enzymatic digestion (3). After washing, the cells were fixed in 1% glutaraldehyde in 0.55 M sorbitol and 1 mM CaCl2 buffered at pH 7.2 with 10 mM sodium cacodylate or HEPES. They were then washed (16 h) in 100 mM sodium cacodylate pH 7.2 and infused with 1 M sucrose in the same buffer for 3 h. A very concentrated suspension of the cells was mounted on a specimen holder and frozen in nitrogen slush (liquid nitrogen at its freezing point).

A New Model of Freeze Etching Apparatus

1972 ◽  
Vol 30 ◽  
pp. 294-294
Author(s):  
Hiroshi Akahori ◽  
Sadahiko Okamura ◽  
Mitsugu Nishiura ◽  
Kenzo Uehira
Keyword(s):  
Freeze Fracture ◽  
Small Hole ◽  
Small Specimen ◽  
Block Method ◽  
New Model ◽  
Specimen Holder ◽  
Lower Block ◽  
Freeze Etching ◽  
Cold Metal ◽  
Metal Block

A new model of freeze etching apparatus has been designed which enabled us to make both freeze fracture and freeze etching replicas of the biological specimens by easy simple procedures.This model is based on the principle of Bullivant's cold metal block method. It is composed of two-wedge-like brass blocks which are combined in a shape of rectangular prism with'a base 5.5 cm x 6. 0 era and a height of 7.5 cm.The lower block has a specimen stage to which a small specimen holder made of copper can be fixed. The specimen is put in a small hole of the specimen holder, and this holder is cooled in Freon 12.

A cryo-SEM technique developed and applied to surfactant liposomes

1993 ◽  
Vol 51 ◽  
pp. 284-285
Author(s):  
Kiran Bhadriraju ◽  
Jayesh Bellare
Keyword(s):  
Sample Preparation ◽  
Liquid Nitrogen ◽  
Freeze Drying ◽  
Freezing Point ◽  
Freeze Fracture ◽  
Depth Of Field ◽  
Particle Sizes ◽  
Copper Sheet ◽  
Freeze Etching ◽  
Liquid Nitrogen Bath

Freeze-fracture replication TEM and Cryo-TEM are developed techniques for studying surfactant dispersions. Application of freeze-fracture cryo-SEM with direct imaging to such systems has the advantages of observing a greater range of particle sizes, large depth of field implying larger tilts together with rotation, and freeze-etching/freeze drying the sample while imaging it. A procedure for cryo-SEM of liquid colloids, which uses a simple sample preparation setup, and its results for liposomal dispersions, are described here.Samples are plunge-frozen by a freezing device (Fig.l) made from a standard desoldering tool (Fig.2) used as a plunging unit. Fracture plates (Fig.3) are made from 0.1 mm thin copper sheet made adhesive to the liquid by 400 mesh TEM grids that are bent over the two edges of the plates and stuck on the non-sample side with a rubber adhesive. The sample is sandwiched between a pair of fracture plates (Fig.3) and plunged into liquid Freon-22 kept at its freezing point (-160°C) in an electrically heated cup (Fig.l) cooled by a liquid liquid nitrogen bath.

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