Comments on ’’Reply to comments on microscope cold stage for the direct observation of vacuum etching and freeze drying’’

The availability of equipment which enables frozen-hydrated material to be viewed over prolonged periods in the SEM has greatly extended the versatility of the instrument and permitted more precise interpretation of the data obtained from it. Artifacts associated with chemical fixation are avoided and soluble components are retained which might otherwise be removed by solvents used for dehydration or critical point drying. In addition the shrinkage which frequently occurs during freeze drying is avoided. Two further advantages of cryo preservation can be invaluable: rapid cooling of motile specimens instantly arrests movement and the immobilization of diffusible compounds and ions enables X-ray microanalysis to be performed with confidence.The speed with which specimens are cooled is not critical if only surface features are to be studied. However, the examination of internal structure demands that ice crystal growth be minimised. Devices are available to maximise cooling rates using a variety of cryogens and specimen transfer to the SEM cold stage is achieved simply and effectively.

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Ca-Histochemistry in slime mold plasmodia

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100081516 ◽

1978 ◽

Vol 36 (2) ◽

pp. 130-131

Author(s):

Ulrich Dierkes

Keyword(s):

Physarum Polycephalum ◽

Carbon Coating ◽

Freeze Drying ◽

Freeze Fracture ◽

Freeze Substitution ◽

Uranyl Acetate ◽

Slime Mold ◽

Staining Procedure ◽

Protoplasmic Streaming ◽

Intracellular Ca

Calcium is supposed to play an important role in the control of protoplasmic streaming in slime mold plasmodia. The motive force for protoplasmic streaming is generated by the interaction of actin and myosin. This contraction is supposed to be controlled by intracellular Ca-fluxes similar to the triggering system in skeleton muscle. The histochemical localisation of calcium however is problematic because of the possible diffusion artifacts especially in aquous media.To evaluate this problem calcium localisation was studied in small pieces of shock frozen (liquid propane at -189°C) plasmodial strands of Physarum polycephalum, which were further processed with 3 different methods: 1) freeze substitution in ethanol at -75°C, staining in 100% ethanol with 1% uranyl acetate, and embedding in styrene-methacrylate. For comparison the staining procedure was omitted in some preparations. 2)Freeze drying at about -95°C, followed by immersion with 100% ethanol containing 1% uranyl acetate, and embedding. 3) Freeze fracture, carbon coating and SEM investigation at temperatures below -100° C.

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The Critical Point Drying Method Applied to Scanning Electron Microscopy of L-929 Cells

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100068382 ◽

1970 ◽

Vol 28 ◽

pp. 278-279

Author(s):

Charles TurnbiLL ◽

Delbert E. Philpott

Keyword(s):

Electron Microscopy ◽

Carbon Dioxide ◽

Surface Tension ◽

Critical Point ◽

Freeze Drying ◽

Attractive Alternative ◽

Crystal Formation ◽

Critical Point Drying ◽

Time Required ◽

Scanning Electron

The advent of the scanning electron microscope (SCEM) has renewed interest in preparing specimens by avoiding the forces of surface tension. The present method of freeze drying by Boyde and Barger (1969) and Small and Marszalek (1969) does prevent surface tension but ice crystal formation and time required for pumping out the specimen to dryness has discouraged us. We believe an attractive alternative to freeze drying is the critical point method originated by Anderson (1951; for electron microscopy. He avoided surface tension effects during drying by first exchanging the specimen water with alcohol, amy L acetate and then with carbon dioxide. He then selected a specific temperature (36.5°C) and pressure (72 Atm.) at which carbon dioxide would pass from the liquid to the gaseous phase without the effect of surface tension This combination of temperature and, pressure is known as the "critical point" of the Liquid.

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Direct Observation of Heat Exchanger Deposits by Cross Sectioning

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100061835 ◽

1967 ◽

Vol 25 ◽

pp. 364-365

Author(s):

R. W. Anderson ◽

D. L. Senecal

Keyword(s):

Electron Microscopy ◽

Transmission Electron Microscopy ◽

Heat Exchanger ◽

Direct Observation ◽

Cross Sections ◽

Preparation Method ◽

Specimen Preparation ◽

Flowing Water ◽

Transmission Electron ◽

Deposit Layer

A problem was presented to observe the packing densities of deposits of sub-micron corrosion product particles. The deposits were 5-100 mils thick and had formed on the inside surfaces of 3/8 inch diameter Zircaloy-2 heat exchanger tubes. The particles were iron oxides deposited from flowing water and consequently were only weakly bonded. Particular care was required during handling to preserve the original formations of the deposits. The specimen preparation method described below allowed direct observation of cross sections of the deposit layers by transmission electron microscopy.The specimens were short sections of the tubes (about 3 inches long) that were carefully cut from the systems. The insides of the tube sections were first coated with a thin layer of a fluid epoxy resin by dipping. This coating served to impregnate the deposit layer as well as to protect the layer if subsequent handling were required.

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Extended Preservation of Iron for Transmission

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100061781 ◽

1967 ◽

Vol 25 ◽

pp. 354-355

Author(s):

E. P. Abrahamson II ◽

M. W. Dumais

Keyword(s):

Acid Solution ◽

Perchloric Acid ◽

Microscopy Study ◽

Perchloric Acid Solution ◽

Transmission Microscopy ◽

Iron Base ◽

Cold Stage

In a transmission microscopy study of iron and dilute iron base alloys, it was determined that it is possible to preserve specimens for extended periods of time. Our specimens were prepunched from 5 to 8 mil sheet to microscope size and annealed for several hours at 700°C. They were then thinned in a glacial acetic-12 percent perchloric acid solution using 10 volts and 20 milliamperes, at a temperature of 8 to 14°C.It was noted that by the use of a cold stage, the same specimen can be observed for periods up to one week without excess contamination. When removal of the specimen from the column becomes necessary, it was observed that a specimen may be kept for later observation in 1,2 dichloroethene or methanol for periods in excess of two weeks.

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SEM Cold Stage Techniques for the Observation of Vegetative and Floral Apices of Oat (Avena sativa L.) Plants

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100080377 ◽

1977 ◽

Vol 35 ◽

pp. 590-591

Author(s):

B. K. Kirchoff ◽

L.F. Allard ◽

W.C. Bigelow

Keyword(s):

Critical Point ◽

Avena Sativa ◽

Substantial Improvement ◽

Fresh Tissue ◽

Cold Stage ◽

Critical Point Drying ◽

Almost All ◽

Cell Collapse ◽

Conductive Paint ◽

Carbon Based

In attempting to use the SEM to investigate the transition from the vegetative to the floral state in oat (Avena sativa L.) it was discovered that the procedures of fixation and critical point drying (CPD), and fresh tissue examination of the specimens gave unsatisfactory results. In most cases, by using these techniques, cells of the tissue were collapsed or otherwise visibly distorted. Figure 1 shows the results of fixation with 4.5% formaldehyde-gluteraldehyde followed by CPD. Almost all cellular detail has been obscured by the resulting shrinkage distortions. The larger cracks seen on the left of the picture may be due to dissection damage, rather than CPD. The results of observation of fresh tissue are seen in Fig. 2. Although there is a substantial improvement over CPD, some cell collapse still occurs.Due to these difficulties, it was decided to experiment with cold stage techniques. The specimens to be observed were dissected out and attached to the sample stub using a carbon based conductive paint in acetone.

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Application of a Simple Cold Stub to the Direct Observation of Frozen Hydrated Sand

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100071508 ◽

1973 ◽

Vol 31 ◽

pp. 212-213

Author(s):

John M. Wehrung ◽

Richard J. Harniman

Keyword(s):

United States ◽

Surface Water ◽

Direct Observation ◽

Controlled Study ◽

Clay Particles ◽

Organic Debris ◽

Rock Formations ◽

Water Tables ◽

Permeable Rock ◽

Natural Means

Water tables in aquifer regions of the southwest United States are dropping off at a rate which is greater than can be replaced by natural means. It is estimated that by 1985 wells will run dry in this region unless adequate artificial recharging can be accomplished. Recharging with surface water is limited by the plugging of permeable rock formations underground by clay particles and organic debris.A controlled study was initiated in which sand grains were used as the rock formation and water with known clay concentrations as the recharge media. The plugging mechanism was investigated by direct observation in the SEM of frozen hydrated sand samples from selected depths.

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