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

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.

Critical Point Drying of Freeze-Fractured Tissue For Scanning Electron Microscopy

1973 ◽  
Vol 31 ◽  
pp. 452-453 ◽  
Author(s):  
Walter J. Humphreys ◽  
Ben O. Spurlock ◽  
Janet S. Johnson
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Surface Tension ◽  
Plastic Deformation ◽  
Critical Point ◽  
Crystal Formation ◽  
Exposed Surface ◽  
Internal Surfaces ◽  
Critical Point Drying ◽  
Scanning Electron

The critical point method eliminates distortion that might have been caused by surface tension during drying of specimens being prepared for scanning electron microscopy. Such preparations can be made rapidly and routinely, and the structural fidelity of surfaces of cells and tissues thus prepared is excellent. But attempts to look inside the tissues or cells with the scanning electron microscope have been less successful. Critical point dried tissue can be broken or cut, and the exposed surface viewed. But plastic deformation of the exposed surface structures caused by the cutting or breaking severely limits the usefulness of this approach. Plastic deformation is minimized when tissue is freeze-fractured to expose internal surfaces, and the water (ice) can be sublimed away from small fragments of tissue without surface tension distortion by freeze-drying the specimen in a vacuum. But freeze damage, resulting mainly from ice crystal formation, is very difficult to avoid.

Preservation of Plant Cell Surfaces For Scanning Electron Microscopy

1973 ◽  
Vol 31 ◽  
pp. 472-473
Author(s):  
Linda M. Sicko ◽  
Thomas E. Jensen
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Critical Point ◽  
Filter Paper ◽  
Freeze Drying ◽  
Cell Surfaces ◽  
Air Drying ◽  
Popular Method ◽  
Critical Point Drying ◽  
Scanning Electron

The use of critical point drying is rapidly becoming a popular method of preparing biological samples for scanning electron microscopy. The procedure is rapid, and produces consistent results with a variety of samples. The preservation of surface details is much greater than that of air drying, and the procedure is less complicated than that of freeze drying. This paper will present results comparing conventional air-drying of plant specimens to critical point drying, both of fixed and unfixed material. The preservation of delicate structures which are easily damaged in processing and the use of filter paper as a vehicle for drying will be discussed.

Delicate Botanical Specimens Preserved for Scanning Electron Microscopy by Critical Point Drying

1971 ◽  
Vol 29 ◽  
pp. 450-451
Author(s):  
Arthur L. Cohen ◽  
Gerald E. Garner
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Critical Point ◽  
Freeze Drying ◽  
Animal Cell ◽  
Pollen Grains ◽  
Scanning Electron Micrographs ◽  
Animal Cells ◽  
Critical Point Drying ◽  
Scanning Electron

The surface forms and structures of animal cells have been strikingly preserved for scanning electron microscopy by freeze-drying and by critical point drying both by the method with CO2 used as the transitional fluid and the later procedure which uses a fluorocarbon (Freon 13) as a medium for the transition from the liquid to the gaseous environment. Freeze-drying is often prolonged (5-12 hours as compared with an hour or less by the critical point method) and in our experience with mold cultures on agar, the substrate shrivels and cracks and hyphal filaments are distorted.Despite, and possibly because of a flexible but inelastic cell wall, plant cells often show greater distortion than do animal cells after evaporative drying or replacement dehydration for mixrotechnical work. The animal cell membrane can contract more or less uniformly on drying - as shown by the numerous micrographs of well-preserved erythrocytes, while plant cell walls often crumple. The many scanning electron micrographs of partially collapsed pollen grains bear witness to this fact.

Scanning Electron Microscopy of Ethanol Infiltrated Freeze-Fractured Cell Pellets

1974 ◽  
Vol 32 ◽  
pp. 98-99
Author(s):  
William G. Henk ◽  
Ben O. Spurlock
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Critical Point ◽  
Freeze Drying ◽  
Cultured Cells ◽  
Resolving Power ◽  
Ice Crystal ◽  
Internal Structures ◽  
Critical Point Drying ◽  
Scanning Electron

The increased depth of focus and superior resolving power of the scanning electron microscope provide advantages over the light microscope in viewing the external morphology of cultured cells and protists. Internal structures have, however, proved more difficult to observe. Freeze drying adequately preserves surface structures but results in poorly preserved cytoplasmic elements due to ice crystal damage. Critical point drying results in good preservation of both surface and cytoplasmic fine structure. Attempts to cut or break critical point dried material, however, result in plastic deformation of the cells. Humphreys, et al, recently introduced freeze fracturing of ethanol infiltrated tissues for biological scanning electron microscopy. We have modified and applied their technique and obtained similar results with Paramecium sp. obtained from mass cultures.

scholarly journals Methods of preparing flower stem samples for scanning electron microscopy

2015 ◽  
Vol 21 (1) ◽  
pp. 17 ◽  
Author(s):  
Poliana Cristina Spricigo ◽  
Jéssica Prada Trento ◽  
Joana Dias Bresolin ◽  
Viviane Faria Soares ◽  
Viviane Faria Soares ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Stainless Steel ◽  
Critical Point ◽  
Freeze Drying ◽  
Osmium Tetroxide ◽  
Cut Flowers ◽  
Critical Point Drying ◽  
Flower Stem ◽  
Scanning Electron

Brazil has great capacity for expansion in the floriculture sector. Studies on postharvest cut flowers contribute to development of the sector, helping to maintain the quality of domestic production. Scanning electron microscopy (SEM) is a powerful tool that allows viewing of flower structures and also microorganisms. The aim of this study was to evaluate methods of preparing flower stem samples for viewing in SEM as a support for studies on postharvest cut flowers. Ways of cutting, fixing, and drying samples were tested. Cutting with a stainless steel blade and through freeze-fracture were tested; fixation was carried out without the use of osmium tetroxide (OsO4); and drying of the samples was performed through freeze-drying and through critical point dryingwithCO2. Cutting with a stainless steel blade proved to be a satisfactory method for stem samples, with low cost and simple application compared to freeze-fracturing. Good fixation and high image contrast were obtained without the use of osmium tetroxide, thus avoiding the use of this toxic compound. Freeze-drying allowed the structure and morphological composition to be viewed, while critical point drying withCO2 preserved the microorganisms present in the samples.

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