Modified gach technique vs critical point drying: Shrinkage analysis for SEM

1987 ◽  
Vol 45 ◽  
pp. 954-955
Author(s):  
B. Thompson ◽  
N. Sculov ◽  
R.E. Crang
Keyword(s):  
Critical Point ◽  
Calf Serum ◽  
Drying Shrinkage ◽  
Specimen Preparation ◽  
Cell Shrinkage ◽  
Whole Cells ◽  
Critical Point Drying ◽  
Prepared Material ◽  
Inverted Microscope ◽  
Phosphate Buffered Saline

The use of co-polymerized glutaraldehyde-carbohydrazide (GACH) was proposed for specimen preparation in scanning electron microscopy (SEM) as a means of avoiding dehydration in organic solvents, and to provide dimensionally stable biological specimens through a process of air-drying. It has been assumed that shrinkage of specimens prepared by the GACH technique should be less than that of conventionally-prepared material by critical point drying (CPD). In a previous study, Bell has reported significant shrinkage of whole cells for SEM. This report compares cell shrinkage in GACH and CPD preparations.Fibroblasts from newborn rats were grown on collagen-coated glass cover-slips (with alpha numeric grids etched onto the surface of the coverslips) in Eagle's minimum essential medium + 10% fetal calf serum for 7 d. (3). Using an inverted microscope with phase-contrast optics, micrographs were taken of the cultures in their live state and 1 h. after fixation with 2.5% glutaraldehyde in Dulbecco's phosphate buffered saline (Figs. 1 and 3).

scholarly journals Microtrabecular lattice of the cytoplasmic ground substance. Artifact or reality.

1979 ◽  
Vol 82 (1) ◽  
pp. 114-139 ◽  
Author(s):  
J J Wolosewick ◽  
K R Porter
Keyword(s):  
Critical Point ◽  
Osmium Tetroxide ◽  
Prolonged Exposure ◽  
Cultured Cells ◽  
Lattice Structure ◽  
Model Systems ◽  
Ground Substance ◽  
Specimen Preparation ◽  
Critical Point Drying ◽  
Cytoplasmic Ground Substance

The cytoplasmic ground substance of cultured cells prepared for high voltage transmission electron microscopy (glutaraldehyde/osmium fixed, alcohol or acetone dehydrated, critical-point dried) consists of slender (3-6 nm Diam) strands--the microtrabeculae (55)--that form an irregular three-dimensional lattice (the microtrabecular lattice). The microtrabeculae interconnect the membranous and nonmembranous organelles and are confluent with the cortices of the cytoplast. The lattice is found in all portions of the cytoplast of all cultured cells examined. The possibility that the lattice structure is an artifact of specimen preparation has been tested by (a) subjecting whole cultured cells (WI-38, NRK, chick embryo fibroblasts) to various chemical (aldehydes, osmium tetroxide) and nonchemical (freezing) fixation schedules, (b) examination of model systems (erythrocytes, protein solutions), (c) substantiating the relaibility of critical-point drying, and (d) comparing images of whole cells with conventionally prepared (plastic-embedded) cells. The lattice structure is preserved by chemical and nonchemical fixation, though alterations in ultrastructure can occur especially after prolonged exposure to osmium tetroxide. The critical-point method for drying specimens appears to be reliable as is the freeze-drying method. The discrepancies between images of plastic-embedded and sectioned cells, and images of whole, critical-point dried cells appear to be related, in part, to the electron-scattering properties of the embedding resin. The described observations indicate that the microtrabecular lattice seen in electron micrographs closely represents the nonrandom structure of the cytoplasmic ground substance of living cultured cells.

Specimen Observation at Large Tilt Angles with a Top-Entry Goniometer Stage

1974 ◽  
Vol 32 ◽  
pp. 558-559
Author(s):  
Mircea Fotino
Keyword(s):  
Electron Microscopy ◽  
Critical Point ◽  
Whole Cells ◽  
Morphological Studies ◽  
Controlled Conditions ◽  
Critical Point Drying ◽  
Cells Cultured

In one of the most resourceful applications of million-volt electron microscopy to biological problems, whole cells cultured directly on grids under controlled conditions and maintained unperturbed in vacuo through critical-point drying are viewed stereoscopically for morphological studies.Great advantage would thus be drawn in this regard from high tilting flexibility that would allow specimen observation under larger tilting angles yielding side stereo views, as it were, in addition to the more routine top views.

Biological specimen preparation for SEM by a method other than critical point drying

1987 ◽  
Vol 45 ◽  
pp. 956-957 ◽  
Author(s):  
J. L. Adams ◽  
C. J. Battjes ◽  
D. A. Buthala
Keyword(s):  
Sample Preparation ◽  
Critical Point ◽  
Biological Samples ◽  
Osmium Tetroxide ◽  
Uranyl Acetate ◽  
Specimen Preparation ◽  
Biological Specimen ◽  
Air Drying ◽  
Critical Point Drying ◽  
Good Preparation

Quality sample preparation for SEM is important to observe fine details without artifacts, and good preparation requires proper fixation, dehydration, drying and coating, An alternative 5 min passage in hexamethyldisilazane (HMDS) can replace critical point crying (CPD) and gives satisfactory results on many biological samples. CPD procedure may take up to 1 h per sample to ensure adequate drying, therefore a brief rinse in HMDS followed by air drying requires less time and equipment yet provides excellent results.Various biological samples were fixed in 3% glutaraldehyde; rinsed 3 times in Millonig's phosphate buffer for 10 min each; post-fixed in 1% osmium tetroxide for 1 h; rinsed as before; fixed again in 1% tannic acid (TA) for 30 min-1 h; rinsed well and partially dehydrated to 70% ethanol; placed in 1% uranyl-acetate (UA) in the dark, overnight: rinsed with 70% ethanol until UA cleared and then dehydrated through 100% ethanol.

Tissue printing for scanning electron microscopy and microanalysis

1993 ◽  
Vol 51 ◽  
pp. 140-141
Author(s):  
Barbara A. Reine
Keyword(s):  
Critical Point ◽  
Chemical Constituents ◽  
Plant Cells ◽  
Plant Morphology ◽  
Specimen Preparation ◽  
Normal Surface ◽  
Tissue Printing ◽  
Critical Point Drying ◽  
Scanning Electron

The study of plant morphology and plant cells in the scanning electron microscope is often compromised by the limitations of specimen preparation techniques. Simple natural dehydration usually results in unacceptable shrinkage and distortion of the normal surface morphology of plant cells. Chemical fixation followed by critical point drying or some substitute for critical point drying such as Peldri II or HMDS (hexamethyldisilazane) improves morphological results but still imparts artifacts, adds chemical constituents to the specimen, and requires the use of toxic chemicals, a hood, and much time.One technique that eliminates many of these disadvantages and is even suitable for specimen preparation in the field is tissue printing. For low magnification imaging and chemical analysis its “elegant simplicity” (2) is compelling. Historically, the application of tissue printing has been in connection with optical microscopy (1,2). However, this technique works very well for low magnification SEM and associated elemental characterization of residues by x-ray microanalysis.

scholarly journals History of High Vacuum and Critical Point Equipment used in EM Specimen Preparation

1993 ◽  
Vol 1 (8) ◽  
pp. 10-11
Author(s):  
Richard A. Denton
Keyword(s):  
New York ◽  
High Pressure ◽  
Critical Point ◽  
High Vacuum ◽  
Specimen Preparation ◽  
New York University ◽  
Vacuum Equipment ◽  
Critical Point Drying ◽  
History Of ◽  
Opening And Closing

An interesting sidelight of building our own 3″ diffusion pumps is that as we built ever larger vacuum equipment, larger pumps were required. Motivated by our success with the 3″ pump, we gradually built a series of larger pumps going up one size at a time to 10″ diameter.Critical point drying was invented by Tom Anderson and described in his fine paper in 1952. He was a bit ahead of his time as the widespread use of the technique was not until the arrival of the SEM in about 1970.I became involved in critical point drying when Dr. Gennaro of New York University asked if I could make him a unit.. He sent me a copy of Tom's original paper and we visited Tom's lab for a demonstration.With Tom's dryer, the chamber opening and closing involved a high pressure threaded seal. Fortunately, he had working with him a man big enough to be a NHL defensive end who manhandled a three foot wrench to seal and unseal the chamber.

The Use of Gach Technique in Stabilizing Biological Specimens For X-Ray Microanalysis

1985 ◽  
Vol 43 ◽  
pp. 452-453
Author(s):  
W.T. Gunning ◽  
R.E. Crang
Keyword(s):  
Surface Morphology ◽  
Critical Point ◽  
Freeze Drying ◽  
Freeze Substitution ◽  
Specimen Preparation ◽  
Preparation Methods ◽  
Bulk Specimen ◽  
X Ray ◽  
Critical Point Drying ◽  
Better Than

An alternative SEM preparative technique is proposed for biological specimens in which cellular diffusible constituents may be retained that are otherwise lost during conventional specimen preparation involving critical point drying. The technique utilizes the copolymerization of glutaraldehyde with carbohy-drazide, and is designated “GACH”. Surface morphology with GACH preparations is as good as, or better than, that preserved using critical point drying. Bulk specimen preparation for elemental analysis has been limited heretofore to frozen-hydrated, freeze drying, freeze substitution, unfixed air dried, and critical point dried specimens. All such techniques offer particular advantages, but with certain limitations. The GACH technique is not being proposed as a superior routine replacement for present biological bulk specimen preparation methods, but rather as a viable alternative technique that allows both the retention of bound ions and acceptable surface morphology without elaborate instrumentation.

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.

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

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.

The Effects of Drying Techniques on Clay-Rich Soil Texture

1974 ◽  
Vol 32 ◽  
pp. 466-467
Author(s):  
T. G. Naymik
Keyword(s):  
Critical Point ◽  
Liquid Nitrogen ◽  
Liquid Nitrogen Temperature ◽  
Drying Methods ◽  
Air Drying ◽  
Freeze Dried ◽  
Critical Point Drying ◽  
Set Up ◽  
The Relationship ◽  
Quartz Grains

Three techniques were incorporated for drying clay-rich specimens: air-drying, freeze-drying and critical point drying. In air-drying, the specimens were set out for several days to dry or were placed in an oven (80°F) for several hours. The freeze-dried specimens were frozen by immersion in liquid nitrogen or in isopentane at near liquid nitrogen temperature and then were immediately placed in the freeze-dry vacuum chamber. The critical point specimens were molded in agar immediately after sampling. When the agar had set up the dehydration series, water-alcohol-amyl acetate-CO2 was carried out. The objectives were to compare the fabric plasmas (clays and precipitates), fabricskeletons (quartz grains) and the relationship between them for each drying technique. The three drying methods are not only applicable to the study of treated soils, but can be incorporated into all SEM clay soil studies.

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