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.

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.

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 The structure of cytoplasm in directly frozen cultured cells. I. Filamentous meshworks and the cytoplasmic ground substance.

1984 ◽  
Vol 99 (5) ◽  
pp. 1655-1668 ◽  
Author(s):  
P C Bridgman ◽  
T S Reese
Keyword(s):  
Cultured Cells ◽  
Ground Substance ◽  
Ice Crystal ◽  
Improved Method ◽  
Intact Cells ◽  
Cultured Fibroblasts ◽  
Cytoplasmic Proteins ◽  
Granular Matrix ◽  
Cytoplasmic Ground Substance ◽  
Xenopus Laevis Embryos

Cultured fibroblasts or epithelial cells derived from Xenopus laevis embryos were directly frozen, freeze-substituted by an improved method, and then either critical-point-dried and viewed as whole mounts, or embedded and thin sectioned. In thin regions of these cells, where ice crystal artifacts are absent, the cytoplasm consisted of a dense, highly interconnected meshwork of filaments, embedded in a finely granular ground substance. The meshwork in directly frozen, intact cells was compared with that in cells that were lysed (physically, with detergents, or with filipin), or fixed with glutaraldehyde before freezing. Although filaments tended to be less numerous in lysed cells, their overall organization was the same as that in intact cells. However, fixation with glutaraldehyde before freezing distorted the meshwork to variable degrees depending on the osmolarity of the fixation buffer, and also obscured the granular ground substance which is obvious in directly frozen cells. With optimal preparative methods, the cytoplasm of these directly frozen cells is shown to consist of a cytoskeleton composed of discrete interwoven filaments interconnected by numerous finer filaments and a readily extractable granular matrix which presumably represents aggregations of cytoplasmic proteins.

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.

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.

Studies on The Cytoplasmic Ground Substance Using High Voltage Electron Microscopy of Whole Cultured Cells

1980 ◽  
Vol 38 ◽  
pp. 818-821
Author(s):  
K.R. Porter ◽  
K.J. Luby
Keyword(s):  
Electron Microscopy ◽  
High Voltage ◽  
Monolayer Culture ◽  
Cultured Cells ◽  
Ground Substance ◽  
High Quality ◽  
High Voltage Electron Microscopy ◽  
Voltage Electron ◽  
Cytoplasmic Ground Substance ◽  
Very High

Cells of several types, when grown and maintained in monolayer culture, will spread on the substrate to be not greater than 1 pm thick in their thinner margins. When fixed with glutaraldehyde and OsO4 and then dried by the critical-point method,these cells can be viewed in the HVEM and stereo images of very high quality can be obtained. Grown directly on formvar-coated gold grids, such cells are quickly and easily prepared for microscopy.

scholarly journals The cytoplasmic filament system in critical point-dried whole mounts and plastic-embedded sections.

1985 ◽  
Vol 100 (5) ◽  
pp. 1474-1487 ◽  
Author(s):  
H Ris
Keyword(s):  
Critical Point ◽  
Cultured Cells ◽  
Three Dimensional ◽  
Intact Cells ◽  
High Voltage Electron Microscopy ◽  
Voltage Electron ◽  
Cytoplasmic Filaments ◽  
Critical Point Drying ◽  
Cytoplasmic Filament ◽  
Whole Mounts

High voltage electron microscopy of intact cells prepared by the critical point drying (CPD) procedure has become an important tool in the study of three-dimensional relationships between cytoplasmic organelles. It has been claimed that critical point-dried specimens reveal a structure that is not visible in sections of plastic-embedded material; it has also been claimed that this structure, in association with known cytoplasmic filaments, forms a meshwork of tapering threads ("microtrabecular lattice"). Alternatively, this structure might be a surface tension artifact produced during CPD. To test possible sources of artifacts during CPD, model fiber systems of known structure were used. It was found that traces of water or ethanol in the CO2 caused distortions and fusion of fibers in pure muscle actin, fibrin, collagen, chromatin, and microtubules that produce a structure very similar to the proposed "microtrabecular lattice." These structures were, however, well preserved if water and ethanol were totally excluded from the CO2. The same results were obtained with whole mounts of cultured cells. A "microtrabecular lattice" was obtained if some water or ethanol was present in the pressure chamber. On the other hand, when water or ethanol were totally excluded from the CO2 during CPD, cytoplasmic filaments were uniform in thickness similar to their appearance in sections of plastic-embedded cells. It is concluded that the "microtrabecular lattice" is a distorted image of the cytoplasmic filament network produced during CPD by traces of water or ethanol in the CO2.

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.

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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