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.

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.

scholarly journals How to Prepare Biological Samples and Live Tissues for Scanning Electron Microscopy (SEM)

2014 ◽  
Vol 3 (2) ◽  
pp. 63-80
Author(s):  
Abolfazl Mehdizadeh Kashi ◽  
Kobra Tahemanesh ◽  
Shahla Chaichian ◽  
Mohammad Taghi Joghataei ◽  
Fateme Moradi ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Freeze Drying ◽  
Specimen Preparation ◽  
Imaging Method ◽  
Tissue Preservation ◽  
Chemical Fixation ◽  
Biological Specimen ◽  
Air Drying ◽  
Critical Point Drying ◽  
Scanning Electron

In this article we review the application and procedures involved in scanning electron microscope (SEM) to observe biological and live tissues through using SEM at high resolution. We discuss practical methods for optimizing tissue preservation to achieve the two principal goals of biological specimen preparation: (a) preserving biological structures as close to their living configuration as possible, and (b) rendering them visible with the desired imaging method. We also review and discuss the relative merits of different fixing (chemical fixation and cryofixation), drying (air-drying, critical point-drying, freeze-drying and chemical-drying) and coating procedures of biological specimens with metals to facilitate visualization in the SEM.

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.

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.

Rapid Critical Point Drying of Tissues in a Parr Bomb

1972 ◽  
Vol 30 ◽  
pp. 400-401
Author(s):  
C.A. Baechler ◽  
W. C. Pitchford ◽  
J. M. Riddle ◽  
C.B. Boyd ◽  
H. Kanagawa ◽  
...  
Keyword(s):  
Critical Point ◽  
Critical Pressure ◽  
Soft Tissues ◽  
Biological Tissues ◽  
Critical Temperatures ◽  
Air Drying ◽  
The Past ◽  
Critical Point Drying ◽  
Great Stress ◽  
Infiltration Techniques

Preservation of the topographic ultrastructure of soft biological tissues for examination by scanning electron microscopy has been accomplished in the past by using lengthy epoxy infiltration techniques, or dehydration in ethanol or acetone followed by air drying. Since the former technique requires several days of preparation and the latter technique subjects the tissues to great stress during the phase change encountered during air-drying, an alternate rapid, economical, and reliable method of surface structure preservation was developed. Turnbill and Philpott had used a fluorocarbon for the critical point drying of soft tissues and indicated the advantages of working with fluids having both moderately low critical pressures as well as low critical temperatures. Freon-116 (duPont) which has a critical temperature of 19. 7 C and a critical pressure of 432 psi was used in this study.

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).

Processing Techniques for Scanning Electron Microscopy Imaging of Giant Cells from Giant Cell Tumors of Bone

2019 ◽  
Vol 25 (6) ◽  
pp. 1376-1382
Author(s):  
Asit Ranjan Mridha ◽  
Indu Barwal ◽  
Abhishek Gupta ◽  
Abdul Majeed ◽  
Adarsh W. Barwad ◽  
...  
Keyword(s):  
Surface Morphology ◽  
Critical Point ◽  
Giant Cell ◽  
Giant Cells ◽  
Giant Cell Tumors ◽  
Microscopy Imaging ◽  
Air Drying ◽  
Critical Point Drying ◽  
Sem Imaging ◽  
Scanning Electron

AbstractGiant cell tumor (GCT) of bone is a common benign lesion that causes significant morbidity due to the failure of modern medical and surgical treatment. Surface ultra-structures of giant cells (GCs) may help in distinguishing aggressive tumors from indolent GC lesions. This study aimed to standardize scanning electron microscopic (SEM) imaging of GC from GCT of bone. Fresh GCT collected in Dulbecco's Modified Eagle Medium was washed to remove blood, homogenized, or treated with collagenase to isolate the GCs. Mechanically homogenized and collagenase-digested GCs were imaged on SEM after commonly used drying methodologies such as air-drying, tetramethylsilane (TMS)-drying, freeze-drying, and critical point-drying (CPD) for the optimization of sample processing. The collagenase-treated samples yielded a greater number of isolated GC and showed better surface morphology in comparison to mechanical homogenization. Air-drying was associated with marked cell shrinkage, and freeze-dried samples showed severe cell damage. TMS methodology partially preserved the cell contour and surface structures, although the cell shape was distorted. GC images with optimum surface morphology including membrane folding and microvesicular structures on the surface were observed only in collagenase-treated and critical point-dried samples. Collagenase digestion and critical point/TMS-drying should be performed for optimal SEM imaging of individual GCs.

Simple desiccation method for SEM using dimethoxypropane

1992 ◽  
Vol 50 (1) ◽  
pp. 760-761
Author(s):  
Frantíšek Weyda
Keyword(s):  
Electron Microscopy ◽  
Freeze Drying ◽  
Simple Procedure ◽  
Aqueous Suspension ◽  
Freeze Substitution ◽  
Biological Specimen ◽  
Air Drying ◽  
Biological Objects ◽  
Critical Point Drying ◽  
Scanning Electron

A number of techniques such as critical point drying (the most commonly used method of drying cells and tissues), freeze-drying or freeze-substitution are available to process various tissues preparations for scanning electron microscopy (SEM). Those techniques are more or less complicated (depending also on expensive equipments) and time consuming. While more simple air-drying from aqueous suspension or organic solvents can be used for some rigid biological specimen, it is generally not satisfactory for most tissues and biological objects. On the opposite, rapid and simple procedure using hexamethyldisilazane (HMDS) and air drying has been successfully applied to insect and mite tissues without observable artifacts. We have developed similar simple and rapid method using dimethoxypropane (DMP) and air drying. DMP has previously been used for chemical dehydration of tissues.

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.

A technique for preparing Beauveria spp. for scanning electron microscopy

1980 ◽  
Vol 58 (15) ◽  
pp. 1700-1703 ◽  
Author(s):  
E. C. Quattlebaum ◽  
G. R. Carner
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Electron Microscope ◽  
Scanning Electron Microscope ◽  
Freeze Drying ◽  
Osmium Tetroxide ◽  
Air Drying ◽  
Preparation Methods ◽  
Critical Point Drying ◽  
Scanning Electron

Vapor fixation for 96 h with 1% osmium tetroxide (OsO4) and 3–4 days air drying produced distortion-free specimens of Beauveria spp. for examination with the scanning electron microscope. A combination of 4 h OsO4 vapor fixation and freeze-drying also reduced disruption satisfactorily but specimens were not as well preserved as with the first method. Preparation methods that were ineffective in preventing collapse of hydrophilic structures were Cling Free® sprayed on specimens prior to examination, freeze-drying, critical-point drying (of unfixed material), and vapor fixation with glutaraldehyde.

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