Specimen preparation, cryotransfer and microscope parameters for cold-stage work in SEM and application for the physical and biological sciences

1986 ◽  
Vol 44 ◽  
pp. 898-901
Author(s):  
J.A. Sargent
Keyword(s):  
Biological Sciences ◽  
Freeze Drying ◽  
Specimen Preparation ◽  
Ice Crystal ◽  
X Ray ◽  
Cold Stage ◽  
Critical Point Drying ◽  
Cryo Preservation ◽  
Precise Interpretation ◽  
Soluble Components

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.

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.

Revealing gross three-dimensional structure in the SEM

1987 ◽  
Vol 45 ◽  
pp. 656-657
Author(s):  
Sterling P. Newberry
Keyword(s):  
Freeze Drying ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Small Object ◽  
Final Solution ◽  
Dimensional Representation ◽  
X Ray ◽  
Three Dimensional Representation ◽  
Freeze Dried ◽  
Critical Point Drying

The beautiful three dimensional representation of small object surfaces by the SEM leads one to search for ways to open up the sample and look inside. Could this be the answer to a better microscopy for gross biological 3-D structure? We know from X-Ray microscope images that Freeze Drying and Critical Point Drying give promise of adequately preserving gross structure. Can we slice such preparations open for SEM inspection? In general these preparations crush more readily than they slice. Russell and Dagihlian got around the problem by “deembedding” a section before imaging. This some what defeats the advantages of direct dry preparation, thus we are reluctant to accept it as the final solution to our problem. Alternatively, consider fig 1 wherein a freeze dried onion root has a window cut in its surface by a micromanipulator during observation in the SEM.

A Versatile High-Vacuum Cryo-Transfer for Cryo-FESEM, Cryo-SPM and other Imaging Techniques

1999 ◽  
Vol 5 (S2) ◽  
pp. 424-425
Author(s):  
Martin Ritter ◽  
Didier Henry ◽  
Stefan Wiesner ◽  
Stephan Pfeiffer ◽  
Roger Wepf
Keyword(s):  
Structural Integrity ◽  
Imaging Techniques ◽  
High Vacuum ◽  
Specimen Preparation ◽  
Ice Crystal ◽  
Structural Water ◽  
Imaging Device ◽  
The Past ◽  
Cold Stage ◽  
Immobilization Techniques

A structure preservation of biological and organic samples, close to native state, can only be reached by cryo immobilization techniques. Cryo immobilization allows not only to preserve the high structural integrity but also to arrest dynamic processes in the μs- ms range.After freezing the sample and preparing the surface of interest, it is important to prevent the sample from ice crystal damage, removal of structural water, condensation of water or other contaminants until imaging. Therefore, ideally the samples are kept below the recrystallisation temperature of water (< 147K) during the transfer from the preparation environment into the imaging chamber.For the transfer of frozen samples several concepts have been followed in the,past: a) the specimen after manipulation/preparation were submersed in liquid nitrogen and transferred to the cold stage of the microscope or b) a preparation chamber was permanently attached to the microscope column allowing the direct transfers between the preparation chamber and the cold stage in the microscope. These concepts allow either a high grade of flexibility combined with a high risk of contamination or to prevent contamination but combined with inflexibility. In addition the later also does not allow using the microscope during the specimen preparation procedure, nor transferring the specimen to an other imaging device.

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.

An Aldehyde/Hydrazide Polymerization Technique for Biological SEM Specimen Preparation

1981 ◽  
Vol 39 ◽  
pp. 644-645
Author(s):  
W. T. Gunning ◽  
R. E. Crang
Keyword(s):  
Electron Microscopy ◽  
Freeze Drying ◽  
Water Soluble ◽  
Specimen Preparation ◽  
Crystal Formation ◽  
Ice Crystal ◽  
Polymer Medium ◽  
Low Contrast ◽  
Transmission Electron ◽  
Limited Application

Two water-soluble embedding techniques for transmission electron microscopy (TEM) utilizing the polymerization of glutaraldehyde were proposed in the early 1970's. Pease and Peterson introduced a glutaraldehyde-urea polymer medium and Heckman and Barrnett proposed a glutaraldehyde-carbohydrazide (GACH) medium. Both permitted the retention of lipids in tissues without the harsh extraction of cellular constituents by fixatives, organic dehydrators and embedding agents. These techniques were of limited application and yielded low contrast or negative-stained images in the TEM. This technique has not been previously considered as potentially useful for scanning electron microscopy (SEM) due to the covering and obliterating of surface morphology. Virtually all SEM techniques for biological and specimen preparation, aside from freeze-drying, replace or extract most of the native elemental compositions of cells and tissues. Freezing and freeze-drying techniques are cumbersome, time-consuming, may require elaborate equipment and can induce artifacts due to ice crystal formation. We propose a new approach of SEM sample preparation using modifications of the GACH technique employed in TEM.

Low-temperature specimen preparation apparatus and techniques for low-temperature scanning electron microscopy (LTSEM) and synchrotron soft x-ray microscopy

1988 ◽  
Vol 46 ◽  
pp. 108-109
Author(s):  
Jacob Bastacky ◽  
Thomas L. Hayes ◽  
Joong Lee ◽  
Charles Lee ◽  
John Goerke ◽  
...  
Keyword(s):  
Low Temperature ◽  
Specimen Preparation ◽  
Ice Crystal ◽  
Tissue Samples ◽  
X Ray ◽  
Specimen Holder ◽  
Ice Crystal Size ◽  
Temperature Scanning ◽  
Scanning Electron

We have developed equipment and techniques for low-temperature scanning electron microscope (LTSEM) and x-ray microscope examinations of animal tissue samples. A cryoprobe for rapidly freezing tissue in vivo, a low-temperature saw for cutting large blocks of frozen tissue without warming, cardboard containers for sample storage in liquid nitrogen (LN2), tools for holding and manipulating frozen specimens, and an under-LN2 specimen viewer are described here. An LTSEM specimen holder for small blocks of tissue and a low-temperature trim saw for precisely cutting such blocks are described elsewhere. We have found such equipment, along with various commercial and specially fabricated dewars, to greatly facilitate our LTSEM experiments.To minimize ice crystal size by freezing as rapidly as possible and to maintain tissue in as close to normal physiologic state as possible during freezing, we use a cryoprobe for freezing 1 cm2 surfaces of tissue in vivo. The cryoprobe (Fig. 1) is a highly-polished 1 mm thick, 10 mm diam.

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 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.

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.

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