Criteria for the evaluation of low-temperature Scanning Electron Microscopy

Low Temperature ◽

Conformational Changes ◽

Chemical Vapour ◽

List Type ◽

Dimensional Changes ◽

Critical Point Drying ◽

Temperature Scanning ◽

Low temperature scanning electron microscopy (LTSEM) has been evaluated with special reference to its application to the study of morphology and development in microorganisms. A number of criteria have been considered and have proved valuable in assessing the standard of results achieved. To further aid our understanding of these results, it has been necessary to compare those obtained by LTSEM with those from more conventional preparatory procedures such as 1) chemical fixation, dehydration and critical point-drying; 2) freeze-drying with or without chemical vapour fixation before hand.The criteria used for assessing LTSEM for the above purposes are as follows: 1)Specimen immobilization and stabilization2)General preservation of external morphology3)General preservation of internal morphology4)Exposure to solvents5)Overall dimensional changes6)Cell surface texture7)Differential conformational changes8)Etching frozen-hydrated material9)Beam damage10)Specimen resolution11)Specimen life

Intracellular structures of rapid frozen biological samples observed by high-resolution low-temperature Scanning Electron Microscopy

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100155657 ◽

1989 ◽

Vol 47 ◽

pp. 736-737

Author(s):

T. Inoué ◽

H. Koike

Keyword(s):

Electron Microscopy ◽

Low Temperature ◽

Liquid Nitrogen ◽

Specimen Preparation ◽

Chemical Fixation ◽

Brown Adipose ◽

Critical Point Drying ◽

Temperature Scanning ◽

Low temperature scanning electron microscopy (LTSEM) is useful to avoid artifacts such as deformation and extraction, because specimens are not subjected to chemical fixation, dehydration and critical-point drying. Since Echlin et al developed a LTSEM, many techniques and instruments have been reported for observing frozen materials. However, intracellular structures such as mitochondria and endoplasmic reticulum have been unobservable by the method because of the low resolving power and inadequate specimen preparation methods. Recently, we developed a low temperature SEM that attained high resolutions. In this study, we introduce highly magnified images obtained by the newly developed LTSEM, especially intracellular structures which have been rapidly frozen without chemical fixation.[Specimen preparations] Mouse pancreas and brown adipose tissues (BAT) were used as materials. After the tissues were removed and cut into small pieces, the specimen was placed on a cryo-tip and rapidly frozen in liquid propane using a rapid freezing apparatus (Eiko Engineering Co. Ltd., Japan). After the tips were mounted on the specimen stage of a precooled cryo-holder, the surface of the specimen was manually fractured by a razor blade in liquid nitrogen. The cryo-holder was then inserted into the specimen chamber of the SEM (ISI DS-130), and specimens were observed at the accelerating voltages of 5-8 kV. At first the surface was slightly covered with frost, but intracellular structures were gradually revealed as the frost began to sublimate. Gold was then coated on the specimen surface while tilting the holder at 45-90°. The holder was connected to a liquid nitrogen reservoir by means of a copper braid to maintain low temperature.

Low Temperature Scanning Electron Microscopy of Soft Bodied Organisms

Microscopy and Microanalysis ◽

10.1017/s1431927600032098 ◽

2001 ◽

Vol 7 (S2) ◽

pp. 1204-1205

Author(s):

Eric F. Erbe ◽

William P. Wergin ◽

Ronald Ochoa ◽

Joseph C. Dickens ◽

William Moser

Keyword(s):

Electron Microscopy ◽

Fine Structure ◽

Low Temperature ◽

Structural Integrity ◽

Mechanical Damage ◽

Critical Point Drying ◽

Temperature Scanning ◽

And Behavior ◽

Resolving the true fine structure and behavior of soft bodied organisms such as arthropod larvae, acari, nematodes, annelids and pupae by conventional scanning electron microscopy has been difficult. Classical preparation techniques, involving immersion fixation, dehydration and critical point drying, cause significant deleterious changes in specimen structure due to effects of surface tension, osmotic stress and mechanical damage. The intact exoskeleton, cuticle or other limiting membranes of these organisms, creates a formidable obstacle to the penetration of fixatives and solvents, which are only effective after a long period of time (hours). During this time, behavioral postures and much of the structural integrity are lost and most organisms detach from their hosts.In previous studies, our laboratory utilized methods in low temperature freeze stabilization, to preserve and observe the undisturbed fine structure of biological samples. Plunge freezing, although more rapid, resulted in many parasitic organisms dislodging from hosts and being lost.

Use of low temperature scanning electron microscopy to observe frozen hydrated seed tissues of Glycine max

Seed Science Research ◽

10.1017/s0960258500002397 ◽

1994 ◽

Vol 4 (3) ◽

pp. 347-353 ◽

Cited By ~ 4

Author(s):

Robert W. Yaklich ◽

William P. Wergin ◽

Eric F. Erbe

Keyword(s):

Electron Microscopy ◽

Low Temperature ◽

Three Dimensional ◽

Developmental Studies ◽

Degree Of Hydration ◽

Quantitative Measurements ◽

Critical Point Drying ◽

Temperature Scanning ◽

AbstractEvidence is provided to show that frozen, hydrated seeds and seed tissues can be observed by low temperature field emission scanning electron microscopy. This technique allows preparation and observation of seed samples that contain 13–60% water without altering their moisture content. The technique provides information about the surface structure of seeds and also allows specimens to be fractured to reveal internal features of tissues. Futhermore, both halves of fractured specimens can be retained, examined and photographed either as two-dimensional micrographs or as stereo pairs when three-dimensional observation (stereology) or quantitative measurements (stereometry) are desired. Use of this technique avoids artefacts associated with chemical fixation, dehydration, and critical point drying—procedures that are usually required to prepare seed tissues for scanning electron microscope examination. This technique does not affect the degree of hydration in specimens; it can be used to localize water in tissues to determine their degree of hydration. This technique should find wide application in developmental studies of seeds, especially during maturation, imbibition and germination.