scholarly journals Cryo-Fracture or Freeze-Fracture, a Method to Expose Internal Tissue Surfaces and Cell Surfaces for Viewing in the Scanning Electron Microscope

2008 ◽  
Vol 16 (4) ◽  
pp. 56-59 ◽  
Author(s):  
Jeannette Taylor
Keyword(s):  
Membrane Surface ◽  
Freeze Fracture ◽  
Internal Tissue ◽  
Cell Surfaces ◽  
Naturally Occurring ◽  
Critical Point Drying ◽  
Tissue Surfaces ◽  
A Cell ◽  
Bowman's Capsule ◽  
Scanning Electron

Cryo-fracture, in conjunction with critical point drying is a method used to prepare biological samples in order to expose, for viewing via scanning electron microscopy, those naturally occurring surfaces which might otherwise remain obscure. For example, the Bowman’s capsule and tubules of a kidney, tiny blood vessels on any organ, inter-cellular spaces in liver or alveoli in the lungs. Also, some surfaces, not normally exposed at all such as the membrane surface of a nuclear envelope, mitochondria or chloroplasts or the cytoplasm of a cell, can be brought to light with this method. Herein is a review of the development of cryo-fracture and how it is currently used at our facility.

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.

Scanning Studies of Cell Surfaces

1973 ◽  
Vol 31 ◽  
pp. 608-609
Author(s):  
Virginia Fonte ◽  
Nancy Weller ◽  
Keith R. Porter
Keyword(s):  
Cell Cycle ◽  
Structural Organization ◽  
Cell Surfaces ◽  
Detailed Characterization ◽  
A Cell ◽  
Relationship Of ◽  
The Relationship ◽  
Scanning Electron

The surfaces of a cell in its topography and anti-genicity expresses subtle variations in the effective genome, as well as the physiology and structural organization of the underlying cytoplasm. Understanding the relationship of these various factors to the surface depends in part on obtaining a detailed characterization of the topography of cells and how this topography changes with phases in the cell cycle, with transformation to malignancy and with the cell's response to such physiologically active agents as cyclic AMP.We have therefore explored the usefulness of the scanning electron microscope in investigations of the cell's topography. Cells grown under favourable in vitro conditions have been fixed in glutaraldehyde, dehydrated in acetone and dried by the critical point method of Anderson.

scholarly journals High-Resolution Imaging of Dried and Living Single Bacterial Cell Surfaces: Artifact or Not?

2011 ◽  
Vol 19 (5) ◽  
pp. 22-25 ◽  
Author(s):  
Dominik Greif ◽  
Daniel Wesner ◽  
Dario Anselmetti ◽  
Jan Regtmeier
Keyword(s):  
Electron Microscope ◽  
High Resolution ◽  
Scanning Electron Microscope ◽  
Metal Coating ◽  
Environmental Scanning ◽  
Cell Surfaces ◽  
Critical Point Drying ◽  
Resolution Imaging ◽  
Sem Imaging ◽  
Scanning Electron

When studying highly resolved scanning electron microscope images of cell surfaces, the question arises, whether the observed patterns are real or just artifacts of the cell preparation process. The following steps are usually necessary for preparation: fixation, drying, and metal coating. Each step might introduce different artifacts. Clever techniques have been developed to dry cells as gently as possible, for example critical point drying with different organic solvents and CO2. Instrument manufacturers also have taken account of this issue, for example, through the realization of the environmental scanning electron microscope (ESEM), operating with a low-vacuum environment saturated with water so that samples might stay hydrated. Another approach is the extreme high-resolution scanning electron microscope (XHR SEM), where the electron beam is decelerated shortly before reaching the sample. This technique requires no metal coating of the sample. Cryo-SEM also may be used, where no sample preparation is required beyond freezing in a high-pressure freezer or other cryo-fixation device. Then the cell can be examined in the frozen, hydrated state using a cryostage. However, at least some kind of preparation is necessary for SEM imaging, and we wanted to find out what changes the preparation makes on the cell surface.

scholarly journals Label-fracture: a method for high resolution labeling of cell surfaces.

1984 ◽  
Vol 99 (3) ◽  
pp. 1156-1161 ◽  
Author(s):  
P Pinto da Silva ◽  
F W Kan
Keyword(s):  
High Resolution ◽  
Cell Shape ◽  
Plasma Membranes ◽  
Freeze Fracture ◽  
Fracture Morphology ◽  
Cell Surfaces ◽  
Surface Distribution ◽  
Membrane Surfaces ◽  
A Cell ◽  
Dense Marker

We introduce here a technique, "label-fracture," that allows the observation of the distribution of a cytochemical label on a cell surface. Cell surfaces labeled with an electron-dense marker (colloidal gold) are freeze-fractured and the fracture faces are replicated by plantinum/carbon evaporation. The exoplasmic halves of the membrane, apparently stabilized by the deposition of the Pt/C replica, are washed in distilled water. The new method reveals the surface distribution of the label coincident with the Pt/C replica of the exoplasmic fracture face. Initial applications indicate high resolution (less than or equal to 15 nm) and exceedingly low background. "Label-fracture" provides extensive views of the distribution of the label on membrane surfaces while preserving cell shape and relating to the freeze-fracture morphology of exoplasmic fracture faces. The regionalization of wheat germ agglutinin receptors on the plasma membranes of boar sperm cells is illustrated. The method and the interpretation of its results are straightforward. Label-fracture is appropriate for routine use as a surface labeling technique.

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.

Ultrastructure of melanocyte-keratinocyte interactions and pigment transfer in vitro

1978 ◽  
Vol 36 (2) ◽  
pp. 650-651
Author(s):  
Raul I. Garcia ◽  
Evelyn A. Flynn ◽  
George Szabo
Keyword(s):  
Direct Injection ◽  
Skin Pigmentation ◽  
Freeze Fracture ◽  
Pigment Granule ◽  
Time Lapse ◽  
Ultrastructural Level ◽  
Lanthanum Tracer ◽  
A Cell

Skin pigmentation in mammals involves the interaction of epidermal melanocytes and keratinocytes in the structural and functional unit known as the Epidermal Melanin Unit. Melanocytes(M) synthesize melanin within specialized membrane-bound organelles, the melanosome or pigment granule. These are subsequently transferred by way of M dendrites to keratinocytes(K) by a mechanism still to be clearly defined. Three different, though not necessarily mutually exclusive, mechanisms of melanosome transfer have been proposed: cytophagocytosis by K of M dendrite tips containing melanosomes, direct injection of melanosomes into the K cytoplasm through a cell-to-cell pore or communicating channel formed by localized fusion of M and K cell membranes, release of melanosomes into the extracellular space(ECS) by exocytosis followed by K uptake using conventional phagocytosis. Variability in methods of transfer has been noted both in vivo and in vitro and there is evidence in support of each transfer mechanism. We Have previously studied M-K interactions in vitro using time-lapse cinemicrography and in vivo at the ultrastructural level using lanthanum tracer and freeze-fracture.

A Scanning Electron Microscopic Study of the Uriniferous Tubules

1973 ◽  
Vol 31 ◽  
pp. 622-623
Author(s):  
Peter M. Andrews
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Renal Glomerulus ◽  
Electron Microscopic ◽  
Vascular Perfusion ◽  
Scanning Electron Microscopic Study ◽  
Scanning Electron Microscopic ◽  
Collecting Tubules ◽  
Bowman's Capsule ◽  
Scanning Electron

Although there have been a number of recent scanning electron microscopic reports on the renal glomerulus, the advantages of scanning electron microscopy have not yet been applied to a systematic study of the uriniferous tubules. In the present investigation, scanning electron microscopy was used to study the ultrastructural morphology of the proximal, distal, thin loop, and collecting tubules. Material for observation was taken from rat kidneys which were fixed by vascular perfusion, sectioned by either cutting or fracturing technigues, and critically point dried.The brush border characterising proximal tubules is first detected on the luminal surface of Bowman's capsule adjacent to the urinary pole orifice. In this region one frequently finds irregular microvilli characterized by broad and flattened bases with occasional bulbous structures protruding from their surfaces.

Remarks on the application of backscattered electron imaging (BEI) to the scanning electron microscopy (SEM) of biological structures

1983 ◽  
Vol 41 ◽  
pp. 484-487
Author(s):  
Etienne de Harven
Keyword(s):  
High Resolution ◽  
Incident Beam ◽  
Spot Size ◽  
Primary Electron ◽  
Critical Point Drying ◽  
Cell Shapes ◽  
High Resolution Images ◽  
Backscattered Electron ◽  
Electron Imaging ◽  
Scanning Electron

Biological ultrastructures have been extensively studied with the scanning electron microscope (SEM) for the past 12 years mainly because this instrument offers accurate and reproducible high resolution images of cell shapes, provided the cells are dried in ways which will spare them the damage which would be caused by air drying. This can be achieved by several techniques among which the critical point drying technique of T. Anderson has been, by far, the most reproducibly successful. Many biologists, however, have been interpreting SEM micrographs in terms of an exclusive secondary electron imaging (SEI) process in which the resolution is primarily limited by the spot size of the primary incident beam. in fact, this is not the case since it appears that high resolution, even on uncoated samples, is probably compromised by the emission of secondary electrons of much more complex origin.When an incident primary electron beam interacts with the surface of most biological samples, a large percentage of the electrons penetrate below the surface of the exposed cells.

Development of the Foramen Secundum in the Chick as Observed by Scanning Electron Microscopy

1976 ◽  
Vol 34 ◽  
pp. 212-213
Author(s):  
M.J.C. Hendrix ◽  
D.E. Morse
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Procaine Hydrochloride ◽  
Cardiac Anomaly ◽  
Chick Embryos ◽  
Congenital Cardiac Anomaly ◽  
Septal Defects ◽  
Critical Point Drying ◽  
Scanning Electron

Atrial septal defects are considered the most common congenital cardiac anomaly occurring in humans. In studying the normal sequential development of the atrial septum, chick embryos of the White Leghorn strain were prepared for scanning electron microscopy and the results were then extrapolated to the human heart. One-hundred-eighty chick embryos from 2 to 21 days of age were removed from their shells and immersed in cold cacodylate-buffered aldehyde fixative . Twenty-four embryos through the first week post-hatching were perfused in vivo using cold cacodylate-buffered aldehyde fixative with procaine hydrochloride. The hearts were immediately dissected free and remained in the fixative a minimum of 2 hours. In most cases, the lateral atrial walls were removed during this period. The tissues were then dehydrated using a series of ascending grades of ethanol; final dehydration of the tissues was achieved via the critical point drying method followed by sputter-coating with goldpalladium.

Forensic Identifications Aided by Scanning Electron Microscopy of Silicone-Epoxy Microreplicas of Calcified and Cornified Structures

1975 ◽  
Vol 33 ◽  
pp. 678-679 ◽  
Author(s):  
R.F. Sognnaes
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Forensic Science ◽  
George Washington ◽  
Epithelial Surface ◽  
Extended Application ◽  
Tissue Surfaces ◽  
Non Destructive ◽  
Scanning Electron ◽  
Surface Changes

Sufficient experience has been gained during the past five years to suggest an extended application of microreplication and scanning electron microscopy to problems of forensic science. The author's research was originally initiated with a view to develop a non-destructive method for identification of materials that went into objects of art, notably ivory and ivories. This was followed by a very specific application to the identification and duplication of the kinds of materials from animal teeth and tusks which two centuries ago went into the fabrication of the ivory dentures of George Washington. Subsequently it became apparent that a similar method of microreplication and SEM examination offered promise for a whole series of problems pertinent to art, technology and science. Furthermore, what began primarily as an application to solid substances has turned out to be similarly applicable to soft tissue surfaces such as mucous membranes and skin, even in cases of acute, chronic and precancerous epithelial surface changes, and to post-mortem identification of specific structures pertinent to forensic science.

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