Transmission Electron Microscopy of Tissue Prepared for Scanning Electron Microscopy by Ethanol-Cryofracturing

Humphreys, W. J.; Spurlock, Ben O.; Johnson, J.

doi:10.3109/10520297509117045

Cited by 38 publications

(12 citation statements)

References 3 publications

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“…Investigators interested in the deep structure of cartilage have used cutting, tearing (breaking wet cartilage), and resin cracking techniques (Takata, 1974) to expose deep structures for SEM examination. Cryofracture after dehydration is superior to other techniques (Clark, 1985;Humphreys et al, 1975). Cryofractures tend to follow planes between the collagen fibers, or, because the frozen fibrils are brittle, break through them with minimal distortion.…”

Section: Considerations In the Preparation Of Cartilage For Semmentioning

confidence: 99%

Scanning electron microscopy of “fibrillated” and “malacic” human articular cartilage: technical considerations

Clark

Simonian

1997

Microsc. Res. Tech.

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Section: Considerations In the Preparation Of Cartilage For Semmentioning

confidence: 99%

Scanning electron microscopy of “fibrillated” and “malacic” human articular cartilage: technical considerations

Clark

Simonian

1997

Microsc. Res. Tech.

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“…In order to facilitate deep-etching, ethanol has been used in combination with freeze-drying or critical point drying for scanning electron microscopy (Boyde & Wood, 1969;Humphreys et al, 1974Humphreys et al, ,1975. Recently Schiller & Taugner (1980) have shown that fractured tissues can be deep-etched and replicated after prolonged soaking in ethanol.…”

Section: P E N E T R a T I N G C R Y O P R O T E C T A N T Smentioning

confidence: 99%

Chemical cryoprotection for structural studies

Skaer¹

1982

Journal of Microscopy

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KEY w o R D s. Cryoprotection, ultrastructure, freeze-fracture, microanalysis, glycerol, polymeric cryoprotectant, frozen sections. S U M M A R YWith the ascendency of techniques for ultrarapid cooling and the successful control of ice crystal damage by purely physical means, it has become necessary to make a case for the continued use of chemical antifreeze agents in any circumstances. These circumstances include the need to explore tissues deeper than those superficial layers, whose morphology can be preserved by ultrarapid cooling, the avoidance of superficial areas of damage inflicted by dissection and tissue slicing, and situations where the growth of ice crystals must be controlled throughout the specimen, as for example for the cutting of frozen sections.The control of ice crystal damage would ideally be by the avoidance of any freezing at all but in practice can be seen in terms of the control of ice crystal size, which in turn depends on the density of nuclei that develop in the specimen and the rate of accretion of water molecules to the crystallites that form on these nuclei. Chemical antifreeze agents act in a variety of ways to increase the density of nuclei and/or to reduce the rate of growth of ice crystals: by promoting nucleation, by enhancing subcooling and by increasing the viscosity of the extracellular medium. In this way, large numbers of small ice crystals are produced. The different types of cryofixative agent each have their own advocates, advantages, special applications and drawbacks and these must be taken into account when considering the range of methods available for the analysis of cryofixed material.

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“…Following this fixation, the tissues were dehydrated in increasing concentrations of ethanol/H,O (30%, 50%, 75%, 90%, and 100%). Each fixed and dehydrated tissue strip was frozen quickly in liquid nitrogen and then fractured manually with a razor blade into two roughly equal longitudinal halves to expose the internal matrix architecture, using a technique similar to that of Hump h r e y s a n d colleagues (20). T h e f r a c t u r e d rectangular strips of tissue were then critical point dried, aligned vertically, mounted, and sprayed with gold-palladium.…”

Section: Methodsmentioning

confidence: 99%

A quantitative analysis of matrix alignment in ligament scars: A comparison of movement versus immobilization in an immature rabbit model

Frank¹,

MacFarlane²,

Edwards³

et al. 1991

Journal Orthopaedic Research

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This investigation quantified the alignment of fibrillar matrix in normal rabbit medial collateral ligaments (MCLs) and in healing MCLs from animals treated with or without knee immobilization. Twenty-four immature female rabbits were given complete midsubstance injuries to their right MCLs. Fifteen of them had that knee pin immobilized in flexion, while the remaining nine were allowed unrestricted cage activity. Animals were sacrificed in groups of three at intervals of 3, 6, or 14 weeks after injury, and both healing MCLs and unoperated contralateral controls were fixed in situ for subsequent removal, freeze-fracture, and preparation for scanning electron microscopy (SEM). A random sampling of SEM photographs followed by automated, statistically validated image processing was used to quantify alignment of matrix in all samples. Results showed that nonimmobilized MCL scars in this model do remodel over 14 weeks of healing, returning to normal alignment values in that time. Surprisingly, MCL scars in immobilized knees were even better, with mean matrix alignments falling statistically within normal MCL limits at all healing intervals studied. If not due to an unknown sampling or fixation artifact, these results suggest that gross knee flexion and extension is not a prerequisite for scar matrix alignment in this immature model of ligament healing.

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Transmission Electron Microscopy of Tissue Prepared for Scanning Electron Microscopy by Ethanol-Cryofracturing

Cited by 38 publications

References 3 publications

Scanning electron microscopy of “fibrillated” and “malacic” human articular cartilage: technical considerations

Scanning electron microscopy of “fibrillated” and “malacic” human articular cartilage: technical considerations

Chemical cryoprotection for structural studies

A quantitative analysis of matrix alignment in ligament scars: A comparison of movement versus immobilization in an immature rabbit model

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