A copper block method for freezing non-cryoprotected tissue to produce ice-crystal-free regions for electron microscopy. I. Evaluation using freeze-substitution
ABSTRACT SUMMARYA wide variety of plant and animal tissues were prepared for electron microscopy by freeze-substitution, after rapid freezing on a liquid nitrogen cooled copper block by the van Harreveld method. Measurements were made of ice crystal size versus depth in tissues that had not been treated with any cryoprotectants. Ice crystal size increased exponentially with depth. It was confirmed that a narrow surface band (approximately 12 μm) was frozen sufficiently rapidly to prevent the formation of electron microscopically-visible ice crystals.
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ABSTRACT: Electronmicroscopicexaminationofepiphysealcartilagetissueprocessed by high pressurefreezing,freezesubstitution, and low temperature embedding revealeda substantial improvement inthe preservationqualityof intracellular organellesby comparison with the resultsobtained under conventional chemical fixationconditions. Furthermore, allcells throughout the epiphysealplate,includingthe terminalchondrocyte adjacentto the region of vascularinvasion,were found to be structurallyintegral .A zone of degenerating cells consistentlyobserved in cartilagetissueprocessed under conventional chemical fixation conditionswas notapparent.Hence, itwould appear thatcelldestructioninthisregionoccurs during chemical processingand isnot a featureofcartilagetissueinthe nativestate.Since thesecellsaresituatedina regionwhere tissuecalcification istakingplace,the implicationis thatthe onsetand progressionofcartilagecalcification are,atleastpartially, controlledby the chondrocytes themselves.The observationthatthe terminalcelladjacentto the zone of vascularinvasionisviablehas important implicationsinrelationto the theory of vascular invasion.Thismay now requirereconceptualizationtoaccommodate thepossibility thatactive celldestructionmay be a preconditionforvascularinvasion.
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ABSTRACT: A freeze-substitution technique is described which enables the ultrastructure of certain types of plant transfer cells to be preserved with minimal ice crystal damage. The ultrastructure of transfer cells fromFunaria, Lonicera, andSenecio after freeze-substitution has been compared with that of glutaraldehyde-osmium fixed material. The irregular clear zone between wall and plasma membrane, present in conventional preparations, is absent in freeze-substituted tissue. It is proposed that this interfacial zone is an artefact caused by expansion of wall ingrowth material during conventional fixation procedures. In transfer cells with a complex wall labyrinth the swelling of wall material severely disrupts the true structure of the wall-membrane apparatus and results in a large decrease in the surface to volume ratio of the protoplast. These findings are supported in the case ofFunaria by a freezefracture study. The reactivity of the plasma-membrane to the PTA/chromic acid stain is enhanced in freeze-substituted material. Use of theThiry silver proteinate reagent in conjunction with freeze-substitution has revealed marked differences between the wall ingrowths ofFunaria sporophyte haustorium transfer cells and those ofLonicera nectary trichomes.Protoplasma 01/1977; 93(1):7-26. DOI:10.1007/BF01276279 · 3.17 Impact Factor
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ABSTRACT: The fine structure of the actomyosin system of Physarum polycephalum was investigated in vitrified specimens after applying a pressure of greater than 2.1 kbar and freezing rates of 500 to 5,000 degrees C/s. The frozen specimens were either freeze-substituted or freeze-fractured and compared with material processed according to conventional methods of freeze-etching preparation. Artifactual alterations, as seen in the form of destroyed areas of the cytoplasm after chemical fixation, were not observed after freeze-substitution. However, small ice crystals formed by recrystallization within most of the cytoplasmic actomyosin fibrils prevented a fine structural analysis. Such a destruction of the fibrillar fine structure was not found after freeze-etching. In replicas of deep-etched objects 10 nm-thick filaments were localized, which could be conclusively identified as F-actin. The actin filaments are located randomly in the peripheral cytoplasm forming the cell cortex. By the process of parallel aggregation, the filaments can be differentiated to fibrils. Thick myosin filaments were not observed. However, structures resembling cross bridges between single actin filaments suggest the existence of oligomeric myosin. The present investigation shows that, in addition to biomembranes, other cytoplasmic differentiations such as components of the groundplasm can be successfully demonstrated employing the deep-etching technique when the freezing methods are improved by avoiding freeze-protection pretreatments.Cell and Tissue Research 02/1981; 217(3):479-95. DOI:10.1007/BF00219359 · 3.33 Impact Factor