A copper block method for freezing non‐cryoprotected tissue to produce ice‐crystal‐free regions for electron microscopy

Journal of Microscopy (Impact Factor: 1.63). 08/2011; 106(3):251 - 260. DOI: 10.1111/j.1365-2818.1976.tb02405.x

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.