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Freeze fracture technique ascertains that sarcoplasmic reticulum vesicles fuse during anisodiametric dehydration which leads to the formation of sheetlike structure of a mean extension of 5000 A. Modification of the membrane lipids by phospholipase A2 digestion or the incorporation of deoxycholate facilitates the coalescence of the vesicles, while it is completely prevented by lipid removal. Membrane fusion during anisodiametric dehydration is considered as resulting from the close contact of highly curved edges from which the membrane proteins have been excluded.
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PMID:Effect of lipid modification on fusion of sarcoplasmic reticulum vesicles. 15 Jan 48

Scanning electron microscopy (SEM) of human peripheral blood lymphocytes (hPBL) processed by two different methods reveals the existence of two main types of cell: villous and smooth. Freeze-fracture of hPBL, also processed by two methods confirms the existence of these morphological types of lymphocyte. Moreover, although the stereo surface replica technique is not a suitable method for the study of hPBL surface due to the lability of the lymphocyte replicas, some smooth cells were also encountered. Qualitative and quantitative analysis of the number, size, length and distribution of microvilli show that variations in the experimental conditions for SEM affect the surface topography of hPBL, indicating the need for studies on the effects of methods of cell collection, fixation and dehydration on the surface morphology of lymphocytes.
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PMID:Freeze-fracture and scanning electron microscopy of lymphocytes: effect of different preparatory techniques on cell surface morphology. 31 37

The effects of several dehydration treatments on the synaptonemal complex (SC), histone solubility in 2.0 M NaCl, and histone-DNA interaction in unfixed rat spermatocytes were evaluated. Freeze substitution with ethanol or dehydration with polyethylene glygol resulted in loss of the SC, preservation of histone solubility and DNA-histone salt linkages. Dehydration with ethylene gylcol or hexylene glycol resulted in preservation of SC with a clear delineation of attachment of the chromatin fibrils to the lateral elements, but a loss of histone solubility and histone-DNA linkages. Dehydration to a fifty percent concentration with glycerol with completion of dehydration with ethylene glycol had the same effect but also resulted in an even distribution of chromatin fibrils. Dehydration with glycerol alone resulted in clumping of chromatin and loss of SC structure, histone solubility and histone-DNA linkages. Partial dehydration to a fifty percent concentration with these three solvents followed by freeze substitution with ethanol resulted in the loss of SC structure and histone solubility but the preservation of histone-DNA linkages. It is likely that these nonaqueous solvents affected the histone hydrophobic groups and thereby altered histone conformation and interactions. These alterations, depending on the treatment used, resulted in the loss or preservation of SC, histone solubility and histone-DNA interactions thereby indicating that the hydrophobic interactions of the histones are crucial for the preservation of these feature of meiotic chromosomes. These results also demonstrate that neither does the preservation of the histone-DNA salt linkages suffice for the preservation of the SC nor does their disruption necessarily result in its loss. The lysine-rich histones, particularly that one unique to meiotic cells, may through their interactions play a crucial role in SC structure.
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PMID:Cytochemical and ultrastructural studies on the synaptonemal complex of rat spermatocytes. 32 69

Three methods usually applied in preparing biological material for the scanning electron microscope were tested by the investigation of two species of amoebae with different content of water (Amoeba proteus, Vannella simplex). Air drying resulted in both the production of cell shrinkage and cell distortion. When the specimens were dryed from media with increasing vapour-pressure, more satisfactory preservation of surface structures could be obtained. The sequence of potency was: Ethanol, chloroform, isopentane, ethyl ether, freon 11 and freon 13. Short drying periods proved to be more favourable than long ones. Critical-point drying provided much better details of cell surface morphology in both amoebae species. However, some arteficial changes were still detectable as small breaks and destruction of the mucous layer. They must be attributed to the fixation and dehydration procedure. Freeze drying turned out to be superior to both air drying and critical-point drying. Specimens prepared by this method showed no visible differences in cell surface morphology compared to living cells. As a consequence of the relatively high content of water the preparation of A. proteus was more difficult than that of V. simplex.
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PMID:[Preparation of biological specimens for the scanning electron microscope (author's transl)]. 79 34

The ultrastructure of bull and boar spermatozoa was investigated following different cryopreparation methods and chemical fixation. Spray-freezing was used for cryofixation in both freeze-etching and freeze-drying studies. Freeze-etching of boar spermatozoa revealed that the arrangement of the postnuclear striations differed from that in the bull. Freeze-drying gave excellent results for structural preservation, which were equal to those of chemical fixation. Some structural details not visible in chemically fixed cells were detected in freeze-dried and vacuum-embedded bull and boar spermatozoa, e.g. the arrangement of the lamellar nuclear contents, known from freeze-fractures, and a fine lamellar structure of the acrosomal contents. Cryofixation by spray-freezing combined with freeze-drying makes any contact of the cells with fixatives, buffer solutions and dehydration media unnecessary, and potentially provides all the advantages of ultrathin sectioning required for histochemical studies.
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PMID:A comparison of the ultrastructure of spray-frozen and freeze-etched or freeze-dried bull and boar spermatozoa with that after chemical fixation. 99

The cell envelope architectures and cytoplasmic structures of Mycobacterium aurum CIPT 1210005, M. fortuitum, M. phlei 425, and M. thermoresistible ATCC 19527 were compared by conventional embedding and freeze-substitution methods. To ascertain the integrity of cells during each stage of the processing regimens, [1-14C]acetate was incorporated into the mycolic acids of mycobacterial walls, and the extraction of labeled mycolic acids was monitored by liquid scintillation counting. Radiolabeled mycolic acids were extracted by both processing methods; however, freeze-substitution resulted in the extraction of markedly less radiolabel. During conventional processing of cells, most of the radiolabel was extracted during the dehydration stage, whereas postsubstitution washes in acetone yielded the greatest loss of radiolabel during freeze-substitution. Conventional embedding frequently produced cells with condensed fibrous nucleoids and occasional mesosomes. Their cell walls were relatively thick (approximately 25 nm) but lacked substance. Freeze-substituted cells appeared more robust, with well-dispersed nucleoids and ribosomes. The walls of all species were much thinner than those of their conventionally processed counterparts, but these stained well, which was an indication of more wall substance; the fabric of these walls, in particular the plasma membrane, appeared highly condensed and tightly apposed to the peptidoglycan. Some species possessed a thick, irregular outer layer that was readily visualized in the absence of exogenous stabilizing agents by freeze-substitution. Since freeze-substituted mycobacteria retained a greater percentage of mycolic acids in their walls, and probably other labile wall and cytoplasmic constituents, we believe that freeze-substitution provides a more accurate image of structural organization in mycobacteria than that achieved by conventional procedures.
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PMID:Reevaluation of envelope profiles and cytoplasmic ultrastructure of mycobacteria processed by conventional embedding and freeze-substitution protocols. 140 Feb 3

We examined the suitability of freeze-substitution and Lowicryl HM20 embedding of aldehyde-fixed rat brain to localize several neural antigens at the ultrastructural level. The following rabbit polyclonal and mouse monoclonal antibodies were used: affinity-purified polyclonal immunoglobulins G raised to B-50/GAP43 (a membrane-anchored, growth-associated protein); affinity-purified polyclonal immunoglobulins G to human glial fibrillary acidic protein (GFAP; a subunit of glial filaments); a polyclonal antiserum raised to adrenocorticotropic hormone[25-39] (a neuropeptide present in dense-core granules); a polyclonal antiserum raised to myelin basic protein (a protein present in compact myelin of the central nervous system); and mouse monoclonal antibodies to synaptophysin (an integral membrane protein of small synaptic vesicles). Rat mesencephalon was fixed by perfusion with buffered 2% glutaraldehyde and 4% paraformaldehyde, cryoprotected, and frozen in liquid nitrogen. Freeze-substitution of tissue was performed with anhydrous methanol and 0.5% uranyl acetate at -90 degrees C. Semi-thin Lowicryl sections were used for light microscopic visualization of B-50 in the ventromedial mesencephalic central gray substance. The procedure preserves well the ultrastructure of this region and the immunoreactivity of the selected antigens. This study shows that dehydration by freeze-substitution, combined with Lowicryl HM20 embedding at sub-zero temperature, provides a successful method of preparation of fixed brain tissue for ultrastructural studies, allowing immunogold localization of several neural antigens by double labeling in the same section and in serial sections.
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PMID:Freeze-substitution and Lowicryl HM20 embedding of fixed rat brain: suitability for immunogold ultrastructural localization of neural antigens. 183 48

Freeze-substitution and more conventional embedding protocols were evaluated for their accurate preservation of eubacterial ultrastructure. Radioisotopes were specifically incorporated into the RNA, DNA, peptidoglycan, and lipopolysaccharide of two isogenic derivatives of Escherichia coli K-12 as representative gram-negative eubacteria and into the RNA and peptidoglycan of Bacillus subtilis strains 168 and W23 as representative gram-positive eubacteria. Radiolabeled bacteria were processed for electron microscopy by conventional methods with glutaraldehyde fixation, osmium tetroxide postfixation, dehydration in either a graded acetone or ethanol series, and infiltration in either Spurr or Epon 812 resin. A second set of cells were simultaneously freeze-substituted by plunge-freezing in liquid propane, substituting in anhydrous acetone containing 2% (wt/vol) osmium tetroxide, and 2% (wt/vol) uranyl acetate, and infiltrating in Epon 812. Extraction of radiolabeled cell components was monitored by liquid scintillation counting at all stages of processing to indicate retention of cell labels. Electron microscopy was also used to visually confirm ultrastructural integrity. Radiolabeled nucleic acid and wall components were extracted by both methods. In conventionally embedded specimens, dehydration was particularly damaging, with ethanol-dehydrated cells losing significantly more radiolabeled material during dehydration and subsequent infiltration than acetone-treated cells. For freeze-substituted specimens, postsubstitution washes in acetone were the most deleterious step for gram-negative cells, while infiltration was more damaging for gram-positive cells. Autoradiographs of specimens collected during freeze-substitution were scanned with an optical densitometer to provide an indication of freezing damage; the majority of label lost from freeze-substituted cells was a result of poor freezing to approximately one-half of the cell population, thus accounting for the relatively high levels of radiolabel detected in the processing fluids. These experiments revealed that gram-positive and gram-negative cells respond differently to freezing; these differences are discussed with reference to wall structure. It was apparent that the cells frozen first (ie., the first to contact the cryogen) retained the highest percentage of all radioisotopes, and the highest level of cellular infrastructure, indicative of better preservation. The preservation of these select cells was far superior to that obtained by more conventional techniques.
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PMID:Evaluation of freeze-substitution and conventional embedding protocols for routine electron microscopic processing of eubacteria. 210 31

Fourier-transform infrared spectroscopy was used to characterize the interaction of stabilizing carbohydrates with dried proteins. Freeze-drying of trehalose, lactose, and myo-inositol with lysozyme resulted in substantial alterations of the infrared spectra of the dried carbohydrates. In the fingerprint region (900-1500 cm-1), there were large shifts in the frequencies of bands, a decrease in absorbance, and a loss of band splitting. These effects mimic those of water on hydrated trehalose. Bands assigned to hydroxyl stretching modes (around 3350 cm-1) were decreased in intensity and shifted to higher frequencies in the presence of the protein. In complementary experiments, it was found that dehydration-induced shifts in the positions of amide I and amide II bands for lysozyme could be partially and fully reversed, respectively, when the protein was freeze-dried in the presence of either trehalose or lactose. In addition, the carboxylate band, which was not detectable in the protein dried without the sugar, was apparent when these sugars were present. myo-Inositol was less effective at shifting the amide bands, and the carboxylate band was not detected in the presence of this carbohydrate. Also tested was the concentration dependency of the carbohydrates' influence on the position of the amide II band for dried lysozyme. The results showed that the ability of a given concentration of a carbohydrate to shift this band back toward the position noted with the hydrated protein coincided, at least in the extreme cases, with the capacity of that same level of carbohydrate to preserve the activity of rabbit skeletal muscle phosphofructokinase during freeze-drying.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:An infrared spectroscopic study of the interactions of carbohydrates with dried proteins. 252 52

Glycerol and spermine prevented the freeze-induced alteration of lipid character in membrane fragments derived from Escherichia coli B logarithmic-phase cells, protecting against loss of membrane function and subsequently maintaining higher cell viability. The membrane specimens derived from the stationary-phase cells exhibited high resistance to freezing-induced alteration of the membrane lipid character, and to freezing injury. Freeze-drying of membrane fragment specimens from both logarithmic- and stationary-phase cells gave rise to alterations in lipid state similar to those shown in freeze-thawing of logarithmic-phase cell membrane specimens. Freeze-drying brought about reduction of cell viability in both growth phase specimens. It is suggested, therefore, that the fundamental cause of the injury induced by freezing of living materials is dehydration of lipid-rich systems such as cellular membranes.
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PMID:Changes in chemical structure and function in Escherichia coli cell membranes caused by freeze-thawing. II. Membrane lipid state and response of cells to dehydration. 264 93


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