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PubMed1965

TRICOMPLEX FIXATION OF PHOSPHOLIPIDS.

A basis for the interpretation of the structure of the cell membrane is often looked for in electron microscope investigations on the structure of lipid models. A difficulty in these investigations is our lack of knowledge of the effect of the preparative treatment on the structure studied. This applies especially to the strongly oxidizing fixatives: osmium tetroxide and potassium permanganate. These agents have, moreover, the drawback that they cannot be used to fix fully saturated lipids. On the basis of existing theories concerning complex colloid systems, a fixation method was developed that allows the electron microscope study of the structure of phospholipid models, irrespective of whether they consist of saturated or unsaturated compounds. With this fixation, namely tricomplex flocculation by means of suitable ions, the structure of the lipid molecules is not altered. Moreover, the site and mode of action of this fixation are well known. The first application of this method to the study of isolated beef brain phospholipids is described.

Biophysical Phenomena↗
PubMed1965

THE FINE STRUCTURE OF CHLOROPLAST STROMA FOLLOWING ALDEHYDE OSMIUM-TETROXIDE FIXATION.

Markedly improved fixation of leaf tissues is obtained by means of a glutaraldehyde (or acrolein)-osmium tetroxide procedure, as compared with the results of potassium permanganate or osmium tetroxide fixation methods. The procedure has proved useful in all species so far examined. Chloroplasts are particularly well preserved. In this paper details of components of the ground-substance of Avena sativa plastids are presented. They include the following:-(i) The "tromacentre" is an area of aggregated fibrils, each 85 A in diameter, and of uncertain length. Individual fibrils may be composed of subunits. The whole aggregate is usually up to 1 micro in diameter, and is visible in thin sections in the light microcope. It is present at all stages of plastid development, and, under conditions of rapid synthesis in the plastid, it may be up to 2 micro in diameter. Evidence that it is proteinaceous is presented. Osmiophilic globules are often associated with it. (ii) Areas which resemble bacterial and blue-green algal nucleoplasms, containing fibrils approximately 30 A wide. These regions are smaller than the stromacentre and, like that structure, they occur in all stages of plastid development. Unlike it, there are several such areas per chloroplast. (iii) Particles which have some of the morphological and staining characteristics of ribosomes. Present at all stages of development, they are approximately two-thirds the size of the cytoplasmic ribosomes. They can occur in groups, thus resembling polyribosomes. (iv) The remaining material is granular, and may include dissociated portions of stromacentre material. The validity of the observations and their significance is discussed.

Aldehydes↗
PubMed1965

A STUDY OF EXTRACELLULAR SPACE IN CENTRAL NERVOUS TISSUE BY FREEZE-SUBSTITUTION.

It was attempted to preserve the water distribution in central nervous tissue by rapid freezing followed by substitution fixation at low temperature. The vermis of the cerebellum of white mice was frozen by bringing it into contact with a polished silver mirror maintained at a temperature of about -207 degrees C. The tissue was subjected to substitution fixation in acetone containing 2 per cent OsO(4) at -85 degrees C for 2 days, and then prepared for electron microscopy by embedding in Maraglas, sectioning, and staining with lead citrate or uranyl acetate and lead. Cerebellum frozen within 30 seconds of circulatory arrest was compared with cerebellum frozen after 8 minutes' asphyxiation. From impedance measurements under these conditions, it could be expected that in the former tissue the electrolyte and water distribution is similar to that in the normal, oxygenated cerebellum, whereas in the asphyxiated tissue a transport of water and electrolytes into the intracellular compartment has taken place. Electron micrographs of tissue frozen shortly after circulatory arrest revealed the presence of an appreciable extracellular space between the axons of granular layer cells. Between glia, dendrites, and presynaptic endings the usual narrow clefts and even tight junctions were found. Also the synaptic cleft was of the usual width (250 to 300 A). In asphyxiated tissue, the extracellular space between the axons is either completely obliterated (tight junctions) or reduced to narrow clefts between apposing cell surfaces.

Animals↗
PubMed1965

AUTORADIOGRAPHIC STUDIES OF INTRACELLULAR CALCIUM IN FROG SKELETAL MUSCLE.

Autoradiographs consisting of a 1000 A thick tissue section and a 1400 A thick emulsion film have been prepared from frog toe muscles labeled with Ca(45). The muscles had been fixed with an oxalate-containing osmium solution at rest at room temperature, at rest at 4 degrees C, during relaxation following K(+) depolarization or after prolonged depolarization. From 6 to 39 per cent of K(+) contracture tension was produced during fixation. The grains in the autoradiographs were always concentrated in the center 0.2 to 0.3 micro of the I band and the region of the overlapping of the thick and thin filaments. The greater the tension produced during fixation, the greater was the concentration in the A band and the smaller the concentration in the I band. Autoradiographs of two muscles fixed by freeze-substitution resembled those of muscles which produced little tension during osmium fixation. Muscles which shortened during fixation produced fewer grains. In the narrow (<2.0 micro) sarcomeres of the shortened muscles, grain density decreased with decreasing sarcomere width. A theoretical analysis of the significance of these grain distributions is proposed and discussed.

Animals↗