Quick-freezing of cultured cardiac cells in situ with special attention to the mitochondrial ultrastructure.

A new method has been developed which allows quick-freezing in situ of primary, cardiac cell cultures grown to confluence on gas-permeable membranes (Petriperm dishes). Small pieces of the growth substratum, with rhythmically beating myocardial cells, were slam-frozen, without cryoprotectants, against the surface of a helium-cooled copper block at approximately 16 K. The quality of the cellular cryopreservation, as judged by ultrastructural criteria, was studied in freeze-substituted specimens processed for transmission electron microscopy. The ultrastructure of cryofixed cardiac cells was compared with that of unfrozen, chemically fixed samples. The severity of cryodistortions increased progressively with increasing distance from the point of first impact. Of particular interest were the dramatic alterations of the mitochondrial ultrastructure. The concept that the reticular and the outer mitochondrial membranes are intimately and strongly associated was clearly demonstrated. Optimally frozen material revealed cryopreserved ultrastructure of high quality. The method described appears to offer an ideal model system for correlating the information gained by phase-contrast microscopy of living cell cultures with the ultrastructure of the same samples fixed in situ by chemical or physical techniques. Cryofixation would be particularly useful for studying dynamic cellular processes associated with physiological and pathophysiological conditions, e.g. metabolic inhibition, anoxia and substrate deprivation.

Fibronectin and laminin in transverse tubules of cardiac myocytes studied by laser confocal microscopy and immunocytochemistry.

The distribution of two non-collagenous glycoproteins of high molecular weight, fibronectin (FN) and laminin (LMN), was investigated in myocardial cells from the ventricle of rats, and from biopsies collected from the auricle of patients undergoing a coronary bypass operation. In order to elucidate the expression of FN and LMN across cells, non-invasive serial sectioning has been carried out by laser scanning confocal microscopy of frozen, immunostained tissue sections. In addition, immunoelectron microscopy was used to study the distribution of these antigens at higher magnifications. These studies show that FN is part of the basement membrane of the surface sarcolemma of both ventricular and atrial cells, in addition to being an abundant protein of the extracellular matrix (ECM). Along transverse tubular(TT)-membranes, FN was only detected in tubules exceeding 200 nm in diameter. Even here, the intensity of labelling varied greatly and was generally low. By contrast, a heavy investment of LMN was organized in the basal lamina along the surface sarcolemma and along ramifications of the entire TT-system in ventricular heart muscle cells. In this way, the network of TT-membrane systems of working heart muscle cells provides a supply of LMN to all depths of the myocardial fibre. In human atrial muscle cells, a regular TT-system appears to be absent. Instead occasional, deep sarcolemmal invaginations occur with diameters of 300-500 nm, the surfaces of which are also invested with LMN. The significance of the present findings has been discussed, with special reference to LMN as a possible component of a series of proteins involved in transmembrane communication between the ECM and the sarcoplasm.

Cryopreservation evaluated with mitochondrial and Z line ultrastructure in striated muscle.

Single, intact, frog skeletal muscle fibres and whole frog hearts were quick-frozen on a polished, liquid-He-cooled copper block and examined in the electron microscope after freeze-substitution and freeze-fracture. In both kinds of striated muscle, collapse of the peripheral and intracristal membrane spaces in mitochondria was found to increase with increasing distance from the point of first impact (PFI) of the muscle cells on the cold copper block. The changes correlated with a previously described gradient of Z line and A band cryodamage occurring with distance from the PFI. The findings in thin sections from freeze-substituted preparations were confirmed by freeze-fracture preparations. It is concluded that, since the mitochondrial membrane changes are concurrent with, and follow the same spatial distribution of, other manifest cryoartefacts, the cryoartefactual nature of the mitochondrial changes must be excluded before functional significance is attributed to them. The collapse of mitochondrial membrane spaces as a sensitive indicator of quality of cryopreservation may apply to non-muscle cells as well.

A correlative study of the freezing patterns in rat myocardium using scanning and transmission electron microscopes.

Rat myocardial tissue, both fresh and chemically fixed, was quench frozen in melting Freon 22 at rates that resulted in in the formation of large ice-crystals. The material was studied with both scanning and transmission electron microscopy. Particular attention was paid to the characteristic freezing patterns within the myofibrils and mitochondria, and to features exposed by such freezing that might otherwise remain obscured. The freezing patterns found in tissue subjected to different processes were compared with the ultrastructure of unfrozen, chemically fixed counterparts. Ice-crystal cavities in the contractile material varied considerably along the length of a given myofiber in which the freezing front had progressed in parallel with the long axis of the myofibers. It is suggested that the intercalated disc functioned as a barrier to the freezing process. Ice generally compressed and distorted the contractile material beyond recognition, although the positions of the Z-bands remained evident and in register. Most single-fixed mitochondria were devoid of visible ice-crystal cavities, and the cristae generally remained intact. On the other hand, ice-crystal cavities were commonly seen between cristae of double-fixed mitochondria. No signs of perforations were seen in lipid bilayer membranes. Numerous strand-like connections between mitochondria as well as between mitochondria and the myofibrillar surface, which were believed to be sarcotubules, were made visible by the slow freezing process. Other strands, transverse to the myofibrils, connecting adjacent Z-bands and joining Z-bands to the sarcolemma, were interpreted as cytoskeletal elements. These strands, together with what apparently were SR tubules, exhibited high resistance to the stresses associated with the formation of ice-crystals.

Influence of plasma proteins on erythrocyte morphology and sedimentation.

The influence of plasma proteins on erythrocytes was studied by interference microscopy, scanning electron microscopy (SEM), and by Westergren erythrocyte sedimentation rate (ESR). Albumin kept erythrocytes dispersed as discoid spheres. Fibrinogen seemed responsible for the rouleaux phenomenon, but needed the co-influence of an immunoglobulin to induce rouleaux type of aggregates and high ESR. IgG, IgA and IgM caused immunologic type of aggregates. Albumin acted synergistically with fibrinogen and immunoglobulins. Normal blood contained a network of rouleaux, which probably explained the low normal ESR. High ESR was either due to rouleaux type aggregates where fibrinogen was dominant, or immunologic type aggregates where IgG, IgA or IgM were dominant proteins. Cold agglutinin disease showed normal blood morphology and normal ESR at 37 degrees C and immunologic type aggregates and high ESR at 25 degrees C.

An ultrastructural study of cryofractured myocardial cells with special attention to the relationship between mitochondria and sarcoplasmic reticulum.

A comparative study of various cryofracturing techniques has been conducted on the mammalian myocardial cell. Quench freezing of fresh or fixed tissue in melting Freon 22 resulted in severe cellular damage due to ice crystallization. Fixation with Karnovsky's fixative prior to quenching had no modifying effect on the size and distribution of the ice crystals. The crystals were orientated primarily in the direction of the long axis of the myofibrils, manifested as empty tube-like structures in the scanning electron microscope (SEM). Regular cross-bridging often seen at the Z-band levels indicated that ice crystals, at least in some portions of the cells, were confined within the sarcomere. Within the same cell the size of the ice crystals could vary considerably. Treatment of the tissue with polyvinylpyrrolidone (PVP) prior to rapid freezing had no noticeable cryoprotective effect. The surface of the thin layer of PVP surrounding the freeze dried tissue appeared amorphous in the SEM. However, the first evidence of ice crystallization was found a few micrometers under the surface. The freezing artefacts were completely circumvented if the cryofracturing was carried out on ethanol-impregnated or on critical point dried material. While the first method resulted in a smooth fracture plane passing through the cell structures, the intracellular fracture plane of the critical point dried material followed the surface of the cell organelles. Separation of the cell organelles caused by freezing or by critical point drying revealed thread-like structures extending from the mitochondrial surface. Re-examination of SEM-processed material in the transmission electron microscope (TEM) revealed that these structures were part of the sarcoplasmic reticulum (SR), and that a close contact between the SR and the outer mitochondrial membrane existed. TEM of conventional prepared material revealed that strands of electron-dense material, here named 'mito-reticular junctional fibres', bridged the narrow gap between the mitochondrial surface and the SR. It is suggested that these fibres have a specific anchoring function.

Transition temperatures in the sensitivity of Escherichia coli B/r to lysozyme.

Lysozyme attacked Escherichia coli B/r in the absence of EDTA or imposed osmotic shocks when the cells were rapidly cooled below specific temperatures. Cells subjected to lysozyme while being cooled to below 20 degrees C began to lose ability to subsequently form colonies. This sensitivity increased with decreasing temperatures and almost all cells cooled to 0 degrees C were affected. Slightly hypertonic solutions did not improve survival. Cells cooled first to as low as 5 degrees C and then subjected to lysozyme while cool did not lose their ability to form colonies subsequent to rewarming. However, 70% of the cells cooled first to 0 degrees C and subjected to lysozyme lost their colony-forming ability. Cell lysis also began when treated near 5 degrees C, but even when treated at 0 degrees C about 50% of the cells maintained their rod shape in the presence of lysozyme. These results are discussed in terms of a possible phase transition in a portion of the cell envelope and/or a transient osmotic swelling as a results of metabolic pumps failing at the low temperatures.

Patterns in quench-frozen, freeze-dried, blood proteins.

Scanning electron micrographs of whole blood prepared with cryotechnics demonstrated patterns assumed to be artifacts of the freezing process. A SEM investigation of the effects of freezing and freeze-drying on columns of purified blood proteins has been carried out using quench-freezing and cryo-fracturing followed by freeze-drying. Correlative studies with a light microscope and a transmission electron microscope are included. The resulting patterns depended on the type and concentration of protein as well as on the position within the sample and the orientation of the fracture plane relative to the column axis.

Large surface blebs on Escherichia coli heated to inactivating temperatures.

Large surface blebs were observed with phase-contrast optics on Escherichia coli B/r and B(s-1) heated to temperatures at which colony-forming ability was lost. Characterization of such blebs was consistent with the view that they were formed by a physical process and were bounded by the outer membrane of the cell. A hypothesis for thermal inactivation of E. coli is presented that places membrane damage near the primary lethal event.

Osmotic pressure in Escherichia coli as rendered detectable by lysozyme attack.

The enhanced susceptibility of plasmolyzed Escherichia coli to lysozyme attack was used to estimate the internal osmotic pressure of these cells under various conditions. Differences were detected between strains, culture media, stages in the growth cycle, and the osmotically active material used to produce plasmolysis. Lysozyme also was found to attack unplasmolyzed cells at 0 C and between 50 and 70 C.

Response of Escherichia coli B-r to high concentrations of sucrose in a nutrient medium.

Escherichia coli B/r was subjected to sucrose concentrations up to 1 m in the presence of Nutrient Broth. Plasmolysis seldom was evident 2 min after this treatment. The subsequent response was characterized by transient decreases in optical density as well as changes in appearance as seen under phase optics. No transient effects were detected in the synthetic rates or in the division of the survivors.

Plasmolysis of Escherichia coli B-r with sucrose.

Escherichia coli B/r cells were plasmolyzed in sucrose solutions and observed under phase contrast. The prevalence of plasmolysis under various conditions was noted, and the degree of plasmolysis was categorized as slight, extensive, or severe. The presence of ions reduced the prevalence of plasmolysis. Survival curves showed that extensive plasmolysis was not lethal to colony-forming ability.