ELECTRON MICROSCOPE OBSERVATIONS ON FROZEN-DRIED CELLS

To explore the problem of artefacts which may be produced during usual fixation, dehydration, and embedding, the authors have examined pancreas, liver, and bone marrow frozen at about -180°C., dried, at -55 to -60°C., embedded in methacrylate, sectioned, and floated on a formol-alcohol mixture. By these treatments the labile structure of living cells can be fixed promptly and embedded in methacrylate avoiding possible artefacts caused by direct exposure to chemical fixatives. Cell structures are ultimately exposed to a fixative when the sections are floated on formol-alcohol, but at this stage artefacts due to chemical fixation are expected to be minimized, as the fixatives act on structures tightly packed in methacrylate polymer. In the central zone of tissue blocks so treated, the cells are severely damaged by ice crystallization but at the periphery of the blocks the cell structure is well preserved. In such peripherally located cells, elements of the endoplasmic reticulum (ER), Palade's granules, homogeneously dense mitochondria, and nuclear envelopes and pores, can be demonstrated without poststaining with OsO₄. The structural organization in the nucleus is distorted by vacuolization. The mitochondrial membranes and cristae, cellular membrane, and the Golgi apparatus, however, are detected only with difficulty. The Golgi region generally appears as a light zone, in which some ambiguous structures are encountered. After staining the sections with OsO₄ or Giemsa solution, an inner mitochondrial structure which resembles the cristae seen in conventional OsO₄-fixed specimens appears, but the limiting membrane is absent. Treatment with OsO₄ or Giemsa solution also renders more distinct the membrane of the ER and Palade's granules but not the Golgi apparatus and cellular membrane. Treatment with ribonuclease results in the disappearance of Palade's granules. On the strength of these observations the authors conclude that OsO₄ fixation gives a satisfactory preservation of such cell structures as the nuclear envelope, endoplasmic reticulum, and Palade's granules, though it may induce slight swelling of these cell components.     

Greasing the wheels of secretory transport

Ever since George Palade's pioneering studies of zymogen secretion from pancreatic acinar cells, the underlying molecular mechanisms of vesicle-mediated protein transport have captivated cell biologists and biochemists. A watershed meeting on "Phosphoinositides and the Golgi", held at the Fogarty International Center of the National Institutes of Health in Bethesda, Maryland (March 13-14, 2001), provided reinterpretation and striking new insights about the functions of this phospholipid class in intracellular protein trafficking.     

The fine structure of the oxytalan fiber

Maraglas-embedded sections of periodontal membranes around continually growing incisors of Sprague-Dawley rats fixed consecutively in cold glutaraldehyde and Palade's 1 % buffered osmium tetroxide were examined under the electron microscope for oxytalan connective tissue fibers. Oxytalan fibers were noted to consist of bundles of filaments approximately 150 A in diameter with an interfilamentous amorphous substance of approximately the same diameter. A periodicity of fibrillar elements was not obvious.     

George Emil Palade: charismatic virtuoso of cell biology

George Palade has created, shared and passed on a multidisciplinary view of the functional organization, biogenesis and dynamics of organelles. His open-mindedness and tenacity, along with his rigour and sense of intellectual elegance, have been remarkable. This focus on the logic of organelles defined a crucial turning point in biomedical science. The following article sketches Palade's research, as part of a larger community that flourished after the Second World War.     

Staining Residual Lipids in Ultrathin Sections of Tissues Embedded in Polyester Resin

Rat suprarenal glands fixed in Palade's 1% OsO₄, buffered at pH 7.7 with veronal-acetate, to which 0.1% MgCl₂ was added, were embedded in Vestopal-W and sectioned at 0.2-1 µ. The sections were attached to slides by floating on water, without adhesive, and drying at 60-80° C, placed in acetone for 1 min and then treated with the following staining procedure: Place the preparation in a filtered solution of oil red O, 1 gm; 70% alcohol, 50 ml; and acetone, C.P., 50 ml; for 0.5-1 hr. Rinse in absolute ethyl alcohol; drain; counterstain with 0.5% aqueous thionin for 5 min; rinse in distilled water; drain; stain in 0.2% azure B in phosphate buffer at pH 9, for 5 min. Dry and apply a drop of immersion oil directly on the section. The preparations are temporary. Ciaccio-positive lipids, rendered insoluble by OsO, fixation, stained red to ochre.     

Ultrastructural study of the neurosecretory granules in the sinus gland of the blue crab,Callinectes sapidus

Summary Seven morphologically different types of neurosecretory granules have been found in the axon terminals of the sinus gland of the blue crab, Callinectes sapidus. They differ from each other in size, shape, staining characteristics, solubility characteristics, core matrix characteristics, axon terminal matrix characteristics, presence or absence of space between the granule membrane and granule core matrix, and frequency of occurrence. Five of the types are segregated in different axon terminals and are believed to represent different hormone-protein complexes. Two of the types, which have lost part or all of their granular contents, are thought to be variants of the other five types. The differences in granular morphology are better revealed by some fixation procedures than others. Palade's acetate-veronal buffered osmium tetroxide, in particular, reveals striking differences. The following observations suggest that different hormone-protein complexes are segregated in different axon terminals and that these complexes may be morphologically distinguished at the level of the electron microscope.Supported by USPHS-NIH Training Grant GM-00669 and Grant GB-7595X from the National Science Foundation.     

Retinal capillary junctions: Ultrastructural tight junction artefacts induced by sodium ions and membrane reduction in streptozotocin diabetes

Summary Retinal capillary junctions were analysed in normal and diabetic rats and in a human retina with the electron microscope. Diabetes mellitus was induced with Streptozotocin. The retinae were fixed in Palade's osmium tetroxide containing sodium or calcium ions and block-stained in uranyl acetate.With Ca-fixation, no significant difference in interendothelial cleft width was detected between retinal layers or between normal and diabetic retinae. Diabetes caused a narrowing of the clefts in the Na-fixed tissue (X±SE, n=375; Normal: 78.6±3.00 Å; Diabetic: 57.7 ±2.42 Å; p0.001). A significant correlation was found between cleft width and the length of the tight junctions or zonulae occludentes (p<0.001). In the nerve fibre layer of the Nadiabetio retina, where cleft narrowing was greatest, there was an increase in length of the zonulae occludentes from 22.8±2.2% to 41.6±3.7% (p<0.001). Ca-fixation prevented these changes, indicating that at least some zonulae occludentes were interendothelial extraction artefacts.In the normal retina, endothelial cell membrane thickness was greater with Ca-than Na-fixation (p<0.001). Diabetes caused a decrease in membrane thickness of Ca-fixed tissue (p<0.001). The diabetic decrease in membrane thickness may explain the increased fragility and increased permeability of diabetic capillaries. Calcium binding by endothelial cell membranes is of primary importance in anticoagulation which is defective in diabetes.Work supported by a grant from the Taverna Estate and The Prince of Wales Hospital, Randwick, N.S.W., Australia. Presented in part at the 8th International Congress on Electron Microscopy held in Canberra, A.C.T., Australia, 1974.     

Phagotrophy and two new structures in the malaria parasite Plasmodium berghei

Blood collected from rats infected with Plasmodium berghei was centrifuged and the pellet was fixed for 1 hour in 1 per cent buffered OsO(4) with 4.9 per cent sucrose. The material was embedded in n-butyl methacrylate and the resulting blocks sectioned for electron microscopy. The parasites were found to contain, in almost all sections, oval bodies of the same density and structure as the host cytoplasm. Continuity between these bodies and the host cytoplasm was found in a number of electron micrographs, showing that the bodies are formed by invagination of the double plasma membrane of the parasite. In this way the host cell is incorporated by phagotrophy into food vacuoles within the parasite. Hematin, the residue of hemoglobin digestion, was never observed inside the food vacuole but in small vesicles lying around it and sometimes connected with it. The vesicles are pinched off from the food vacuole proper and are the site of hemoglobin digestion. The active double limiting membrane is responsible not only for the formation of food vacuoles but also for the presence of two new structures. One is composed of two to six concentric double wavy membranes originating from the plasma membrane. Since no typical mitochondria were found in P. berghei, it is assumed that the concentric structure performs mitochondrial functions. The other structure appears as a sausage-shaped vacuole surrounded by two membranes of the same thickness, density, and spacing as the limiting membrane of the body. The cytoplasm of the parasite is rich in vesicles of endoplasmic reticulum and Palade's small particles. Its nucleus is of low density and encased in a double membrane. The host cells (reticulocytes) have mitochondria with numerous cristae mitochondriales. In many infected and intact reticulocytes ferritin was found in vacuoles, mitochondria, canaliculi, or scattered in the cytoplasm.     

Phagotrophy and Two New Structures in the Malaria Parasite Plasmodium berghei

Blood collected from rats infected with Plasmodium berghei was centrifuged and the pellet was fixed for 1 hour in 1 per cent buffered OsO₄ with 4.9 per cent sucrose. The material was embedded in n-butyl methacrylate and the resulting blocks sectioned for electron microscopy. The parasites were found to contain, in almost all sections, oval bodies of the same density and structure as the host cytoplasm. Continuity between these bodies and the host cytoplasm was found in a number of electron micrographs, showing that the bodies are formed by invagination of the double plasma membrane of the parasite. In this way the host cell is incorporated by phagotrophy into food vacuoles within the parasite. Hematin, the residue of hemoglobin digestion, was never observed inside the food vacuole but in small vesicles lying around it and sometimes connected with it. The vesicles are pinched off from the food vacuole proper and are the site of hemoglobin digestion. The active double limiting membrane is responsible not only for the formation of food vacuoles but also for the presence of two new structures. One is composed of two to six concentric double wavy membranes originating from the plasma membrane. Since no typical mitochondria were found in P. berghei, it is assumed that the concentric structure performs mitochondrial functions. The other structure appears as a sausage-shaped vacuole surrounded by two membranes of the same thickness, density, and spacing as the limiting membrane of the body. The cytoplasm of the parasite is rich in vesicles of endoplasmic reticulum and Palade's small particles. Its nucleus is of low density and encased in a double membrane. The host cells (reticulocytes) have mitochondria with numerous cristae mitochondriales. In many infected and intact reticulocytes ferritin was found in vacuoles, mitochondria, canaliculi, or scattered in the cytoplasm.     

Sur la signification des vacuoles intraneuronales du noyau supra-optique de la souris en surcharge osmotique

Résumé La fixation osmiée et l'étude ultrastructurale permettent de préciser que les grosses vacuoles intraneuronales des cellules du N.S.O. de la Souris, qui apparaissent après une surcharge osmotique prolongée, ont un contenu lipidique.

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Summary Fixation with Palade's fluid and ultrastructural study reveal that after prolonged ingestion of hypertonic saline, the vacuoles in the Cells of the supraoptic nuclei of the Mouse contain lipid droplets.

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Dédié amicalement à Madame le Professeur Berta Scharrer, avec nos très respectueux hommages.

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Attachée de Recherches au C.N.R.S.

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Collaboratrice technique. Travail réalisé dans le cadre de la R.C.P. No 39 du C.N.R.S.     

A STUDY OF THE STRUCTURE OF SPLENIC SINUSES IN MAN AND IN THE ALBINO RAT WITH THE LIGHT MICROSCOPE AND THE ELECTRON MICROSCOPE

The splenic sinuses in the spleens of 5 human beings and 7 albino rats have been studied in the light microscope and electron microscope after fixation in Dalton's fluid and Palade's fluid and embedding in n-butyl methacrylate. Splenic sinuses are tortuous vascular channels of large but variable diameter which represent the first venous vessels in the spleen and make up almost the entire red pulp in man and in rats. These vessels are composed of reticulo-endothelial cells flattened to endothelial form and sheathed by a netted reticulum. The luminal surface of the endothelium is made highly irregular by delicate and variable cytoplasmic protrusions, slender corridors separating adjacent endothelial cells, anastomotic openings to other sinuses, bulgings of entire cells, and even thrusts of endothelium spanning the sinai lumen. The supporting reticulum presents a well developed latticed appearance in tangential sections of sinuses, but in most cuts is punctate or linear. The reticulum is composed of strands without limiting membranes, which, in substance, are amorphous and resemble basement membrane. Material identical in appearance to the substance of the reticulum may be present in the endothelium, suggesting that the reticulum is formed by endothelial cells. The endothelium also contains deposits of presumed ferritin and hemosiderin. The extreme luminal bulgings of endothelium suggest production of circulating monocytes or lymphocytes by detachment of endothelial cells. Sinuses are patent and collapsed to varying degrees. Patent sinuses are separated by collapsed sinuses and these collapsed sinuses appear to constitute splenic (Billroth) cords.     

The Influence of Electric Field and Confinement on Cell Motility

The ability of cells to sense and respond to endogenous electric fields is important in processes such as wound healing, development, and nerve regeneration. In cell culture, many epithelial and endothelial cell types respond to an electric field of magnitude similar to endogenous electric fields by moving preferentially either parallel or antiparallel to the field vector, a process known as galvanotaxis. Here we report on the influence of dc electric field and confinement on the motility of fibroblast cells using a chip-based platform. From analysis of cell paths we show that the influence of electric field on motility is much more complex than simply imposing a directional bias towards the cathode or anode. The cell velocity, directedness, as well as the parallel and perpendicular components of the segments along the cell path are dependent on the magnitude of the electric field. Forces in the directions perpendicular and parallel to the electric field are in competition with one another in a voltage-dependent manner, which ultimately govern the trajectories of the cells in the presence of an electric field. To further investigate the effects of cell reorientation in the presence of a field, cells are confined within microchannels to physically prohibit the alignment seen in 2D environment. Interestingly, we found that confinement results in an increase in cell velocity both in the absence and presence of an electric field compared to migration in 2D.     

Cell poration and cell fusion using an oscillating electric field.

It has been shown in previous studies that cell poration (i.e., reversible permeabilization of cell membrane) and cell fusion can be induced by applying a pulse (or pulses) of high-intensity DC (direct current) electric field. Recently we suggested that such electro-poration or electro-fusion can also be accomplished by using an oscillating electric field. The DC field relies solely on the dielectric breakdown of the cell membrane to induce cell fusion. The oscillating field, on the other hand, can produce not only a dielectric breakdown, but also a sonicating motion in the membrane that could result in a structural fatigue. Thus, a combination of a DC field and an oscillating field is expected to enhance the efficiency of cell poration and cell fusion. This study is an experimental test of such an idea. Here, pulses of high-intensity, DC-shifted RF (radio frequency) electric field were used to induce cell poration and cell fusion. The fusion experiments were done on human red blood cells. The poration experiments were done on a fibroblast cell line using a molecular probe (which is a DNA plasmid containing the marker gene chloramphenicol acetyltransferase, CAT) and assayed by a gene transfection technique. It was found that the pulsed RF field is highly efficient in both cell fusion and cell poration. Also, in comparison with electro-poration using a DC field, the RF field results in a higher percentage of cells surviving the exposure to the electric field.     

Modification of electrokinetic properties of nuclei in human buccal epithelial cells by electric fields

The influence of an alternating (50 Hz) electric field (5--110 V/cm) on the state of human buccal epithelium cells was studied by the methods of intracellular microelectrophoresis, heterochromatin staining with orcein, and indigo carmine staining for viability and membrane integrity evaluations. Electric field exposure induced an increase in electrophoretic mobility of cell nuclei, decreased numbers of heterochromatin granules near the inner membrane of cell nucleus, and induced cell membrane damage; but cell viability was conserved. Nuclear and cell membrane properties varied with electric field strength and age of the donors. The data obtained are interpreted as evidence of electric field induced activation of the functional state of nuclei.     

Magnetic field direction differentially impacts the growth of different cell types

Magnetic resonance imaging (MRI) machines have horizontal or upright static magnetic field (SMF) of 0.1–3 T (Tesla) at sites of patients and operators, but the biological effects of these SMFs still remain elusive. We examined 12 different cell lines, including 5 human solid tumor cell lines, 2 human leukemia cell lines and 4 human non-cancer cell lines, as well as the Chinese hamster ovary cell line. Permanent magnets were used to provide 0.2–1 T SMFs with different magnetic field directions. We found that an upward magnetic field of 0.2–1 T could effectively reduce the cell numbers of all human solid tumor cell lines we tested, but a downward magnetic field mostly had no statistically significant effect. However, the leukemia cells in suspension, which do not have shape-induced anisotropy, were inhibited by both upward and downward magnetic fields. In contrast, the cell numbers of most non-cancer cells were not affected by magnetic fields of all directions. Moreover, the upward magnetic field inhibited GIST-T1 tumor growth in nude mice by 19.3% (p < 0.05) while the downward magnetic field did not produce significant effect. In conclusion, although still lack of mechanistical insights, our results show that different magnetic field directions produce divergent effects on cancer cell numbers as well as tumor growth in mice. This not only verified the safety of SMF exposure related to current MRI machines but also revealed the possible antitumor potential of magnetic field with an upward direction.     

Behavior of Cells in Rotating Electric Fields with Account to Surface Charges and Cell Structures

The behavior of a single biological cell in a rotating electric field is investigated both theoretically and experimentally. The torque acting on the cell is calculated. The dependence of the torque on electric cell properties (the dielectric constants, the conductivities, and the surface charges of the cell components) and the field frequency is discussed. The dependence of the rotation velocity on the field frequency shows a typical resonance behavior. It is discussed in which manner the single rotation extrema are related to the different homogeneous cell compartments (cytoplasm, cell membrane, and cell wall). It is shown that the cell surface charge shifts the resonance frequency and influences the absolute value of rotation velocity.     

Calculation of Externally Applied Electric Field Intensity for Disruption of Cancer Cell Proliferation

It is already known that electrostatic, magnetostatic, extremely low-frequency electric fields, and pulsed electric field could be utilized in cancer treatment. The healing effect depends on frequency and amplitude of electric field. In the present work, a simple theoretical model is developed to estimate the intensity of electrostatic field that damages a living cell during division. By this model, it is shown that magnification of electric field in the bottleneck of dividing cell is enough to break chemical bounds between molecules by an avalanche process. Our model shows that the externally applied electric field of 4?V/cm intensity is able to hurt a cancer cell at the dividing stage.     

No effect of extremely low-frequency magnetic field observed on cell growth or initial response of cell proliferation in human cancer cell lines

An effect on the tumor promotion process, as represented by accelerated cell growth, has been indicated as one example of areas that demonstrate the possibility of biological effects of extremely-low frequency magnetic fields. We, therefore, exposed the five cell lines (HL-60, K-562, MCF-7, A-375, and H4) derived from human tumors to a magnetic field for 3 days to investigate the effects on cell growth. Prior to exposure or sham exposure, the cells were precultured for 2 days in low serum conditions. The number of growing cells was counted in a blind manner. To investigate the effect on the initial response of cell proliferation, two cell lines were synchronized in G1 phase by serum starvation and then exposed to a magnetic field for 18 h (H4 cells) or 24 h (MCF-7 cells), both with and without serum stimulation. The rate of DNA synthesis, taken as a measure of the cell proliferation, was determined by following the incorporation of [(3)H]-thymidine into the DNA. Three different magnetic field polarizations at both 50 and 60 Hz were used: linearly polarized (vertical); circularly polarized; and an elliptically polarized field. Magnetic field flux densities were set at 500, 100, 20 and 2 microT (rms) for the vertical field and at 500 microT (rms) for the rotating fields. No effect of magnetic field exposure was observed on either cell growth or the initial response of cell proliferation.     

Morphologic responses of osteoblast-like cells in monolayer culture to ELF electromagnetic fields

Osteoblast-like cells (MC 3T3-E1) were exposed for 24 h, immediately after plating, to a 60 Hz, 0.7 mT rms magnetic flux density, sufficient to induce an electric field of 0.5 mV/m rms, in order to investigate the influence of ELF field exposure on cell morphology. Using phase contrast images of the live cells, computerized image-analysis permitted rapid and objective quantification of cell length, width, area, perimeter, circularity and angular orientation. While the field-exposed cells were consistently smaller than sham treated cells, the morphologic alterations were not significantly different in the exposed cell population when cell orientation was not considered. When analyzed with respect to cell orientation, cells oriented parallel to the induced electric field (orthogonal to the applied magnetic field) demonstrated a significant decrease in cell length and an increase in roundness. These results confirm and extend previous studies on the morphologic adaptation of cells to low level ELF electromagnetic fields. The results suggest that the observed responses most likely depend on the induced electric field, with a field intensity threshold well below 1 mV/m. Further, these results provide important clues to the specific mechanism by which such low level fields may be capable of influencing cell behavior, and help to explain some of the difficulties in obtaining robust responses in in vitro EMF experiments.