Time-lapse Cinematography Research Articles

Quantitative cytochemical studies on interphase growth: I. Determination of DNA, RNA and mass content of age determined mouse fibroblasts in vitro and of intercellular variation in generation time

A combination of quantitative cytochemistry and time-lapse cinematography has been used for studying the DNA, RNA and protein (dry mass) synthesis during interphase of mouse fibroblasts in vitro. This method is mainly associated with two error factors, i.e., variation in generation time and variation in the amount of DNA, RNA and protein of cells in the same stage of interphase. The magnitude of these factors were compared and were discussed in connection with the relation between growth and division. The cellular DNA, RNA and mass content doubled during interphase; there was no evidence for loss of material during mitosis. There was an error in RNA and, to greater extent, in mass distribution to the daughter cells at mitosis, while the error of DNA distribution was of the same order as the error of measurements. The G 1 (preceding DNA synthesis), the S (DNA synthesis) and the G 2 (following DNA synthesis) periods were 8, 6 and 5 hr, respectively, and the rate of DNA synthesis was constant. RNA and mass increased throughout the entire interphase. The rate of mass synthesis was higher in the latter part of interphase. It was not possible to judge whether the rate of RNA synthesis was constant or changed during interphase. DNA synthesis seemed to be initiated at a critical cellular mass. The importance of such a critical mass was discussed with regard to maintenance of the size homogeneity of the cell population, intercellular variations in generation times and the “trigger” mechanism for mitosis.

Studies of Diurnal Changes in Cherry Root Growth and Nutational Movements of Apple Root Tips by Time-lapse Cinematography

Studies of Diurnal Changes in Cherry Root Growth and Nutational Movements of Apple Root Tips by Time-lapse Cinematography

Studies of cleavage and development of isolated sea urchin blastomeres

Abstract Isolated blastomeres from young larvae of Echinocyamus pusillus and Psammechinus miliaris were studied by means of time-lapse cinematography. The isolated mesomeres and macromeres cleave at about the same rate as in situ in intact larvae. It was found that single meso- and macromeres were able to develop into blastulae. Isolated micromeres cleave somewhat more slowly than the micromeres in situ and as isolated cells they were not able to pass through more than three cleavages. They are characterized by the sending out of cytoplasmic protrusions. The rate of cleavage is different for meso-, macro- and micromeres and the meso- and macromeres cleave, both in situ and as isolated cells, considerably faster than the micromeres. Owing to the different rates of cleavage during the blastula stage the cells of the prospective ectoderm multiply more rapidly than the cells, which later form the endoderm and mesoderm and therefore the early development is characterized by an “animal” trend. Even minor change...

TOXOPLASMA GONDII IN CELL CULTURES FROM RAT RETINA.

Retinal cultures from young rats were inoculated with the RH strain of Toxoplasma gondii, and observed by phase contrast microscopy and time lapse cinematography. Parasite multiplication followed by destruction of the host cell was demonstrated in ganglion cells, bipolar nerve cells, rods, neuroglial cells, retinal pigment epithelium and cells of mesenchymal origin. The retinal pigment epithelium cells and the cells of mesenchymal origin were filled with a large number of Toxoplasma parasites and showed no degenerative changes, while the nerve cells, rods and neuroglial cells contained only a few parasites and showed terminal degenerative changes both in the cytoplasm and the nucleus. The parasites invaded the ganglion cell soma also through the dendrites. The binary division of the parasite resulted in the formation of two daughter parasites inside the ultimately destroyed mother individual. During mitosis of the host cell a clone might be divided into two parts, each with its own membrane, or may reappear intact in one of the daughter cells. Evidence is presented indicating that the membrane surrounding the Toxoplasma clone is formed by the host cell.

THE CELL--'EMPTY JELLY' OR A COMPLEX SERIES OF STRUCTURES WITH DEFINITE FORMS AND FUNCTIONS?

What is a cell? How does it function? Of what is it made? Ever since man has been able to pose these questions, the answers have eluded him. Then, with the development of the electron microscope a decade ago, a new era began and today scientists are able to make studies which could not have been thought of a few years ago. Reporting on "Cell Pathology, 1964" at the 45th annual meeting of the American College of Physicians in Atlantic City, April 6-10, Marcel Bessis, MD, director of the research laboratories at the National Transfusion Center in Paris, presented a glimpse of intracellular life and intercellular relationships that the session moderator classed as "one of the most exciting experiences in the facts of life that we've ever had," and drew an ovation from the capacity audience. Utilizing, in addition to the electron microscope, the phase contrast microscope, time-lapse cinematography, microsurgical

Cellular mechanisms in the morphogenesis of the sea urchin embryo: Cell contacts within the ectoderm and between mesenchyme and ectoderm cells

The contacts between various pseudopodal elements and the ectoderm in the sea urchin gastrula have been investigated by means of time-lapse cinematography and interference contrast microscopy on slightly compressed larvae, where the ectoderm is more or less extended and the cells tend to separate to a varying extent. In zones of the ectoderm where cell contact is moderate, the cells are joined up by points of attachment which are restricted to the margin of the inner and outer end of the cells. Where the contact is low, only the outer ends are involved in these mutual attachments, whereas in the animal plate, where the contact is strongest, the cells are attached to each other along the whole of their length. An increased packing of the cells can thus be correlated with the occurrence of inner contact points or broad contacts. The pseudopods of the primary mesenchyme, the archenteron tip, and the free secondary mesenchyme cells only attach at the points where the ectoderm cells attach to each other, and particularly to the inner points of attachment. The distribution of the inner points of attachment explains the correlation between the location of the primary mesenchyme cells and the thickened zones within the ectoderm. The inability of pseudopods to make strong contacts with the animal plate and the tendency of the archenteron tip pseudopods to attach to the dorsal side during gastrulation are tentatively explained in terms of availability of free contact surface within the ectoderm, but other factors may also be involved. The basis of the apparent differences between the pattern of primary and secondary mesenchyme is discussed. The inner and the outer contact points appear to be structurally different. The nature and origin of the cell contacts within the ectoderm is discussed, and it is suggested that the mutual adhesion between the cells is rather low, but that the cells tend to spread at the hyaline membrane and an inner layer which promotes a direct adhesion between the cells. The broad contact within the animal plate, however, indicates a high mutual adhesiveness of the cell surface as a whole. The results suggest that many of the complex morphological features of the young sea urchin embryo have a simple background.

Studies on the cellular basis of morphogenesis in the sea urchin embryo: Formation of the coelom, the mouth, and the primary pore-canal

The cellular basis of the morphogenesis of the coelom, mouth, and the primary pore-canal in the sea urchin larva (Psammechinus miliaris) has been studied by means of time-lapse cinematography. The purpose of this work is to see to what extent it may be possible to discuss these apparently complicated morphogenetic events in the same terms as we have used with regard to earlier and more simple morphogenetic events, e.g. gastrulation, namely as result of changes in contacts between cells, of pseudopod activity, and simple geometrical relations. The archenteron is brought in contact with the oral ectoderm by means of pseudopod activity, particularly from the unpaired coelom rudiment at the archenteron tip. The coelom rudiment rounds up and is delineated from the oesophagus by means of a constriction, a process which may, at least partly, reflect changes in contact between the cells. For geometrical reasons it is the anterior coelom surface close to the oesophagus that becomes attached to the oral ectoderm. The contact surface increases as a result of pseudopodal activity of the coelom cells. The non-attached part of the coelom becomes pulled out into a bi-lobed sac with a narrow isthmus. The formation of this shape appears to be explicable mainly in terms of graded differences in the strength of the contacts that the coelom pseudopods make with adjacent structures and as a consequence of the broad attachment of the coelom to the oral ectoderm. The coelom sacs are pulled out at the expense of the isthmus that becomes very narrow. The upper edge of the oesophagus therefore comes very close to the oral ectoderm. The coelom pseudopods attaching to the oesophagus pull out the coelomic sacs backwards and anchor the coelom sacs to the sides of the oesophagus. Many of the pseudopod forming cells are then released from the coelom and form a mesenchyme, which spins around the oesophagus. The left coelomic sac is larger than the right, and some of its pseudopods extend the coelom towards the dorsal ectoderm so that a canal is formed, the primary pore-canal, which fuses with the ectoderm. Some factors that may determine the course of the canal are discussed. The slow contractility of the pseudopods, both those attached to the coelom and those of the released mesenchyme spun around the oesophagus, later changes into a rapid periodic contractile activity which brings about peristaltic and other movements of the oesophagus. It is not known if also the oesophagus wall itself has such a contractile ability. The cells of the oral ectoderm reduce their contact with each other and pulsate on their inner surface, and the oral region therefore invaginates in a manner very similar to the primary invagination of the archenteron. The bi-lobed coelom is pulled apart into two separate sacs, and the edge of the oesophagus therefore comes in direct contact with the oral invagination with which it fuses. The perforation of the oral invagination appears to be brought about by the mechanical action of the coelomic pseudopods and by the tensions which are associated with the beginning peristaltic contractions of the oesophagus. A postulated chemical factor, probably released from the archenteron tip, must weaken the hyaline layer to make a rupture of the oral ectoderm possible. Only a few cells in the oral ectoderm are released during this process. The liberated cells show an amoeboidic activity. The initially small perforation widens as a result of the contractile activity of the coelom-oesophagus complex. The relation between the cellular activities concerned and the postulated morphogenetic gradients is briefly discussed. It is concluded that a complex set of structures may arise from a succession of a few basic cellular events organized in a rather simple manner.

Activity manifestations of Hassall's corpuscles in vitro as revealed by cinematography.

Thymic explants from newborn rats were cultivated in Rose chambers under dialysis membranes. With the use of phase contrast, time-lapse cinematography, the following activity manifestations were observed: 1. Several corpuscles showed rotatory movements; 2. nuclear rotation was observed in some cells of the corpuscles; 3. some individual cells in the 15-day culture showed pulsatile activity which might be involved in the mechanism of the rotatory movements of the cellular aggregates; 4. the corpuscles increased in size by the addition of cells and/or by mitosis; 5. the elements at the periphery of the more mature corpuscles (7- and 9-day cultures) were viable, while the central area appeared to be composed of necrotic tissue or hyalinized material.

A cell line from trypsinized adult rabbit brain tissue.

Trypsinized cerebral hemispheres, cerebellum or pial material from young adult rabbits were cultivated through many passages and for more than 6 months. Longterm cultures of the various tissues were undistinguishable and ultimately demonstrated a clearcut shift to hyperploidy. However, primary cultures and those in the first few passages showed a number of morphologically different cell types. Some were clearly recognizable as fibrous astrocytes, while others exhibited many characteristics of protoplasmic astrocytes. With the aid of time-lapse cinematography individual elements were found to undergo morphological transformation, even to losing their characteristic features. A third type of cell, clearly distinguishable from the others by its epithelial arrangement, probably was of endothelial origin. Ultimately the cell line obtained from all three sources bore a closer resemblance to the endothelial than to the astrocytic elements.

Rat pineal parenchymal cells as observed in tissue culture.

The pineal gland from normal adult male rats has been grown in tissue culture and the findings with phase-contrast, time-lapse cinematography have been correlated with observations made with stained cultures and paraffin sections. At least three distinct parenchymal cells were observed: 1. The chief cell with characteristic processes exhibiting pinocytosis. 2. A fat-laden cell which showed rapid nuclear rotation. 3. A pineal basophile with distinct intracytoplasmic granules.

Antigen-antibody reactions in relation to pinocytosis: I. Cell injury and repair following Staphylococcus toxin and antiserum

Time-lapse cinematography of cells in tissue culture is shown to provide a sensitive means of observing alterations under the action of toxic filtrates of a bacterial pathogen. Such changes are shown to be reversible under appropriate conditions by the action of immune serum. We postulate that large molecules, both of toxin and of antitoxin, may be taken into the cells by pinocytosis.

Cellular mechanisms in the morphogenesis of the sea urchin larva: Change in shape of cell sheets

The cellular basis of the morphogenesis of cell sheets has been studied in sea urchin larvae ( Psammechinus miliaris) by means of time-lapse cinematography. Particular attention has been focussed on the role of localized increases in adhesion between cells in the ectoderm sheet, without loss of contact with the hyaline membrane, bringing about thickening and a resulting increase in curvature in such sheets. It seems as if the major changes in external form of the larva from the blastula stage up to the time of extension of the arms can be largely explained in such terms. The time-sp⊙ace pattern of such changes appear to be simple — they start in a field or ring-like zone around the vegetal pole, which results in the pear-like form of the early gastrula, and later appear in another ring-like zone around the ventral side, including the ventral surface of the animal pole, which produces the ventral flattening of the gastrula. The resulting thickenings in the latter case form the ventral ciliated band and the animal plate. The thickenings are sometimes associated with the formation of spaces between the cells in the adjacent ectoderm. The ectoderm cells bordering the spaces become active, and close the spaces by means of a pseudopodal mechanism. The relationship between these changes in the ectoderm and the other early morphogenetic processes is discussed.

Studies on the cellular basis of morphogenesis of the sea urchin embryo: The formation of the blastula

The formation of the blastula in the sea urchin Psammechinus miliaris has been investigated from the 16-cell stage to hatching by time-lapse cinematography. It was found that: 1. 1. At successive cleavages the volume of both blastocoel and blastula increase, and then decrease following cleavage, but leaving a net increase. 2. 2. The cells adhere firmly to the hyaline layer. 3. 3. The cleavages are radial. The theory postulating osmotic mechanisms for increasing blastocoel volume does not easily account for the observations. It is suggested that blastula formation may be regarded as a problem in packing of cells and may be accounted for in mainly geometrical and mechanical terms. General considerations of the effect of uniform changes in adhesion and tension in the cell membrane, on the form of a tube or sphere made up of a single sheet of cells, indicate that the changes in cell shape at cleavage, the radial plane of cleavage, and the attachment of the cells to the hyaline layer are sufficient to account for the main features of blastula formation. The increase in blastocoel volume is due to the reduction in thickness of the blastula wall at successive cleavages.

Studies on the cellular basis of morphogenesis of the sea urchin embryo: Development of the skeletal pattern

A study has been made of the determination of pattern formation in a biological system at the cellular and supracellular level, i.e. of the skeletal pattern in sea urchin larvae. Such larvae are particularly suitable for this purpose, since their entire development can easily be recorded by means of time-lapse cinematography. The results, which in part are an extension of those of v. Ubisch indicate that two main mechanisms operate in this case, one “crystallographic”, and one “pseudopodal”. The “crystallographic” mechanism is responsible for the crystalline structure of the skeleton, the formation of its initial triradiate shape, and the tendency of its branches to grow straight when not preceded by pseudopods. The pseudopods of the skeleton-forming mesenchyme are responsible for the reorientation of the initial triradiate structure, which is laid down with random orientation, but whose branches subsequently become directed in a characteristic way. Pseudopods are also responsible for all bends and branches, including spine formation. The direction of the pseudopods and the distribution of the primary mesenchyme cells are determined—as in the stage before skeletal growth starts—by random exploration of the ectoderm by the pseudopods, and preferential attachment to points of strongest adhesiveness. The ectoderm thus determines, via the mesenchyme cells, the major features of the skeletal pattern which, in other words, reflects the changing pattern of adhesiveness of the ectoderm. The properties of the mesenchyme can also affect the pattern. The problem of precision of developmental events is discussed.

Studies on synchronous division of FL cells by chilling

1. 1. Growth curves of FL strain cells were determined by two methods; in replicate cultures from which samples were counted every 3 or more hours, and in plate culture by photographing the culture once a day. The growth curves obtained by both methods showed good parallelism. The growth of cells in culture plate was also followed by time-lapse cinematography, but in this method, growth was not as good when compared to the tube culture or the other plate culture method. 2. 2. FL cells in replicate culture were chilled at 4 °C for one hour 24 or more hours after subculture, when growth was in logarithmic phase. Half of the cells continued to divide after placing them again at 37 °C, but half appeared to be delayed to divide just one generation time after chilling.

Some Aspects of Time-Lapse Cinematography and Related Arts

Time-lapse photography has many applications including medical research, law, industry and agriculture. A discussion of medical research describes studies of normal and malignant cells of tissue and blood; human capillaries and studies on human eyes with an automatic “eye movement” camera. Time-lapse photography has also played an important part in a patent litigation. Microscopic studies at low and high temperatures (freezing of motor oil, and baking) are discussed. This technique is also used in the study of plant growth.

Osservazioni cinematografiche sullo sviluppo in vitro del polmone embrionale del topo in condizioni normali e dopo irradiazione con raggi gamma

Summary The authors studied the development in vitro of embryonic mouse lung, by means of time-lapse cinematography, in normal conditions and after gamma rays irradiation. The growth and branching of the buds already formed at explantation time and the formation of new buds and of tertiary branches are described. In irradiated lung anlagen the bronchial tree is invaded by opaque material derived from degenerated, cells of the bronchial epithelium. Later the opaque material gradually disappears and the lung anlagen show their previous transparency again: contraction movements of parts of the bronchial tree contribute to this process. The ramification process in irradiated lungs is strongly showed down.

Studies of Gliomas in Tissue Culture, Using Time-Lapse Cinematography

Studies of Gliomas in Tissue Culture, Using Time-Lapse Cinematography

Effect of irradiation on cell division and nucleic acid synthesis in strain U-12 fibroblasts

The sequence of events following an irradiation of 500 R was investigated in strain U-12 fibroblasts. The irradiation caused a rapid and almost complete inhibition of cell multiplication, as measured by nuclear counts, the mitotic index, and the number of colonies produced from single cells. Although mitotic figures reappeared and increased in frequency to abnormally high levels 13–20 h after irradiation, a recovery in cell multiplication did not occur. Time-lapse cinematography showed that the increased incidence in mitotic figures was very probably caused by the increased time spent by these cells in abortive mitoses. In contrast to the immediate inhibition of mitosis, DNA synthesis was reduced at a later time, falling to less than 50% of the control 18–24 h after irradiation. In this system, therefore, the immediate inhibition of mitosis cannot be the result of a deficiency in DNA synthesis, as measured by the number of individual cells incorporating [ 3H]thymidine or by the specific activity of DNA after incorporation of 32P. On the other hand, it is possible that the reduction in the number of cells synthesizing DNA results entirely from the inhibition of mitosis and subsequent failure of the orderly progression of cells through the mitotic cycle.

Multinucleation of skeletal muscle in vitro.

Healthy, mature, spontaneously contracting muscle was cultivated from explants of 13-day chick embryos for periods up to 4 months in the multipurpose chamber (Rose, 1954) using cellophane-strip technique (Rose et al., 1958) with silicone gaskets, Eagle's medium including 10 per cent horse serum reinforced with 300 mg-per cent of glucose, and the teased type of explant. This method provided optically ideal conditions for the study of muscle fibers with oil immersion, phase contrast time-lapse cinematography at 1 frame per minute without apparent damage for periods as long as 10 days. In no case was mitosis, amitosis, or nuclear "budding" observed in the course of muscle development. Multinuclear muscle fibers have been shown with cine technique to result from both myoblast fusion and polar extension of preformed (explanted) muscle tissue. Myoblast fusion was the only demonstrable way of giving rise to multinucleation. Nuclear membrane "wrinkling" was shown to be merely a temporary distortion that occurred during nuclear migration and rotation. It is suggested that this phenomenon may be responsible for numerous reports of amitosis in the genesis of muscle fibers. The histological development of new straps resulted from an orderly sequence of events. Included in these were polar extension, nuclear migration, rotation, and fixation. Following these events there was increased mitochondrial activity, myofibril formation, and cross-banding. Spontaneous contractions were seen throughout the entire course of differentiation in vitro but became more regular and stronger in the later stages.