Time-lapse Techniques Research Articles

Cell cycle arrest and morphological alterations following microinjection of NIH3T3 cells with Pur alpha.

Levels of Pur alpha, a protein implicated in control of both DNA replication and gene transcription, fluctuate during the cell cycle, being lowest in early S phase and highest just after mitosis. Here we have employed a new video time-lapse technique enabling us to determine the cell cycle position of each cell in an asynchronous culture at a given time and to ask whether introduction of Pur alpha protein at specific times can affect cell cycle progression. Approximately 80% of all NIH3T3 cells injected with Pur alpha were inhibited from passing through mitosis. Cells injected with Pur alpha during S or G2 phases were efficiently blocked with a 4N (G2 phase) DNA level, as determined by quantitative DNA photometry of individual cells. Of the cells injected with Pur alpha during G1 phase, 40% experienced a rapid cell death characterized by extreme cellular fragmentation. Of those G1 injected cells which remained viable, approximately equal numbers were arrested with either 2N or 4N DNA levels. Cells arrested by Pur alpha in G2 phase grew to cover a large surface area. These results link fluctuations in Pur alpha levels to aspects of cell cycle control.

Fibroblast movements during contraction of collagen lattices--a quantitative study using a new three-dimensional time-lapse technique with phase-contrast laser scanning microscopy.

In this study we assessed the behavior of fibroblasts during contraction of collagen lattices. We applied a new technique for three-dimensional time-lapse studies of movements of living cells using phase-contrast laser scanning microscopy. Five anchored and five floating collagen lattices were studied regarding the activity of cells during a 7-h period of active contraction. Three-dimensional reconstructions of the fibroblasts and their extensions were made from datasets of 16-26 "optical sections" 5 microm apart recorded hourly during the period of measurements. The distance between fibroblast nuclei in the floating lattices decreased by a mean of 6.8 microm, but remained constant in the anchored group. Only minor variations were found in the angle between a line connecting any two nuclei and the tangent of the lattice margin. The lengths of the cellular extensions continuously changed by shortening and extending, and an increasing number of intercellular contacts were established with time. The angle between the extensions and the periphery of the lattice varied continually, and no distinct pattern of arrangement of the extensions was seen. In conclusion, we have shown in living cells in vitro that fibroblasts do not appear to move around within lattices during contraction but rather send out and withdraw cellular extensions continuously. This speaks against cellular locomotion or movement as a main feature of contraction. Time-lapse scanning laser microscopy has also been shown to be a suitable method to study cellular behavior quantitatively in three dimensions during lattice contraction.

Thermotaxis and protoplasmic oscillations inPhysarum plasmodia analysed in a novel device generating stable linear temperature gradients

The application of sublethal temperature gradients offers a simple, non-invasive means for in vivo studies of thermotaxis and other temperature-dependent processes in various organisms. Development, for instance, can be dramatically desynchronized, and the resulting development gradients allow to analyze physiological inter-dependencies between locally separated subsystems. For this purpose a simple device has been developed, by which a stable linear gradient of 8 °C/cm is established on an inert metal sheet with the aid of Peltier elements. The effects of linear temperature gradients on fusion, growth, and migration of plasmodia of the slime moldPhysarum polycephalum was filmed by 16 mm film time-lapse technique, and their local contraction—relaxation cycles analysed by “multistrip kymography”, which represents a graphic documentation of the spatio-temporal pattern of protoplasmic movements that occur along well-defined regions within the giant cell.Physarum plasmodia preferentially fuse, and grow, in the range of 24–26 °C. Different parts of a single macroplasmodium can simultaneously show positive and negative thermotaxis. The contraction—relaxation cycles generating the protoplasmic shuttle streaming within the network of veins essentially depend on local temperatures and are instantaneously desynchronized by the temperature gradient. Thus they cannot be controlled by a central pacemaker or an overall electric signal. However, there is a strong tendency to locally synchronize the various oscillation frequencies present within the giant cell if temperature differences do not exceed 2 °C.

Time-lapse Video Photography: A Low-tech Way to Make Plants Dance

The growth processes of most horticultural crops are too slow to be visually interesting to students. Time lapse photography has been used for years to speed up the action and make plants “come alive.” With the advent of video technology, time lapse techniques have become convenient, easy, and affordable. The system which we have found satisfactory consists of a time lapse video cassette recorder, linked by optical fiber cable to a closed circuit color video camera in a ventilated housing. Typically, the camera has been set up in a greenhouse compartment, monitoring growth processes of vegetable crops, and linked by cable to the VCR in an office 80 m away. Equipment costs with one camera are less than $3000. Two cameras can be set up to do comparative growth studies, with two images side-by-side, using a screen splitter. Costs of the latter system is about $4500. Growth processes such as cabbage head formation, curd growth in cauliflower, and weed-crop competition of mustard and peas have been the subjects so far. The technique lends itself to increasing the visual impact of teaching, and gaining a better understanding of plant growth processes in research.

Technical Notes: Sampling Rate for Measurement of Piglet Thermoregulatory Behavior

Diurnal piglet behavior of heat lamp usage was recorded by photographic time lapse technique at 15 min intervals for 24 h and expressed as the percentage of litter mates using the heat lamp. The 15 min behavioral data was then divided into subsets with a sampling interval of 30, 45, 60, 75, 90, or 120 min; and data series comprised of the averages of 15 min data corresponding to each of the augmented sampling intervals. Correlation analyses between the subsets and the average data series indicated that a sampling interval of up to 60 min results in reliable measurement of the dynamic heat lamp usage of the piglets.

Developmental asynchrony caused by steep temperature gradients does not impair pattern formation in the wasp, Pimpla turionellae L.

Generating developmental gradients by temperature gradients established within a developing organism is an easy, non-invasive technique to study physiological interdependencies between locally separated subsystems. A linear temperature gradient of about 10° C/mm was maintained up to 5 h in either direction along the long axis of a long-germ-type hymenopteran egg, which was simultaneously filmed by the 16 mm timelapse technique. The result was a dramatic desynchronization of development, which between the egg poles could reach up to 9.3 h relative to normal development. Within the same egg, up to seven mitotic waves (i.e. eight different nuclear generations) were observed at the same time, and the subsequent cellularization process was extremely asynchronous. The "initial regions" of the mitotic waves, the "fountain flow" of the ooplasm, and the gastrulation process were shifted towards the egg pole kept at higher temperatures. Developmental processes occurring successively in normal development now took place simultaneously, with either direction of the temperature gradient. For instance, while gastrulation had started in the warm region, midblastula transition and cellularization were in progress in the middle of the egg, and intravitelline nuclear multiplication occurred at the cold pole, by rapid and still biphasic cell cycles. In some respects, development resembled that of a short-germ-type insect egg. Nevertheless, the developmental processes were resynchronized after the temperature gradient was switched off. Surprisingly, the extreme desynchronization during early development did not affect the segment pattern of the resulting embryos. The technique of inducing well-defined developmental asynchronies might be applied in Drosophila to analyse the subtle interplay between maternal and zygotic gene activities described in this species.

Target Cell Selection and Specific Synapse Formation By Identified Ly'Mnaea Neurons in Vitro

Our research is aimed at determining both cellular and molecular mechanisms that underlie target cell selection and specific synapse formation in the nervous system. We have developed an in vitro experimental system which utilizes identified pre and postsynaptic neurons from the mollusc Lymnaea stagnalis. A giant presynaptic neuron, the right pedal dorsal 1 (RPeD1), re-establishes its specific synapses in culture with only postsynaptic target cells (such as visceral J, I), but not with non target cells (VF, RPB). In an attempt to begin to elucidate mechanisms that determine the specificity of synaptogenesis between Lymnaea neurons in vitro, we studied the behaviour of both target and non-target cell growth cones as they encountered RPeD1's growth cones for the first time. Utilizing time-lapse video imaging techniques, we demonstrate that RPeD1 growth cones attract and repel target and non-target cell growth cones respectively. We suggest that the specificity of synaptogenesis between RPeD 1 and both target and non-target cells is determined at the growth cone level and may involve release of a diffusible substance from the growth concs of RPeD 1.

The Parasitic Behaviour of Second-Stage Juveniles of Meloidogyne Incognita in Roots of Arabidopsis Thaliana

The parasitic behaviour of second-stage juveniles (J2) of Meloidogyne incognita in roots of Arabidopsis thaliana was recorded at 25 ± 1 °C with the aid of video-enhanced contrast light microscopy and time lapse techniques. The J2 invaded the root primarily in the region of elongation, close to the meristematic zone. Invasion was achieved, in most cases, by destroying epidermal and subepidermal cells, while intercellular invasion between epidermal cells was less frequent. Inside the root the J2 oriented themselves always in the direction of the root-tip and migrated towards it between cortical and meristematic cells without causing any damage. Occasional attempts at intracellular migration through cortical cells were observed but these failed due to insurmountable barriers. When the J2 reached the apex of the root, they turned round and migrated backwards between the cells towards the differentiating vascular cylinder. Within the differentiated cylinder migration eventually stopped and giant-cell induction was initiated. After a period of induction, the J2 became surrounded by young multinucleate giant-cells within about 24 h. A characteristic behavioural pattern, composed of continuous head and stylet movements, interspersed by periods of stylet-tip protrusion and metacorpal bulb pumping, was maintained throughout all phases of parasitism, also when the J2 fed from the young giant-cells.

Holographic methods for component manufacture and evaluation

The paper gives a brief introduction to holographic interferometry and reviews inspection procedures such as real-time and time lapse techniques applied to continuous wave and pulsed laser set-ups. The examples presented here represent some of the holographic work performed by the author on composites and metal components, demonstrating the detection of debonds, cracks external and internal, the visualisation of erosion, corrosion and vibration, lack of penetration in welded steel pipes, etc. Current work is aimed at quantifying artificially flawed models of pressure vessels by observing surface deformations. The images are being captured and digitised for further analysis by computer, parallel to finite element verification.

Cinematographic analysis of vascular smooth muscle cell interactions with extracellular matrix

The interactions of vascular smooth muscle cells with growth modulators and extracellular matrix molecules may play a role in the proliferation and migration of these cells after vascular injury and during the development of atherosclerosis. Time-lapse cinematographic techniques have been used to study cell division and migration of bovine carotid artery smooth muscle cells in response to matrix molecules consisting of solubilized basement membrane (Matrigel) and type I collagen. When cells were grown adjacent to Matrigel, both migration and cell proliferation were increased and interdivision time was shortened. Cells grown in Matrigel or in type I collagen had markedly reduced migration rates but interdivision time was not altered. Further, diffusible components of the Matrigel were found to stimulate proliferation of the smooth muscle cells.

Partition of protein and DNA during cytokinesis in human breast cancer cell lines

Three human breast cancer cell lines (HTB-126, MDA-231, and HTB-122) with DNA index (DI) values between 1.26 and 1.72 were analysed together with a diploid mouse embryonal carcinoma cell line (PCC3) by a TV-video time-lapse technique (pedigree analysis). Cytochemical parameters (DNA and proteins) were studied in individual cells in a rapid scanning microspectrophotometer. Post-mitotic sister cell pairs were analysed after Feulgen-naphthol-yellow staining. The DI values of the cell lines were selected to reflect various well-known clinical ploidy entities differing in malignancy potentials. A mitotic disturbance of the partition of DNA and protein to daughter cells was found in particular in MDA-231 closest to the triploid DNA modal value (DI = 1.37). Duration of mitosis was considerably longer in the near triploid line compared to the other lines. The MDA-231 line was also least sensitive to suboptimal growth conditions. This report calls attention to a possible causality between mitotic error and intraclonal genotype and cell mass heterogeneity.

A Hyperbaric Chamber for Observation of Cell Cultures at Pressure

A chamber system has been constructed for the study of hydrostatic and gas pressure effects on cells in culture. The chamber is fitted with a phase contrast light microscope allowing direct observation and video recording of the cells at pressure. Cell differentiation over a period of hours to days may thus be recorded and taped from as many as 24 cell samples in the same experiment, using a time-lapse recording technique. Solutions may be introduced and removed through stainless steel tubing via an injection pump, thus providing the additional possibility of biochemical and pharmacological studies.

Permeability changes in the egg-shell of hookworms during development and eclosion.

The development and hatching of Ancylostoma ceylanicum and Ancylostoma tubaeforme eggs in a range of potentially inimical solutions has been examined using time-lapse video micro-recording techniques. In all compounds tested the larvae developed to the active pretzel stage. The inception of activity resulted in an increase in shell permeability to water and small molecules. In anthelmintics of molecular weight (Mr) 400 development continued normally until about the time that control eggs hatched, when there was a marked decrease in larval activity and eclosion did not occur. The addition of a low Mr surface-active agent potentiated the effect of the anthelmintic and caused larval activity to be lost earlier. The calcium inhibitor lanthanum chloride affected larval development rather than eclosion. Large Mr compounds did not affect either development or hatching and the results suggest that the egg-shell may act as a form of sieve, allowing the essential oxygen to reach the developing larva while excluding large potentially dangerous molecules. No appreciable differences were found between the two species of hookworm examined.

The selective inhibition of growth cone extension by specific neurites in culture

We have found that neurites from embryonic chick retinal and sympathetic explants do not mix in culture, while retinal neurites mix with retinal neurites, and sympathetic neurites mix with sympathetic neurites. These results confirm those obtained with embryonic rat tissues by Bray et al. (1980). We have also used video time-lapse techniques to examine the behavior of individual retinal and sympathetic growth cones as they attempt to cross retinal and sympathetic neurites. We have found that retinal growth cones cross retinal neurites without delay, retinal growth cones usually retract from sympathetic neurites but often cross them on a second advance, sympathetic growth cones repeatedly retract from retinal neurites, rarely managing to cross them at all, and sympathetic growth cones cross sympathetic neurites but are sometimes delayed. Our results indicate that retinal and sympathetic growth cones can distinguish between specific labels associated with retinal and sympathetic neurites. They also suggest that active avoidance could play an important role in growth cone navigation.

Cinematographic analysis of endothelial cell proliferation in culture

A method is described for the application of time-lapse cinematographic techniques (TLC) to analysis of the behavior of individual cells and clones in endothelial cultures. Using TLC, detailed and precise information on cell cycle time, proliferative state, and migration activity can be obtained on several hundred individual cells in a given filmed sequence of proliferating endothelial cultures.

Role of fibronectin in primary mesenchyme cell migration in the sea urchin.

We studied the effect of fibronectin (FN) on the behavior of primary mesenchyme cells isolated from sea urchin mesenchyme blastulae in vitro using a time-lapse technique. The migration of isolated primary mesenchyme cells reconstituted in seawater and horse serum is dependent on the presence or absence of exogenous FN in the culture media. The cells in FN, 4 and 40 micrograms/ml, show a high percentage of migration and migrate long distances, whereas a higher concentration of FN at 400 micrograms/ml tends to inhibit migration.

Well Logging and Its Applications in Cased Holes

Summary Workovers and recompletions of old wells, evaluation and monitoring of reservoir behavior during primary, secondary, and ternary recovery, and exploration for bypassed oil in cased wells make the proper selection of cased-hole logs and/or optimal logging suites a necessity. Required information includes the integrity of the cement bond between casing and the formation, lithology identification, porosity, type and distribution of reservoir fluids, formation permeability, and anticipated watercut estimates. These data can be obtained from vitrious log responses, which include natural total and/or spectral gamma ray radioactivity measurements, radiation-type logs (density, neutron), pulsed neutron logs. acoustic measurements, etc. In addition. special time-lapse logging techniques monitor reservoir behavior and state of hydrocarbon depletion, whereas log-inject-log (LIL) techniques enhance the accuracy of residual oil saturation (ROS) estimates. Introduction Cased-hole logging capabilities and associated quantitative interpretation techniques have become very important in several applications, including, cased-hole exploration, completion, workovers. and the monitoring of hydrocarbon reservoirs under primary, secondary, or tertiary recovery schemes. Cased-hole well logging concepts are reviewed which are currently used (1) to explore for bypassed hydrocarbons in abandoned or old workover wells; (2) to work in fresh, brackish, or unknown formation water salinities; (3) to evaluate hydrocarbons in new wells in which openhole logs could not be run, (4) to monitor production behavior and depletion in reservoirs under primary driving mechanisms, breakthrough in waterfloods, chemical and micellar projects, C02, and steam- and fireflood projects; (5) for residual oil saturation based on LIL techniques, etc. Several field case examples and references are presented. Reservoir Characterization Lithology. Natural radioactivity measurements have phyed an important role in wireline logging operations since about 1935. Most of the natural gamma ray logging techniques applied in formation evaluation measure the total and/or individual contribution of gamma rays from potassium, uranium, and thorium series by means of standard gamma ray and/or spectral gamma ray logging. The fact that natural gamma-ray intensities vary as a function of lithology was recognized as early as 1939. Gamma rays are the radiations originating within an atomic nucleus. A nucleus gives off excessive energy (gamma rays) as the result of radioactive decay or an induced nuclear reaction. Radioactive decay consists of the emission or capture of elementary or composite particles with consequent transformations into daughter nuclei characterized by different atomic numbers and, in some cases, by different mass numbers. As early as the 1950's, field tests in boreholes were carried out to study the feasibility of detecting some of these nuclides by gamma ray spectroscopy techniques that identify characteristic gamma rays. Of particular interest are those of potassium and the uranium and thorium series. Both uranium and thorium are characterized by specific decay series. Potassium consists of three isotopes that exhibit masses of 39, 40, and 41 with abundames of 9318, 0.0119, and 6.9%. The only unstable isotope of potassium is the nuclide potassium-40, the major contributor, which emits a single, easily identifiable gamma ray at 1.46 MeV. Hence, in addition to total gamma ray counts, the Spectralog TM measures and records the gamma rays emitted by potassium-40 (40K) at 1.46 MeV, the uranium series nuclide bismuth-214 (214Bi) emanating gamma rays at 1.764 MeV and the thorium series nuclide thallium-208 ( 208Th) emanating gamma rays at 2.614 MeV. These nuclides are of particular interest to the oil industry since, in various amounts, all are found in subsurface formations as constituents of potential reservoir rocks. Table 1 illustrates the distribution of potassium (K, %), uranium (U, ppm), and thorium (Th, ppm) for several formation constituents detrimental to optimal reservoir rock conditions. Application of such spectral gamma ray data may be made either qualitatively or quantitatively. As is extensively documented in logging literature, natural spectral gamma ray logging assists greatly in geological studies (lithology identification, recognition of depositional environment, stratigraphic correlation, source rock evaluation, etc.), complex reservoir rock analysis (heavy minerals, mica, feldspar, glauconite, fractures, silt. etc.), shaliness estimates, in-situ clay typing, cation exchange capacity (CEC) estimates, and radioactivity buildup under dynamic fluid conditions (channeling behind pipe, high-permeability streaks, fractures. caverns, watered-out zones, etc.) and/or around perforations. JPT P. 249^

Improvement of the microcinematography technic for the study of cell cycles

An improvement of time-lapse microcinematography technique is described. It consists in directly printing the time on the microscopical frame, at the moment of the shooting. The time (digital watch), as well as other relevant parameters (temperature etc.) are displayed on a "parameter board", the image of which is encrusted into the microscopical frame by means of an auxiliary two-component lens system. These lenses (current type of microscopical and photographical objectives) are centered on an axis perpendicular to the microscope-camera axis and provide a reduced image of the "parameter board", which is projected on the film edge after deflection by a 45 degree mirror. The latter (aluminized perspex sheet) is located above the photographical eyepiece; it is pierced at the place of the eyepoint in order to give way to the light rays coming out of the cellular culture.

Image and Stereotype: Bluegrass Sound Tracks

sound of bluegrass music, especially the five-string banjo, is heard frequently in contemporary audio-visual media. On television it ripples along behind advertisements or in situation comedies. In movies it accompanies chases, slapstick comedy routines, country themes, or some combination of these. use of a certain kind of music to accompany a certain range of visual images indicates that it is one of the sound track conventions of the film and video industries. Historical descriptions of bluegrass music note that the spread and success of bluegrass music as a popular genre resulted, in part, from its popularity in the visual media.' Sound tracks from successful movies and television shows often sell well as records. There have been three such hits with bluegrass music in the past two decades: The Ballad of Jed Clampett from television's Beverly Hillbillies (1962), Foggy Mountain Breakdown from Bonnie and Clyde (1967), and Dueling Banjos from Deliverance (1972). These three recordings are the best-selling records in the history of bluegrass. success of each reinforced and expanded the use of such music as a dramatic convention in visual media, giving added cultural meaning to the music. development of this convention, with its various connotations, began in the early 1960s. In the summer of 1961 Joe Anderson, a film director in the department of photography at Ohio State University in Columbus, put together a short movie consisting of time-lapse shots taken at Ohio State football games.2 In the time-lapse technique a movie camera is run at a slow speed so that fewer frames per second are exposed. When the film is projected at standard speed, the visual action appears rapid but jerky. Time has been compressed, motion fragmented. technique is familiar to most of us from classroom biology films, which show how seeds sprout and buds flower. During the early days of silent film, movies were shot at a smaller number of frames per second, and these, when played back on modern projectors, give a time-lapse effect to human motion that often seems strange, even funny.

Pulsed Neutron Capture Log Evaluation Using Synthetic Base Logs (includes associated papers 10920 and 10939 )

The evaluation of recompletion opportunities using pulsed neutron logs ismade easier by preparing predictive synthetic logs using openhole log data.Wellsite personnel can assess drainage quickly by comparing the actual logwith the synthetic expected response, thus avoiding time-consuming trialcalculations. Introduction The pulsed neutron capture (PNC) log has proved tobe a valuable production surveillance tool for theU.S. gulf coast environment since its introduction in1962. In this geographic area, more than 900 PNClogging runs have been made in producing Shell wellsalone. We have come to rely on this tool as a reliableaid for basing many of our remedial andrecompletion decisions.In conventional PNC log evaluation, watersaturation (Sw) calculated from openhole logs iscompared with Sw computed from the cased-holePNC log. Drainage is indicated when a statisticallysignificant difference in Sw is observed. Althoughthis technique is a practical procedure, the calculations can be time-consuming and subject tohidden errors.A nearly foolproof method of assessing drainagefrom PNC log data is the time-lapse technique. Inthis method, a base PNC log is run early in the life of the well, preferably before water production begins.When subsequent drainage requires assessment, asecond log is run and compared with the first. If thewellbore environment (production equipment, annular fluids) has not changed greatly, differencesbetween the logs can be caused only by reservoir fluidchange. The problem with this approach, of course, is the expense and practicality of building a completebase-log inventory. It usually is not justifiable.This paper presents a simple computer techniquefor creating synthetic base PNC logs using theoriginal openhole log information. Offering many ofthe advantages of actual base-log data, the computer technique is easy to use and surprisingly accurate. Inaddition, synthetics can indicate quickly whether aPNC log can be interpreted reliably under adverseconditions. This can help the engineer choose morewisely among evaluation alternatives before a loggingcommitment is made. Example Fig. 1 shows an example of a synthetic pulsedneutron log made using this technique. An openholegamma-ray induction log is displayed on the left;cased-hole PNC curves (synthetic and actual) areshown on the right. Note the capture cross sectioncurve of a Schlumberger TDT-K (TM) log ("Sigma log"-the dashed curve) run 1 year after the well wasdrilled and cased. This curve is flanked by two logtraces, Sigma i and Sigma d, generated by the computer process usingopenhole logs. The Sigma i curve represents a "synthetic"PNC sigma curve at initial conditions. It shows whatan actural PNC base log would have looked likebefore the zone was drained. The d curve representsthe fully drained condition, in which the zone is totally water saturated. This curve represents the endpoint of drainage.(Actually, this condition will neverquite be reached since some residual hydrocarbonsaturation always remains after waterflood inwater-wet reservoirs.) JPT P. 667＾