Laser light-scattering analysis of protoplasmic streaming in the slime mold Physarum polycephalum

Laser light scattering is shown to be an effective means of obtaining a rapid, objective assessment of dynamic changes in the intact plasmodium of the myxomycete Physarum polycephalum during bidirectional (shuttle) streaming. The motion of material in a 100 mum diameter region of a plasmodial vein was studied by following changes in the autocorrelation function of the fluctuations in the scattered light intensity. The autocorrelation function was recorded at 10 s intervals and analyzed to follow changes in the flow velocity of protoplasm associated with shuttle streaming. Rhythmic velocity changes and a "beating" pattern of velocity maxima were readily observed. In an attempt to locate the site of underlying structural changes in the vein responsible for the changing pattern of flow, the average scattered intensity was separated into components derived from moving and stationary scatterers. Periodic variations in the light intensity due to stationary scatterers are related to the streaming cycle and indicate the occurrence of important structural changes in the vein walls. Two possible interpretations of the data are offered; one involving gross dynamic changes in vein structure, the other involving the formation, contraction, or breakdown of fibrillar material in the vein wall during the streaming cycle.     

Photon correlation analysis of cytoplasmic streaming

Laser light scattering has been used to investigate particle movements in a plant cell. Intensity autocorrelation functions are obtained by digital photon correlation of laser light scattered from cells of Nitella opaca both during cytoplasmic streaming and during the transitory cessation of streaming induced by electrical stimulation. The average velocity computed from the periodic oscillation in the intensity autocorrelation function during streaming corresponds to the velocity estimated using light microscopy. An estimate of the distribution of streaming velocities has been obtained from the decay in the amplitude of the envelope of the autocorrelation function derived from a streaming cell.     

THE CHANGING PATTERN OF BIREFRINGENCE IN PLASMODIA OF THE SLIME MOLD, PHYSARUM POLYCEPHALUM

Plasmodia of the acellular slime mold, Physarum polycephalum, reveal a complex and changing pattern of birefringence when examined with a sensitive polarizing microscope. Positively birefringent fibrils are found throughout the ectoplasmic region of the plasmodium. In the larger strands they may be oriented parallel to the strand axis, or arranged circularly or spirally along the periphery of endoplasmic channels. Some fibrils exist for only a few minutes, others for a longer period. Some, particularly the circular fibrils, undergo changes in birefringence as they undergo cyclic deformations. In the ramifying strand region and the advancing margin there is a tendency for fibrils of various sizes to become organized into mutually orthogonal arrays. In some plasmodia the channel wall material immediately adjacent to the endoplasm has been found to be birefringent. The sign of endoplasmic birefringence is negative, and its magnitude is apparently constant over the streaming cycle. The pattern of plasmodial birefringence and its changes during the shuttle streaming cycle of Physarum are considered in the light of several models designed to explain either cytoplasmic streaming alone or the entire gamut of plasmodial motions. The results of this and other recent physical studies suggest that both streaming and the various other motions of the plasmodium may very likely be explained in terms of coordinated contractions taking place in the fibrils which are rendered visible in polarized light.     

A study of protoplasmic streaming in Nitella by laser Doppler spectroscopy.

Laser light scattered from particles in the streaming protoplasm of a living cell is shifted in frequency by the Doppler effect. The spectrum of the scattered light can be measured and interpreted to infer details of the velocity distribution in the protoplasm. We have developed this approach to study the protoplasmic streaming in the fresh-water alga Nitella. Our results indicate a characteristic flow pattern to which diffusion makes a negligible contribution. No difference in the velocity of particles of different size is indicated. The streaming velocity linearly with temperature with a supraoptimal temperature of 34 degrees C, and the velocity distribution becomes narrower at high temperatures. The protoplasmic streaming can be inhibited by laser light, and this effect has been used to study the photoresponse of the algae. Using beam diameters of about 50 mum, we have shown that the inhibition is very local, becoming minimal at a displacement of about 200 mum in the upstream direction and 400 mum in the downstream direction. Prolonged exposure produces a bleached area free of chloroplasts, which is three orders of magnitude less sensitive to photoinhibition.     

A study of protoplasmic streaming inPhysarum by laser Doppler spectroscopy

Summary Protoplasmic streaming in the slime moldPhysarum polycephalum has been characterized using laser Doppler spectroscopy. Measurement of the spectrum of scattered laser light permits simultaneous determination of the velocities of all particles in the laser beam, with the relative intensity from each particle proportional to its light scattering cross-section. Simple experimental modifications allow the tracking of the oscillations of the streaming velocities. Rhythmic wall contractions can be monitored simultaneously with the flow velocities. Interpretation of the Doppler spectra shows that a small fraction of the particles in the flowing protoplasm are moving with velocities two to four times greater than the characteristic velocities reported by optical microscopy. Transverse velocities in the tubes are nearly as great as the longitudinal velocities. The shape of the Doppler spectrum at the maximum of the oscillation cycle is consistent with a spatial velocity profile which is sharper than parabolic, presumably because of a viscosity gradient from the center to the walls of the plasmodial tubes. The shape of the Doppler spectrum of depolarized scattered light is of approximately the same form. The response of the plasmodium to increased temperature is an increase in the frequency of the velocity oscillations with little change in the magnitude of the velocities. The response of the plasmodium to very high intensities of laser light is to gel at the point of incidence.     

Investigation of the temperature dependence of the cross bridge parameters for attachment,force generation and detachment as deduced from mechano-chemical studies in glycerinated single fibres from the dorsal longitudinal muscle of Lethocerus maximus

Birefringence change during excitation was studied by using Nitellopsis obtusa. The velocity change of cytoplasmic streaming during an action potential was measured simultaneously by fluctuation analysis of transmitted light intensity. The origin of the retardation change was discussed by comparing optical retardation change to the time course of the action potential, the cytoplasmic streaming velocity change and the cell contraction.By the time course analysis of retardation change, we concluded that the change of the birefringence might be the sum of the changes of cytoplasmic flow and that of the size of length and diameter of the cell. But it is still difficult to separate the change to its components.     

Protoplasmic streaming during the cell cycle of Physarum polycephalum

It has been reported that protoplasmic streaming stops during the synchronous mitosis exhibited by growing plasmodia of P. polycephalum. Our data reveal that at no time during the mitotic cycle did streaming stop. However, during a 3–5 min period at anaphase the percent of each oscillation period accounted for by an outward flow was precisely equal in duration to the corresponding inward flow. At all other periods the duration of outward flow exceeded that of inward flow. Plasmodial migration or locomotion was briefly arrested at telophase, although shuttle streaming persisted.     

Dynamic light scattering studies of the aggregation of lysozyme under crystallization conditions.

The intensity autocorrelation functions of light scattered by lysozyme solutions under pre-crystallization conditions in NaCl-containing media were recorded at scattering angles from 20 degrees to 90 degrees. The measurements, conducted on freshly prepared protein solutions supersaturated more than 3-fold, indicate the simultaneous presence of two scatterer populations which can be assigned to individual protein molecules and to large particles. When solutions are undersaturated, or slightly supersaturated, light scattering only reveals the presence of the small scatterers. In the supersaturated medium, where aggregates were detected, lysozyme crystals grew in a time-span of 1-3 days after the scattering experiments. These results are medium, where aggregates were detected, lysozyme crystals grew in a time-span of 1-3 days after the scattering experiments. These results are correlated with the nucleation step during protein crystallization.     

Volume phase transition of bovine vitreous body in vitro and determination of its dynamics

The phase equilibrium property and structural and dynamical properties of bovine vitreous body was studied by macroscopic observation of swelling behavior and dynamic light scattering under various conditions. It was found that the vitreous body collapses into a compact state isotropically or anisotropically depending on the external conditions. The vitreous body collapses while maintaining the shape when the pH (相似文献   

Localized dynamic light scattering: a new approach to dynamic measurements in optical microscopy.

We present a new approach to probing single-particle dynamics that uses dynamic light scattering from a localized region. By scattering a focused laser beam from a micron-size particle, we measure its spatial fluctuations via the temporal autocorrelation of the scattered intensity. We demonstrate the applicability of this approach by measuring the three-dimensional force constants of a single bead and a pair of beads trapped by laser tweezers. The scattering equations that relate the scattered intensity autocorrelation to the particle position correlation function are derived. This technique has potential applications for measurement of biomolecular force constants and probing viscoelastic properties of complex media.     

Cytoplasmic streaming inParamecium

Summary Certain species ofParamecium demonstrate rotational cytoplasmic streaming, in which most cytoplasmic particles and organelles flow along permanent route, in a constant direction. By means of novel methods of immobilization, observation and recording, some dynamic properties of cytoplasmic streaming have been described. It was found that the velocity profiles of coaxial layers of cytoplasm have a (parabolic) paraboidal shape and the mean output of cytoplasm flow in different examined zones of streaming is constant. As the consequence of randomly distributed elementary propulsion units within the cytoplasm, particles, which serve as markers of movement, exhibit movements of a saltatory nature; this form of movement is seen inParamecium streaming only in cases of error due to polarization of the saltating particles. Interaction of actin filaments and myosin is likely to occur under specific conditions in microcompartments of cytoplasm where local solations are generated eventually leading to contractions which might propagate on gelated neighbouring areas. Places of elementary contractions are scattered. Therefore the motile effect appears as streaming. Rotational cytoplasmic streaming inParamecium may serve as a convenient model for the study of the dynamics and function of cytoplasmic motility.     

Dynamic light scattering microscopy. A novel optical technique to image submicroscopic motions. I: theory

The theoretical basis of an optical microscope technique to image dynamically scattered light fluctuation decay rates (dynamic light scattering microscopy) is developed. It is shown that relative motions between scattering centers even smaller than the optical resolution of the microscope are sufficient to produce significant phase variations resulting in interference intensity fluctuations in the image plane. The timescale and time dependence for the temporal autocorrelation function of these intensity fluctuations is derived. The spatial correlation distance, which reports the average distance between constructive and destructive interference in the image plane, is calculated and compared with the pixel size, and the distance dependence of the spatial correlation function is derived. The accompanying article in this issue describes an experimental implementation of dynamic light scattering microscopy.     

Structural dynamics of frog muscle during isometric contraction

Intensity fluctuation autocorrelation functions of laser light scattered by actively contracting muscle were measured at points in the scattered field. They were reproducible and showed characteristics which depended on the physiological state of the muscle and the parameters of the scattering geometry. The autocorrelation functions had large amplitudes and decay rates that varied significantly with the phase of the contraction-relaxation cycle. The dependence of the autocorrelation function on scattering geometry indicated many elements with diameters on the order of 0.5 mum (presumed to be myofibrillar sarcomeres or their A bands or I bands) undergo independent random changes in their axial positions and their internal distribution of optical polarizability during the plateau of an isometric tetanus. The experimental results are interpreted in terms of a model in which most of the scattering elements in isometrically contracting muscle have random fluctuating axial velocities of average magnitude 20 nm/ms that persist for a few milliseconds at least. In addition to these axial motions there are local fluctuations in polarizability. Similar intensity fluctuation autocorrelation functions were observed throughout the active state on two muscle preparations, whole sartorius muscle and small bundles of single fibers (three to eight) of semitendinosus muscle. These results imply that the tension developed during an isometric tetanus contains a fluctuating component as well as a constant component.     

Rotational and translational motions of human spermatozoa: angle dependence of dynamic laser light scattering

We have studied how the dynamic components of laser light scattered from human spermatozoa depend on the scattering angle. This was done by investigating the halfwidth of the intensity autocorrelation function. A model of the spermatozoa as freely rotating and translating linear objects was adequate to describe the scattered light. Rotational motions determined the halfwidth of the intensity autocorrelation function at very small scattering angles and contribution from translational motions was dominant at scattering angles larger than 20 degrees. The contribution from translational motions increased with increasing scattering angle. We found a nearly linear relationship between the translation speed and the rotation frequency. However, the ratio between the two properties varied more than expected from the methodological error. Therefore we introduced a propelling efficacy as a concept to describe the swimming efficiency. This property might contain important information about the swim characteristics.Abbreviations ACF Autocorrelation function - _1/2 halfwidth - RGD Rayleigh-Gans-Debye - SD Standard deviation Correspondence to: P. Thyberg     

Autocorrelation and spectrum of the modulated retinal discharge: A comparison of theoretical and experimental results

The theoretical autocorrelation of the cat ganglion cell discharge under stationary and dynamic conditions of light stimulus is compared with the autocorrelation determined experimentally. To obtain the theoretical autocorrelation, the stationary discharge is described by a stationary random point process of independent intervals equally distributed according to a gamma function, and the dynamic behaviour is described by a model defined in a previous paper. Comparison shows that the model predicts the experimental results. Finally, the power density spectrum is analysed and the relevance of the results to signal transmission by the retinal system is discussed.     

Velocity distributions of the streaming protoplasm in Nitella flexilis.

Laser light is Doppler-shifted in frequency by the streaming endoplasm of living cells of Nitella flexilis. The frequency spectrum of the scattered light can be interpreted as the histogram of velocities within the organism, with the exception of the intense low-frequency portion of the spectrum. We demonstrate that the lowest-frequency component is the result of amplitude modulation of the scattered light by the array of chloroplasts in the cell. Measurement of the streaming endoplasm in a photobleached "window" region allows correction of the frequency distribution for the modulation component. The complete velocity histogram for the streaming endoplasm is calculated directly from the corrected frequency distribution. Measurements of vacuolar and endoplasmic motions show that the tonoplast, the membrane separating the vacuole and the endoplasm, seems to be flowing along with the endoplasm and vacuolar sap. Placing the cell in medium containing ATP in concentrations greater than 10(-3) M greatly increases the contribution of low velocities to the velocity histogram. Cytochalasin B at high dosages (10-50 mug/ml) does not noticably change the shape of the velocity histogram, while at low dosages (1 mug/ml) there is an increase in the contribution of low velocities to the velocity histogram. Colchicine in high concentrations (1%) has no observable effect on the velocity histogram.     

Haemodynamics of coronary artery - saphenous vein bypass

The velocity distribution of a suspension of red blood cell ghosts in an idealized model of the coronary artery-saphenous vein bypass has been investigated with the aid of laser Doppler anemometry. Pulsatile flow simulated pressure variations in the ascending aorta and ghost cell velocities were determined by the Doppler shift of scattered laser light. Using four different model bypasses it was demonstrated that turbulent flow at the graft-coronary intersection can be delayed by decreasing the discontinuity in diameter between the bypass vein and coronary artery, and also by reducing the bypass vein and host coronary artery intersection angle.     

Identification of the scattering elements responsible for lens opacification in cold cataracts.

Using both quasi-elastic light scattering spectroscopy and angular dissymmetry in the intensity of the scattered light, we examined the onset of turbidity for intact calf lenses and for isolated nuclear cytoplasm. In the case of the nuclear cytoplasm these measurements demonstrate the presence of two kinds of scatterers: small units of approximately 100-A radius and larger elements whose size is distributed around 1,500 A. As the temperature is decreased towards the cold cataract temperature, the intensity of light scattered by the small units stays almost constant while the intensity scattered by the large elements increase very strongly. The opacification of the lens cytoplasm produced by decreasing the temperature results principally from an increase in the concentration of the large scattering elements. For the intact nucleus the situation is qualitatively similar, but the mean size of the large scattering elements shows a more substantial increase than in the isolated cytoplasm as temperature is lowered towards the cold cataract temperature.     

Repair of functional and ultrastructural alterations after thermal injury of Physarum polycephalum

Repair of thermal injury of Physarum polycephalum Schw. plasmodia has been studied by light and electron microscopy. As a result of heating the plasmodia for 10 min at 42°C both the unordered and shuttle protoplasmic streaming were arrested; the outer plasmodial membrane showed alterations at sites of contact with water; the onset of the next mitosis was considerably delayed. The plasmodial ultrastructure was markedly disturbed, including disappearance of the granular component of the nucleoili and a compact, almost fibrillar structure of the latter. The mitochondria became distorted and their intracristal spaces enlarged while the outer and inner membranes appeared in some places to be separated. Glycogen particles disappeared from the cytoplasm. Recovery of both types of protoplasmic streaming of the motility of the plasmodium, of the resistance of its membrane to contact with water, and of the ability of the organism to go through the cell cycle went all hand in hand with the normalization of the structure of nucleoli, mitochondria and cytoplasm. All of the functional and structural characteristics are normalized within ca. 9 h following heating.