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.     

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.     

Light-scattering studies of bull spermatozoa. II. Interaction and concentration effects.

The complete autocorrelation function of the intensity fluctuations of laser light scattered from motile bull spermatozoa is shown to depend upon several factors not previously considered. Samples of bull spermatozoa generally contain a substantial proportion of dead cells, which give rise to slowly decaying components of the autocorrelation function. Whereas previous work has concentrated on the form of the fast decaying autocorrelation component, we are concerned here with the relative amplitude and shape of the slow autocorrelation component and the general form of the composite function. In principle, the relative amplitudes of the fast and slow components of the autocorrelation function can be used as an assay of the proportion of swimming cells. We show that this amplitude ratio depends upon cell concentration, scattering cell geometry, and scattering angle. A simple model is developed to explain these results on the basis of the asymmetry of light scattered from these cells, motile/immotile cell interactions, wall-swimming effects, and geotactic reorientation of dead cells.     

Microscope laser light scattering spectroscopy of single biological cells

A microscope laser light scattering setup was developed, allowing us to do intensity autocorrelation spectroscopy on the light scattered from a volume as small as (2 μm)³. This non-invasive technique makes cytoplasmic studies possible inside single live biological cells. The effect of osmotic swelling and shrinking on the diffusion coefficient of hemoglobin inside intact red blood cells is shown as an illustrative example of the applicability and sensitivity of this new experimental method.     

Cytoplasmic actomyosin fibrils in tissue culture cells

Summary A special cell line derived from a rat mammary adenocarcinoma (RMCD cells) displays a distinct pattern of actomyosin fibrils (AM fibrils) visible with phase contrast, Nomarski interference and polarized light optics. It was shown that the cytoplasmic AM fibrils are arranged as bundles of highly parallel F-actin filaments. The chemical nature of the filaments was identified by incubation with heavy meromyosin from rabbit skeletal muscle.These cytoplasmic actomyosin fibrils actively contract under isotonic conditions. This was shown by contraction experiments under polarized light optics, by cinematographic analysis and by direct proof of the contractility of AM fibrils isolated by laser micro-dissection. Thus, cytoplasmic AM fibrils can be assumed to represent structures essential for motive force generation in contraction processes in non-muscle cells.We thank Dr. W. Meier-Ruge and Mr. W. Bielser (Basic Medical Research Departments, Sandoz AG, Basle, Switzerland) for making the laser equipment available to us and for their kind cooperation during this investigation. Supported by the Deutsche Forschungsgemeinschaft, Bonn-Bad Godesberg.     

Microscope laser light scattering spectroscopy of single biological cells

A microscope laser light scattering setup was developed, allowing us to do intensity autocorrelation spectroscopy on the light scattered from a volume as small as (2 micron)3. This non-invasive technique makes cytoplasmic studies possible inside single live biological cells. The effect of osmotic swelling and shrinking on the diffusion coefficient of hemoglobin inside intact red blood cells is shown as an illustrative example of the applicability and sensitivity of this new experimental method.     

The structure of cytoplasm in directly frozen cultured cells. II. Cytoplasmic domains associated with organelle movements

The relationship between organelle movement and cytoplasmic structure in cultured fibroblasts or epithelial cells was studied using video-enhanced differential interference contrast microscopy and electron microscopy of directly frozen whole mounts. Two functional cytoplasmic domains are characterized by these techniques. A central domain rich in microtubules is associated with directed as well as Brownian movements of organelles, while a surrounding domain rich in f-actin supports directed but often intermittent organelle movements more distally along small but distinct individual microtubule tracks. Differences in the organization of the cytoplasm near microtubules may explain why organelle movements are typically continuous in central regions but usually intermittent along the small tracks through the periphery. The central type of cytoplasm has a looser cytoskeletal meshwork than the peripheral cytoplasm which might, therefore, interfere less frequently with organelles moving along microtubules there.     

A comparison of methods for morphological studies of cultured cells

Summary A number of fixation methods for different types of cells in culture were compared, and the best preservation of nuclear and cytoplasmic details was obtained by fixation with Bouin's solution for 15 min, prior to staining with hematoxylin and eosin. All of the fixatives, including Bouin's solution, damaged various structures, notably the peripheral glas-attached cytoplasm and the intercellular connections. Micrographs obtained by bright field, phase contrast, and interference contrast (Nomarski) microscopy are presented. Much more realistic pictures, bringing out details not observed after fixation and staining, were obtained by Nomarski microscopy of living, unfixed cultures. Most conspicuous were numerous thin, cytoplasmic, cilia-like extensions, concentrated on the glass-attached peripheral margins, which were also visible on other cell surfaces and as intercellular connections. These structures were most characteristic of SV40-transformed human amnion cells. Although fixation and staining emphasize certain cell components (for example, inclusion bodies), many aspects of cellular morphology are better demonstrated by observing living cells by interference microscopy or by Nomarski interference contrast microscopy. Surface features of unfixed cells, seen by Nomarski interference contrast microscopy, were similar to the surface features of glutaraldehyde-osmium tetroxide-fixed cells studied as metallic replicas in the electron microscope. Supported in part by National Cancer Institute Research Grant CA-08748 and contributions from the Albert Soiland Cancer Foundation.     

Dynamic light scattering microscopy. A novel optical technique to image submicroscopic motions. II: Experimental applications

An experimental verification of an optical microscope technique to create spatial map images of dynamically scattered light fluctuation decay rates is presented. The dynamic light scattering microscopy technique is demonstrated on polystyrene beads and living macrophage cells. With a slow progressive scan charge-coupled device camera employed in a streak-like mode, rapid intensity fluctuations with timescales the order of milliseconds can be recorded from these samples. From such streak images, the autocorrelation function of these fluctuations can be computed at each location in the sample. The characteristic decay times of the autocorrelation functions report the rates of motion of scattering centers. These rates show reasonable agreement to theoretically expected values for known samples with good signal/noise ratio. The rates can be used to construct an image-like spatial map of the rapidity of submicroscopic motions of scattering centers.     

Observation of flagellation of spermatozoa by depolarized laser light scattering.

Depolarized laser light-scattering theory was applied to derive the autocorrelation function of laser light scattered by motile spermatozoa, assuming that each spermatozoon is a chain of rotatable rigid ellipsoids of revolution and also that the rotational velocity about an axis perpendicular to the symmetry axis of the ellipsoid is constant for times of the order of the characteristic decay time of the autocorrelation function. The rotations are produced by flagellar movements of the spermatozoa. The correlation function thus obtained was related to the second-order coefficient of a Legendre polynomial expansion of the rotation of the direction angle of the ellipsoidal axis. The experimental fact that the correlation function for dead spermatozoa of sea urchin resembled that for flagella mechanically separated from spermatozoa indicated to us that the depolarized light was scattered mainly by flagella. The rotational velocity distribution of the flagella was determined by comparing the theoretical analysis with the experimentally obtained correlation functions for the motile and dead spermatozoa. The value of the average velocity caused by the flagellation, 230 rad/s, was in good agreement with that measured under an optical microscope.     

Multiangle light scattering flow photometry of cultured human fibroblasts: comparison of normal cells with a mutant line containing cytoplasmic inclusions.

Multi-angle light scattering flow photometry was used to study the light scattering properties of normal cultured fibroblasts and a mutant fibroblast line containing cytoplasmic lysosomal inclusions. The effect of glutaraldehyde fixation on the light scattering properties of the cells was also examined and correlated with their ultrastructure. Normal fibroblasts showed uniform organelle distribution with few vacuoles or dense bodies in the cytoplasm while the mutant line showed abnormal cytoplasmic inclusions of varying morphology, density and lucency. As predicted by light scattering theory, the mutant cells containing the cytoplasmic inclusions scattered more light at large angles (greater than theta = 1.85 degrees) than did the normal cells. Glutaraldehyde fixation decreased light scattering at small angles (less than theta = 1.85 degrees), increased light scattering at larger angles (greater than theta = 1.85 degrees) in both normal and mutant cells and enhanced resolution of the light scattering signatures. The mutant line scattered 2-3 times more light at a wide angle (greater than theta = 12.74 degrees) than did the normal cells. These data suggest that abnormal lysosomal storage inclusion bodies in the cytoplasm of the cells can be detected by differential light scattering methods.     

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.     

Visualization of the Peroxisomal Compartment in Living Mammalian Cells: Dynamic Behavior and Association with Microtubules

Peroxisomes in living CV1 cells were visualized by targeting the green fluorescent protein (GFP) to this subcellular compartment through the addition of a COOH-terminal peroxisomal targeting signal 1 (GFP–PTS1). The organelle dynamics were examined and analyzed using time-lapse confocal laser scanning microscopy. Two types of movement could be distinguished: a relatively slow, random, vibration-like movement displayed by the majority (~95%) of the peroxisomes, and a saltatory, fast directional movement displayed by a small subset (~5%) of the peroxisomes. In the latter instance, peak velocities up to 0.75 μm/s and sustained directional velocities up to 0.45 μm/s over 11.5 μm were recorded. Only the directional type of motion appeared to be energy dependent, whereas the vibrational movement continued even after the cells were depleted of energy. Treatment of cells, transiently expressing GFP–PTS1, with microtubule-destabilizing agents such as nocodazole, vinblastine, and demecolcine clearly altered peroxisome morphology and subcellular distribution and blocked the directional movement. In contrast, the microtubule-stabilizing compound paclitaxel, or the microfilament-destabilizing drugs cytochalasin B or D, did not exert these effects. High resolution confocal analysis of cells expressing GFP–PTS1 and stained with anti-tubulin antibodies revealed that many peroxisomes were associated with microtubules. The GFP–PTS1–labeled peroxisomes were found to distribute themselves in a stochastic, rather than ordered, manner to daughter cells at the time of mitosis.     

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.     

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 μm 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.     

A study of the dynamic properties of the human red blood cell membrane using quasi-elastic light-scattering spectroscopy.

A quasi-elastic light-scattering (QELS) microscope spectrometer was used to study the dynamic properties of the membrane/cytoskeleton of individual human red blood cells (RBCs). QELS is a spectroscopic technique that measures intensity fluctuations of laser light scattered from a sample. The intensity fluctuations were analyzed using power spectra and the intensity autocorrelation function, g(2)(tau), which was approximated with a single exponential. The value of the correlation time, Tcorr, was used for comparing results. Motion of the RBC membrane/cytoskeleton was previously identified as the source of the QELS signal from the RBC (R. B. Tishler and F. D. Carlson, 1987. Biophys. J. 51:993-997), and additional data supporting that conclusion are presented. Similar results were obtained from anucleate mammalian RBCs that have structures similar to that of the human RBC, but not for morphologically distinct, nucleated RBCs. The effect of altering the physical properties of the cytoplasm and the membrane/cytoskeleton was also studied. Osmotically increasing the cytoplasmic viscosity led to significant increases in Tcorr. Increasing the membrane cholesterol content and increasing the intracellular calcium content both led to decreased deformability of the human RBC. In both cases, the modified cells with decreased deformability showed an increase in Tcorr, demonstrating that QELS could measure biochemically induced changes of the membrane/cytoskeleton. Physiological changes were measured in studies of age-separated RBC populations which showed that Tcorr was increased in the older, less deformable cells.     

Histological and Ultrastructural Studies on the Conjunctiva of the Barred Owl (Strix varia)

This report is the first characterization of the histology and ultrastructure of the barred owl conjunctiva. The inferior eyelid was dominated by a large disk-shaped plate covered by a non-keratinized stratified squamous or cuboidal epithelium of variable thickness. The apical surface of the plate epithelium varied from flat to long microvilli or even short cytoplasmic extensions similar to those seen in the third eyelid. All specimens had a few goblet cells filled with mucous secretory granules in the plate region. The underlying connective tissue was a dense fibroelastic stroma. Eosinophils were surprisingly common in the epithelial layer and underlying connective tissue in the plate and more distal orbital mucosal region. The orbital mucosa contained goblet cells with heterogeneous glycosylation patterns. The leading edge and marginal plait of the third eyelid are designed to collect fluid and particulate matter as they sweep across the surface of the eye. The palpebral conjunctival surface of the third eyelid was covered by an approximately five-cell-deep stratified squamous epithelium without goblet cells. The bulbar surface of the third eyelid was a bilayer of epithelial cells whose superficial cells have elaborate cytoplasmic tapering extensions reaching out 25 μm. Narrow cytofilia radiated outwards up to an additional 15–20 μm from the cytoplasmic extensions. Lectin labeling demonstrated heterogeneous glycosylation of the apical membrane specializations but only small amounts of glycoprotein-filled secretory granules in the third eyelid.     

Computer-assisted analysis of particulate axoplasmic flow in organized CNS tissue cultures

Using organized nervous system tissue cultures of embryonic mouse spinal cord, time-lapse motion pictures of intra-axonal particle movements were made using Nomarski optics. The paths of 272 particles were followed and analyzed using digital computer methods. Particles had a bidirectional saltatory motility with net anterograde and retrograde velocities which were shown to be the same (about 1 micron per second). When anterograde moving and retrograde moving particles were analyzed separately for anterograde and retrograde saltations, the velocities of saltations in the direction of net movement were found to be 1.5 times greater than saltations against the direction of net movement. These differences were statistically significant. In addition, it was shown that there were regions within the axon where variations in particle motion were similar from particle to particle as they passed, and that more disturbed motion was found to occur near intra-axonal objects which were judged to be mitochondria. No decrease of particle velocity was noted in regions away from mitochondria nor was an increase in velocity noted near them. Computer-drawn particle plots illustrate the various facets of particle motion.     

Computer-assisted analysis of particulate axoplasmic flow in organized CNS tissue cultures.

Using organized nervous system tissue cultures of embryonic mouse spinal cord, time-lapse motion pictures of intra-axonal particle movements were made using Nomarski optics. The paths of 272 particles were followed and analyzed using digital computer methods. Particles had a bidirectional saltatory motility with net anterograde and retrograde velocities which were shown to be the same (about 1 micron per second). When anterograde moving and retrograde moving particles were analyzed separately for anterograde and retrograde saltations, the velocities of saltation in the direction of net movement were found to be 1.5 times greater than saltations against the direction of net movement. These differences were statistically significant. In addition, it was shown that there were regions within the axon where variations in particle motion were similar from particle to particle as they passed, and that more disturbed motion was found to occur near intra-axonal objects which were judged to be mitochondria. No decrease of particle velocity was noted in regions away from mitochondria nor was in increase in velocity noted near them. Computer-drawn particle plots illustrate the various facets of particle motion.     

Targeting of secretory vesicles to cytoplasmic domains in AtT-20 and PC- 12 cells

Organelles are not uniformly distributed throughout the cytoplasm but have preferred locations that vary between tissues and during development. To investigate organelle targeting to cytoplasmic domains we have taken advantage of the mouse pituitary cell line, AtT-20, which, when induced to extend long processes, accumulates dense core secretory granules at the tips of the processes. During mitosis, these secretory granules accumulate along the plane of division. Protein synthesis is not mandatory for such redistribution of secretory granules. To explore the specificity of the redistribution we have used transfected AtT-20 cells that express the immunoglobulin kappa light chain. While the endogenous hormone ACTH is found in secretory granules, the kappa chain is a marker for organelles involved in constitutive secretion. By immunofluorescence, kappa also accumulates at the tips of growing processes, and along the midline of dividing cells, suggesting that the redistribution of vesicles is not specific for dense-core secretory granules. Since there is evidence for selective organelle transport along processes in neuronal cells, the rat pheochromocytoma cell PC-12 was transfected with DNA encoding markers for regulated and constitutive secretory vesicles. Again regulated and constitutive vesicles co-distribute, even in cells grown in the presence of nerve growth factor. We suggest that at least in the cells studied here, cytoskeletal elements normally carry exocytotic organelles to the surface; when the cytoskeletal elements coalesce in an extending process, exocytotic organelles of both the constitutive and regulated pathway are transported nonselectively to the tips of the cytoskeletal elements where they accumulate.