Flow sorting of mouse pancreatic B cells by forward and orthogonal light scattering

Mouse islet cell suspensions were separated into subpopulations based on forward low angle and orthogonal scattered light. Separation fractions were analyzed for hormone content by radioimmunoassays. The pancreatic B-cells had the highest orthogonal light scattering, while they overlapped in forward low angle light scattering with other endocrine (glucagon, pancreatic polypeptide and somatostatin containing cells) and nonendocrine cells. Living and paraformaldehyde fixed islet cell suspensions showed similar distributions of combined light scattering.     

Particle motion in single acinar cells observed by microscope laser light scattering spectroscopy

We report here the direct observation of the motion of cytoplasmic subcellular organelles and macromolecules within single, metabolically active, pancreatic acinar cells using microscope laser-light scattering spectroscopy (MLLSS). The relative amount and the effective diffusion coefficient of the mobile particles shows a marked difference in magnitude between the apical and basal regions of the cell. Secretory stimulation evoked by the cholinergic agonist bethanechol induces changes in the relative motion in the cytoplasm of approximately one fifth of the acinar cells. This study demonstrates the feasibility and utility of a novel technique, MLLSS, for the analysis of intracellular events in regions as small as (2 m)³ in single, granule secreting cells. It also shows MLLSS to be a powerful tool for the detection and measurement of altered motion in disrete subcellular regions of small mammalian cells after biochemical and pharmacological manipulations.     

Measurement of inherent particle properties by dynamic light scattering: introducing electrorotational light scattering.

Common dynamic light scattering (DLS) methods determine the size and zeta-potential of particles by analyzing the motion resulting from thermal noise or electrophoretic force. Dielectric particle spectroscopy by common microscopic electrorotation (ER) measures the frequency dependence of field-induced rotation of single particles to analyze their inherent dielectric structure. We propose a new technique, electrorotational light scattering (ERLS). It measures ER in a particle ensemble by a homodyne DLS setup. ER-induced particle rotation is extracted from the initial decorrelation of the intensity autocorrelation function (ACF) by a simple optical particle model. Human red blood cells were used as test particles, and changes of the characteristic frequency of membrane dispersion induced by the ionophore nystatin were monitored by ERLS. For untreated control cells, a rotation frequency of 2 s-1 was induced at the membrane peak frequency of 150 kHz and a field strength of 12 kV/m. This rotation led to a decorrelation of the ACF about 10 times steeper than that of the field free control. For deduction of ERLS frequency spectra, different criteria are discussed. Particle shape and additional field-induced motions like dielectrophoresis and particle-particle attraction do not significantly influence the criteria. For nystatin-treated cells, recalculation of dielectric cell properties revealed an ionophore-induced decrease in the internal conductivity. Although the absolute rotation speed and the rotation sense are not yet directly accessible, ERLS eliminates the tedious microscopic measurements. It offers computerized, statistically significant measurements of dielectric particle properties that are especially suitable for nonbiological applications, e.g., the study of colloidal particles.     

Clearance of apoptotic cells by phagocytes

Phagocytic clearance of apoptotic cells may be considered to consist of four distinct steps: accumulation of phagocytes at the site where apoptotic cells are located; recognition of dying cells through a number of bridge molecules and receptors; engulfment by a unique uptake process; and processing of engulfed cells within phagocytes. Here, we will discuss these individual steps that collectively are essential for the effective removal of apoptotic cells. This will illustrate our relative lack of knowledge about the initial attraction signals, the specific mechanisms of engulfment and processing in comparison to the extensive literature on recognition mechanisms. There is now mounting evidence that clearance defects are responsible for chronic inflammatory disease and contribute to autoimmunity. Therefore, a better understanding of all aspects of the clearance process is required before it can truly be manipulated for therapeutic gain.     

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.     

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.     

Cell discrimination by multiangle light scattering.

Measurement of the light scattered by biological cells as a function of scattering angle provides information that can be correlated with cell type. Two flow systems that provide multiangle scattering data from cells have been constructed and tested. The first utilizes two narrow-aperture detectors positioned at different angles; the second utilizes the motion of the cell to generate complete scatter patterns of individual cells over a 67 degrees range of scattering angle.     

Depolarized light scattering by amides and peptides

The Rayleigh ratios R_H have been measured for the depolarized scattering dilute solutions of N-ethylacetamide (EA), N-methylpropionamide (MP), and N-acetyl-pyrrolidine (AP) in P-dioxane, and dilute aqueous solutions of N-methylacetamide (MA), N,N-dimethylacetamide (DMA), EA, and MP. Squares of the optical anisot-ropies γ of the amides are obtained through extrapolation of these mesurements to infinite dilution. Values of γ² found for EA and MP in dioxane are in good agreement with calculations based principally on the previously evaluated polarizability tensor of the amide group in conjunction with C? C and C? H bond polarizabilities. The calculations also involve averaging over all conformations, each being weighted according to the estimated conformational energy. The mean-squared optical anisotropies (γ²) of the oligoglycines and oligoalanines are calculated by similarly averaging over all skeletal conformations. The anistropic polarizability tensor \documentclass{article}\pagestyle{empty}\begin{document}$ \hat \alpha $\end{document} for the prolyl structural unit is derived for γ² for AP. The much larger optical anisotropies exhibited by the amides when dissolved in water as compared with those observed in dixane are discussed.     

Continuous assay for DNA polymerization by light scattering

The time course of DNA polymerization, catalyzed by DNA polymerase I, can be monitored by following changes in light scattering that result from increases in molecular weight of the polymer. Increases in mass of 10% result in an easily measurable change in light scattering. It has been shown that the light scattering change is correlated with the incorporation of radioactive nucleotide, as measured by the filter binding method. Continuous monitoring of the time dependence of polymerization should prove useful in kinetic analysis of the polymerase reaction and related enzyme-DNA interactions.     

A theory of diffuse light scattering by lungs

We present a theoretical treatment of backscattered light from the interior of a lung illuminated by a thin beam of light normally incident on the pleural surface. An approximate formula is developed describing how the backscattered intensity varies with distance from the point of light entry. This is shown to depend markedly on the optical mean free path and on the effective extinction coefficient. We attempt to relate the optical mean free path to the mean alveolar size. This relationship is found to depend primarily on septal reflection and refraction. Reflection is treated quantitatively. Refraction is much more difficult and may have to be approached empirically. We present here the rudiments of a technique with implications for the possibility of dynamic stereology.     

Dynamic light scattering by kappa- and lambda-carrageenan solutions

The intensity correlation functions of kappa- and lambda-carrageenan in various salt solutions and at different concentrations have been determined with the help of dynamic light scattering. From the first cumulant of these correlation functions the values of the translational diffusion coefficients D have been derived. They increase with macromolecular concentration. The extrapolated values to infinite dilution of the diffusion coefficients increase with increasing salt concentration as expected from the salt concentration dependence of the r.m.s. radii of gyration determined previously by static light scattering. The translational diffusion coefficient of lambda-carrageenan in 0.1 M NaCl is smaller than the corresponding value for the kappa species. This is consistent with the difference in contour length and linear charge density of the two samples used. No satisfactory interpretation for the concentration dependence of the diffusion coefficient seems to be possible at present. Although current theories for the macromolecular and salt concentration dependence of D, taking into account charge effects, seem to be applicable, they do not allow for a consistent interpretation of the data. No specific difference between the solution behaviour of kappa- and lambda-carrageenan has been detected.     

Characterization of acid-denatured DNA by low-angle light scattering

Low-angle light scattering results reported previously demonstrated that measurements on high molecular weight native DNA must be made at angles below 30° in order to obtain correct molecular weights. Earlier light-scattering data obtained on denaturated DNA at angles above 30° showed no change in molecular weight upon denaturation, even though other techniques clearly showed that strand separation occurred. This paper reports low-angle measurements on solutions of calf thymus and T7 DNA denatured under acidic conditions. The results demonstrate that a halving of molecular weight consistent with strand separation is detected by light scattering only when low-angle data are used to obtain correct molecular weights for native material. As expected from theoretical considerations, the scattering from denatured DNA is a linear function of sin²(θ/2), where θ is the angle of observation. This result shows that anticipated experimental artifacts have no significant effect on the low-angle measurements and demonstrates that the curvature in the scattering envelope observed for native DNA below 30° is an inherent property of the native molecule.     

Erythrocyte aggregation: approach by light scattering determination

Erythro-aggregometer is a Couette viscometer which was developed to measure aggregation parameters of red blood cells. The system is based on the analysis of the light intensity backscattered by a blood suspension. It allows to approach aggregation phenomenon in terms of kinetics, structural and rheological parameters. The measurement system designed for use with a microcomputer is suitable for both research and clinical investigations.     

Self-assembly of melanin studied by laser light scattering

The unknown molecular weight and chemical structure of melanin place the study of these pigments outside the range of the classical biochemical techniques; thus in this paper the problem of characterizing these heterogeneous biopolymers was approached by means of light scattering techniques, static and dynamic. The static technique allowed us to identify the macromolecular properties (MW and R(g)(2)(1/2)) of melanin extracted from sepia inksac and of two synthetic analogues: L-Dopa melanin obtained by autooxidation and by enzymatic oxidation by Tyrosinase. By dynamic light scattering (DLS), the hydrodynamic radius R(h) was measured to monitor the temporal behaviour of the polymerization and aggregation processes and R(h) variation by changing the chemical constraints of the polymerization medium, such as pH and ionic strength. The fractal dimension d of the aggregates of melanin, both natural and synthetic, in the past only recognized during the aggregation of the synthetic one by lowering the pH of the medium, was a useful parameter to further investigate and compare the structure of melanin granules of differing origins, revealing for the natural sample, a structure with clusters that are spherical, not largely hydrated and self-assembled, following a reaction limited aggregation kinetics (d=2.38).     

Desmin filaments studied by quasi-elastic light scattering.

We studied polymers of desmin, a muscle-specific type III intermediate filament protein, using quasi-elastic light scattering. Desmin was purified from chicken gizzard. Polymerization was induced either by 2 mM MgCl(2) or 150 mM NaCl. The polymer solutions were in the semidilute regime. We concluded that the persistence length of the filaments is between 0.1 and 1 microm. In all cases, we found a hydrodynamic diameter of desmin filaments of 16-18 nm. The filament dynamics exhibits a characteristic frequency in the sense that correlation functions measured on one sample but at different scattering vectors collapse onto a single master curve when time is normalized by the experimentally determined initial decay rate.     

Discrimination of myogenic and nonmyogenic cells from embryonic skeletal muscle by 90 degrees light scattering

A myogenic cell suspension was isolated from the breast muscles of 10-day-old chicken embryos by trypsin digestion. The cell preparation was subjected to Percoll density centrifugation to reduce the number of fibroblast-like cells present. The Percoll-isolated, predominantly myogenic cell population was then fractionated by flow cytometry using 90 degrees light scattering as the parameter for sorting. Cells exhibiting lower scatter, with a peak of 45 units, produced cultures containing myotubes and gave rise only to myogenic clones. Cells exhibiting higher scatter (120-200 units) produced nonmyogenic cultures and gave rise to nonmyogenic clones. Cells with intermediate light scatter were also detected. The latter produced both myogenic and nonmyogenic clones. The differences in light scatter presumably reflect higher cytoplasmic complexity of the nonmyogenic cells compared with the myogenic cells. Moreover, the differences in light scattering properties of the different cell types offer a means for the isolation of pure populations of myogenic cells directly from the intact muscle.     

Discrimination of early and late apoptotic cells by NBD-phosphatidylserine-labelling and time-lapse observation of phagocytosis of apoptotic cells by macrophages

Since free apoptotic cells are not detected in normal tissues, it is generally believed that apoptotic cells are removed as soon as they appear in vivo. A fluorescent derivative of phosphatidylserine, 1-palmitoyl-2-[6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl]-sn-glycero-3-phospho-L-serine (NBD-PS) is known to be incorporated into living cells, and thereafter gradually absorbed into either fatty acid-free bovine serum albumin or fetal calf serum from the outer leaflet of the cell membrane. When thymocytes were irradiated with X-ray and cultured in the presence of NBD-PS, cells became less fluorescent as apoptosis advanced, but early apoptotic cells were still positive for NBD-PS. We then co-cultured such early apoptotic thymocytes with resident peritoneal macrophages. Upon examination under a time-lapse fluorescence microscope, it was found that the attachment of early apoptotic cells to macrophages does not cause rapid phagocytosis, as compared with late apoptotic cells, suggesting the possibility that, in contrast to the widely held view, early apoptotic cells may not be quickly removed by phagocytes in vivo.     

The small angle light scattering of biological cells. Theoretical considerations

The light scattered by living biological cells (assumed homogeneous spheres with a relative refractive index, m = 1.05) at small angles has been calculated by the Hodkinson approximation and the more rigorous Mie theory. Both methods predict that relative volume distributions may be estimated from low angle scattering measurements on each cell in a population. Under conditions of short wavelength illumination or strong absorption, absolute volume information may also be obtained.