The correlation of human erythrocyte shape with dark-field light scattering intensity]

This study was concerned with the quantitative evaluation of dark field light scattering by sedimented erythrocytes of banked human blood samples. Due to considerable variability of both appearance and amount of scattered light the discocyte group had to be subdivided into discocyte I and discocyte II. The mean intensity of scattered light increased about three fold from discocyte II to echinocytes I, II, III, sphaeroechinocyte, and sphaerocyte. On the other hand the average light scattering intensity of discocytes I exceeded that of discocytes II about 2.5 times, with individual data varying over a wide range. There was a rapid disappearing of discocytes I correlated with time of storage. Therefore it is concluded that discocytes I represent the initial stage of erythrocytes transforming under banking conditions.     

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.     

Optical characteristics of flowing blood

The transmitted light strength (TS) through a thin blood layer changes with variation in blood flow, such as positive streaming transparency for low hematocrits and negative streaming transparency for high hematocrits. These phenomena are examined theoretically and experimentally. Maxwell’s equations are solved assuming that erythrocytes are oblate spheroids to investigate these phenomena due to flowing blood. The theoretical results reveal that the scattering and absorption cross sections for flowing blood are larger than those for stagnant blood. Experimental results indicate that the TS for both oxygenated and deoxygenated flowing blood, with a hematocrit of up to approximately 20%, was stronger than that for stagnant blood. The TS decreased for flowing blood with a hematocrit of approximately 20% or greater. Applying the theoretical scattering and absorption cross sections to the absorption and multiple scattering theory of Victor Twersky, the changes in the TS due to flowing blood are obtained theoretically. From the theoretical and experimental results, the positive streaming transparency phenomenon of flowing blood with a low hematocrit and the negative streaming transparency phenomenon with a high hematocrit are found to result from increased scattering and absorption cross sections because of the orientation of flowing erythrocytes.     

Effects of hemolysis, hematocrit, RBC swelling, and flow rate on light scattering by blood in a 0.26 cm ID transparent tube

The intensity of light scattering by blood in a tube of diameter 0.26 cm was measured with an apparatus devised by us at different angles on an incident cross-sectional plane. Changes in angular distribution of light intensity associated with hemolysis, and changes in hematocrit (Ht), red blood cell (RBC) swelling, and flow rate were plotted on polar coordinates. The dyssymmetry index, defined as the ratio of the intensity of light at 45 degrees to that at 135 degrees, was used to characterize the shape of the diagrams of light scattering. The index decreased with Ht, flow rate, but increased with RBC swelling. It is concluded that light scattering by blood requires intactness of the RBC membrane. Even when the cell membrane is intact, light scattering is subject to changes with the flow rate of blood, presumably due to RBC aggregation.     

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.     

生物组织光散射等效颗粒模型及Mie相函数计算

为研究生物组织的散射特性,将其从散射效果上等效为离散的球形散射体的集合,结合经典的Mie散射理论对生物组织散射相函数进行数值计算。计算结果表明:Mie散射相函数能够描述生物组织后向(大角度)散射光强振荡特性与等效粒径的对应关系,可为基于后向散射光的无创伤或微创伤诊疗提供理论依据;Mie散射相函数能够解释生物组织散射光空间分布与波长的相关性,为医学诊疗上入射光波长选择提供参考;合理选择集群散射体粒度分布参数,可实现对复杂生物组织散射相函数的精确描述。     

Sialic acid content and IgG binding of the glycocalyx of preserved erythrocytes]

Fractions of light and heavy erythrocytes were separated by centrifugation from blood samples banked in ACD-AG medium at 4 degrees C for periods up to 6 weeks. Both light and heavy erythrocytes have lost about 4,9% of their content of sialic acids during banking for 6 weeks. This reduction is in accord with a 6%-decrease of their agglutination by means of alcian blue. It is, however, a variance with the inhibition of agglutination by anti-IgG sera. The present findings provide evidence for the role alterations of the red cell membrane may play in the process of recognition and phagocytosis of banked erythrocytes. With regard to these alterations we suggest two types of rapid elimination of transfused banked erythrocytes: a) Primary elimination refers to cells primarily loaded with immunoglobulins such as to get recognized and phagocytized by macrophages. b) Secondary elimination accounts for rigid erythrocytes suffering from additional degradation while retained in the spleen prior to their loading with immunoglobulins and ensuing phagocytosis. Secondary elimination is considered a process more relevant to reutilisation of banked blood.     

Relationship of turbidity to the stages of platelet aggregation

The process of platelet aggregation as detected by turbidity changes in the platelet aggregometer was studied relative to light scattering by large particles. For latex beads a plot of light scattering intensity/unit mass versus particle size gave increased light scattering intensity for small particle sizes but decreased scattering at large particle size. This behavior is predicted by Rayleigh-Gans theory. These results were related to the platelet aggregometer, an optical instrument used to measure the association of small particles (monomeric platelets) to large particles (platelet aggregates). Formalin-fixed platelets do not show changes in light transmission due to energy-requiring processes, such as shape change, so that turbidity changes in the presence of aggregating agents could be attributed to a change in platelet aggregation state. Small platelet aggregates showed increased turbidity compared to a similar mass of monomeric platelets. In fact, very large platelet aggregates that were visible to the unaided eye were needed to produce a decrease in light scattering intensity. Thus, turbidity can either increase or decrease with platelet aggregation depending on the size of the aggregates. Studies of platelet aggregation that show no initial increase in turbidity must be characterized by dominance of large platelet aggregates and monomeric platelets.     

A study of the kinetics and mechanism of ADP-triggered platelet aggregation

The time-course of ADP-triggered aggregation of human blood platelets has been followed by sensitive right-angle light scattering intensity measurements as a function of the platelet and fibrinogen concentrations. Rayleigh-Gans light scattering theory has been combined with the Smoluchowski aggregation model to predict the dependence of the right-angle scattering intensity on particle size and concentration as well as the time-dependent changes during aggregation. The validity of the calculations was confirmed by measuring the scattering intensity with suspensions of polystyrene microspheres of known radius, as well as the time-dependent changes in the 90 degrees scattering intensity during aggregation of these particles. However, in contrast to the predictions of the model, the time-course of the scattering intensity changes during platelet aggregation was characterized by single exponential decay with a rate constant which reached a limiting value of 0.017 s-1 at high platelet concentrations. The value of kagg was also independent of the fibrinogen concentration over a 30-fold range. Covalently cross-linked fibrinogen dimers and Fragment D-inhibited fibrin protofibrils yielded aggregation rates that agreed with those measured with fibrinogen. The results indicate that the rate of platelet aggregation is not limited by either the rate of fibrinogen binding or the frequency of platelet-platelet collisions under these conditions.     

Measurement of intrinsic optical backscattering characteristics of cells using fiber-guided near infrared light

Background  

Intrinsic optical signals (IOS), which reflect changes in transmittance and scattering light, have been applied to characterize the physiological conditions of target biological tissues. Backscattering approaches allow mounting of the source and detector on the same side of a sample which creates a more compact physical layout of device. This study presents a compact backscattering design using fiber-optic guided near-infrared (NIR) light to measure the amplitude and phase changes of IOS under different osmotic challenges.     

Proposal for the simultaneous measurement of light absorption and backscattering by aquatic particulates

The paper presents a new experimental method that measures lightabsorption and backscattering by aquatic particles, combiningthe innovative features of the ‘modified filter–transfer–freeze’(FTF) and ‘transmittance–reflectance’ (T-R)techniques, namely no path-length amplification and accuratecorrectionfor scattering loss, respectively. This method (in short: theFTF/T-R method) measures both the transmittance and the reflectanceof particles deposited on a glass slide, using a double-beamspectrophotometer equipped with an integrating-sphere attachment.The data are interpreted by an algorithm that yields both theabsorption and the backscattering coefficients of the particlesample. The paper includes a summary of results of validationtests, that have been performed using samples ranging from purephytoplankton (low light scattering) to pure detrital particles(high light scattering), as well as a detailed analysis of theexperimental error. The FTF/T-R measurement is somewhat morelaborious than the standard transmittance measurement of particlesretained on glass fibre filters. However, it has the importantasset of permitting the simultaneous determination of lightabsorption and backscattering and it yields more accurate absorptiondata in situations where the magnitude of path amplificationby glass fibre filters is uncertain and light scattering bythe particles is high.     

Aggregation of red blood cells studied by ultrasound backscattering

The erythrocyte aggregation phenomenon is an important factor in capillary circulation. This phenomenon can be evaluated by a number of methods (microscopic observations, viscometry, light measurements) which cannot be applied simply to in vivo measurements. In contrast, ultrasound which propagates through soft tissues allows measurement of the mechanical properties of red blood cell (RBC) suspensions which depend on the aggregation phenomenon. We devised an apparatus in order to measure in vitro the ultrasonic backscattering intensity of RBC suspensions. First, with latex particles of different sizes, the ultrasonic backscattering coefficient has been measured in order to evaluate the apparatus response. Then, the ultrasonic backscattering coefficient of different aggregated erythrocyte suspensions has been measured and correlated with the erythrocyte sedimentation rate. Finally, the size of RBC aggregates of different suspensions has been evaluated.     

Light scattering and cell volumes in osmotically stressed and frozen-thawed cells

Recent reports, indicating that under some conditions the intensity of light scattering from cells is a nonlinear function of cell volume, have led to the widespread generalization that intensity of low-angle light scattering indicates cell size. This study was performed to measure the relationships between light scattering and cell volumes in an-isotonic solutions and after a freeze-thaw stress. Cell volumes in isolated human lymphocytes, human granulocytes, and hamster fibroblasts were deliberately altered by exposure to anisotonic solutions. Boyle-vant Hoff plots of cell volume as a function of inverse osmotic pressure showed that the cells behaved as osmometers. Similar plots of right-angle and low-angle light scattering showed that the intensity of light scattering varied inversely with cell volume. In other experiments where cells were frozen without cryoprotectant at various sub zero temperatures to -25 degrees C and then thawed rapidly, cell viability decreased progressively with decreasing temperature, as did the intensity of both low-angle and right-angle light scattering, although cell volumes remained relatively constant. The intensity of both low- and high-angle light scattering varied inversely with cell volumes in hypertonic and hypotonic solutions, but cell damage induced by freezing and thawing resulted in significant reductions in the intensity of low-angle light scattering with little change in cell volume. These observations show that light scattering and cell volumes can vary independently, and they underline the need for a better understanding of the phenomenon of light scattering from living cells.     

Four-parameter white blood cell differential counting based on light scattering measurements

Measurement of the depolarized orthogonal light scattering in flow cytometry enables one to discriminate human eosinophilic granulocytes from neutrophilic granulocytes. We use this method to perform a four-parameter differential white blood cell analysis. A simple flow cytometer was built equipped with a 5-mW helium neon laser that measures simultaneously four light scattering parameters. Lymphocytes, monocytes, and granulocytes were identified by simultaneously measuring the light scattering intensity at angles between 1.0 degrees and 2.6 degrees and angles between 3.0 degrees and 11.0 degrees. Eosinophilic granulocytes were distinguished from neutrophilic granulocytes by simultaneous measurement of the orthogonal and depolarized orthogonal light scattering. Comparison of a white blood cell differentiation of 45 donors obtained by the Technicon H-6000 and our instrument revealed good correlations. The correlation coefficients (r2) found were: 0.99 for lymphocytes, 0.76 for monocytes, 0.99 for neutrophilic granulocytes, and 0.98 for eosinophilic granulocytes. The results demonstrate that reliable white blood cell differentiation of the four most clinically relevant leukocytes can be obtained by measurement of light scattering properties of unstained leukocytes.     

Effect of change in concentration upon lens turbidity as predicted by the random fluctuation theory.

Theoretical calculations were performed to predict how the light scattering intensity would change with changes in concentration in the gel state. The theory of light scattering was applied to a random distribution of hard spheres. The spherical particles with constant diameter were embedded in a medium having a different refractive index. The light-scattering intensities obtained as a function of concentration showed that in dilute solutions the scattered light intensity increases with concentration. However, in concentrated solution greater than 0.1 or 0.2 volume fraction, the light-scattering intensity decreases with increase in concentration.     

Translational motion of actin filaments in the presence of heavy meromyosin and MgATP as measured by Doppler broadening of laser light scattering

Intensity fluctuations of laser light scattering were utilized in order to follow enhancement of translational motion of the actin-heavy meromyosin (HMM) complex in extremely dilute solutions accompanied by the hydrolysis of MgATP. Such enhancement was anticipated on the basis of the idea that active streaming along actin filaments should be associated with their mechanochemical reactivity. Native tropomyosin was added in order to stabilize actin in its filamentous form, thus allowing the reduction of actin concentration below 50 micrograms/ml to enable free movement of neighboring filaments and yet give a reliable signal. Analysis of the data in terms of Doppler broadening led to an approximate evaluation of the average velocity of translation of the mobile filaments. This velocity was found to increase with increasing HMM concentration up to a maximum attained at a molar ratio HMM/actin of 1:2, and then decreased. Total intensity measurements indicate that the mobile scatterer is actually a complex of HMM with an isolated actin filament. HMM subfragment-1 was found to be ineffective. These results suggest that cooperation between the two myosin heads is necessary for efficient induction of active streaming along isolated actin filaments.     

Light scattering from suspensions of membrane fragments derived from sonication of beef heart mitochondria.

The intensity of light scattering from suspensions of membrane fragments prepared by sonication of beef heart mitochondria in the presence of EDTA at alkaline pH (ESMP) was determined at 45, 90, and 135 degrees with light of wavelength 546 nm. The dissymmetry ratio Z = I45 degrees c/I135 degrees c, where I45 degrees c and I135 degrees c are the scattering intensities at 45 and 135 degrees extrapolated to zero particle concentration and corrected for reflectance effects, was used to calculate particle size from the Rayleigh-Gans-Debye theory. An average particle diameter D of 184-190 nm was obtained, within the range of particle diameter 50-300 nm determined previously by electron microscopy. This average diameter determined by light scattering is a useful parameter for characterization of ESMP particle size. We propose the term: light scattering average particle diameter, DLS, for this parameter. The refractive index of ESMP was determined to be 1.443 by measurement of scattering intensity in buffer solutions of varying sucrose concentration. The value of Z was independent of sucrose concentration in this determination, showing that the particles are osmotically inactive toward sucrose. The values of average particle diameter DLS and of refractive index fall within the range of validity of the Rayleigh-Gans-Debye theory, for which light scattering changes are attributable solely to dimension change, rather than to change in particle refractive index. Uptake of water accompanying energy-linked salt uptake in ESMP was calculated from light scattering changes to be 0.18 mul of H2O/mg of protein, compared with 0.49 mul of H2O/mg of protein measured by dextran inaccessibility. Measurement of light scattering changes provides a rapid and sensitive method for determining volume changes of ESMP. The magnitude of the volume change observed during energy-linked water and salt uptake and the initial degree of hydration suggests that ESMP are analogous to polyelectrolyte gels with regard to sorption of strong electrolytes and that the Donnan formulation for ion exchange equilibria may be usefully applied to these processes in ESMP.     

内陆湖泊主要藻种散射特性

通过室内培养4种主要淡水藻种--铜绿微囊藻(Microcystis aeruginosa)、普通小球藻(Chlorella vulgaris)、梅尼小环藻(Cyclotella meneghiniana)以及卵形隐藻(Cryptomonas ovata), 测定其散射和后向散射特征参数、用单位叶绿素a浓度的散射值和后向散射值来表征4种藻的散射和后向散射能力。结果表明, 铜绿微囊藻散射和后向散射能力最强, 其次为梅尼小环藻, 普通小球藻的能力最弱。通过计算后向散射概率, 显示铜绿微囊藻和梅尼小环藻的后向散射概率值较高, 普通小球藻和卵形隐藻的后向散射概率值较低。后向散射特性影响因子分析显示, 影响后向散射值的主要因素有叶绿素a浓度及藻蓝蛋白色素比例。当叶绿素a浓度不断增加时, 其后向散射值不断增大; 当藻类所含叶绿素a比重不断上升时, 其后向散射值也不断增大。而细胞粒径与后向散射值之间未表现出很好的相关性。因此, 通过单位叶绿素a散射和后向散射概率特征可以辨别出藻细胞形态较为接近的铜绿微囊藻和普通小球藻。     

Quasi-elastic light scattering studies of membrane motion in single red blood cells.

Studies of red blood cells (RBCs) and RBC ghosts, using a quasi-elastic light scattering (QELS) microscope spectrometer, have identified the membrane as the primary source of the light scattering signal. This is the first report in which motion of the cell membrane has been demonstrated to be the primary source of the QELS signal from a cell. Cytoplasmic changes induced in the RBC by varying the osmotic strength of the medium were also detected using this technique. Comparison of the data from white blood cells (WBCs) with the RBC data demonstrated significant differences between different types of cells.