Light-scattering Profiles Research Articles

Scanning flow cytometer modified to distinguish phytoplankton cells from their effective size, effective refractive index, depolarization, and fluorescence

A laser flow cytometer based on scanning flow cytometry has been assembled. The unpolarized and linearly polarized light-scattering profiles, as well as the side emitted light in different spectral bands, were measured, allowing the simultaneous and real-time determination of the effective size and the effective refractive index of each spherelike particle. Additionally, each particle could be identified from depolarization and fluorescence measured simultaneously. The tests with aqueous samples of polystyrene spheres, fluorescent or nonfluorescent, and phytoplankton cells demonstrate that the system is able to retrieve size and refractive index with an accuracy of 1% and that the depolarization and fluorescence measurements allow the classification of particles otherwise indistinguishable.

Flow cytometric quantification of bacteria in vaginal swab samples self-collected by adolescents attending a gynecology clinic

Bacterial vaginosis (BV) is an important risk factor in reproductive health outcomes, such as pre-term birth and sexually transmitted infections including HIV. However, its etiology, diagnosis and treatment remain poorly defined. We evaluated flow cytometry as a tool to quantify total bacterial cells in vaginal specimens self-collected longitudinally by adolescents. BV was diagnosed by Gram-stain (criteria of Hay and Ison). Average flow cytometric counts of bacterial cell-units (BCU) was log 10 8.04 per gram sample and was found to correlate with sample weight ( p < 0.0001). BV was frequently observed in this group, with 22 of 32 participants (69%) diagnosed with BV for at least one timepoint. Surprisingly, increased BCU was associated with normal Hay–Ison score ( p = 0.0003), even when adjusting for sample weight ( p = 0.02). Since presence and quantity of Lactobacillus defines normal vaginal microbiology (ie. absence of BV), this result indicates a possible bias towards dominance of Lactobacillus cells in measurements of “total” BCU. Increased BCU per gram was associated in multivariate analysis with longer self-reported time since last menstruation ( p = 0.004) and last sexual intercourse ( p = 0.007). Sperm was detected in 3 samples provided by those reporting sexual intercourse in the previous 24 h. Light-scattering profiles of bacteria and vaginal cells in samples collected over time from an individual were often identical and distinct from other individuals. To our knowledge, this is the first description of flow cytometry for analysis of commensal bacteria in vaginal specimens. Further development may help to illuminate the complex dynamics of vaginal microbial communities underlying BV.

Phase separation and dewetting in polymer blend thin films

We have studied morphology and kinetics of phase separation and dewetting in thin films of polystyrene and poly(vinyl methyl ether) blend on glass substrate using light scattering (LS) and optical microscope (OM) techniques as well as using atomic force microscopy (AFM). As the film thickness decreases from several tens μm, a peak in LS profiles characteristic to phase separation once disappears at the film thickness of ∼1μm, which corresponds to the characteristic wavelength of phase separation. Below this film thickness, we found dewetting is triggered in addition to the phase separation. In thin films below about 100 nm a scattering peak is again observed, which has been assigned to the characteristic wavelength of the dewetting. The results are discussed in terms of the characteristic wavelength of phase separation in relation to the film thickness.

Characterization of spherical particles using high-order neural networks and scanning flow cytometry

We retrieve the radius R, real n and imaginary k parts of the refractive index of homogeneous spherical particles using angular distribution of the light-scattering intensity. To solve the inverse light-scattering problem we use a high-order neural-network technique. The effect of network parameters on optimization is examined. The technique is evaluated for noise-corrupted input data at 0.6 μm< R<10.6 μm, 1.02< n<1.38, and 0< k<0.03. The errors of retrieval for nonabsorbing particles do not exceed 0.05 μm for radius and 0.015 for refractive index. The experimental verification is fulfilled by experimental data retrieved by means of a scanning flow cytometer. The light-scattering profiles of polystyrene beads and spherized red blood cells are processed with the high-order neural networks and a non-linear regression at Mie theory. The parameters retrieved by the high-order neural networks correlate well with the parameters retrieved by the least-square method.

A study of light scattering of mononuclear blood cells with scanning flow cytometry

This study describes the measurement of light scattering of human mononuclear blood cells, the development of an appropriate optical model for those cells, and solution of the inverse light-scattering problem. The angular dependency of light-scattering intensity of mononuclear blood cells was experimentally measured by means of scanning flow cytometry. A sphere consisting of several concentric homogeneous layers with different refractive indices was tested as an optical model for mononuclear blood cells. A five-layer model has given the best agreement between experimental and theoretical light-scattering profiles. The inverse light-scattering problem was solved for a five-layer model with an optimization procedure that allows one to retrieve cell parameters: cell size relates to the outer diameter of the fifth layer; size of the nucleus relates to the outer diameter of the third layer. Mean values of cell size, nuclear size, refractive indices of nucleus and cellular cytoplasm were determined for blood monocytes and lymphocytes.

Concentration Effect on the Aggregation of a Self-Assembling Oligopeptide

Concentration is a key parameter in controlling the aggregation of self-assembling oligopeptides. By investigating the concentration effects, an aggregation mechanism of EAK16-II is proposed. Depending on the critical aggregation concentration (CAC) of EAK16-II, the oligopeptide aggregates into protofibrils through seeding and/or a nucleation process. Protofibrils then associate with each other to form fibrils. The CAC was found to be ∼0.1 mg/ml by surface tension measurements. The nanostructures of aggregates were imaged and analyzed by atomic force microscopy. Globular and fibrillar aggregates were observed, and their dimensions were further quantified. To ensure that the aggregates were formed in bulk solution, light scattering (LS) measurements were conducted to monitor the fibril formation with time. The LS profile showed two different rates of aggregation depending on whether the peptide concentration was above or below the CAC. At high concentrations, the LS intensity increased strongly at early times. At low concentrations, the LS intensity increased only slightly. Our study provides information about the nature of the oligopeptide self-assembly, which is important to the understanding of the fibrillogenesis occurring in conformational diseases and to many biomedical engineering applications.

Effect of factor VIII on tissue factor-initiated spatial clot growth

Using time-lapse videomicroscopy, we studied the role of coagulation factor VIII (fVIII) in tissue factor-initiated spatial clot growth on fibroblast monolayers in a thin layer of non-stirred recalcified plasma from healthy donors or patients with severe Haemophilia A. Analysis of temporal evolution of light-scattering profiles from a growing clot revealed existence of two phases in the clot growth-initiation phase in a narrow (0.2 mm) zone adjacent to activator surface and elongation phase in plasma volume. While the initiation phase did not differ in normal and haemophilic plasmas, the rate of clot growth in the elongation phase in haemophilic plasma constituted only 30% of that in normal plasma. Supplementation of haemophilic plasma with 0.05 U/ml fVIII restored the normal clot growth rate (44.9 +/- 2.5 microm/min) at high but not at low fibroblast density. Our results indicate that the functioning of the intrinsic tenase complex is critical for normal spatial clot growth.

Monitoring by laser-flow-cytometry of the polycyclic aromatic hydrocarbon-degrading Sphingomonas sp. strain 107 during biotreatment of a contaminated soil.

A flow cytometric method (FCM) was used to detect and accurately enumerate a polycyclic aromatic hydrocarbon-degrading bacterial strain, Sphingomonas sp. 107, inoculated into a soil sample artificially contaminated with pyrene. To compare the FCM method with colony forming unit (CFU) assays, a rifampicin-resistant Sphingomonas sp. 107 was obtained which could be distinguished from the indigenous microflora, since there was no organism resistant to rifampicin in the soil that could transform indole to indigo (naphthalene dioxygenase activity). By combining light-scattering profiles (i.e., morphological properties), ethidium bromide influx (i.e., cell wall permeability), and fluorescence in situ hybridization against the 16S rRNA (i.e., detection specificity), we could enumerate the bacterial population of interest from the indigenous microflora and soil debris during the biotreatment. The FCM technique revealed that the number of inoculated Sphingomonas cells decreased gradually for 15 days of incubation before reaching a steady level of 7 to 12 x 10(5) cells.g-1 of soil. Similar values were obtained with the CFU assay. During this period, pyrene concentration decreased from 632 to 26 mg.kg-1 of dry soil. The FCM detection was improved by adding blocking reagent to the hybridization buffer to minimize the non-specific attachment of the fluorescent probe to soil particles. Combined with the improvements in probe technology, FCM detection was shown to be a good alternative to the conventional culture methods for the analysis of bacterial populations in environmental samples. This technique could be potentially useful for the detection of microorganisms that grow poorly in culture.

Structure development in the phenolic resin/poly(methyl methacrylate-co-ethyl acrylate) copolymer blends

Single-phase mixtures of phenolic derivatives with three poly(methyl methacrylate-co-ethyl acrylate) copolymers [P(MMA-co-EA)s] were cured in the presence of hexamethylenetetramine and the reaction kinetics and phase-separation processes during cure were investigated by differential scanning calorimetry (DSC), light scattering, and optical microscopy (OM). DSC measurements revealed that the higher the fraction of multifunctional phenols in the total phenols the lower the conversion at gelation in the phenolic-rich phase. The time variation of the light-scattering profile during cure demonstrated the characteristic feature for spinodal decomposition. OM observation revealed that a cocontinuous two-phase structure appeared after a certain time lag and coarsened to a spherical domain structure consisting of phenolic resin particles dispersed in the P(MMA-co-EA)s matrix. The periodic distances, Λm, in the phase-separated structure changed with curing time: (1) The higher the component of MMA in P(MMA-co-EA)s and (2) the higher the fraction of multifunctional phenols in the total phenols, the smaller the Λm. These results may imply that the phase separation is suppressed by the faster gelation in the phenolic-rich phase so that the phase-separated structure can be fixed by the network formation in the phenolic-rich phase at the early stage of spinodal decomposition. This results in a two-phase structure with a shorter periodic distance. © 1998 John Wiley & Sons, Inc. J Appl Polym Sci 68: 1829–1837, 1998

Measurement of Glycoprotein llb/llla Blockade by Flow Cytometry with Fluorescein Isothiocyanate-conjugated Crotavirin, a Member of Disintegrins

The blockade of platelet membrane glycoprotein IIb/IIIa by a monoclonal antibody, 7E3, was measured by flow cytometry using a fluorescein isothiocyanate-conjugated disintegrin, FITC-crotavirin, as the probe. After treatment of platelets with 7E3 or 7E3 F(ab')2, there is a good correlation between the inhibition of platelet aggregation and the blockade of FITC-crotavirin's binding to platelets. The content of glycoprotein IIb/IIIa for the subsequent binding of FITC-crotavirin to the 7E3-pretreated platelets highly correlated to the extent of glycoprotein IIb/IIIa, remaining available. It was evidenced by the observation that the sum of glycoprotein IIb/IIIa occupation by 7E3 and that of FITC-crotavirin approached the total amount of glycoprotein IIb/IIIa expressed on the platelet membrane. This indicates that the percentage inhibition of FITC-crotavirin's binding at the saturation dose reflects the extent of glycoprotein IIb/IIIa blockade by 7E3. At the saturation binding concentration (5 micrograms/ml), FITC-crotavirin did not displace platelet bound 7E3. Gating the light-scattering profile for platelets, the binding of FITC-crotavirin to platelet glycoprotein IIb/IIIa could be easily determined in diluted whole blood by direct stain method. The available unoccupied glycoprotein IIb/IIIa of platelets in the 7E3 or 7E3 F(ab')2-pretreated whole blood were measured by flow cytometry at the saturation binding dose of FITC-crotavirin (4 micrograms/ml) and the data showed that the higher deconcentration of antibody added into whole blood, the lower debinding of FITC-crotavirin to platelets. This technique may provide an alternative rapid method for measuring the blockade of glycoprotein IIb/IIIa by 7E3, a promising anti-thrombotic agent, thus providing a monitoring method for adjusting the therapeutic dose of 7E3 or its related derivatives.

Polymerization-induced spinodal decomposition of [formula omitted] methacrylate mixture and the influence of incorporating poly(vinyl acetate) macromonomer

The structural development during radical polymerization of a mixture of methyl methacrylate (MMA) and poly(ethylene- co-vinyl acetate) (EVA) was investigated by light scattering and optical microscopy. In the early stage of polymerization, the mixture of MMA/EVA was a single phase. As the polymerization of MMA proceeded, phase separation took place via spinodal decomposition (SD). This was supported by the characteristic change in light-scattering profile with reaction time. Analysis by the linearized and scaling theories showed that the reaction-induced phase separation behaviour was different from the familiar thermally induced SD during isothermal annealing after temperature jump. The scaling analysis showed that the final phase-separated structure may be formed by the interruption of a co-continuous percolation structure. Moreover, to investigate the effect of graft copolymer on SD, a poly(vinyl acetate) (PVAc) macromonomer with a methacrylate end-group was added to the MMA/EVA mixture to yield PMMA-PVAc graft copolymer during the polymerization. The incorporation of PVAc macromonomer increased the dominant wavenumber of the final frozen phase-separated structure (smaller periodic distance). The reasons seem to be that the concentration fluctuation growth is suppressed until the system reaches a deep quench depth at which the fluctuation should have a large wavenumber, and that the coarsening is retarded at the late stage by the graft copolymer formed in situ.

Interfacial Interaction of Polymer/Liquid Crystal Molecules and Electrooptical Properties of their Composite Systems

Polymer/liquid crystal composite films were prepared from a solution of polymer and nematic liquid crystal (LC) by a solvent casting method. The phase-separated structure of the composite film was controlled by the solvent evaporation rate. The light-scattering profile of a poly(diisopropyl fumarate)/LC: 40/60 w/w solution during solvent evaporation exhibited a periodic structure, indicating that the phase-separated structure was formed by spinodal decomposition. The aggregation structure of the composite film was investigated with a scanning electron microscope (SEM). SEM observation of the composite film suggested the presence of periodicity and dual connectivity of polymer and LC phases. The faster the solvent was evaporated, the smaller the LC channel (domain) size in the composite film. The composite film, composed of poly(methyl methacrylate) (PMMA) and a nematic LC (E44) with a positive dielectric anisotropy, exhibited remarkable and reversible light-scattering-light-transmission switching, under t...

Characterization of bacteria by multiparameter flow cytometry

An arc-lamp based flow cytometer was used to obtain high resolution measurements of the light scattering characteristics and DNA contents of eight different bacteria. Light scatter profiles of bacteria are a useful first step when flow cytometry is used to characterize organisms. Scanning and transmission electron microscopy of the bacterial samples demonstrate that the structural basis of the light scattering profiles is not always clear, i.e. some organisms appear to have anomalous light scattering characteristics. The use of a third measurement parameter, DNA content, allowed much better discrimination of the organisms. Flow cytometry shows great promise as a method for the rapid discrimination and identification of bacterial populations.

Preliminary studies for an immunotherapeutic approach to the treatment of human myeloma using chimeric anti-CD38 antibody

Multiple myeloma is a disease in which conventional chemotherapy has only limited value, but which may be ideal for treatment with passive antibody against a suitable cell surface antigen on the neoplastic plasma cell. The CD38 antigen is known to be present on the majority of neoplastic plasma cells, and this was confirmed by detailed examination of bone marrow aspirates from three patients. Strong expression of CD38 was confined to cells which, by the criteria of light-scattering profiles and possession of cytoplasmic Ig, were plasma cells. The vast majority of neoplastic plasma cells appeared to be involved. Using a cell line as a model, it was found that the CD38 antigen acts as a target for a chimeric antibody prepared from the antibody OKT10. The chimeric antibody consists of the Fab portion of the mouse monoclonal antibody linked by a stable thioether bond to an Fc molecule derived from human IgG1, thereby forming mouse Fab-human Fc. In contrast to the parent antibody, the chimeric molecule mediates antibody-dependent cellular cytotoxicity (ADCC) very efficiently with human blood mononuclear effector cells, and is effective at low concentration. Also, even though the CD38 antigen is present on natural killer cells, there appears to be little deleterious action of the antibody on effector cell function. The antibody also failed to affect the growth of progenitor cells of the granulocyte/macrophage or erythroid lineages present in normal bone marrows, despite the suspicion that these cells express the antigen. Other advantages of the CD38 molecule are that it is not found in the serum of patients with myeloma, and it does not appear to modulate in vitro. Fourteen patients with florid myeloma and on various chemotherapeutic regimes had an undiminished capacity to mediate ADCC with the chimeric antibody, when compared with normal individuals. The maintenance of ADCC activity, coupled with the known suppression of the antibody response in these patients, augers well for treatment with chimeric antibody.

Preliminary Studies for an Immunotherapeutic Approach to the Treatment of Human Myeloma Using Chimeric Anti-CD38 Antibody

Multiple myeloma is a disease in which conventional chemotherapy has only limited value, but which may be ideal for treatment with passive antibody against a suitable cell surface antigen on the neoplastic plasma cell. The CD38 antigen is known to be present on the majority of neoplastic plasma cells, and this was confirmed by detailed examination of bone marrow aspirates from three patients. Strong expression of CD38 was confined to cells which, by the criteria of light-scattering profiles and possession of cytoplasmic Ig, were plasma cells. The vast majority of neoplastic plasma cells appeared to be involved. Using a cell line as a model, it was found that the CD38 antigen acts as a target for a chimeric antibody prepared from the antibody OKT10. The chimeric antibody consists of the Fab portion of the mouse monoclonal antibody linked by a stable thioether bond to an Fc molecule derived from human IgG1, thereby forming mouse Fab-human Fc. In contrast to the parent antibody, the chimeric molecule mediates antibody-dependent cellular cytotoxicity (ADCC) very efficiently with human blood mononuclear effector cells, and is effective at low concentration. Also, even though the CD38 antigen is present on natural killer cells, there appears to be little deleterious action of the antibody on effector cell function. The antibody also failed to affect the growth of progenitor cells of the granulocyte/macrophage or erythroid lineages present in normal bone marrows, despite the suspicion that these cells express the antigen. Other advantages of the CD38 molecule are that it is not found in the serum of patients with myeloma, and it does not appear to modulate in vitro. Fourteen patients with florid myeloma and on various chemotherapeutic regimes had an undiminished capacity to mediate ADCC with the chimeric antibody, when compared with normal individuals. The maintenance of ADCC activity, coupled with the known suppression of the antibody response in these patients, augers well for treatment with chimeric antibody.

Structure development in epoxy resin modified with poly(ether sulphone)

Structure development during the curing reaction in a ternary mixture of diglycidyl ether of bisphenol A (epoxy), poly(ether sulphone) (PES) and diaminodiphenylmethane (curing agent) was investigated by light scattering, scanning electron microscopy (SEM) and differential scanning calorimetry. In the early stage of curing, the ternary blend was a single-phase mixture. As the cure reaction proceeded, phase separation took place via the spinodal decomposition induced by the increase in the molecular weight of epoxy. This was supported by the characteristic change in light-scattering profile with curing time. SEM observation revealed the formation of a unique two-phase structure: connected globules of epoxy-rich phase arranged regularly (typical periodic distance, 0.5 μm) in a matrix of a PES-rich phase. The unique structure is assumed to be formed under a specific situation: competitive progress of phase separation and of the crosslink reaction. That is, once a loose network is established at the epoxy-rich region in the phase-separated system, further coarsening to a droplet-type structure could be suppressed, eventually resulting in the interconnected globule structure. Compared with neat epoxy resin, the cured ternary resin exhibited higher adhesive strength, in both peel and shear modes. The excellent adhesive property seems to originate from the characteristic connected-globule morphology.

Cell lines derived from avian lymphomas exhibit two distinct phenotypes

Lymphoid cell lines were derived from three avian leukosis virus (ALV)-induced lymphomas. These cell lines contained proviral DNA sequences integrated upstream from the c- myc proto-oncogene, expressed increased levels of c- myc RNA, and were tumorigenic in syngeneic animals. While cell surface immunoglobulin (IgM) was expressed by all three cell lines, only one of the lines secreted IgM into the culture medium. Further, analysis by light microscopy and flow cytometry demonstrated that these cell lines exhibited two distinct morphological and light-scattering profiles. The two nonsecreting lines exhibited a lymphoblastoid phenotype, whereas, the secreting line possessed a more differentiated plasmacytoid phenotype. These findings implicate the activation of c- myc in the pathogenesis of tumors representing two distinct stages of B-cell differentiation within a single animal species.

Quantitative introduction of a given macromolecule into cells by fusion with erythrocyte ghosts using a fluorescence activated cell sorter.

FITC-conjugated bovine serum albumin (FITC-BSA) molecules were quantitatively introduced into human erythrocyte ghosts by gradual hemolysis. When the ghosts and L cells were fused with UV-inactivated HVJ (Sendai virus), FITC-BSA was introduced into the cytoplasm of the L cells and fluorescence could be observed inthe cells with a fluorescence microscope. A mixture of L cells and ghosts was introduced into a fluorescence activated cell sorter (FACS), which could separate the mononuclear cells on the basis of their light-scattering profile. Four distinct populations of mononuclear cells were found by fluorescence analysis. These populations were separated from the cell mixture and found to correspond to cells fused with one, two and three ghosts and unfused cells. After separation, the cells from each population could form colonies in culture. As a given macromolecule can be quantitatively introduced into erythrocyte ghosts with the FITC-BSA, after fusion of these ghosts with cells, this sorting method is useful for separating cells containing a definite number of macromolecules.

Temporal variability of suspended matter in Astoria Canyon

Suspended matter in Astoria canyon was monitored by means of an in situ nephelometer and by means of light-scattering and particle concentration measurements performed aboard ship on water samples. Nephelometer profiles obtained along the axis of the canyon in February and April 1973 indicate that the canyon is divided into two distinct zones: a nearshore zone in which the suspensoid distribution undergoes large changes and an offshore zone in which the distribution varies to a much lesser degree. A 15-hour time series of light-scattering and particle concentration profiles at a depth of 1100 m in the canyon shows extensive and rapid changes in suspended matter concentration at several depths. The effect of nonsteady state distributions of suspended matter on calculations of the coefficient of eddy diffusivity is examined and shown to be an important consideration in a submarine canyon. The extent to which observed distributions of suspended matter vary with time is important relative to their roles as indicators of abyssal circulation and sediment dispersal, and at the present time, information on the time variability of suspended particulates, especially in the deep ocean, is limited. In estuaries and on the continental shelf a great deal of work has been done concerning the distribution and transport of sediments [Swift et al., 1972]. However, observations of changing distributions of suspended matter, which are an integral part of the problem of sediment transport, have been rare. The response of suspended sediment to tidal effects has been studied by Schubel [1969, i971], Wasowski [1974], and others. Over the cohtinental shelf, Rodolfo et al. [1971] have observed the responses of suspended matter to a hurricane, and Harlett [1972], in a study of sediment transport on the shelf off Oregon, reported several time-series measurements of light transmission in depths of 100-200 m. The data presented in this report resulted from our efforts to determine how the observed distributions of suspended particulates respond to the regimes of wind, surface and internal waves, and tides and currents in deep oceanic waters. Our initial efforts in this study were to determine .the extent in time and space of changes in the concentration of suspended matter in the nearshore region and to extend these measurements offshore. It was felt that Astoria canyon would be an advantageous site for this study for several reasons. It has been suggested often [Moore, 1969; Lyall et al., 1971] that submarine canyons are sites of 'channelization' of sediment transport and perhaps of bottom water flow, and there have been numerous studies of bottom currents in submarine canyons [Fenner et al., 1971; Shepard and Marshall, 1973; Keller et al., 1973], all of which have shown that bottom currents of significant magnitude exist in canyons and that flow reversals are common. One /

A Study of the Relationship Between Geometry and Light Scattering Profiles of Single Wool Fibers

A study of relationship between fiber geometry and the light scattering profiles of single wool fibers, obtained by drawing the fibers through a light beam and continuously recording the far field interference pattern, has shown that the spacing of the first minimum in the profile can be closely related to the fiber diameter via the Fraunhofer far field diffraction equation for an opaque strap.