A proposal for optical diagnostics through the enhancement of diffraction patterns using thin-film interference filters

Coarse clumping of solid materials within diseased biological cells can have a marked influence on the light scattering pattern. Perturbations in refractive index lead to distinct variations in the cytometric signature, especially apparent over wide scattering angles. The large dynamic range of scattering intensities restricts collection of data to narrow angular intervals believed to have the highest potential for medical diagnosis. We propose the use of an interference filter to reduce the dynamic range. Selective attenuation of scattering intensity levels is expected to allow simultaneous data collection over a wide angular interval. The calculated angular transmittance of a commercial shortwave-pass filter of cut-off wavelength 580 nm indicates significant attenuation of scattering peaks below ∼10°, and reasonable peak equalization at higher angles. For the three-dimensional calculation of laser light scattered by cells we use a spectral method code that models cells as spatially varying dielectrics, stationary in time. However, we perform preliminary experimental testing with the interference filter on polystyrene microspheres instead of biological cells. A microfluidic toolkit is used for the manipulation of the microspheres. The paper intends to illustrate the principle of a light scattering detection system incorporating an interference filter for selective attenuation of scattering peaks.     

Mie-type scattering and non-Beer-Lambert absorption behavior of human cells in infrared microspectroscopy

We report infrared microspectral features of nuclei in a completely inactive and contracted (pyknotic) state, and of nuclei of actively dividing cells. For pyknotic nuclei, the very high local concentration of DNA leads to opaqueness of the chromatin and, consequently, the absence of DNA signals in the IR spectra of very small nuclei. However, these nuclei can be detected by their scattering properties, which can be described by the Mie theory of scattering from dielectric spheres. This scattering depends on the size of the nucleus; consequently, quite different scattering cross-sections are calculated and observed for pyknotic and mitotic nuclei.     

Light scattering from nucleated biological cells.

The light scattered from nucleated biological cells has been investigated by using four different theoretical models: an opaque disk, a homogeneous sphere, an opaque ring, and a coated sphere. By comparing these four models, diffraction at the edges of the cell and the nucleus has been found to be the predominate scattering mechanism for nucleated biological cells at low angles. The scattering patterns of nucleated cells are found to have a fine lobe (high-frequency) structure dependent on whole cell size, and an envelope lobe (low-frequency) structure dependent on relative nucleus size. The models indicate that the present technique for measuring cell size with a single low-angle light detector is highly dependent on the nucleus to cell diameter ratio. Whole cell size is better estimated by the ratio of the outputs from two low-angle detectors.     

Correction to: Three-dimensional quantification of twisting in the Arabidopsis petiole

Organisms have a variety of three-dimensional (3D) structures that change over time. These changes include twisting, which is 3D deformation that cannot happen in two dimensions. Twisting is linked to important adaptive functions of organs, such as adjusting the orientation of leaves and flowers in plants to align with environmental stimuli (e.g. light, gravity). Despite its importance, the underlying mechanism for twisting remains to be determined, partly because there is no rigorous method for quantifying the twisting of plant organs. Conventional studies have relied on approximate measurements of the twisting angle in 2D, with arbitrary choices of observation angle. Here, we present the first rigorous quantification of the 3D twisting angles of Arabidopsis petioles based on light sheet microscopy. Mathematical separation of bending and twisting with strict definition of petiole cross-sections were implemented; differences in the spatial distribution of bending and twisting were detected via the quantification of angles along the petiole. Based on the measured values, we discuss that minute degrees of differential growth can result in pronounced twisting in petioles.

     

Separation and characterisation of light scattering transients from rod outer segments of vertebrate photoreceptors: design and performance of a Multi Angle Flash Photolysis Apparatus (MAFPA)

A device was built for the simple computer-controlled routine determination of the angular dependence of light scattering transients obtained from biological material. It was called Multi Angle Flash Photolysis Apparatus (MAFPA). The MAFPA allows the simultaneous registration of rapid, light-induced light scattering transients at eight scattering angles between 0 degree and 28 degrees. In typical applications changes in scattered light intensity as small as delta I/I = 4 X 10(-5) can be resolved at scattering angles less than 24 degrees, while at 28 degrees the resolution drops to delta I/I = 2 X 10(-4). The time resolution is 32 microseconds. The MAFPA was designed for high accuracy, ease of use and ruggedness. It is made from relatively inexpensive parts and can be copied fairly easily by a good machine/electronics shop. In this communication we describe the design of the MAFPA and how it was used for the characterisation of four structurally distinct light-induced light scattering signals from photoreceptor rod outer segments. These signals are known as P (or binding) signal, G- (or dissociation) signal, N (or rhodopsin) signal and as the ATP-dependent signal AL. The signals have been separated by means of their different angular dependence, their different saturation behavior and nucleotide requirement. A great number of detailed studies will have to be carried out before one can fully understand the physical and biochemical origin of these signals. At this point, however, it can be stated that the so-called 'dissociation signal', showing an angular dependence indicative of a change in refractive index or scattering mass, is not merely an inversion of the preceding 'binding signal', the latter clearly reflecting a gross structural change, i.e. a shrinkage of the disks. Moreover, there are conditions where P signals are observed to persist even after the completion of the subsequent dissociation signals. The two remaining signals N and AL show a pronounced angular dependence which is not easily interpreted. The fact that both exhibit a maximal amplitude at relatively small angles seems to indicate the participation of rather large structural domains.     

A miniaturized wide-angle 2D cytometer.

BACKGROUND: We present an optical waveguide based cytometer that is capable of simultaneously collecting the light scattered by cells over a wide range of solid angles. Such comprehensive scattering data are a prerequisite for the microstructural characterization of cells. METHODS: We use latex beads as cell mimics, and demonstrate the ability of this new cytometer to collect back-scattered light in two dimensions (2D). This cytometer is based on a liquid-core optical waveguide, excited by prism coupling, that also serves as the microfluidic channel. In principle, our use of a hemispherical lens allows the collection of scattered light from 0 to 180 degrees in 2D. RESULTS: The experimentally observed positions of the intensity peaks of the back-scattered light agree well with theoretical prediction of scattering from both 4.0- and 9.6-mum diameter latex beads. The position of the bead, relative to the axes of the hemispherical lens and the microchannel, strongly affects the scattering pattern. We discuss a computational method for determining these offsets. CONCLUSIONS: We show that wide-angle 2D light scattering patterns of cell-sized latex beads can be observed in a microfluidic-based optical cytometer that uses leaky waveguide mode excitation. This chip-based system is compatible with emerging chip-based technologies.     

Equilibrium gel permeation: measurement of solute partitioning by direct fluorescence scanning

The feasibility of using fluorescence detection in quantitative gel permeation measurements has been explored. It is shown that the effect of scattering by the gel matrix can be evaluated in terms of pathlength-dependent turbidity functions for excitation and emission wavelengths. Experimental studies were carried out to evaluate these functions in cross-linked dextran gels (Sephadexes) and in agarose gels (Sepharoses). Empirical turbidity functions derived for these gels have a simple form, leading to accurate simplifying approximations for the scattering correction required in a fluorescence gel permeation measurement. Using this approach, partition cross-sections were estimated for dansyl-conjuaated β-gamma globuline and for dansyl-conjugated bovine serum albumin. The results establish feasibility of the method and clearly indicate the instrumentation requirements for its accurate implementation.     

A hybrid image processing system for X-ray images of an external fixator

In the field of orthopaedics, treatment of extremity deformities can be realised by means of external fixators. However, control of such biomedical system is very difficult. Some different mathematical models have been developed to improve quality of this service. Most of the parameters, which are used in these models, have been obtained from two orthogonal X-ray images: one from anteroposterior, AP, direction and the other from a lateral, L, direction. The quality of the results of this model is dependent on the accuracy of the input parameters. Measuring these parameters is a time-consuming issue, and the accuracy of the results is also low. To increase the quality of the measurement, the reference points should be chosen from the edges of the biomedical system, and it is important to find the edges without noise. To achieve this purpose, Sobel edge detector, binary large object analysis, thresholding and inverting are applied as image processing steps. The results are compared with manual measurement values which have been obtained earlier. The results show that semi-automatic measurement of the parameters is more accurate and faster than manual measurement. It shows that the efficiency of the fixator method has been improved.     

Conformation of peptides in lipid membranes studied by x-ray grazing incidence scattering

Although the antimicrobial, fungal peptide alamethicin has been extensively studied, the conformation of the peptide and the interaction with lipid bilayers as well as the mechanism of channel gating are still not completely clear. As opposed to studies of the crystalline state, the polypeptide structures in the environment of fluid bilayers are difficult to probe. We have investigated the conformation of alamethicin in highly aligned stacks of model lipid membranes by synchrotron-based x-ray scattering. The (wide-angle) scattering distribution has been measured by reciprocal space mappings. A pronounced scattering signal is observed in samples of high molar peptide/lipid ratio which is distinctly different from the scattering distribution of an ideal helix in the transmembrane state. Beyond simple models of ideal helices, the data is analyzed in terms of models based on atomic coordinates from the Brookhaven Protein Data Bank, as well as from published molecular dynamics simulations. The results can be explained by assuming a wide distribution of helix tilt angles with respect to the membrane normal and a partial insertion of the N-terminus into the membrane.     

The measurement of bacterial translation by photon correlation spectroscopy.

Photon correlation spectroscopy is shown to be a practical technique for the accurate determination of translational speeds of bacteria. Though other attempts have been made to use light scattering as a probe of various aspects of bacterial motility, no other comprehensive studies to establish firmly the basic capabilities and limitations of the technique have been published. The intrinsic accuracy of the assay of translational speeds by photon correlation spectroscopy is investigated by analysis of synthetic autocorrelation data; consistently accurate estimates of the mean and second moment of the speed distribution can be calculated. Extensive analyses of experimental preparations of Salmonella typhimurium examine the possible sources of experimental difficulty with the assay. Cinematography confirms the bacterial speed estimates obtained by photon correlation techniques.     

Cell cycle studies of murine leukemia cell L5l78Y by polarization effects of light scattering

The structural changes during the life cycle of a synchronized population of mouse leukemia cell line L5l78Y have been described by polarized light scattering measurements. Exponentially growing cells were synchronized by an automatic excess thymidine-colcemid treatment technique. Samples were removed from the suspension culture and fixed with glutaraldehyde at hourly intervals throughout the life cycle. The effect these cell samples had in changing right-hand circularly polarized light to 45° linearly polarized light during the scattering process was measured at angles 6–l60° to the incident beam. The reproducibility of the light scattering signals for each time interval was statistically evaluated and found to have good intertrial correlation for each time period in the angular range 6–60° to the incident beam. Statistically significant changes were seen between cell samples during the synchronous life cycle. Therefore, the system developed has applications as an extremely sensitive measure of cell structure, and of structural changes caused by low-level chemical, physical or biological agents.     

Molecular dynamics decomposition of temperature-dependent elastic neutron scattering by a protein solution

Molecular dynamics simulations are performed of bovine pancreatic trypsin inhibitor in a cryosolution over a range of temperatures from 80 to 300 K and the origins identified of elastic dynamic neutron scattering from the solution. The elastic scattering and mean-square displacement calculated from the molecular dynamics trajectories are in reasonable agreement with experiments on a larger protein in the same solvent. The solvent and protein contributions to the scattering from the simulation model are determined. At lower temperatures (< approximately 200 K) or on shorter timescales ( approximately 10 ps) the scattering contributions are proportional to the isotopic nuclear scattering cross-sections of each component. However, for T > 200 K marked deviations from these cross-sections are seen due to differences in the dynamics of the components of the solution. Rapid activation of solvent diffusion leads to the variation with temperature of the total elastic intensity being determined largely by that of the solvent. At higher temperatures (>240 K) and longer times ( approximately 100 ps) the protein makes the only significant contribution to the scattering, the solvent scattering having moved out of the accessible time-space window. Decomposition of the protein mean-square displacement shows that the observed dynamical transition in the solution at 200-220 K involves activation of both internal motions and external whole-molecule rotational and translational diffusion. The proportion that the external dynamics contributes to the protein mean-square displacement increases to approximately 30 and 60% at 300 K on the 10- and 100-ps timescales, respectively.     

Analysis of fractal properties of globular protein surfaces by means of small-angle x-ray scattering]

The "cube method" [M. Yu. Pavlov, B. A. Fedorov, Biopolymers, 22, 1507, 1983] has been used to calculate the intensity of the large-angle X-ray scattering from the volumes of several globular proteins. In the logarithmic plots of the scattered intensity curves from three of these proteins, there is a linear region at scattering angles corresponding to Bragg distances of from about 6.3 A to 21 A. This linear region possibly may be due to the fractal properties of the surfaces of these proteins on length scoles from 6.3 A to 21 A, and the fractal dimensions corresponding to the power-law scattering at these scattering angles have been evaluated.     

Shortest path analysis using partial correlations for classifying gene functions from gene expression data

MOTIVATION: Gaussian graphical models (GGMs) are a popular tool for representing gene association structures. We propose using estimated partial correlations from these models to attach lengths to the edges of the GGM, where the length of an edge is inversely related to the partial correlation between the gene pair. Graphical lasso is used to fit the GGMs and obtain partial correlations. The shortest paths between pairs of genes are found. Where terminal genes have the same biological function intermediate genes on the path are classified as having the same function. We validate the method using genes of known function using the Rosetta Compendium of yeast (Saccharomyces Cerevisiae) gene expression profiles. We also compare our results with those obtained using a graph constructed using correlations. RESULTS: Using a partial correlation graph, we are able to classify approximately twice as many genes to the same level of accuracy as when using a correlation graph. More importantly when both methods are tuned to classify a similar number of genes, the partial correlation approach can increase the accuracy of the classifications.     

Individual Escherichia coli cells studied from light scattering with the scanning flow cytometer

BACKGROUND: Flow cytometry is a powerful tool for the analysis of individual particles in a flow. Differential light scattering (an indicatrix) was used for many years to obtain morphologic information about microorganisms. The indicatrices play the same role for individual particle recognition as a spectrum for substance characterization. We combined two techniques to analyze the indicatrix of the cells for the purpose of developing a database of light-scattering functions of cells. METHODS: The scanning flow cytometer (SFC) allows the measurement of the entire indicatrix of individual particles at polar angles ranging from 5 degrees to 100 degrees. In this work, light-scattering properties of Escherichia coli have been studied both experimentally and theoretically with the SFC and the T-matrix method, respectively. The T-matrix method was used because of the nonspherical shape of E. coli cells, which were modeled by a prolate spheroid. RESULTS: The indicatrices of E. coli cells were stimulated with T-matrix method at polar angles ranging from 10 degrees to 60 degrees. The absolute cross-section of light scattering of E. coli has been determined comparing the cross section of polystyrene particles modeled by a homogeneous sphere. The E. coli indicatrices were compared for logarithmic and stationary phases of cell growth. CONCLUSIONS: The indicatrices of E. coli were reproducible and could be used for identification of these cells in biologic suspensions. The angular location of the indicatrix minimum can be used in separation of cells in logarithmic and stationary phases. To use effectively the indicatrices for that purpose, the light-scattering properties of other microorganisms have to be studied.     

Kinematics of human arm reconstructed from spatial tracking system recordings

The kinematics of the human arm in terms of angles of rotations in the joints is reconstructed from the spatial tracking system (Fastrack() Polhemus) recordings. The human arm is modeled by three rigid bodies (the upper arm, the forearm and the hand) with seven degrees of freedom (three in the shoulder, two in the elbow and two in the wrist). Joint geometry parameters (orientations of the axes relative to the arm segments, the angles and the distances between the axes) have been calculated on the basis of passive rotations in the joints. The calculated parameters have been used to solve the direct kinematics problem for the reaching movements in different directions. The difference between calculated and recorded positions and accelerations of the hand has been used to assess the accuracy of the proposed method of kinematics reconstruction. The error analysis showed that spatial tracking system recordings and human arm kinematics reconstruction could reliably be used to accurately analyze multijoint movement in humans.     

Real-time PCR to assess the Leishmania load in Lutzomyia longipalpis sand flies: screening of target genes and assessment of quantitative methods

Visceral Leishmaniasis is an endemic disease in Brazil caused by Leishmania infantum chagasi and its main vector species is the sand fly Lutzomyia longipalpis. Epidemiological studies have used conventional PCR techniques to measure the rate of infection of sand flies collected in the field. However, real-time PCR can detect lower parasite burdens, reducing the number of false negatives and improving the quantification of Leishmania parasites in the sand fly. This study compared genes with various copy numbers to detect and quantify L. infantum chagasi in L. longipalpis specimens by real-time PCR. We mixed pools of 1, 10 and 30 male sand flies with various amounts of L. infantum chagasi, forming groups with 50, 500, 5000 and 50,000 Leishmania parasites. For the amplification of L. infantum chagasi DNA, primers targeting kDNA, polymerase α and the 18S ribosome subunit were employed. Parasites were measured by absolute and relative quantification. PCR detection using the amplification of kDNA exhibited the greatest sensitivity among the genes tested, showing the capacity to detect the DNA equivalent of 0.004 parasites. Additionally, the relative quantification using these primers was more accurate and precise. In general, the number of sand flies used for DNA extraction did not influence Leishmania quantification. However, for low-copy targets, such as the polymerase α gene, lower parasite numbers in the sample produced inaccurate quantifications. Thus, qPCR measurement of L. infantum chagasi in L. longipalpis was improved by targeting high copy-number genes; amplification of high copy-number targets increased the sensitivity, accuracy and precision of DNA-based parasite enumeration.     

Simultaneous quantification of components of neoglycolipid-coated liposomes using high-performance liquid chromatography with evaporative light scattering detection

Liposomes composed of dipalmitoylphosphatidylcholine (DPPC), cholesterol and a neoglycolipid, mannopentaose-conjugated dipalmitoylphosphatidylethanolamine (Man5-DPPE), have been shown to have a strong adjuvant effect in inducing the antigen-specific cellular immunity. In this study, a rapid and simple analytical method using a HPLC system with an evaporative light scattering detector was developed for simultaneous quantification of the liposome components Man5-DPPE, cholesterol and DPPC. The chromatographic separation of these components was performed using a trimethylsilane column with an isocratic mobile phase of chloroform–methanol–water (1:33:6, v/v) after disrupting the liposomes with chloroform–methanol–water (10:10:3, v/v). This HPLC method provided sufficient reproducibility and linearity of calibration curves for the quantification of the liposome constituents. In addition, this method can be used for the quantification of various neoglycolipids with different carbohydrate structures.     

Sodium as nutrient and toxicant

Background and Scope

Because of the crucial role coarse roots (>2 mm diameter) play in plant functions and terrestrial ecosystems, detecting and quantifying the size, architecture, and biomass of coarse roots are important. Traditional excavation methods are labor intensive and destructive, with limited quantification and repeatability of measurements over time. As a nondestructive geophysical tool for delineating buried features in shallow subsurface, ground penetrating radar (GPR) has been applied for coarse root detection since 1999. This article reviews the state-of-knowledge of coarse root detection and quantification using GPR, and discusses its potentials, constraints, possible solutions, and future outlooks. Some useful suggestions are provided that can guide future studies in this field.

Conclusions

The feasibility and accuracy of coarse root investigation by GPR have been tested in various site conditions (mostly in controlled conditions or within plantations) and for different plant species (mostly tree root systems). Thus far, single coarse root identification and coarse root system mapping have been conducted using GPR, including roots under pavements in urban environment. Coarse root diameter and biomass have been estimated from indexes extracted from root GPR radargrams. Coarse root development can be observed by repeated GPR scanning over time. Successful GPR-based coarse root investigation is site specific, and only under suitable conditions can reliable measurements be accomplished. The best quality of root detection by GPR is achieved in well-drained and electrically-resistive soils (such as sands) under dry conditions. Numerous factors such as local soil conditions, root electromagnetic properties, and GPR antenna frequency can impact the reliability and accuracy of GPR detection and quantification of coarse roots. As GPR design, data processing software, field data collection protocols, and root parameters estimation methods are continuously improved, this noninvasive technique could offer greater potential to study coarse roots.     

Narrow-angle forward light scattering from individual algal cells: implications for size and shape discrimination in flow cytometry

Single-cell forward light scattering patterns have been examinedfor four algal species (one pennate diatom, two green flagellatesand one filamentous cyanobacterium), mounted statically in afocused laser beam. In all cases, the distribution of lightintensity at narrow angles (within the first scattering lobe)is well described by diffraction theory. Narrow-angle forwardscattering measurements can therefore be used in principle todeduce the size and approximate shape of algal cells. The feasibilityof using this technique in flow cytometry has been tested usingan instrument which orientates elongated cells uniformly inthe flow stream, and uses fibre optics to make azimuthally resolvedforward scatter measurements at sub-degree polar angles. Withthis instrument it is possible to discriminate between specieswith similar volume and fluorescence characteristics using forwardlight scattering as a shape-sensitive parameter.