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.     

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.     

Quantification of microbial productivity via multi-angle light scattering and supervised learning

This article describes the use of chemometric methods for prediction of biological parameters of cell suspensions on the basis of their light scattering profiles. Laser light is directed into a vial or flow cell containing media from the suspension. The intensity of the scattered light is recorded at 18 angles. Supervised learning methods are then used to calibrate a model relating the parameter of interest to the intensity values. Using such models opens up the possibility of estimating the biological properties of fermentor broths extremely rapidly (typically every 4 sec), and, using the flow cell, without user interaction. Our work has demonstrated the usefulness of this approach for estimation of yeast cell counts over a wide range of values (10(5)-10(9) cells mL-1), although it was less successful in predicting cell viability in such suspensions.     

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.     

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

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

Porous Nanomaterials for Ultrabroadband Omnidirectional Anti‐Reflection Surfaces with Applications in High Concentration Photovoltaics

Materials for nanoporous coatings that exploit optimized chemistries and self‐assembly processes offer capabilities to reach ≈98% transmission efficiency and negligible scattering losses over the broad wavelength range of the solar spectrum from 350 nm to 1.5 µm, on both flat and curved glass substrates. These nanomaterial anti‐reflection coatings also offer wide acceptance angles, up to ±40°, for both s‐ and p‐polarization states of incident light. Carefully controlled bilayer films have allowed for the fabrication of dual‐sided, gradient index profiles on plano‐convex lens elements. In concentration photovoltaics platforms, the resultant enhancements in the photovoltaics efficiencies are ≈8%, as defined by experimental measurements on systems that use microscale triple‐junction solar cells. These materials and their applications in technologies that require control over interface reflections have the potential for broad utility in imaging systems, photolithography, light‐emitting diodes, and display technologies.     

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.     

Time-resolved angular dependent measurement of triggered light scattering changes in biological suspensions

We describe a photometer for time-resolved measurements of small changes in light scattering suited for suspensions of biological material. The time resolution is 35 μs, the amplitude resolution for bovine rot outer segments is typically ΔI/I = 5 · 10^?4 at a scattering angle of ? = 20°. The use of the apparatus is demonstrated by recording the near infrared scattering of bovine rod outer segments after excitation with flashes of green light.Semiconductor detector arrays are arranged centrosymmetrically around a hemispherical cuvette. The optical characteristics of a hemispherical cuvette and the resulting geometry of cuvette and detection are discussed.Calculations of optimal signal transfer and noise of the detectors led to the following arrangement for each scattering angle: pairs of parallel connected photodiodes are fed into several current-to-voltage converters, whose output voltages are summed up by a summing amplifier.For the test of the device so-called N signals of fresh and liquid N₂-frozen and thawed ROS samples were measured at four scattering angles simultaneously. A strong angular dependence (difference scattering curve) of the relative light scattering change is seen for fresh ROS which is transformed into a flat curve by freezing and thawing. It is concluded that the competence of the fresh sample to extend the light-induced local events — presumably rhodopsin conformational changes — into the gross-structural range is terminated by freezing.     

Quantification of electron energy-loss spectra with K and L shell ionization cross-sections

Quantitative EELS microanalysis in the analytical electron microscope involves the use of partial scattering cross-sections to relate measured edge intensities of the elements to their composition.To test the accuracy of the presently available quantification procedures for K and L edges, we have quantified a wide range of oxidic compounds by using cross-sections which have been calculated by the efficiency factor method and the hydrogenic approximation.An accuracy for element ratios of the order 10–30% can be obtained with these calculated cross-sections for small collection angles (e.g. 5.9 mrad), whereas quantification of spectra recorded with very large collection angles can result in higher errors.Furthermore, partial scattering cross-sections for L edges have been determined by the use of stoichiometric oxide standards and these cross-sections have been found to provide more accurate quantification results when compared to the calculated cross-section.     

Structural changes and aggregation of human influenza virus

The pH-induced change in the structure and aggregation state of the PR-8 and X-31 strains of intact human influenza virus has been studied in vitro. Reducing the pH from 7.4 to 5.0 produces a large increase in the intensity of light scattered to low angles. A modest increase in the polydispersity parameter from cumulants fits to the dynamic light scattering correlograms accompanies the increase, as does a change in how that parameter varies with scattering angle. These trends imply that the virus particles are not uniform, even at pH 7.4, and tend to aggregate as pH is reduced. The scattering profiles (angular dependence of intensity) never match those of isolated, spherical particles of uniform size, but the deviations from that simple model remain modest at pH 7.4. At pH 5.0, scattering profiles calculated for aggregates of uniformly sized spheres come much closer to matching the experimental data than those computed for isolated particles. Although these observations indicate that acid-induced aggregation develops over a period of minutes to hours after acidification, a nearly instantaneous increase in hydrodynamic size is the first response of intact virus particles to lower pH.     

Whole-body angular momentum in incline and decline walking

Angular momentum is highly regulated over the gait cycle and is important for maintaining dynamic stability and control of movement. However, little is known regarding how angular momentum is regulated on irregular surfaces, such as slopes, when the risk of falling is higher. This study examined the three-dimensional whole-body angular momentum patterns of 30 healthy subjects walking over a range of incline and decline angles. The range of angular momentum was either similar or reduced on decline surfaces and increased on incline surfaces relative to level ground, with the greatest differences occurring in the frontal and sagittal planes. These results suggest that angular momentum is more tightly controlled during decline walking when the risk of falling is greater. In the frontal plane, the range of angular momentum was strongly correlated with the peak hip and knee abduction moments in early stance. In the transverse plane, the strongest correlation occurred with the knee external rotation peak in late stance. In the sagittal plane, all external moment peaks were correlated with the range of angular momentum. The peak ankle plantarflexion, knee flexion and hip extension moments were also strongly correlated with the sagittal-plane angular momentum. These results highlight how able-bodied subjects control angular momentum differently on sloped surfaces relative to level walking and provide a baseline for comparison with pathological populations that are more susceptible to falling.     

Polarized light scattering for rapid observation of bacterial size changes.

The effect of changing growth conditions on the diameter of rod-shaped bacteria was studied in vivo with the use of polarized light scattering. The value of a ratio of scattering matrix elements was measured as a function of scattering angle at various times after nutritional "upshift" for two strains of Escherichia coli cells. The peak locations of the scattering function were calibrated against the diameter for rod-shaped bacteria. The peaks moved toward smaller angles as a function of time after upshift, indicating that the diameter was increasing. Under special conditions, substantial peak shifts occurred within a few minutes of growth condition change, indicating a rapid onset of growth in diameter. The rate of increase of the diameters after upshift was obtained from the angular shift of peak location. This rate was approximately 14 nm/min for E. coli K12 and approximately 9 nm/min for E. coli B/r at 37 degrees C. The rate of diameter increase is smaller at lower temperatures. Experiments with Bacillus megaterium showed that any diameter change after nutritional upshift at 37 degrees C is limited to at most a very small increase, at least for the strain and medium tested.     

Observation of correlated X-ray scattering at atomic resolution

Tools to study disordered systems with local structural order, such as proteins in solution, remain limited. Such understanding is essential for e.g. rational drug design. Correlated X-ray scattering (CXS) has recently attracted new interest as a way to leverage next-generation light sources to study such disordered matter. The CXS experiment measures angular correlations of the intensity caused by the scattering of X-rays from an ensemble of identical particles, with disordered orientation and position. Averaging over 15 496 snapshot images obtained by exposing a sample of silver nanoparticles in solution to a micro-focused synchrotron radiation beam, we report on experimental efforts to obtain CXS signal from an ensemble in three dimensions. A correlation function was measured at wide angles corresponding to atomic resolution that matches theoretical predictions. These preliminary results suggest that other CXS experiments on disordered ensembles—such as proteins in solution—may be feasible in the future.     

An approach for time-resolved x-ray scattering.

Recent biological optical spectroscopic studies have correlated discrete spectroscopic states with biological function in several systems. One of the challenges of molecular biophysics is to correlate structural changes with these spectroscopic states. From small-angle x-ray scattering one can obtain a key structural parameter, the radius of gyration of solubilized proteins. The method described in this paper would permit determination small changes in the radius using polychromatic synchrotron radiation. The high flux of the storage ring combined with an enhancement factor of approximately 10(4), obtained by removing the requirement for monochromatic radiation, will permit determining the radius on a millisecond time scale. Unlike energy-dispersive methods, this method would use all available energies over a wide range of angles.     

Fingerprint‐to‐CH stretch continuously tunable high spectral resolution stimulated Raman scattering microscope

Stimulated Raman scattering (SRS) microscopy is a label‐free method generating images based on chemical contrast within samples, and has already shown its great potential for high‐sensitivity and fast imaging of biological specimens. The capability of SRS to collect molecular vibrational signatures in bio‐samples, coupled with the availability of powerful statistical analysis methods, allows quantitative chemical imaging of live cells with sub‐cellular resolution. This application has substantially driven the development of new SRS microscopy platforms. Indeed, in recent years, there has been a constant effort on devising configurations able to rapidly collect Raman spectra from samples over a wide vibrational spectral range, as needed for quantitative analysis by using chemometric methods. In this paper, an SRS microscope which exploits spectral shaping by a narrowband and rapidly tunable acousto‐optical tunable filter (AOTF) is presented. This microscope enables spectral scanning from the Raman fingerprint region to the Carbon‐Hydrogen (CH)‐stretch region without any modification of the optical setup. Moreover, it features also a high enough spectral resolution to allow resolving Raman peaks in the crowded fingerprint region. Finally, application of the developed SRS microscope to broadband hyperspectral imaging of biological samples over a large spectral range from 800 to 3600 cm^?1, is demonstrated.     

Simple multi-filter sensors for determining relative radiation in the sea and in freshwater

The design, construction, testing and use of an underwater relative radiation sensor are described. The sensor incorporates four narrow band interference filters and a broad band (400–700 nm) filter so that variation in spectral quality as well as visible light attenuation can be determined. Comparison with a proprietary, calibrated sensor, a Macam SD 101, indicates the reliability of the relative radiation sensor of the range 1 to >300 Wm^–2 P.A.R. and to a depth of at least 20 m. Brief reference is made to selected data from a variety of aquatic habitats. A general table for the comparison of radiation and illumination units is presented.     

Molecular weight of T7 and calf thymus DNA by low-angle light scattering

A low-angle light-scattering instrument has been used to measure molecular weights of native calf-thymus and T7 DNA. Molecular weights obtained by extrapolation of angular data to 0° from measurements above 30° are less than molecular weights from extrapolation of data taken at low angles (below 30°). The low-angle molecular weights determined for calf-thymus DNA and for T7 DNA agree well with estimates of weight-average molecular weight obtained with other techniques. The low-angle light-scattering molecular weight for calf-thymus DNA is higher than previously reported values by light scattering at angles above 30°. A concentration dependence in the scattering from DNA solutions is also observed.     

Low-angle light-scattering instrument for DNA solutions

A light-scattering instrument capable of measurements on native DNA at angles as low as 10° is described. The major features of the instrument, which make it capable of low-angle measurements, are the use of an intense light source in which the incident beam is monitored directly and the use of a long, rectangular sample cell in which the scattered light can be measured at low angles with no interference from the incident beam. Methods for calibrating the instrument and for determining scattering correction factors are described. Procedures for the preparation and use of various calibration standards are given.     

C-phycocyanin hydration water dynamics in the presence of trehalose: an incoherent elastic neutron scattering study at different energy resolutions

We present a study of C-phycocyanin hydration water dynamics in the presence of trehalose by incoherent elastic neutron scattering. By combining data from two backscattering spectrometers with a 10-fold difference in energy resolution we extract a scattering law S(Q,omega) from the Q-dependence of the elastic intensities without sampling the quasielastic range. The hydration water is described by two dynamically different populations--one diffusing inside a sphere and the other diffusing quasifreely--with a population ratio that depends on temperature. The scattering law derived describes the experimental data from both instruments excellently over a large temperature range (235-320 K). The effective diffusion coefficient extracted is reduced by a factor of 10-15 with respect to bulk water at corresponding temperatures. Our approach demonstrates the benefits and the efficiency of using different energy resolutions in incoherent elastic neutron scattering over a large angular range for the study of biological macromolecules and hydration water.