Light scattering measurement in an arc lamp-based flow cytometer

The epi-illumination optics employed in most arc lamp-based flow cytometers may be modified so as to produce a dark-field configuration which facilitates highly sensitive detection of both forward and large angle light scattering in an instrument with a "jet on open surface" flow chamber. Forward scattering is detected at angles upwards from about 2 degrees, while large angle scattering includes angles above 18 degrees. Theoretical considerations suggest that large angle scattering measured around 20 degrees may be as efficient as that measured at 90 degrees for the purpose of distinguishing cells on the basis of intracellular structure. This was supported by the finding that dual parameter light scattering histograms of leukocyte suspensions obtained with the arc lamp-based instrument were closely similar to such histograms recorded with a laser-based instrument with the large angle detector at 90 degrees. Different species of bacteria could be distinguished by means of the dual parameter light scattering device, as could different species of sea algae. The sensitivity of the device is sufficient to measure 0.2 microns polystyrene particles in both forward and large angle scattering.     

Development of instrumentation to allow the detection of microorganisms using light scattering in combination with surface plasmon resonance

This paper describes work carried out to develop a biosensor which allows two separate detection principles to operate simultaneously at the same surface. A prototype device was constructed that provided Kretschmann-configuration surface plasmon resonance (SPR) measurement of refractive index (RI) changes using an 820 nm LED light source, whilst a 635 nm diode laser was used to produce light scattering signals from bacterial spores. Both effects occurred at a gold-coated surface. The RI changes were measured conventionally from the side of the gold layer nearer to the light sources. The scattered light was imaged from the opposite face which was in contact with the aqueous sample. Specific detection of bacterial spores through the light scattering mode using antibody capture was investigated. The flow dynamics and interactions with the surface of individual spores were observed. A comparison with SPR for detection using the same antibody/antigen pair was made. Spore suspensions that were readily detectable by light scattering at 10(7) ml(-1) did not provide significant responses by SPR. The potential for future developments is discussed.     

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.     

Rotational and translational motions of human spermatozoa: angle dependence of dynamic laser light scattering

We have studied how the dynamic components of laser light scattered from human spermatozoa depend on the scattering angle. This was done by investigating the halfwidth of the intensity autocorrelation function. A model of the spermatozoa as freely rotating and translating linear objects was adequate to describe the scattered light. Rotational motions determined the halfwidth of the intensity autocorrelation function at very small scattering angles and contribution from translational motions was dominant at scattering angles larger than 20 degrees. The contribution from translational motions increased with increasing scattering angle. We found a nearly linear relationship between the translation speed and the rotation frequency. However, the ratio between the two properties varied more than expected from the methodological error. Therefore we introduced a propelling efficacy as a concept to describe the swimming efficiency. This property might contain important information about the swim characteristics.Abbreviations ACF Autocorrelation function - _1/2 halfwidth - RGD Rayleigh-Gans-Debye - SD Standard deviation Correspondence to: P. Thyberg     

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.     

Low angle light scattering studies on whole, half, and quarter molecules of T2 bacteriophage DNA.

Static light scattering measurements have been made at angles as low as 8 degrees on whole, half, and quarter molecules of native, T2 bacteriophage DNA in 0.195 M Na+. The fragments were obtained by high-speed stirring of the native DNA, and fractionated on methylated-albumin-kieselguhr columns. Accompanying measurements of sedimentation coefficients and intrinsic viscosities were made. Because linear extrapolations of light scattering data above 8 degrees for these samples were suspect, the measurements were analyzed by fitting curves calculated from the theory of wormlike coils to experimental curves at c = 0. Results showed that the excluded volume parameter, epsilon, must be used in analyzing the scattering curves; a reasonable value of epsilon was 0.08, in agreement with that found for T7 DNA (Harpst, J. A. 1980. Biophys. Chem. 11:295-302). The persistence length of all three DNAs in this paper was 50 +/- 5 nm, showed no dependence on molecular weight, but was somewhat below that reported previously for T7 DNA (60 nm). Theoretical curves calculated with the preceding parameters had a clear upward curvature in scattering envelopes below 8 degrees for quarter and half molecules, but such curvature was minimal for whole T2 DNA, so that linear extrapolations of experimental data above 8 degrees gave a molecular weight and root-mean-square radius which were nearly the same as those from theory. The molecular weight and radius for whole T2, derived from the comparison of theory and experiment, were 115 X 10(6) and 1,224 nm, respectively. The measurements on T2 DNA were clearly at the upper limit of current techniques.     

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.     

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.     

Development of a charged-coupled device-based light-scattering instrument for the detection of C-reactive protein using particle-enhanced immunoassay

A novel light-scattering instrument has been developed for rapid detection of immunoreactions in test latex particle-enhanced immunoassays. The detector consists of a flat-field grating and a charge-coupled device mounted on a rotating platform, and the detector measures a continuous spectrum from 350 nm to 735 nm at 440 polar angles with a resolution of 0.5 degrees. Optimal detection for rates of immunoreaction were determined by intensity of scattered light at different angles. Instrumental precisions were all shown to fall within 5% of the target relative standard deviation limits. The accuracy of the instrument was confirmed using monodispersed latex particles of known size and shape. The initial results showed the possibility of a sensitive and accurate detection of C-reactive protein throughout the range of clinical interest, thus demonstrating a significant potential for biomedical applications.     

Effect of incident angle of light on sensitivity and detection limit for layers of antibody with surface plasmon resonance spectroscopy

The effect of the incident angle of light on sensitivity and the detection limit for surface-plasmon resonance spectroscopy were examined. The sensitivities and the detection limit were experimentally measured using an antibody as a modeled analyte in the incident angles of a light region of 66-76 degrees. The results showed that the sensitivity of a smaller incident angle was higher than that of a larger one. For instance, the sensitivity of a 66 degree incident angle was three times higher than that of a 76 degree incident angle. The detection limit with a 66 degree incident angle was one-tenth of that with a 76 degree incident angle. These sensitivities and detection limits were compared with those of a commercially produced surface-plasmon resonance instrument. This comparison demonstrated that a wavelength resolution of the order of less than 10(-2) nm was necessary to obtain satisfactory sensitivities and detection limits. In addition, the refractive index and thickness of the antibody layer formed on a sensor surface was proposed by experimental results and theoretical calculation.     

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.     

Differential of light-scattering detection in an arc-lamp-based epi-illumination flow cytometer

Light-scattering histograms of blood cell suspensions were recorded for various ranges of scattering angles by means of an arc-lamp-based flow cytometer (AL-FCM). The results were compared with those obtained with a conventional, laser-based flow cytometer (L-FCM) with forward scattering (2-20 degrees) and scattering at right angles. Measuring with the AL-FCM in the angle range upward from 13 degrees, the relative light-scattering intensities of lymphocytes, monocytes, and granulocytes were essentially independent of scattering angle and closely similar to the values measured as right-angle scattering in the L-FCM. With a range of scattering angles upward from 2 degrees the AL-FCM yielded histograms similar although not identical to that of the forward-scattering detector in the L-FCM. Differentiation between live and dead cells in this mode of operation was similar in the two instruments.     

Methodology for assaying recombinant interleukin-2 associated with liposomes by combined gel exclusion chromatography and fluorescence

A simple methodology based on fluorescence and gel exclusion chromatography (GEC) has been developed to assay recombinant Interleukin-2 (rIL-2) associated with vesicles. A Sephadex G75 column was used to separate the liposomes from non-entrapped rIL-2. The elution of the rIL-2 liposomes was monitored by coupling fluorescent and light scattering detection. The solubilisation of the vesicles with octylglucoside (OG) before the assay was necessary to avoid interference from light scattering. This methodology can be automated to yield an on-line system that can separate, solubilise and quantify rIL-2 in liposome samples. It can be extended to any protein associated with vesicles provided that the former can be detected by fluorescence.     

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.     

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.     

The small angle light scattering of biological cells. Theoretical considerations

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

Lab-on-a-chip immunoassay for multiple antibodies using microsphere light scattering and quantum dot emission

Double detection of microsphere light scattering and quantum dot emission was demonstrated for lab-on-a-chip immunoassay without using stationary support. We conjugated quantum dots (QDs) onto microspheres to enable multiplex assays as well as to enhance the limit of detection (LOD). We named this configuration "nano-on-micro" or "NOM". Upon radiation with UV light (380nm), a stronger light scattering signal is observed with NOMs than QDs or microspheres alone. Additionally, NOMs are easier to handle than QDs. Since QDs also provide fluorescent emission, we are able to utilize an increase in light scattering for detecting antigen-antibody reaction and a decrease in QD emission to identify which antibody (or antigen) is present. Two types of NOM combinations were used. One batch of microspheres was coated with QDs emitting at 655 nm and mouse IgG (mIgG); the other with QDs emitting at 605 nm and bovine serum albumin (BSA). A mixture of these two NOMs was used to identify either anti-mIgG or anti-BSA. NOM particles and target solutions were mixed in a microfluidic device (using highly carboxylated microspheres as previously demonstrated by our group) and on-chip detection was performed using proximity optical fibers. Forward light scattering at 380 nm was collected. With the positive target, the scattering signal was increased. The LOD was as low as 50 ng ml(-1) (330 pM) with p<0.05. Fluorescent emission (655 or 605 nm) was simultaneously collected. With the positive target, the emission signal was attenuated. Therefore, we were able to detect two different antibodies simultaneously with two different detection protocols. We believe this NOM bioassay has the ability to screen for and detect multiple antibodies with minimal sample processing and handling (one-step lab-on-a-chip immunoassay).     

DETECTION OF ESCHERICHIA COLI O157 IN RAW BEEF USING A CCD BASED LIGHT SCATTERING INSTRUMENT

A sensitive and easy-to-perform instrumentational method for the detection of Escherichia coli O157 in raw minced beef is described. The detection is based on a light scattering immunoassay and a charge-coupled device (CCD) direct readout spectrometer measuring the scattered light spectral signals at an optimized angle of 20° to the axis of transmitted light. Using latex particles coated with antibodies for E. coli O157, the method sensitivity has significantly improved comparing with the visual immunoassay assessment method when detecting the presence of this bacterium in spiked beef samples. The method is capable of detecting E. coli O157 at the level of 10³ cfu mL^-1 after 6 h of incubation of the spiked samples. This study has demonstrated a faster technique (within 8 h) for the detection of E. coli O157 in raw beef and a possible new application for the CCD based light scattering instrument.     

Dynamic light scattering studies of the aggregation of lysozyme under crystallization conditions.

The intensity autocorrelation functions of light scattered by lysozyme solutions under pre-crystallization conditions in NaCl-containing media were recorded at scattering angles from 20 degrees to 90 degrees. The measurements, conducted on freshly prepared protein solutions supersaturated more than 3-fold, indicate the simultaneous presence of two scatterer populations which can be assigned to individual protein molecules and to large particles. When solutions are undersaturated, or slightly supersaturated, light scattering only reveals the presence of the small scatterers. In the supersaturated medium, where aggregates were detected, lysozyme crystals grew in a time-span of 1-3 days after the scattering experiments. These results are medium, where aggregates were detected, lysozyme crystals grew in a time-span of 1-3 days after the scattering experiments. These results are correlated with the nucleation step during protein crystallization.     

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.