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.     

Simultaneous separate detection of low angle and large angle light scattering in an arc lamp-based flow cytometer

A device is described for simultaneous separate detection of the light scattering of cells at low and large scattering angles in an arc lamp-based flow cytometer with epi-illumination through an oil immersion microscope objective. Light scattering was measured in a dark field configuration that allows separate detection of light scattering greater than 2 degrees and 15 degrees, respectively. Dual parameter light scattering histograms of a blood cell suspension containing various types of leukocytes were closely similar to that obtained with a commercial laser-based instrument with light scattering detection at forward and right angles. The sensitivity of the device was sufficient to measure polystyrene particles with 0.25-micron diameter. A potential application may be differentiation of bacteria.     

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.     

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.     

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.     

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.     

On the effect of parent-aneurysm angle on flow patterns in basilar tip aneurysms: towards a surrogate geometric marker of intra-aneurismal hemodynamics

As a part of previous computational fluid dynamic (CFD) validation studies, particle image velocimetry (PIV) of two anatomically realistic basilar artery tip aneurysm models revealed two distinct types of flow (one of which has yet to be reported in the literature), characterized by the location and strength of the intra-aneurismal vortex. We hypothesized that these distinct "hemodynamic phenotypes" could be anticipated by a simple geometric parameter: the angle of the aneurysm bulb relative to the parent artery. An idealized basilar tip aneurysm model was constructed to allow independent control of this angle, and CFD simulations were carried out for angles ranging from 2 degrees to 30 degrees , these extremes corresponding to the angles measured from the two anatomically realistic models. The gross hemodynamics predicted by the idealized model for 2 degrees and 30 degrees were consistent with those seen in the corresponding anatomically realistic models. For the idealized model, the flow type switched at an angle between 8 degrees and 12 degrees . Sensitivity studies suggested that, near these angles, the hemodynamic phenotype was sensitive to inflow momentum. Outside this range, however, the parent-bulb angle appeared to be a robust predictor of hemodynamic phenotype. Our findings suggest that blood flow dynamics in basilar artery tip aneurysms fall into one of the two broad phenotypes, each subject to distinct hemodynamic forces. That the general features of these flow types may be anticipated by a relatively simple-to-measure geometric parameter could help ease the introduction of hemodynamic information into routine clinical decision-making.     

Analysis of ploidy in a planarian by flow cytometry

Flow cytometry (flow microfluorimetry) provides a quick means for analysis of ploidy in planarians. Nuclei from homogenized tissues of the freshwater planarian Dugesia japonica japonica Ichikawa et Kawakatsu were stained with propidium iodide and measured with an argon-laser flow cytometer to produce histograms of DNA content. Tissues from sexually mature individuals produced histograms with a 1n (haploid) peak but no 3n peak (triploid peak), whereas those from asexual individuals showed a 2n peak or a 3n peak or both, but no 1n peak. Thus, the 1n peak distinguished sexual individuals. Mixoploid individuals, i.e., mosaics with both diploid and triploid tissues, were identified by the presence of both a 2n peak and a 3n peak. The ratios of the heights of the 2n and 3n peaks from tissues in different parts of a single mixoploid individual were similar, suggesting that the diploid and triploid cells are homogeneously distributed.     

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.     

Two color light scattering identifies physical differences between lymphocyte subpopulations

Forward angle light scattering of two different wavelengths by cells in a flow cytometer was used to investigate physical differences between lymphocytes of different lineage, functional subclass and developmental stage. Correlation of the ultraviolet (UV: 351 nm and 364 nm) and 488 nm light scattering signals produced by lymphoid cells demonstrated that the two signals were not equivalent and that they placed different emphasis on the physical parameters characterizing lymphocytes. Both small T and B lymphocytes from peripheral lymphoid tissues and mitogenically activated large T and B lymphocyte blasts were discriminated by both wavelengths. Differences between the Lyt-2 negative and Lyt-2 positive T lymphocyte subsets were also apparent. Two color light scattering could also discriminate between immature thymocytes and mature peripheral T cells and between small bone marrow cells and mature peripheral B cells. In bone marrow an increase in UV light scattering coincided with the appearance of cell surface immunoglobulin on small cells. These data establish that two color light scattering is a sensitive probe for distinguishing cells of apparently similar morphology and that it can be used to study the physical changes that occur during lymphoid cell differentiation.     

Immunofluorescence analysis of bacillus spores and vegetative cells by flow cytometry

A commercially available flow cytometer (Cytofluorograf) was used for the immunofluorescence (IF) analysis of spores of Bacillus anthracis, Bacillus cereus, and Bacillus subtilis, using fluorescein-labelled antispore conjugates. The cytometer was modified to allow analysis of known numbers of bacteria. In attempting to identify the region of the cytometer fluorescence histogram associated with the presence of stained spores, evidence was produced for signal components due to antibody bound to extracellular antigens. Under some reaction conditions these components were large enough partially or completely to obscure the fluorescence distribution imputed to the spores. The results support the hypothesis that the fluorescence histogram for a bacterial suspension can be modified by subtracting the histogram of the cell-free centrifugation supernatant to provide a fluorescence distribution more representative of the bacteria themselves. Spore and vegetative forms of B. anthracis could be differentiated in the flow IF assay by comparing the peak and area (integral) values of the photomultiplier output. The 90 degrees scatter histograms of the stained spores and their cell-free supernatants were so alike in shape that it was not possible to ascribe a unique peak to the spores themselves. Overall, these results confirm the considerable potential of flow cytometry for the rapid and quantitative IF assay of bacterial populations.     

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.     

Variance within homogeneous phytoplankton populations, II: Analysis of clonal cultures

In part I of this series of articles, a framework was presented for interpreting histograms of volume or fluorescence as measured by a flow cytometer on homogeneous phytoplankton populations. In this paper, the analytical framework is applied to flow cytometric histograms from laboratory experiments involving clonal phytoplankton cultures. The density function derived in part I was modified to include a third parameter representing a linear shift of the origin. This modified density function was fitted to chlorophyll fluorescence histograms for populations believed to be asynchronous (grown in continuous light) and also to histograms from populations grown on a 14:10 (h:h) light/dark cycle. Near-synchronous subpopulations sorted from an asynchronous population were also analyzed. In populations in which underlying assumptions (asynchronous divisions, constant growth) are valid, curve fits provide estimates of the inherent variability among cells at age 0. The implication of fitting the density function to populations in which these assumptions are not valid is discussed.     

Particle flow behavior in models of branching vessels. II. Effects of branching angle and diameter ratio on flow patterns

To further elucidate the role of fluid mechanical factors in the localization of atherogenesis and thrombogenesis, we have studied the 3-dimensional flow patterns in square T-junctions with branching angles theta from 30 degrees to 150 degrees and diameter ratios d/D (side: main tube) from 1.05/3.0 to 1.0. Cine films of the motions of tracer microspheres in dilute suspensions were taken at inflow Reynolds numbers from 15 to 400 and flow ratios (main: side tube) from 0.1 to 4.0. Flow patterns with suspension entering through the main tube were similar to those previously described in uniform 3 mm diameter T-junctions: paired vortices (spiral secondary flows) symmetrical about the common median plane formed at the entrances of the main and side daughter tubes. Particles circulated through the main vortex, some crossing above and below the mainstream into and through the side vortex. At the geometrical flow ratio, the main vortex became smaller and smaller as the branching angle (theta less than 90 degrees) and diameter ratio decreased, and was confined to a thin side tube was a minimum. In obtuse angle T-junctions the stagnation point shifted from the flow divider into the side tube, enhancing the flow disturbance there. The velocity distributions in main and side tubes were skewed towards the inner walls close to the flow divider. When flow entered through the side tube, a pair of recirculation zones formed in the main tube at the inner wall of the bend with a sharper angle.     

The resolution of aneuploid DNA stem lines by flow cytometry: limitations imposed by the coefficient of variation and the percentage of aneuploid nuclei

Factors important in the resolution of cell sub-populations with differing DNA contents were investigated using an EPICS C flow cytometer. Software is available for the EPICS C which permits data from any two histograms to be superimposed or added together before display. Samples of fresh and archival thyroid tissue, stained with propidium iodide, were analysed on the flow cytometer and the peak channel number noted. The photomultiplier (PMT) voltage was increased and the sample analysed again producing a second histogram with a higher peak channel number. The two histograms were added together to simulate a cell suspension with two sub-populations with a different DNA content. By systematically altering the PMT voltage and the number of nuclei included in each analysis, it was possible to examine the importance of DNA index and the percentage of tumor cells with an aneuploid DNA content for both fresh and paraffin-embedded thyroid nuclei. The crucial importance of achieving a low coefficient of variation (CV) was demonstrated and consequently the reservations that pertain when archival material is studied, particularly in tumours where DNA aneuploidy is frequently expressed with a low DNA index.     

Prey catching in the archer fish: angles and probability of hitting an aerial target

When trying to squirt down aerial arthropods, archer fishes (Toxotidae) have to cope with the displacement of the image of the target by refraction of light at the water surface. It has been suggested, and accepted, that Toxotes jaculatrix would avoid the effect of refraction by squirting vertically. In our previous study, however, Toxotes chatareus was observed to squirt under a wide range of angles, including angles much smaller than 90 degrees. The aim of the present study was to asses in T. chatareus (N=15), the frequency distribution of angles used, the relation between angle and probability to hit, the relation between target height and angle, and the smallest angles the fishes can use. The outcome was a distribution with a range of 102-58 degrees and a median of 74 degrees, no relation between angle and probability to hit, no relation between target height and angle and a smallest angle of 45 degrees. The results clearly indicate that T. chatareus does not evade the refraction effect by squirting only vertically (90 degrees ), but masters a considerable refraction effect thanks to a mechanism that is not investigated here. The capacity to hit prey under a wide range of angles is seen as an important adaptation to the natural habitat where the visibility of prey is restricted by vegetation. The constraints of the range of angles are discussed.     

Measurement of fibre pennation using ultrasound in the human quadriceps in vivo.

Real-time ultrasound scanning was used to measure the angles of fibre pennation of vastus lateralis (VL) and vastus intermedius (VI) of the human quadriceps (n = 12) in vivo. The maximum isometric force and cross-sectional area of the quadriceps were also measured. With the knee at right-angles the mean fibre angles for VL and VI respectively were 0.133 (0.021) rad [7.6 degrees (1.2 degrees)] and 0.143 (0.028) rad [8.2 degrees (1.6 degrees)] [mean (SD)], which is within the range of angles measured on cadavers. The mean angle decreased in going from the contracted [VL, 0.244 rad (14 degrees); VI, 0.279 rad (16 degrees)] to the stretched [VL, 0.105 rad (6 degrees); VI, 0.122 rad (7 degrees)] position. There was a significant positive correlation between fibre angle and muscle cross-sectional area but no relationship between fibre angle and force per cross-sectional area. No increase in fibre angle was detected after 3 months strength training. We conclude that ultrasound can be used to measure pennation angles of superficial muscle groups but we could not demonstrate a relationship between pennation and force-generating capacity.     

Method for analysis of cellular DNA content of paraffin-embedded pathological material using flow cytometry

A method has been developed that allows flow cytometry to be used for measuring the cellular DNA content of paraffin-embedded human tumors. Thick (i.e., 30 micron) sections were cut from tissue blocks using a microtome and dewaxed in xylene. The sections were then rehydrated by sequentially immersing them in 100, 95, 70, and 50% ethanol before finally washing in distilled water. Single cell suspensions were then prepared by incubation in 0.5% pepsin, pH 1.5, at 37 degrees C for 30 min. The cells were counted, washed, and stained with 1 microgram/ml 4',6'-diamidino-2-phenylindole for 30 min, and DNA content was measured using an ICP 22 flow cytometer. There was a good correlation between the DNA histograms produced using this method and those obtained using unfixed tissue from the same tumor stained with ethidium bromide plus mithramycin. This method allows the retrospective study of archival material where the clinical outcome is already known, and it should, therefore, be particularly useful for determining the prognostic significance of abnormal DNA content measured by flow cytometry.     

Structural studies of the tRNA domain of tmRNA

tmRNA is a small, stable prokaryotic RNA. It rescues ribosomes that have become stalled during the translation of mRNA fragments lacking stop codons, or during periods of tRNA scarcity. It derives its name from the presence of two separate domains, one that functions as a tRNA, and another that serves as an mRNA. We have carried out modeling and transient electric birefringence studies to determine the angle between the acceptor stem and anticodon stem of the tRNA domain of Eschericia coli tmRNA. The results of the modeling studies yielded an interstem angle of 110 degrees, in agreement with the lower end of the range of angles (111 degrees -137 degrees ) determined experimentally for various solution conditions. The range of experimental angles is greater than the angles observed for any of the tRNA crystal structures, in line with the presence of a shortened D stem. The secondary structure of the tRNA domain is conserved for all known tmRNA sequences, so we propose that the angle is also conserved. These results also suggest that the region of tmRNA between P2a and P2b may interact with the decoding site of the ribosome.     

A unified procedure for conservative (morphology) and integral (DNA and immunophenotype) cell staining for flow cytometry

BACKGROUND: Current methods for multiparameter DNA flow cytometry suffer from several limitations. These include significant modifications of cell morphological parameters, the impossibility to counterstain cells with certain fluorochromes, and laborious tuning of the instrument that, for some procedures, must be equipped with an ultraviolet (UV) laser. To overcome these problems, we developed a novel method for the simultaneous analysis of morphological parameters, four-color immunophenotyping, and stoichiometric DNA labeling using a bench-top flow cytometer. METHODS: The method consists of a mild permeabilization/fixation treatment at room temperature, followed by labeling with fluorochrome-conjugated monoclonal antibodies (mAbs) and with the DNA dye 7-aminoactinomycin D (7-AAD) at 56 degrees C. RESULTS: Using this method, we analyzed resting peripheral blood mononucleated cells (PBMC), proliferating T cells cultured in the presence of interleukin-2 (IL-2), and lymphoblastoid B cells. Lymphocytes, monocytes, and lymphoblasts treated by this procedure retained differential light scattering (DLS) characteristics virtually identical to those of untreated cells. This allowed regions to be drawn on forward scatter (FSC) and side scatter (SSC) cytograms resolving different cell populations. DLS were preserved well enough to distinguish large lymphoblasts in the S or G2/M phases from small G0/G1 cells. Also, stainability with fluorescein-isothiocyanate (FITC), R-phycoerythrin (PE), allophycocyanin (APC)-conjugated mAbs was generally preserved. DNA labeling with 7-AAD was of quality good enough to permit accurate cell cycle analysis. CONCLUSIONS: The method described here, which we called integral hot staining (IHS), represents a very simple, reproducible, and conservative assay for multiparameter DNA analysis using a bench-top flow cytometer. Last but not least, the cytometer tuning for multiparameter acquisition is straightforward.