Experimental findings on gynecologic cell orientation and dynamics for three flow nozzle geometries.

Gynecologic cell orientation and dynamics in sheath flow for three flow nozzle geometries are studied and compared. Cell orientations are classified into four categories based upon visual inspection of dark field laser stroboscopic photographs of cells in flow. Results are discussed in the context of slit-scan cytofluorometry.     

Local overall volumetric gas-liquid mass transfer coefficients in gas-liquid-solid reversed flow jet loop bioreactor with a non-Newtonian fluid

The local overall volumetric gas-liquid mass transfer coefficients at the specified point in a gas-liquid-solid three-phase reversed flow jet loop bioreactor (JLB) with a non-Newtonian fluid was experimentally investigated by a transient gassing-in method. The effects of liquid jet flow rate, gas jet flow rate, particle density, particle diameter, solids loading, nozzle diameter and CMC concentration on the local overall volumetric gas-liquid mass transfer coefficient (K(L)a) profiles were discussed. It was observed that local overall K(L)a profiles in the three-phase reversed flow JLB with non-Newtonian fluid increased with the increase of gas jet flow rate, liquid jet flow rate, particle density and particle diameter, but decreased with the increase of the nozzle diameter and CMC concentration. The presence of solids at a low concentration increased the local overall K(L)a profiles, and the optimum of solids loading for a maximum profile of the local overall K(L)a was found to be 0.18x10(-3)m(3) corresponding to a solids volume fraction, varepsilon(S)=2.8%.     

EMOSS: an epiillumination microscope objective slit-scan flow system

An epiillumination microscope objective slit-scan flow system has been fabricated utilizing two dimensional slit scanning with hydrodynamic sample stream focussing. Low resolution (4 micron) analysis of cellular fluorescence is facilitated by the definition of a stabilized flow plane through hydrodynamic focussing. Coincidence of the region of stabilized flow with the focal plane of the microscope objective will allow for the collection and subsequent imaging of fluorescence from cells oriented along this plane. Two orthogonal slit-scan contours are generated as a cell traverses the excitation region. It is hoped that the need for a three dimensional system will be precluded by preferential orientation of the cells in the region of stabilized flow. Cellular fluorescence is collected by a high numerical aperture epiillumination optical system and imaged onto two orthogonal slits. Two photomultiplier tubes are used to detect fluorescence. It is anticipated that the epiillumination microscope objective slit-scan flow system will be used with a variety of fluorescent stains and markers, as well as extended to the research of light scattered by cells. (Steen, H.B., Cytometry 1:26-31, 1980.     

Interaction forces between red cells agglutinated by antibody. I. Theoretical.

A general method of calculating forces, torques, and translational and rotational velocities of rigid, neutrally buoyant spheres suspended in viscous liquids undergoing a uniform shear flow has been given by Arp and Mason (1977). The method is based on the matrix formulation of hydrodynamic resistances in creeping flow by Brenner and O'Neill (1972). We describe the solution of the Brenner-O'Neill force-torque vector equation in terms of the particle and external flow field coordinates and derive expressions for the normal force acting along, and the shear force acting perpendicular to, the axis of the doublet of spheres, the latter explicitly given for the first time. The equations consist of a term comprising force and torque coefficients obtained from the matrices of the hydrodynamic resistances (functions of the distance h between sphere surfaces which have been computed), and terms comprising the orientation of the doublet axis relative to the coordinates of the external flow field and the shear stress (which can be experimentally determined). We have applied the theory to a system of doublets of sphered, hardened human red cells of group A or B antigenic type cross-linked by the corresponding antibody at a fixed interparticle distance. Working from studies of the breakup of doublets of red cells in an accelerating Poiseuille flow, given in the succeeding paper, we are able to compute the hydrodynamic force required to separate the two spheres. Previous work has shown that the theory can be applied to doublets in a variable shear, Poiseuille flow, provided the ratio of particle to tube diameter is small. In calculating the force-torque coefficients it was assumed that the cells are crosslinked by antibody with h = 20 nm.     

A variational calculus approach to the suspension flow problem using the minimal force hypothesis

Analytic expressions for the velocity profile and distribution of neutrally buoyant particles in laminar flow were obtained as functions of the radial distance. A modified Einstein viscosity model and the hypothesis that the total force on all the particles flowing in the tube is a minimum were used. The methods of the variational calculus were used in the mathematical development. A velocity profile differing only slightly from the parabolic form of that for Hagan-Poiseuille flow was obtained. For particle distribution the equations developed predict a maximum concentration along the center-line for some flows and a maximum concentration in a ring some distance from the center line in other flows. Both of these concentration profiles have been observed experimentally. Quantitative predictions from the equations derived must await further experimental work to permit calculation of the parameters included in the equations.     

Slit-scan flow cytometry of mammalian chromosomes.

A flow cytometer has been constructed which measures total fluorescence and the distribution of fluorescence along isolated, stained mammalian chromosomes. In this device, chromosomes flow lengthwise at 4 m/sec through a 1-micrometer thick laser beam. The fluorescence from each chromosome is recorded at 10 nsec intervals; the sequence of recorded values represents the distribution of fluorescence along the chromosome and is stored in the memory of a waveform recorder. The total fluorescence of each chromosome is also measured and recorded. Preliminary studies show that doublets of 1.83 micrometers diameter microspheres flow with their long axes parallel to the direction of flow and that the two microspheres are resolved in the slit-scan profile. Ethidium bromide stained Muntjac and Chinese hamster chromosomes have also been slit-scanned. Centromeres were resolved in many of the Nos. 1 and 2 Chinese hamster chromosomes and the Nos. 1 and X + 3 Muntjac chromosomes.     

Imaging in flow.

Imaging in flow has been valuable in investigating discrepancies in flow cell measurements due to cell orientation and flow dynamics. This paper discusses optical consideration in flow imaging, slit and full field imaging systems and various cell motion arresting techniques from the standpoint of image plane exposure and suitable detector choices. It concludes with an explanation of the slit-imaging techniques employed in a multidimensional slit-scan flow system and slit-scan correlation system.     

Dye exclusion artifact in flow cytometers

BACKGROUND: Cells exclude their own volume of dye solution in the sample flow which carries them through the flow chamber of the flow cytometer, thereby affecting the otherwise constant signal arising from the fluorescence of this solution. Under certain conditions, this phenomenon may significantly influence the fluorescence signal of the cells. MATERIALS AND METHODS: Using the slit scan technique, we studied this phenomenon as observed for monodisperse polystyrene particles in fluorescein solution. RESULTS: The measurements show that dye solution accumulates just in front of the particle and just behind it, with a relative void in between. This phenomenon is most likely caused by the rapid constriction of the flow as it enters the orifice of the nozzle or flow chamber, giving rise to a pulse of fluorescence which adds to that of the particle or cell itself. The magnitude of this artifact depends on the design and dimensions of the nozzle/flow chamber as well as on the rate of sample flow. CONCLUSIONS: The dye exclusion artifact may affect measurements of cells when they are in a dye solution having a fluorescence per unit volume which is significant compared to that of the cells, especially at low sample flow rates.     

A freeze-capture method for the study of platelet-sized particle distributions

A rapid freezing method was developed to study the distributions of fluorescent platelet-sized particles in flows of blood suspensions through thin-walled capillary tubes. Segments of frozen tubes were mounted in a refrigerated microtome on the stage of an epifluorescence microscope. Sections of tube were cut away, images of newly exposed cross-sections were recorded on video tape, and distances of the particles from the wall were measured from recorded images. The distance data were used to construct histograms that were proportional to the local concentration. Results indicated that this method is suitable for the study of the distribution of platelet-sized particles over a wide range of hematocrit, that the basic profile is reproducible to within 15%, and that the non-uniform profile is not a result of events at the tube entrance.     

Liquid circulation velocity and overall gas holdup in a modified jet loop bioreactor with low density particles

Effect of low density particles on the apparent liquid circulation velocity and overall gas holdup was studied in a modified reversed flow jet loop bioreactor. Experiments were conducted using polyurethane beads, polystyrene particles which are comparable to bioparticles found in biological applications and glass beads. Influence of gas and liquid flow rates, draft tube to reactor diameter ratio and solids loading on these hydrodynamic properties were studied. The liquid circulation velocity was found to increase with an increase in liquid flow rate but decrease with an increase in gas flow rate or solids loading. The overall gas holdup increased with an increase in gas or liquid flow rate but decreased with an increase in solids loading. The range of optimum draft tube to reactor diameter ratio was found to be 04–0.5. The results obtained with low density particles were comparatively better than those with glass beads. Correlations were proposed to evaluate liquid circulation velocity and overall gas holdup in terms of operational and geometrical variables.     

Improved flow cytometric sorting of X‐ and Y‐chromosome bearing sperm: Substantial increase in yield of sexed semen

The yield of flow cytometric sorted X‐ and Y‐chromosome‐bearing sperm in a given time period is an important factor in the strategies used for fertilization and the production of sex‐preselected offspring. This yield is dependent on the efficiency with which the modified flow cytometer/cell sorter analyzes the DNA of spermatozoa. The efficiency is directly related to the number of sperm with the correct orientation during DNA analysis. Currently, the efficiency of flow cytometric sperm sorting is low since orientation of the sperm head to laser excitation is rate limiting. To overcome this problem, a new nozzle was designed to enhance sperm orientation and tested under flow cytometric sorting conditions. The degree of orientation improvement was determined with different sample rates using viable sperm and dead sperm of several different species. There was at minimum, a two‐fold increase in the proportion of oriented sperm when comparing the new nozzle with the currently used modified flow cytometer/cell sorter employing a beveled needle. More than 60% of intact bull sperm and boar sperm were correctly oriented compared with 25% to 30% using the beveled needle system. A unique characteristic of the novel nozzle was that the proportion of oriented sperm was independent of sample rate and of sperm motility. The accuracy of DNA measurement together with high purity sorting was tested using the novel nozzle. The novel nozzle was unique in that accuracy of measurement and sorting performance were not diminished. Using the new nozzle, samples of 88% purity of sorted X‐sperm and Y‐sperm were obtained for viable bull and boar sperm. The yield of flow cytometric sorted X‐ and Y‐chromosome‐bearing sperm using the novel nozzle was, on average, twice that obtained by using the beveled needle system in conjunction with a standard equipment nozzle for orientation. Mol. Reprod. Dev. 52:50–56, 1999. Published 1999 Wiley‐Liss, Inc.     

On the Correlation of the Volume of a Prolate-Spheroidal Cell, Tetrahymena as Determined by Electronic Particle Counters and by Morphometry

ABSTRACT. The cell volume provided by electronic particle counters may be incorrect. As a particle, or cell, passes the counting device, its volume is calculated as a sphere. The electronically derived, mean cell volume (electronic MCV) of a population of Tetrahymena (prolate spheroid) is smaller than the volume (morphometric MCV) calculated from measured cell length and width. This discrepancy was studied using a Coulter Multisizer particle counter and cell morphometry. The electronic MCV averaged 0.70 of the morphometric MCV (1.00) but changed from 0.72 (fast growth) to 0.63 and 0.76 (slow or no growth) for cells having a mean length/width of 2.05, 2.33, and 1.61, respectively. The measured diameter of latex particles (used for calibration) was identical to that stated, but the diameter of the electronic MCV was larger than the width of the cells which related to wehther the length/width of the cells was above, or below, 2.00. Hence, electron particle counters register primarily the width of a prolate-spheroidal cell, oriented with its long axis in the direction of flow, and uses this value as diameter for the calculated sphere, whereas for more spherical cells, tumbling without any orientation, a mean of the axes is used. Factors for correction of the electronic MCV of Tetrahymena are provided.     

Computational analysis of confined jet flow and mass transport in a blind tube.

A computational analysis of confined nonimpinging jet flow in a blind tube is performed as an initial investigation of the underlying fluid and mass transport mechanics of tracheal gas insufflation. A two-dimensional axisymmetric model of a laminar steady jet flow into a concentric blind-end tube is put forth and the governing continuity, momentum, and convection-diffusion equations are solved with a finite element code. The effects of the jet diameter based Reynolds number (Re(j)), the ratio of the jet-to-outer tube diameters (epsilon), and the Schmidt number (Sc) are evaluated with the determined velocity and contaminant concentration fields. The normalized penetration depth of the jet is found to increase linearly with increasing Re(j) for epsilon = O(0.1). For a given epsilon, a ring vortex that develops is observed to be displaced downstream and radially outward from the jet tip for increasing Re(j). The axial shear stress profile along the inside wall of the outer tube possesses regions of fixed shear stress in addition to a local minimum and maximum in the vicinity of the jet tip. Corresponding regions of axial shear stress gradients exist between the fixed shear stress regions and the local extrema. Contaminant concentration gradients develop across the ring vortex indicating the inward diffusion of contaminant into the jet flow. For fixed epsilon and Sc and Re(j) approximately 900, normalized contaminant flow rate is observed to be approximately twice that of simple diffusion. This model predicts modest net axial contaminant transport enhancement due to convection-diffusion interaction in the region of the ring vortex.     

Fringe-scan flow cytometry

We describe the development of a scanning flow cytometer capable of measuring the distribution of fluorescent dye along objects with a spatial resolution of 0.7 micron. The heart of this instrument, called a fringe-scan flow cytometer, is an interference field (i.e., a series of intense planes of illumination) produced by the intersection of two laser beams. Fluorescence profiles (i.e., records showing the intensity of fluorescence measured at 20 ns intervals) are recorded during the passage of objects through the fringe field. The shape of the fringe field is determined by recording light scatter profiles as 0.25 micron diameter microspheres traverse the field. The distribution of the fluorescent dye along each object passing through the fringe field is estimated from the recorded fluorescence profile using Fourier deconvolution. We show that the distribution of fluorescent dye along microsphere doublets and along propidium iodide stained human chromosomes can be determined accurately using fringe-scan flow cytometry. The accuracy of fringe-scan shape analysis was determined by comparing fluorescence profiles estimated from fringe-scan profiles for microspheres and chromosomes with fluorescence profiles for the same objects measured using slit-scan flow cytometry.     

Application of Fraunhofer diffraction theory to feature-specific detector design.

Light scatter from epithelial cells in a slit-scan flow system is modeled using the Fraunhofer condition of scalar diffraction theory. Power spectra are calculated for successive positions of model cells in the line focus of a laser beam with a Fourier transform computer program. Using the calculated power spectra, detector configurations are designed to detect specific cell structures of interest. Detector configurations are tested in a static slit-scan scatter apparatus. Data indicating the ability to detect boundaries and cell orientation are discussed.     

Asymmetric flows of spherical particles in a cylindrical tube

To study the rheological behavior of blood cells in various flow patterns through narrow vessels, we analyzed numerically the motion of blood cells arranged in one row or two rows in tube flow, at low Reynolds numbers. The particles are assumed to be identical rigid spheres placed periodically along the vessel axis at off-axis positions with equal spacings. The flow field of the suspending fluid in a circular cylindrical tube is analyzed by a finite element method applied to the Stokes equations, and the motion of each particle is simultaneously determined by a force-free and torque-free condition. In both cases of single- and two-file arrangements of the particles, their longitudinal and angular velocities are largely affected by the radial position and the axial spacing between neighboring particles. The apparent viscosity of the asymmetric flows is higher than that of the symmetric flow where particles are located on the tube centerline, and this is more pronounced when particles are placed farther from the tube centerline and when the axial distance between neighboring particles is reduced.     

A protocol for Papanicolaou staining of cytologic specimens following flow analysis

A protocol has been developed for restaining cytologic specimens that have been analyzed on a multidimensional slit-scan flow system. The technique involves Papanicolaou staining of cells on a membrane filter that has been previously stained with acridine orange and fixed with glutaraldehyde buffer. The specimen and staining solutions were sequentially added to a 5-micrometers pore size, 47-mm diameter Gelman "Metricel" filter while it remained in a glass filtration apparatus. The practice of retaining the filter in the filtration apparatus throughout the staining procedure minimizes cell loss and eliminates specimen cross contamination when compared with conventional filter dip staining. The availability of this postflow specimen Papanicolaou staining protocol permits accurate determination of the performance characteristics of a multidimensional slit-scan flow system and should be useful whenever staining of a limited number of cells with minimal cell loss is desired.     

Aerosol deposition and flow limitation in a compliant tube

Studies in intact dogs have suggested that aerosol deposition is enhanced in the proximity of a flow-limiting segment (FLS) formed during cough. The mechanism for that observation was investigated using a monodisperse (geometric SD less than or equal to 1.15) fluorescent aerosol produced in a condensation generator. The aerosol was passed through a compliant tube (Penrose) that had been mounted vertically in a two-chamber box. The surrounding pressure (Ps) in the upstream chamber was controlled independent of the surrounding pressure in the downstream chamber, thus allowing development of an FLS near the exit of the upstream chamber. At fixed inlet pressure (P1) and Ps, flow limitation was achieved over a range of 0.1-0.5 l X s-1 by lowering downstream pressure alone (P2). The influence of the FLS cross-sectional geometry on the site of peak deposition was examined because area of an FLS is a function of transmural pressure (Ptm = Px - Ps). For those constriction geometries that did not involve opposing wall contact, the deposition distribution was characterized by a single peak immediately downstream of the constriction. In the most compressed geometries the peak in deposition was diminished and shifted further downstream. Total aerosol deposition was found to be characterized by a dimensionless particle inertia parameter formed as the ratio of particle stopping distance and the minor radius of the elliptical tube cross section. The deposition of small particles with an inertial parameter less than 0.01 was found to be independent of geometry and constriction velocity.(ABSTRACT TRUNCATED AT 250 WORDS)     

On transport of suspended particulates in tube flow.

Blood cells suspended in shear flows exhibit much larger dispersive motions than those predicted by the Stokes-Einstein formula for Brownian diffusion. The lateral migration and the erratic motions of the 8 microns red blood cells (RBC) is thought to be analogous to a diffusive process. It is shown that the often cited convective-diffusion theory may not be an adequate model for describing the transverse migration of suspended cells in blood flow. A comprehensive review of both the classical theory and of contemporary work in particle transport is presented, with particular emphasis on low Reynolds number tube flows. The mechanisms of Taylor dispersion, the effects of Brownian perturbations on translational and rotational motions of the suspended particles in shear fields, and the influence of integratable and chaotic advections, are individually examined. The classical experiment by Segre and Silberberg (1962) lead us to believe that particle hydrodynamics may play an important role in transverse migrations. In this light, we have further examined the hydrodynamic aspects of the so-called "tubular pinch" effect, the lateral migration of rigid spheres. We have also discussed the transverse motions of liquid drops, and the reversibility of the organization of suspensions in transport. The convective accelerations in the entrance region of a tube can produce relative velocities between fluid medium and various type of particulates if there is a difference in density. The deformable RBC, an "active-type" particle, can provide feedback to the flow from both mass and momentum considerations; the more rigid platelet, a "passive-type" particle, will experience a much smaller relative velocity as compared to the RBC. We may expect that particles of different densities are transported to different equilibrium annular positions before entering the fully developed flow region. The erratic, lateral movement of suspended particulates in steady laminar tube flow can be described by the usual Lagrangian coordinates.     

Force acting on spheres adhered to a vessel wall

To evaluate the force and torque acting on leukocytes attached to the vessel wall, we numerically study the flow field around the leukocytes by using rigid spherical particles adhered to the wall of a circular cylindrical tube as a model of adherent leukocytes. The adherent particles are assumed to be placed regularly in the flow direction with equal spacings, in one row or two rows. The flow field of the suspending fluid is analyzed by a finite element method applied to the Stokes equations, and the drag force and torque acting on each particle, as well as the apparent viscosity, are evaluated as a function of the particle to tube diameter ratio and the particle arrangements. For two-row arrangements of adhered particles where neighboring particles are placed alternately on opposite sides of the vessel, the drag and the torque exerted on each particle are higher than those for single-row arrangements, for constant particle to tube diameter ratio and axial spacing between neighboring particles. This is enhanced for Larger particles and smaller axial spacings. The apparent viscosity of the flow through vessels with adhered particles is found to be significantly higher than that without adhered particles or when the particles are freely floating through the vessels.