Model of Red Blood Cell Rotation in the Flow toward a Cell Sizing Orifice: Application to Volume Distribution

The rotation of human red blood cells (RBC) as they flow in the shear field established by a Coulter type orifice is modeled. This model, based on hydrodynamics of ellipsoid rotation in laminar creeping flow, is used to calculate the probability of the cells entering the orifice with a specific orientation. The electrical resistance change produced by a cell passing through the orifice of an electronic cell volume detector is the product of an orientation-dependent shape factor and the cell volume. This paper presents a method to calculate the shape factor probability distribution which can be used to predict its effect on the cell volume distribution. Experimental results confirm the theoretical prediction that the right skewness of resistance change distributions is in part a result of the nonspherical shape of red cells.     

The resolution of the ultrasound pulsed Doppler for blood velocity measurements

Pulsed ultrasound Doppler velocity meters (PUDVM) permit noninvasive blood velocity measurements. The emitted ultrasound beam characteristics primarily determine the resolution of the instrument when recording velocity profiles. The sample volume, the small region over which velocity information data are detected, was found to be > 2·3 mm³ depending on the transducer disk dia., distance in front of the disk, sampling time increment, and pulse length. The shape of the sample volume approximates a cylinder in the near field and a frustrum of a cone in the far field. The end surfaces of the sample volume were affected by the emitted pulse shape. Ultrasonic beam cross-sections were found to be smaller than predicted by theory due to the finite threshold levels of the PUDVM. The variation of the sample volume with range was illustrated by steady laminar flow velocity profile measurements in rigid tubes. The accuracy of velocity measurements was within 5 per cent with slightly larger deviations occurring near the walls due to the finite sample volume.     

Development of a pH gradient within a biofilm is dependent upon the limiting nutrient

Monospecies Citrobacter sp. biofilms were grown in a laminar flow cell using a carbon-limiting medium. Microelectrode measurements showed no change in pH between the bulk fluid and biofilm when the flow cell was supplied with the carbon-limiting medium under static or flowing conditions. When the biofilm was supplied with a phosphate-limiting medium the biofilm became more acidic than the bulk fluid and developed a gradient within. The implications for metals-bioremediation processes are discussed.     

Cell volume and plasma membrane osmotic water permeability in epithelial cell layers measured by interferometry.

The development of strategies to measure plasma membrane osmotic water permeability (Pf) in epithelial cells has been motivated by the identification of a family of molecular water channels. A general approach utilizing interferometry to measure cell shape and volume was developed and applied to measure Pf in cell layers. The method is based on the cell volume dependence of optical path length (OPL) for a light beam passing through the cell. The small changes in OPL were measured by interferometry. A mathematical model was developed to relate the interference signal to cell volume changes for cells of arbitrary shape and size. To validate the model, a Mach-Zehnder interference microscope was used to image OPL in an Madin Darby Canine Kidney (MDCK) cell layer and to reconstruct the three-dimensional cell shape (OPL resolution < lambda/25). As predicted by the model, a doubling of cell volume resulted in a change in OPL that was proportional to the difference in refractive indices between water and the extracellular medium. The time course of relative cell volume in response to an osmotic gradient was computed from serial interference images. To measure cell volume without microscopy and image analysis, a Mach-Zehnder interferometer was constructed in which one of two interfering laser beams passed through a flow chamber containing the cell layer. The interference signal in response to an osmotic gradient was analyzed to quantify the time course of relative cell volume. The calculated MDCK cell plasma membrane Pf of 6.1 x 10(-4) cm/s at 24 degrees C agreed with that obtained by interference microscopy and by a total internal reflection fluorescence method. Interferometry was also applied to measure the apical plasma membrane water permeability of intact toad urinary bladder; Pf increased fivefold after forskolin stimulation to 0.04 cm/s at 23 degrees C. These results establish and validate the application of interferometry to quantify cell volume and osmotic water permeability in cell layers.     

Sarcomeric domain organization within single skinned rabbit psoas fibers and its effects on laser light diffraction patterns.

Total intensity and fine structure of first-order laser light diffraction maxima from single skinned rabbit psoas fibers were studied. Total intensity of the diffraction maxima was measured as a function of the incidence angle (omega-scan). In the most homogeneous fibers, most of the intensity in the diffraction maxima is confined to a rather narrow range of incidence angles. Fibers with less homogeneous striation patterns, apparently composed of several regions of distinct sarcomere length and tilt of striation (domains), give rise to several narrow intensity peaks in their omega-scans. Left and right first-order diffraction lines produce omega-scans of almost identical shape, composed of one or more intensity peaks, with each pair of corresponding peaks separated by about the same angle. The data indicated that in single skinned rabbit psoas fibers, light diffraction is dominated by Bragg diffraction and that the peaks within omega-scans can be directly correlated with domains within the illuminated fiber segment. In the most homogeneous fiber segments the diameter of domains, estimated from the width of the corresponding maxima in the omega-scans, could almost be as large as the fiber diameter. On average, from the number of peaks in the omega-scans two to three domains with an average length of approximately 250-350 microns can be identified in a fiber cross-section. Therefore, on average only a small number of domains (8 per mm) are found within skinned rabbit psoas fiber segments. In contrast, the number of substructural lines within the diffraction maxima is large even for microscopically homogeneous fibers. Substructural lines appear to be present only when several domains are illuminated simultaneously. Separation and width of these substructural lines are approximately inversely proportional to the length of the illuminated region of the fiber. These data suggest that the substructural lines are due to interference between domains, illuminated simultaneously by a light source with a high degree of spatial coherence (laser). The relevance of these findings for measurements of sarcomere length by laser light diffraction is discussed.     

A numerical analysis of forces exerted by laminar flow on spreading cells in a parallel plate flow chamber assay

Exposure of spreading anchorage-dependent cells to laminar flow is a common technique to measure the strength of cell adhesion. Since cells protrude into the flow stream, the force exerted by the fluid on the cells is a function of cell shape. To assess the relationship between cell shape and the hydrodynamic force on adherent cells, we obtained numerical solutions of the velocity and stress fields around bovine aortic endothelial cells during various stages of spreading and calculated the force required to detach the cells. Morphometric parameters were obtained from light and scanning electron microscopy measurements. Cells were assumed to have a constant volume, but the surface area increased during spreading until the membrane was stretched taut. Two-dimensional models of steady flow were generated using the software packages ANSYS (mesh generation) and FIDAP (problem solution). The validity of the numerical results was tested by comparison with published results for a semicircle in contact with the surface. The drag force and torque were greatest for round cells making initial contact with the surface. During spreading, the drag force and torque declined by factors of 2 and 20, respectively. The calculated forces and moments were used in adhesion models to predict the wall shear stress at which the cells detached. Based upon published values for the bond force and receptor number, round cells should detach at shear stresses between 2.5 and 6 dyn/cm(2), whereas substantially higher stresses are needed to detach spreading and fully spread cells. Results from the simulations indicate that (1) the drag force varies little with cell shape whereas the torque is very sensitive to cell shape, and (2) the increase in the strength of adhesion during spreading is due to increased contact area and receptor densities within the contact area. (c) 1993 John Wiley & Sons, Inc.     

Modeling of shear stress experienced by endothelial cells cultured on microstructured polymer substrates in a parallel plate flow chamber

The application of physical stimuli to cell populations in tissue engineering and regenerative medicine may facilitate significant scientific and clinical advances. However, for the most part, these stimuli are evaluated in isolation, rather than in combination. This study was designed to combine two physical stimuli. The first being a microstructured tissue culture polystyrene substrate, known to produce changes in cell shape and orientation, and the second being laminar shear stress in a parallel plate flow chamber. The combined effects of these stimuli on endothelial cell monolayers cells were evaluated in a parallel plate flow chamber and using a computational fluid dynamics (CFD) model. The topography of the cell monolayers cultured on different microstructured surfaces was determined using confocal laser scanning microscopy (CLSM), and this topographic information was used to construct the CFD model. This research found that while the specific underlying structures were effectively planarized by the cell monolayer, significant differences in cell shape and orientation were observed on the different microstructured surfaces. Cells cultured on grooved substrates aligned in the direction of the grooves and showed higher retention after 1-h LSS conditioning than those cultured on pillars. The modeled shear stress distributions also showed differences. While minor differences in the magnitude of shear stress were noted, aligned cell monolayers experienced significantly lower spatial gradients of shear stress when compared with cells that were not pre-aligned by surface features. The results presented here provide an analysis of how one form of physical stimulus can be moderated by another and also provide a methodology by which the understanding of cell responses to topographic and mechanical stimuli can be further advanced.     

A low-cost polyacrylamide gel scanning densitometer

A simple, low-cost, home-built acrylamide gel scanning densitometer is described. The instrument was designed to provide gel scans for the purpose of comparing the relative rates of mobility of bands. It does not include provisions for large volume, integration of peaks, or micrometer resolution. The key to the simplicity of the device was through the use of a rotary rather than a transverse gel carriage. A turntable was attached directly to the shaft of a synchronous clock motor with a slow rate of rotation. A curved gel holder, placed on the turntable, carries the gel through the light beam. A commercial strip chart recorder is used with the scanner.     

Estimating volume flow rates through xylem conduits

We discuss the errors in common approximations of the volume flow rate for laminar flow through conduits with noncircular transverse sections. Before calculating flow rates, ideal geometric shapes are chosen to represent the noncircular transverse sections. The Hagen–Poiseuille equation used with hydraulic diameter underestimates the volume flow rate for laminar flow through conduits even with such ideal shapes. Correction factors that have been proposed for the Hagen–Poiseuille equation also lead to underestimates of the volume flow rate for those shapes. The exact solutions are sometimes difficult to attain, but rates calculated using the exact solutions for the ideal shapes may be as much as five times higher than the approximated rates for common transversely elongated shapes. Either the exact solutions or the approximations may be used to calculate volume flow rates through the xylem of plants. Both of these methods actually approximate flow through the original conduits because the shapes used are approximations of the conduits’ transverse sections. We recommend using the exact solutions whenever possible; they should be closer to the real solution than other approximations. We give tables of correction factors for use in the cases where calculating volume flow rate from the approximate solution, the Hagen–Poiseuille equation, is more feasible. Obtaining theoretical volume flow rates that are larger than previously thought highlights the need to clarify the causes of differences between the theoretical rates and the smaller measured volume flow rates in plant xylem.     

Modification of a vibrating-tube density meter for precise temperature scanning

We have devised a method of temperature scanning with a vibrating-U-tube density meter in which temperature fluctuations are much reduced compared to those using a constant or programmable thermostat. The standard error of a density measurement is 5 × 10^?7 g/ml. Volume changes associated with conformational changes of macromolecular systems can be precisely measured. Using this instrument the volume expansion-melting curves of lipid dispersions have been obtained. The curves are similar in shape and resolution to the excess heat-capacity curves derived from differential scanning calorimetry performed on the same sample. Temperature scanning allows measurements of expansivity as well as apparent volume throughout a temperature range of interest.     

Endothelial actin cytoskeleton remodeling during mechanostimulation with fluid shear stress

Fluid shear stress stimulation induces endothelial cells to elongate and align in the direction of applied flow. Using the complementary techniques of photoactivation of fluorescence and fluorescence recovery after photobleaching, we have characterized endothelial actin cytoskeleton dynamics during the alignment process in response to steady laminar fluid flow and have correlated these results to motility. Alignment requires 24 h of exposure to fluid flow, but the cells respond within minutes to flow and diminish their movement by 50%. Although movement slows, the actin filament turnover rate increases threefold and the percentage of total actin in the polymerized state decreases by 34%, accelerating actin filament remodeling in individual cells within a confluent endothelial monolayer subjected to flow to levels used by dispersed nonconfluent cells under static conditions for rapid movement. Temporally, the rapid decrease in filamentous actin shortly after flow stimulation is preceded by an increase in actin filament turnover, revealing that the earliest phase of the actin cytoskeletal response to shear stress is net cytoskeletal depolymerization. However, unlike static cells, in which cell motility correlates positively with the rate of filament turnover and negatively with the amount polymerized actin, the decoupling of enhanced motility from enhanced actin dynamics after shear stress stimulation supports the notion that actin remodeling under these conditions favors cytoskeletal remodeling for shape change over locomotion. Hours later, motility returned to pre-shear stress levels but actin remodeling remained highly dynamic in many cells after alignment, suggesting continual cell shape optimization. We conclude that shear stress initiates a cytoplasmic actin-remodeling response that is used for endothelial cell shape change instead of bulk cell translocation. atherosclerosis; cytoskeletal dynamics; endothelial cells; mechanotransduction     

Real-time direct observation of single-molecule DNA hydrolysis by exonuclease III

Single-molecule DNA digestion by exonuclease III, which has 3' to 5' exonuclease activity, was analyzed using a micro-channel with two-layer laminar flow. First, a DNA-bead complex was optically trapped in one layer in the absence of exonuclease III permitted the DNA to be stretched by the laminar flow. The exonuclease III reaction was initiated by moving the trapped DNA-bead complex to another layer of flow, which contained exonuclease III. As the reaction proceeded, the fluorescently-stained DNA was observed to shorten. The process was photographed; examination of the photographs showed that the DNA molecule shortened in a linear fashion with respect to the reaction time. The digestion rate obtained from the single-molecule experiment was compared to that measured from a bulk experiment and was found to be ca. 28 times higher than the bulk digestion rate.     

Microstructure of cell wall-associated melanin in the human pathogenic fungus Cryptococcus neoformans

Melanin is a virulence factor for many pathogenic fungal species, including Cryptococcus neoformans. Melanin is deposited in the cell wall, and melanin isolated from this fungus retains the shape of the cells, resulting in hollow spheres called "ghosts". In this study, atomic force, scanning electron, and transmission electron microscopy revealed that melanin ghosts are covered with roughly spherical granular particles approximately 40-130 nm in diameter, and that the melanin is arranged in multiple concentric layers. Nuclear magnetic resonance cryoporometry indicated melanin ghosts contain pores with diameters between 1 and 4 nm, in addition to a small number of pores with diameters near 30 nm. Binding of the antibodies to melanin reduced the apparent measured volume of these pores, suggesting a mechanism for their antifungal effect. We propose a model of cryptococcal melanin structure whereby the melanin granules are held together in layers. This structural model has implications for cell division, cell wall remodeling, and antifungal drug discovery.     

Nanosecond fluorescence microscopy. Emission kinetics of fura-2 in single cells.

A microscope based time-correlated single photon counting instrument has been constructed to measure fluorescence intensity and emission anisotropy decays from fluorophores in single cells on a nanosecond time scale. The sample is excited and the emission collected using epi-illumination optics with frequency-doubled pulses from the cavity-dumped output of a synchronously pumped dye laser serving as an excitation source. Collection of decays from a single cell is possible due to the presence of an iris in the emission path that can be reduced to less than the diameter of a single cell. Using the instrument the decay of 60 nM 1,6-diphenyl-1,3,5-hexatriene was measured, demonstrating that adequate data for lifetime analysis can be recorded from fewer 10(3) molecules of the fluorophore in an illuminated volume of 23 fl. In addition, the intensity and anisotropy decays of fura-2 in single adherent cells and in suspensions of fura-2 loaded cells in suspension, although the relative amplitudes and decay constants vary somewhat from cell to cell. The results indicate that a significant but variable fraction of fura-2 is bound to relatively immobile macromolecular components in these cells.     

Shear‐induced diffusion of red blood cells measured with dynamic light scattering‐optical coherence tomography

Quantitative measurements of intravascular microscopic dynamics, such as absolute blood flow velocity, shear stress and the diffusion coefficient of red blood cells (RBCs), are fundamental in understanding the blood flow behavior within the microcirculation, and for understanding why diffuse correlation spectroscopy (DCS) measurements of blood flow are dominantly sensitive to the diffusive motion of RBCs. Dynamic light scattering‐optical coherence tomography (DLS‐OCT) takes the advantages of using DLS to measure particle flow and diffusion within an OCT resolution‐constrained three‐dimensional volume, enabling the simultaneous measurements of absolute RBC velocity and diffusion coefficient with high spatial resolution. In this work, we applied DLS‐OCT to measure both RBC velocity and the shear‐induced diffusion coefficient within penetrating venules of the somatosensory cortex of anesthetized mice. Blood flow laminar profile measurements indicate a blunted laminar flow profile and the degree of blunting decreases with increasing vessel diameter. The measured shear‐induced diffusion coefficient was proportional to the flow shear rate with a magnitude of ~0.1 to 0.5 × 10^?6 mm². These results provide important experimental support for the recent theoretical explanation for why DCS is dominantly sensitive to RBC diffusive motion.      

A computer-aided measuring system for the characterization of yeast populations combining 2D-image analysis, electronic particle counter, and flow cytometry

An integrated measuring system was developed that directly compares the shape of size distributions of Saccharomyces cerevisiae populations obtained from either microscopic measurements, electronic particle counter, or flow cytometer. Because of its asymmetric mode of growth, a yeast population consists of two different subpopulations, parents and daughters. Although electronic particle counter and flow cytometer represent fast methods to assess the growth state of the population as a whole, the determination of important cell cycle parameters like the fraction of daughters or budded cells requires microscopic observation. We therefore adapted a semiautomatic and interactive 2D-image processing program for rapid and accurate determination of volume distributions of the different sub-populations. The program combines the capacity of image processing and volume calculation by contour-rotation, with the potential of visual evaluation of the cells. High-contrast images from electron micrographs are well suited for image analysis, but the necessary air drying caused the cells to shrink to 35% of their hydrated volume. As an alternative, hydrated cells overstained with the fluorochrome calcofluor and visualized by fluorescence light microscopy were used. Cell volumes calculated from length, and diameter measurements with the assumption of an ellipsoid cell shape were underestimated as compared to volumes derived from 2D-image analysis and contour rotation, because of a deviating cell shape, especially in the older parent cells with more than one bud scar. The bimodal volume distribution obtained from microscopic measurements was identical to the protein distribution measured with the flow cytometer using cells stained with dansylchloride, but differed significantly from the size distribution measured with the electronic particle counter. Compared with the flow cytometer, 2-D image analysis can thus provide accurate distributions with important additional information on, for instance, the distributions of subpopulations like parents, daughters, or budded cells.     

Growth kinetics ofPseudomonas fluorescens microcolonies within the hydrodynamic boundary layers of surface microenvironments

Computer-enhanced microscopy (CEM) was used to study the growth kinetics of bacterial microcolonies attached to the wall of a continuous-flow slide culture. Image processing increased effective microscope resolution and quantitated colony growth at 10 min intervals. Three growth parameters were used to determine growth rate: the time required for cell fission, the specific rate of increase in cell number, and the specific rate of increase in cell area. Growth rate was initially constant regardless of colony size, as assumed previously in deriving colonization kinetics. However, at low substrate concentrations growth rate varied depending on laminar flow velocity. Growth was flow-dependent at a glucose concentration of 100 mg/liter and flow-independent at a concentration of 1 g/liter. This indicated that the surface microenvironment became substrate-depleted in the absence of sufficient laminar flow velocities and that glucose rather than oxygen was rate limiting.     

Enhanced thermal stability and mismatch discrimination of mutation-carrying DNA duplexes and their kinetic and thermodynamic properties in microchannel laminar flow

This article reports the enhancement of thermal stability involving normal duplex and mutation-carrying DNA duplexes in microchannel laminar flow. The application of an in-house temperature-controllable microchannel-type flow cell is demonstrated for improved discrimination of mismatch base pairs such as A-G and T-G that are difficult to distinguish due to the rather small thermal destabilizations. Enhancement in thermal stability is reflected by an increased thermal melting temperature achieved in microchannel laminar flow as compared with batch reactions. To examine the kinetics and thermodynamics of duplex-coil equilibrium of DNA oligomers, denaturation-renaturation hysteresis curves were measured. The influence of microchannel laminar flow on DNA base mismatch analysis was described from the kinetic and thermodynamic perspectives. An increasing trend was observed for association rate constant as flow rate increased. In contrast, an apparent decrease in dissociation rate constant was observed with increasing flow rate. The magnitudes of the activation energies of dissociation were nearly constant for both the batch and microchannel laminar flow systems at all flow rates. In contrast, the magnitudes of activation energies of association decreased as flow rate increased. These results clearly show how microchannel laminar flow induces change in reaction rate by effecting change in activation energy. We anticipate, therefore, that this approach based on microchannel laminar flow system holds great promise for improved mismatch discrimination in DNA analyses, particularly on single-base-pair mismatch, by pronouncedly enhancing thermal stability.     

Characterization of rat bone marrow lymphoid cells. I. A study of the distribution parameters of sedimentation velocity, volume and electrophoretic mobility

Various cell populations in rat bone marrow were characterized by means of a two dimensional separation using velocity sedimentation and free flow electrophoresis and by electrical sizing of the separated cells. Up to 4.5 mm/hr five different populations with discrete distributions in volume (coefficient of variation 10% to 13%) and sedimentation velocity (coefficient of variation 6% to 10%) were observed. Three of the small sized populations represented lymphocytes and small normoblasts and two of the larger sized populations represented myeloid cells. Almost all of these cells were in the G0/G1 cycle phase. In the faster sedimenting fractions which contained immature myeloid, erythroid and undefined blast cells and two S phase populations, discrete volume distributions were not evaluated. The cell populations with homogeneous volume (particularly the small lymphocytes) showed high density variations which condiserably impair the separation resolution. The cells sedimenting slower than 3.5 mm/hr were further separated by means of free flow electrophoresis into three peaks differing in electrophoretic mobility (EPM). The peaks of low and high EPM contained two populations and the peak of medium EPM contained three populations all characterized by normal volume distributions of uniform coefficient of variation between 11% and 14%. The small cells in the peaks of high and medium EPM were normolblasts and the other cells were lymphocytes. The biological significance of these results is discussed.