Quantification of cellular properties from external fields and resulting induced velocity: magnetic susceptibility.

An experimental technique is discussed in which the magnetic susceptibility of immunomagnetically labeled cells can be determined on a cell-by-cell basis. This technique is based on determining the magnetically induced velocity that an immunomagnetically labeled cell has in a well-defined magnetic energy gradient. This velocity is determined through the use of video recordings of microscopic images of cells moving in the magnetic energy gradient. These video images are then computer digitized and processed using a computer algorithm, cell tracking velocimetry, which allows larger numbers (>10(3)) of cells to be analyzed.     

虚拟细胞模型研究进展

虚拟细胞是20世纪末在国外兴起的一种利用现代信息技术和计算机模拟进行细胞研究的全新手段。主要是通过计算机建立人工细胞模型,模拟细胞内外环境,进行生物学的研究和探索。综述了国外主要的虚拟细胞模型的研究概况。     

A simple apparatus for measuring cell settling velocity

Accurate cell settling velocity determination is critical for perfusion culture using a gravity settler for cell retention. We have developed a simple apparatus (a "settling column") for measuring settling velocity and have validated the procedure with 15-μm polystyrene particles with known physical properties. The measured settling velocity of the polystyrene particles is within 4% of the value obtained using the traditional Stokes' law approach. The settling velocities of three hybridoma cell lines were measured, resulting in up to twofold variation among cell lines, and the values decreased as the cell culture aged. The settling velocities of the nonviable cells were 33-50% less than the corresponding viable cells. The significant variation of settling velocities among cell populations and growth phases confirms the necessity of routine measurement of this property during long-term perfusion culture.     

Determining particle density distribution of expanded bed adsorbents

This study presents an experimental approach to measure the density distribution of expanded bed adsorption (EBA) matrices. We report on the use of a series of solutions of caesium trifluoroacetate (CsTFA) of varying density spun in a laboratory centrifuge so as to separate representative matrix samples on the basis of bead density. Mass data was used to plot a decumulative density distribution for the matrix. By performing laser light scattering-based measurements on the same samples of matrix the variation in particle size with density was determined. Particle settling velocity distributions were then calculated using these data and compared with a settling velocity distribution calculated on the basis of an assumed constant bead density. The study demonstrates a reliable and simple method for the characterisation of matrix density distribution. For the case of the Streamline matrices tested the particle size distribution is constant with varying bead density. Bead densities varied from 1.5 to 2.1 g/cm3 in the CsTFA solutions. These were then adjusted using bead porosity to give a density range of 1.11-1.33 g/cm3 in aqueous buffer (assumed 1.0 g/cm3) The differences in resultant settling velocity distributions when based upon measured density distribution than when based upon an assumed mean density value were shown to be insignificant. This result confirms experimentally that an assumption of a single constant mean density for EBA particles is acceptable for hydrodynamic modelling and performance prediction purposes.     

Effect of accounting for interfractional CTV shape variations in PTV margins on prostate cancer radiation treatment plans

PurposeThe aim of this study was to account for interfractional clinical target volume (CTV) shape variation and apply this to the planning target volume (PTV) margin for prostate cancer radiation treatment plans.MethodsInterfractional CTV shape variations were estimated from weekly cone-beam computed tomography (CBCT) images using statistical point distribution models. The interfractional CTV shape variation was taken into account in the van Herk’s margin formula. The PTV margins without and with the CTV shape variation, i.e., standard (PTV_ori) and new (PTV_shape) margins, were applied to 10 clinical cases that had weekly CBCT images acquired during their treatment sessions. Each patient was replanned for low-, intermediate-, and high-risk CTVs, using both margins. The dose indices (D98 and V70) of treatment plans with the two margins were compared on weekly pseudo-planning computed tomography (PCT) images, which were defined as PCT images registered using a deformable image registration technique with weekly CBCT images, including contours of the CTV, rectum, and bladder.ResultsThe percentage of treatment fractions of patients who received CTV D98 greater than 95% of a prescribed dose increased from 80.3 (PTV_ori) to 81.8% (PTV_shape) for low-risk CTVs, 78.8 (PTV_ori) to 87.9% (PTV_shape) for intermediate-risk CTVs, and 80.3 (PTV_ori) to 87.9% (PTV_shape) for high-risk CTVs. In most cases, the dose indices of the rectum and bladder were acceptable in clinical practice.ConclusionThe results of this study suggest that interfractional CTV shape variations should be taken into account when determining PTV margins to increase CTV coverages.     

Erythrocyte membrane alterations induced by Plasmodium simium infection in Saimiri sciureus: relation to Schüffner's dots.

The nature of erythrocyte membrane alterations in Plasmodium simium infections was determined employing light microscopy, carbon replication and transmission electron microscopy. Light microscopy of Giemsa stained preparations shows that infected cells initially acquire a faint stippling (schuffnerization) which becomes pronouced with subsequent parasite development. Enlargement of the host cell usually accompanied stippling. Both phenomena appear to depend on host cell age since infected mature erythrocytes were neither stippled nor enlarged. Carbon replicas show numerous indentations over the outer membrane surface of most infected cells. Their distribution suggests that they account for Schuffner's granules. The surface indentations are manifest as small infundibular which open to the infected cell's surface. Cytoplasmic microvesciles in the infected cell's stroma frequently are observed adjacent or catenated to the surface infundibula. Images suggest their funsion with the surface infundibula thus adding membrane to the cell's surface and accounting for host cell enlargement.     

Quantification of both the presence, and oxidation state, of Mn in Bacillus atrophaeus spores and its imparting of magnetic susceptibility to the spores

Bacillus atrophaeus spores were previously reported to have significant magnetic susceptibility in a magnetic field due to the presence of Mn. However, relatively little is known about the total amount and distribution of the oxidation state of Mn associated with this specific strain's spores. Using the instrument, cell tracking velocimetry (CTV) both magnetically induced velocity and settling velocity was quantitatively measured. Visual observations, and calculated diameter using previously reported densities, indicate that the spores are present in the form of clusters of approximately 3–6 µm. Treatment of these clusters with EDTA or pH of 2.0 or below resulted in not only the disruption of the spore clusters, but also a significant decrease in magnetic susceptibility, in some cases by almost two orders of magnitude. Since the magnetic susceptibility of Mn varies significantly between the three typically reported valance states of Mn, Mn(II), Mn(III), and Mn(IV); X‐Ray Photoelectron Spectroscopy, XPS, was used to determined the valance states of Mn in the spores. This XPS analysis, which penetrates up to 10 nm into the spore, returned the following fractions: 0.41, 0.38, and 0.21 for the valance states: Mn(II), Mn(III), and Mn(IV), respectively. The total mass of Mn associated with each spore cluster was determined by ICP‐MS. A second, completely independent estimate of Mn mass associated with each spore cluster was made, by mathematically solving for the amount of Mn per spore cluster using the experimentally measured magnetophoretic mobility and the magnetic susceptibility of each of the three valence states from the XPS analysis. IPC‐MS returned a value of 3.28 × 10⁻¹¹ g of Mn per spore cluster while the calculated estimation from mobility and XPS analysis retuned a value of 1.16 × 10⁻¹¹ g, which given the complexity of the two techniques, is a reasonable agreement. Finally, a discussion of potential applications of the magnetic properties of these spores is presented. Bioeng. 2011; 108:1119–1129. © 2010 Wiley Periodicals, Inc.     

Functional analysis of spontaneous cell movement under different physiological conditions

Cells can show not only spontaneous movement but also tactic responses to environmental signals. Since the former can be regarded as the basis to realize the latter, playing essential roles in various cellular functions, it is important to investigate spontaneous movement quantitatively at different physiological conditions in relation to a cell's physiological functions. For that purpose, we observed a series of spontaneous movements by Dictyostelium cells at different developmental periods by using a single cell tracking system. Using statistical analysis of these traced data, we found that cells showed complex dynamics with anomalous diffusion and that their velocity distribution had power-law tails in all conditions. Furthermore, as development proceeded, average velocity and persistency of the movement increased and as too did the exponential behavior in the velocity distribution. Based on these results, we succeeded in applying a generalized Langevin model to the experimental data. With this model, we discuss the relation of spontaneous cell movement to cellular physiological function and its relevance to behavioral strategies for cell survival.     

The fine structure of secretion in Hyalophysa chattoni: formation of the attachment peduncle and the chitinous phoretic cyst wall.

The settling tomite stage of the apostome Hyalophysa chattoni secretes a phoretic cyst wall composed of chitin, mucopolysaccharides, and protein. Within 1 1/2 h after settling, an electron-dense proteinaceous cyst layer (the outer layer) is formed from secretions originating at the base of the kineties and from the thick pellicular layer between the kineties. The inner cyst layer, composed primarily of chitin (acidic and neutral polysaccharides are also present), is secreted across the entire cell surface. Cyst wall formation is completed within 6 h. The fine structure of endocyst secretion resembles stages in the secretion of chitin by fungi, yeasts, and arthropods. A proteinaceous attachment peduncle is secreted to anchor the cell to a shrimp host and is formed by the release of electrondense dense secretory bodies from the cell's ventral surface.     

An information-theory model of cellular survival, mutation and transformation and the relevance of this model to chemical carcinogenesis

A model of cellular survival, mutation and transformation is presented in accordance with information theory. A cellular system is considered to be stable with respect to its environment when the vital information the cell expresses at least equals the information requirements of the environment. Environmental agents, such as mutagens, perturb the cell's expression of information such that an imbalance occurs between the cell's information requirement and the cell's ability to express vital information. This imbalance, which is interpreted as the intrinsic entropy of the cell, serves as a measure of biological cell death. If the cell compensates for the altered ability to express information by adapting to a less restricted set of information requirements, then one may view the cell as having undergone a "transformation" to a less restricted phenotype. This paper will elucidate the mathematical inter-relationships of cellular survival, mutation and transformation and will relate these mathematical concepts to chemical carcinogenesis.     

Comparison of OPS imaging and conventional capillary microscopy to study the human microcirculation.

Orthogonal polarization spectral (OPS) imaging is a new clinical technique for observation of the microcirculation of organ surfaces. For validation purposes, we compared OPS images of the nailfold skin with those obtained from conventional capillary microscopy at rest and during venous occlusion in 10 male volunteers. These images were computer analyzed to provide red blood cell velocity and capillary diameters of the same nailfold capillaries at rest and during venous occlusion. Results showed that OPS images provided similar values for red blood cell velocity and capillary diameter as those obtained from capillary microscopy images. OPS imaging, however, provided significantly better image quality, as shown by comparison of image contrast between OPS imaging and capillary microscopy. This made image analysis better and easier to perform. It is anticipated, therefore, that OPS imaging will become a new and powerful technique in the study of the human microcirculation in vivo because it can be used on human internal organs.     

Mapping the cellular distribution of labelled molecules by SIMS microscopy.

We took advantage of one of the main possibilities of ion microscopy, ie isotopic analysis, to study the cellular distribution of molecules labelled either with carbon 14 or with stable isotopes of low natural abundance such as nitrogen 15 and deuterium. The surface of the sample is bombarded with an ion beam (O2+, Cs+ etc). Secondary ions emitted from the sample are filtered by a mass spectrometer and the distribution of the labelling isotope is recorded. In this way, we obtained images showing the characteristic distribution of 14C-thymidine and D-arginine in human fibroblasts, and of 15N-adenine in organotypic cultures of human breast cancer cells. The spatial resolution on the acquired images was close to 0.1 micron when using the UPS-ONERA ion microprobe. The sensitivity of the method for detecting carbon 14 is far greater than that of autoradiography and the technique is both fast and quantitative. On the other hand, the capacity of ion microscopy for studying the tissular distribution of molecules labelled with stable isotopes, opens the way for biological and pharmacological tracer studies of human diseases.     

Magnetophoretic mobilities correlate to antibody binding capacities

METHODS: A methodology and a mathematical theory have been developed, which allow quantitation of the expression levels of cellular surface antigens using immunomagnetic labels and cell tracking velocimetry (CTV) technology. RESULTS: Quantum Simply Cellular (QSC) microbeads were immunomagnetically labeled with anti-CD2 fluorescein isothiocyanate (FITC) antibodies and anti-FITC MACS paramagnetic nanoparticles. Magnetophoretic mobility has been defined as the magnetically induced velocity of the labeled cell or microbead divided by the magnetophoretic driving force, proportional to the magnetic energy density gradient. DISCUSSION: Using computer imaging and processing technology, the mobility measurements were accomplished by microscopically recording and calculating the velocity of immunomagnetically labeled QSC microbeads in a nearly constant magnetic energy gradient. A calibration curve correlating the measured magnetophoretic mobility of the immunomagnetically labeled microbeads to their antibody binding capacities (ABC) has been obtained. CONCLUSION: The results, in agreement with theory, indicate a linear relationship between magnetophoretic mobility and ABC for microbeads with less than 30,000 ABC. The mathematical relationships and QSC standardization curve obtained allow determination of the number of surface antigens on similarly immunomagnetically labeled cells.     

Phototropic Curvature in Phycomyces

The distribution of curvature and of bending speed along the cell's growing region are studied during steady state phototropic bending. At the start, elemental bending speed parallels the known axial distribution of growth rate. Hence regional phototropic sensitivity is initially determined by the local growth rate, and unilateral visible light acts proportionally at all levels of the growth zone. In the later course of bending, the bending speed distribution shifts downward instead of progressing upward in step with the cell's elongation. Furthermore, during phototropic inversion reversed bending begins high in the growth zone and progresses downward while normal bending continues below. These spatial and temporal changes in the distribution of differential growth are considered to be due to a fixed rate of supply of material used in growth that is transported from lower regions of the cell and asymmetrically distributed within the growth zone.     

The use of magnetite-doped polymeric microspheres in calibrating cell tracking velocimetry

Continuous magnetic separation, in which there is no accumulation of mass in the system, is an inherently dynamic process, requiring advanced knowledge of the separable species for optimal instrument operation. By determining cell magnetization in a well-defined field, we may predict the cell trajectory behavior in the well-characterized field environments of our continuous separators. Magnetization is determined by tracking the migration of particles with a technique known as cell tracking velocimetry (CTV). The validation of CTV requires calibration against an external standard. Furthermore, such a standard, devoid of the variations and instabilities of biological systems, is needed to reference the method against day-to-day shifts or trends. To this end, a method of synthesizing monodisperse, magnetite-doped polymeric microspheres has been developed. Five sets of microspheres differing in their content of magnetite, and each of approximately 2.7 microm diameter, are investigated. An average gradient of 0.18 T/mm induces magnetic microsphere velocities ranging from 0.45 to 420 microns/s in the CTV device. The velocities enable calculation of the microsphere magnetization. Magnetometer measurements permit the determination of magnetization at a flux density comparable to that of the CTV magnet's analysis region, 1.57 T. A comparison of the results of the CTV and magnetometer measurements shows good agreement.     

Computational and experimental investigation of flow and particle settling in a roller bottle bioreactor

It is shown that cell settling is a key factor affecting the performance of roller bottle bioreactors. The two-dimensional cross-sectional flow at the center of a roller bottle is simulated using a finite difference method, and the settling behavior of cells is simulated using particle dynamics algorithms and validated experimentally using fluorescent particles. The settling behavior of particles in the roller bottle flow is studied using both steady and time dependent rotation rates. Under steady flow conditions the flow is divided into two regions: one where the particles settle to the wall and one where the particles remain suspended indefinitely. The relative size of these two regions depends on the ratio of the settling velocity to the rotation rate of the bottle. For unsteady flows generated by periodic changes of the bottle rotation direction, the settling of cells is accelerated significantly, leading to complete deposition in just a few turns of the bottle.     

Three-dimensional elemental mapping of phosphorus by quantitative electron spectroscopic tomography (QuEST)

We describe the development of quantitative electron spectroscopic tomography (QuEST), which provides 3-D distributions of elements on a nanometer scale. Specifically, it is shown that QuEST can be applied to map the distribution of phosphorus in unstained sections of embedded cells. A series of 2-D elemental maps is derived from images recorded in the energy filtering transmission electron microscope for a range of specimen tilt angles. A quantitative 3-D elemental distribution is then reconstructed from the elemental tilt series. To obtain accurate quantitative elemental distributions it is necessary to correct for plural inelastic scattering at the phosphorus L(2,3) edge, which is achieved by acquiring unfiltered and zero-loss images at each tilt angle. The data are acquired automatically using a cross correlation technique to correct for specimen drift and focus change between successive tilt angles. An algorithm based on the simultaneous iterative reconstruction technique (SIRT) is implemented to obtain quantitative information about the number of phosphorus atoms associated with each voxel in the reconstructed volume. We assess the accuracy of QuEST by determining the phosphorus content of ribosomes in a eukaryotic cell, and then apply it to estimate the density of nucleic acid in chromatin of the cell's nucleus. From our experimental data, we estimate that the sensitivity for detecting phosphorus is 20 atoms in a 2.7 nm-sized voxel.     

Possible role of contact following in the generation of coherent motion of Dictyostelium cells

After aggregation by chemotaxis, cells of the cellular slime mold Dictyostelium discoideum form a multicellular structure and show coherent motion such as vortices. Here, we present a mathematical model to explain both aggregation and coherent motion of cells in two-dimensional space. The model incorporates chemotactic response of cells and the cell's property, called "contact following", to follow the other cells with which they are in contact. Analytical study and computer simulation using the model show that with contact following, cells form circular clusters within which cell rotation occurs. Unidirectional cell motion in a long belt of cells is another type of solution of the model. Besides, contact following has an effect to accelerate cell cluster merging. By considering the mechanism of cell movement, possible explanations of contact following are proposed.     

Experimental Measurement of Settling Velocity of Spherical Particles in Unconfined and Confined Surfactant-based Shear Thinning Viscoelastic Fluids

An experimental study is performed to measure the terminal settling velocities of spherical particles in surfactant based shear thinning viscoelastic (VES) fluids. The measurements are made for particles settling in unbounded fluids and fluids between parallel walls. VES fluids over a wide range of rheological properties are prepared and rheologically characterized. The rheological characterization involves steady shear-viscosity and dynamic oscillatory-shear measurements to quantify the viscous and elastic properties respectively. The settling velocities under unbounded conditions are measured in beakers having diameters at least 25x the diameter of particles. For measuring settling velocities between parallel walls, two experimental cells with different wall spacing are constructed. Spherical particles of varying sizes are gently dropped in the fluids and allowed to settle. The process is recorded with a high resolution video camera and the trajectory of the particle is recorded using image analysis software. Terminal settling velocities are calculated from the data.The impact of elasticity on settling velocity in unbounded fluids is quantified by comparing the experimental settling velocity to the settling velocity calculated by the inelastic drag predictions of Renaud et al.¹ Results show that elasticity of fluids can increase or decrease the settling velocity. The magnitude of reduction/increase is a function of the rheological properties of the fluids and properties of particles. Confining walls are observed to cause a retardation effect on settling and the retardation is measured in terms of wall factors.     

Deconvolution of the cellular force-generating subsystems that govern cytokinesis furrow ingression

Cytokinesis occurs through the coordinated action of several biochemically-mediated stresses acting on the cytoskeleton. Here, we develop a computational model of cellular mechanics, and using a large number of experimentally measured biophysical parameters, we simulate cell division under a number of different scenarios. We demonstrate that traction-mediated protrusive forces or contractile forces due to myosin II are sufficient to initiate furrow ingression. Furthermore, we show that passive forces due to the cell's cortical tension and surface curvature allow the furrow to complete ingression. We compare quantitatively the furrow thinning trajectories obtained from simulation with those observed experimentally in both wild-type and myosin II null Dictyostelium cells. Our simulations highlight the relative contributions of different biomechanical subsystems to cell shape progression during cell division.