Boundaries in gravitational and magnetic activation of cells for sorting.

Standard deviations in the distribution of radii of cells and particles are considered to arrive at realistic limits in the use of gravitational and magnetic activation of cells for sorting. Using a specific fractionation design, it is shown that the radius of particles (or cells) may be fractionated down to a precision of +/- 0.76%. Although higher precisions could be obtained with other designs, the number of particles available per fraction is inversely proportional to the precision desired. Thus, one would prefer to keep the precision as moderate as permissible by the experiments.     

Heavy charged particles produce a bystander effect via cell-cell junctions.

Radiation-induced damage to living cells results from either a direct hit to cellular DNA, or from indirect action which leads to DNA damage from radiation produced radicals. However, in recent years there is evidence that biological effects such as cell killing, mutation induction, chromosomal damage and modification of gene expression can occur in a cell population exposed to low doses of alpha particles. In fact these doses are so low that not all cells in the population will be hit directly by the radiation. Using a precision alpha-particle microbeam, it has been recently demonstrated that irradiated target cells can induce a bystander mutagenic response in neighboring "bystander" cells which were not directly hit by alpha particles. Furthermore, these results suggest that gap-junction mediated cell-to-cell communication plays a critical role in this bystander phenomenon. The purpose of this section is to describe recent studies on bystander biological effects. The recent work described here utilized heavy charged particles for irradiation, and investigated the role of gap-junction mediated cell-cell communication in this phenomenon.     

Determination of Mammalian Cell Counts,Cell Size and Cell Health Using the Moxi Z Mini Automated Cell Counter

Particle and cell counting is used for a variety of applications including routine cell culture, hematological analysis, and industrial controls^1-5. A critical breakthrough in cell/particle counting technologies was the development of the Coulter technique by Wallace Coulter over 50 years ago. The technique involves the application of an electric field across a micron-sized aperture and hydrodynamically focusing single particles through the aperture. The resulting occlusion of the aperture by the particles yields a measurable change in electric impedance that can be directly and precisely correlated to cell size/volume. The recognition of the approach as the benchmark in cell/particle counting stems from the extraordinary precision and accuracy of its particle sizing and counts, particularly as compared to manual and imaging based technologies (accuracies on the order of 98% for Coulter counters versus 75-80% for manual and vision-based systems). This can be attributed to the fact that, unlike imaging-based approaches to cell counting, the Coulter Technique makes a true three-dimensional (3-D) measurement of cells/particles which dramatically reduces count interference from debris and clustering by calculating precise volumetric information about the cells/particles. Overall this provides a means for enumerating and sizing cells in a more accurate, less tedious, less time-consuming, and less subjective means than other counting techniques⁶.Despite the prominence of the Coulter technique in cell counting, its widespread use in routine biological studies has been prohibitive due to the cost and size of traditional instruments. Although a less expensive Coulter-based instrument has been produced, it has limitations as compared to its more expensive counterparts in the correction for "coincidence events" in which two or more cells pass through the aperture and are measured simultaneously. Another limitation with existing Coulter technologies is the lack of metrics on the overall health of cell samples. Consequently, additional techniques must often be used in conjunction with Coulter counting to assess cell viability. This extends experimental setup time and cost since the traditional methods of viability assessment require cell staining and/or use of expensive and cumbersome equipment such as a flow cytometer.The Moxi Z mini automated cell counter, described here, is an ultra-small benchtop instrument that combines the accuracy of the Coulter Principle with a thin-film sensor technology to enable precise sizing and counting of particles ranging from 3-25 microns, depending on the cell counting cassette used. The M type cassette can be used to count particles from with average diameters of 4 - 25 microns (dynamic range 2 - 34 microns), and the Type S cassette can be used to count particles with and average diameter of 3 - 20 microns (dynamic range 2 - 26 microns). Since the system uses a volumetric measurement method, the 4-25 microns corresponds to a cell volume range of 34 - 8,180 fL and the 3 - 20 microns corresponds to a cell volume range of 14 - 4200 fL, which is relevant when non-spherical particles are being measured. To perform mammalian cell counts using the Moxi Z, the cells to be counted are first diluted with ORFLO or similar diluent. A cell counting cassette is inserted into the instrument, and the sample is loaded into the port of the cassette. Thousands of cells are pulled, single-file through a "Cell Sensing Zone" (CSZ) in the thin-film membrane over 8-15 seconds. Following the run, the instrument uses proprietary curve-fitting in conjunction with a proprietary software algorithm to provide coincidence event correction along with an assessment of overall culture health by determining the ratio of the number of cells in the population of interest to the total number of particles. The total particle counts include shrunken and broken down dead cells, as well as other debris and contaminants. The results are presented in histogram format with an automatic curve fit, with gates that can be adjusted manually as needed.Ultimately, the Moxi Z enables counting with a precision and accuracy comparable to a Coulter Z2, the current gold standard, while providing additional culture health information. Furthermore it achieves these results in less time, with a smaller footprint, with significantly easier operation and maintenance, and at a fraction of the cost of comparable technologies.     

Quantification of rotavirus-like particles by gel permeation chromatography

There is a lack of accurate and practical methods that require only small amounts of sample for quantifying virus-like particles (VLP). In this work, gel permeation (GP) HPLC was used to quantify double-layered rotavirus-like particles (dlRLP) produced in insect cells. The proposed methodology utilized two columns in series (pore sizes of 200 and 50 nm) and had a high precision (relative standard deviation below 5%). GP-HPLC not only allowed the routine quantification of dlRLP, but also of assembly intermediaries and other viral structures present in the samples. For the first time, kinetics of dlRLP accumulation could be followed. This methodology is valuable for designing new production processes and for optimizing dlRLP monitoring.     

Monosized, magnetic polymer particles: their use in separation of cells and subcellular components, and in the study of lymphocyte function in vitro

By employing the principles of "activated swelling", monosized, superparamagnetic polymer particles have been prepared ranging in size from 1-100 microns. Both during and after the swelling process, the particles can be modified to meet a series of specific demands making them potentially very interesting for many separation and assay purposes. Using monoclonal antibodies to direct the magnetic beads to their targets, immunomagnetic separation has turned out to be one of the most specific, reliable and, above all, the fastest technique available today to isolate particulate material for further studies. So far, most efforts have been concentrated on methodology for fractionation of cells in suspension, such as removal of tumour cells from bone marrow or isolation of lymphoid cells from peripheral blood. These studies have both established the parameters necessary for optimal performance and at the same time laid the groundwork for future developments making immunomagnetic separation an exciting new tool in many research areas. High speed and specificity are the most conspicuous features of immunomagnetic cell separation. These properties have been exploited in the successful development of a new technique for tissue typing of cells directly from peripheral blood specimens. Both higher sensitivity and specificity have been obtained. The same principles can be used for fast and safe quantification of cell populations and subpopulations in blood and cell suspensions. The functions of, and interactions between, peripheral blood cell populations or subpopulations in the immune response have also been studied with high precision. The significance of direct cell contact on the one hand, and soluble factors on the other, can now be established in detail. Immunomagnetic beads have also been used to study the interaction between various T lymphocyte membrane molecules in the early phases of the activation process. Finally, the usefulness of specially developed particles for the fractionation of subcellular components is described.     

Fusion from without directed by human immunodeficiency virus particles.

Fusion from without is the process through which particles of some enveloped viruses can direct fusion of target cells in the absence of viral replication. We demonstrate here that human immunodeficiency virus (HIV) particles can efficiently promote fusion from without. Using HeLa-CD4 cells carrying a Tat-inducible lacZ gene, we observed syncytia as early as 6 h after exposure to HIV particles, before HIV gene expression could be detected. Efficient syncytium formation could be obtained when cells were treated with zidovudine, which prevented HIV replication and expression but not cell-cell fusion. Fusion was also observed when cells were exposed to particles of a replication-defective HIV integrase mutant. Fusion from without by HIV particles could be blocked by a monoclonal antibody specific for the V3 loop of the HIV-1 envelope glycoprotein and by soluble CD4. This mechanism of cytopathicity, which can involve cells that do not actively replicate HIV and can be directed by replication-defective particles, could participate in the pathogenicity of the CD4 cell depletion that characterizes HIV infection.     

Use of the FlowCAM for semi-automated recognition and enumeration of red tide cells (Karenia brevis) in natural plankton samples

Early detection is the most effective way to mitigate the effects of harmful algal blooms (HAB). Cell counts based on examination of microplankton samples using settling chambers and visual inspection with an inverted microscope are tedious and time consuming, and counting precision is generally poor at low cell densities. The FlowCAM is a continuous imaging flow cytometer designed to characterize particles in the microplankton size range (20–200 μm diameter). In this study we examined the ability of the FlowCAM to improve routine monitoring protocols for HAB species by automatically recording information on size and fluorescence per cell. This will eliminate the need to examine cells outside the ranges of these measurements for our target species, Karenia brevis. We also tested the ability of image comparison software to match images of cells in mixed assemblages to images of the target species. For simple mixtures of cultured dinoflagellates, the ability of the image matching software to discriminate target cells varied greatly depending on how similar the two species were in size and shape. When target cells were added to natural plankton samples, the image recognition software correctly identified 80–90% of the target cells, but misidentified 20–50% of non-target cells in the size range of the target species. We conclude that the FlowCAM is less tedious and time-consuming than microscopy, allowing for examination of more cells for greater counting precision. The cell recognition software helps reduce the numbers of cells that must be screened, but images must still be examined by a trained operator to identify the HAB species of interest.     

Sizing-up finite fluorescent particles with nanometer-scale precision by convolution and correlation image analysis

Determining the positions, shapes and sizes of finite living particles such as bacteria, mitochondria or vesicles is of interest in many biological processes. In fluorescence microscopy, algorithms that can simultaneously localize such particles as a function of time and determine the parameters of their shapes and sizes at the nanometer scale are not yet available. Here we develop two such algorithms based on convolution and correlation image analysis that take into account the position, orientation, shape and size of the object being tracked, and we compare the precision of the two algorithms using computer simulations. We show that the precision of both algorithms strongly depends on the objects size. In cases where the diameter of the object is larger than about four to five times the beam waist radius, the convolution algorithm gives a better precision than the correlation algorithm (it leads to more precise parameters), while for smaller object diameters, the correlation algorithm gives superior precision. We apply the convolution algorithm to sequences of confocal laser scanning micrographs of immobile Escherichia coli bacteria, and show that the centroid, the front end, the rear end, the left border and the right border of a bacterium can be determined with a signal-to-noise-dependent precision down to ~5 nm.     

Tracking of fluorescence nanoparticles with nanometre resolution in a biological system: assessing local viscosity and microrheology

The aim of the study was to establish a user-friendly approach for single fluorescence particle 3D localization and tracking with nanometre precision in a standard fluorescence microscope using a point spread function (PSF) approach, and to evaluate validity and precision for different analysis methods and optical conditions with particular application to microcirculatory flow dynamics and cell biology. Images of fluorescent particles were obtained with a standard fluorescence microscope equipped with a piezo positioner for the objective. Whole pattern (WP) comparison with a PSF recorded for the specific set-up and measurement of the outermost ring radius (ORR) were used for analysis. Images of fluorescent particles were recorded over a large range (about $7\,\upmu \text{ m }$ ) of vertical positions, with and without distortion by overlapping particles as well as in the presence of cultured endothelial cells. For a vertical range of $6.5\,\upmu \text{ m }$ , the standard deviation (SD) from the predicted value, indicating validity, was 9.3/8.7 nm (WP/ORR) in the vertical and 8.2/11.7 nm in the horizontal direction. The precision, determined by repeated measurements, was 5.1/3.8 nm in the vertical and 2.9/3.7 nm in the horizontal direction. WP was more robust with respect to underexposure or overlapping images. On the surface of cultured endothelial cells, a layer with 2.5 times increased viscosity and a thickness of about $0.8\,\upmu \text{ m }$ was detected. With a validity in the range of 10 nm and a precision down to about 3–5 nm obtained by standard fluorescent microscopy, the PSF approach offers a valuable tool for a variety of experimental investigations of particle localizations, including the assessment of endothelial cell microenvironment.     

Centrifugal Sedimentation of Virus Particles for Electron Microscopic Counting

Centrifuge cells with conical chambers were provided by using special inserts for the stainless-steel tubes that fit the Spinco SW-39 rotor. Particulate material, centrifuged in these cells, was collected on carbon-coated glass discs. These discs were exposed to OsO(4) vapor, dehydrated in graded alcohols, air-dried, and metal-shadowed. The metal-shadowed carbon film was floated from the glass, mounted on a grid, and examined. A knowledge of cell geometry and microscope magnification allowed correlation of the number of particles observed to a volume of the original suspension. A precision of +/-6% at the 95% confidence level was attained when counting approximately 100 particles per 10,000 x field. Applications and advantages of the method are discussed.     

Gold nanoparticle-protein arrays improve resolution for cryo-electron microscopy

Cryo-electron microscopy single particle analysis shows limited resolution due to poor alignment precision of noisy images taken under low electron exposure. Certain advantages can be obtained by assembling proteins into two-dimensional (2D) arrays since protein particles are locked into repetitive orientation, thus improving alignment precision. We present a labeling method to prepare protein 2D arrays using gold nanoparticles (NPs) interconnecting genetic tag sites on proteins. As an example, mycobacterium tuberculosis 20S proteasomes tagged with 6x-histidine were assembled into 2D arrays using 3.9-nm Au NPs functionalized with nickel-nitrilotriacetic acid. The averaged top-view images from the array particles showed higher resolution (by 6-8A) compared to analysis of single particles. The correct 7-fold symmetry was also evident by using array particles whereas it was not clear by analysis of a comparable number of single particles. The applicability of this labeling method for three-dimensional reconstruction of biological macromolecules is discussed.     

Precise particle tracking against a complicated background: polynomial fitting with Gaussian weight

We present a new particle tracking software algorithm designed to accurately track the motion of low-contrast particles against a background with large variations in light levels. The method is based on a polynomial fit of the intensity around each feature point, weighted by a Gaussian function of the distance from the centre, and is especially suitable for tracking endogeneous particles in the cell, imaged with bright field, phase contrast or fluorescence optical microscopy. Furthermore, the method can simultaneously track particles of all different sizes, and allows significant freedom in their shape. The algorithm is evaluated using the quantitative measures of accuracy and precision of previous authors, using simulated images at variable signal-to-noise ratios. To these we add new tests: the error due to a non-uniform background, and the error due to two particles approaching each other. Finally the tracking of particles in real cell images is demonstrated. The method is made freely available for non-commercial use as a software package with a graphical user-interface, which can be run within the Matlab programming environment.     

The Use of Carbon and Carmine Particles in Cell Fusion Experiments to Distinguish Hybrids from Parental Cells

The phagocytosis of inert particles, a long-known in vivo phenomenon among cells of the reticuloendothelial system, has more recently been found to be a widespread capability of cells in vitro (Gropp 1963) and can be utilized as a marking system when colored particles are employed. Carbon particles (black) were used by Stoker (1964) as cellular markers and later carmine particles (red) were used as markers in cell transformation studies (Stoker 1967; Rabinowitz and Sachs 1968).     

Efficient insertion from an internal long terminal repeat (LTR)-LTR sequence on a reticuloendotheliosis virus vector is imprecise and cell specific.

To examine the fidelity and efficiency of integration from a covalently closed long terminal repeat (LTR)-LTR sequence in vivo, we isolated individual spleen necrosis virus proviruses that arose following infection of chicken embryo fibroblasts (CEFs) and sequenced the provirus-cell DNA junctions. Some but not all CEF preparations allowed efficient insertion from the internal sequence. Moreover, in contrast to integration from the normal ends of the viral DNA, which occurs with precision with respect to the viral DNA, insertion from the internal sequence was not precise. In particular, there were short deletions of variable size from the viral DNA and these proviruses were not flanked by short direct repeats. Although this imprecise insertion can be efficient in CEFs, such integration is very inefficient in two other cell types (D17 and QT47) that support the replication of reticuloendotheliosis viruses. Thus, it is possible that there is a cell-specific factor(s) in CEFs required for efficient but imprecise insertion or, alternatively, D17 and QT47 cells contain a factor that abrogates integration from an internal LTR-LTR junction. Virus particles released from CEFs do not efficiently use the LTR-LTR junction following infection of D17 cells. Therefore, if there is a CEF-specific factor required for insertion, it does not appear to be transferred through particles.     

Effect of Host Cell Wall Material on the Adsorbability of Cofactor-requiring T4

The adsorbability of T4 on host cells was determined as a function of time after their liberation from infected cells. Freshly liberated (nascent) particles are readily adsorbed but lose their adsorbability with a half-time of about 2 days at 5 C, but only about 20 min at 37 C. They can be made adsorbable again with an alpha-amino acid cofactor like l-tryptophan, and this state of adsorbability can be stabilized by cell wall material from Escherichia coli. Such stabilized particles lose their adsorbability at a rate similar to that at which nascent particles lose theirs. Most freshly liberated particles are observed by means of electron microscopy to have "debris" attached to their baseplates and to have most of their six, long tail fibers free, whereas "old" particles that have lost their adsorbability appear relatively "clean" with most of their tail fibers wrapped around their sheaths. Nascent particles have densities that are lower than those of old particles. The material responsible for nascent adsorbability seems to be a fragment of the host's cell wall, for nascent adsorbability is destroyed by lysozyme. Furthermore, nascent T4 particles liberated from host cells with radioactively labeled walls carry the label in density gradients but lose it as they lose adsorbability. In addition, only a small proportion of particles liberated from infected spheroplasts are nascently adsorbable, whereas most particles liberated from intact cells are adsorbable.     

The Use of Carbon and Carmine Particles in Cell Fusion Experiments to Distinguish Hybrids from Parental Cells

The phagocytosis of inert particles, a long-known in vivo phenomenon among cells of the reticuloendothelial system, has more recently been found to be a widespread capability of cells in vitro (Gropp 1963) and can be utilized as a marking system when colored particles are employed. Carbon particles (black) were used by Stoker (1964) as cellular markers and later carmine particles (red) were used as markers in cell transformation studies (Stoker 1967; Rabinowitz and Sachs 1968).     

Cytopathology of satellite tobacco mosaic virus and its helper virus in tobacco

Ultrastructural responses of tobacco cells infected with a newly discovered satellite virus (STMV) that has an isometric morphology and is associated with rigid rodshaped tobacco mosaic virus (TMV) were studied in situ. In cells infected with TMV alone,TMV particles occurred as crystalline arrays in the cytoplasm and were usually associated with TMV-characteristic X bodies. In cells infected with both TMV and STMV, particles of STMV occurred only in cells that contained TMV particles, which suggests a correlation between the satellite and helper virus presence. However, the replication and/or accumulation sites of STMV appear to be independent from its helper virus. Unlike TMV particles, STMV particles were associated with several cytopathic structures such as granular inclusions, membranous vesicles of 50–80 nm, and myelin-like bodies which were all bounded by a single common membrane, No X bodies occurred in cells containing STMV particles, and the mitochondria possessed abnormal tubular structures containing flocculent material.     

Precise nanometer localization analysis for individual fluorescent probes

Calculation of the centroid of the images of individual fluorescent particles and molecules allows localization and tracking in light microscopes to a precision about an order of magnitude greater than the microscope resolution. The factors that limit the precision of these techniques are examined and a simple equation derived that describes the precision of localization over a wide range of conditions. In addition, a localization algorithm motivated from least-squares fitting theory is constructed and tested both on image stacks of 30-nm fluorescent beads and on computer-generated images (Monte Carlo simulations). Results from the algorithm show good agreement with the derived precision equation for both the simulations and actual images. The availability of a simple equation to describe localization precision helps investigators both in assessing the quality of an experimental apparatus and in directing attention to the factors that limit further improvement. The precision of localization scales as the inverse square root of the number of photons in the spot for the shot noise limited case and as the inverse of the number of photons for the background noise limited case. The optimal image magnification depends on the expected number of photons and background noise, but, for most cases of interest, the pixel size should be about equal to the standard deviation of the point spread function.     

Transfer of murine intracisternal A particle phenotype in chloramphenicol-resistant cytoplasts

Murine intracisternal A particles have a number of properties which are common to known RNA tumor viruses, but horizontal transmission has not been previously demonstrated. The apparent absence of infectivity may be related to the failure of these particles to be released from cisternae of endoplasmic reticulum. Previous biological studies using isolated, purified A particles have been compromised by the fact that the isolation procedure requires small amounts of nonionic detergent.Using some techniques of somatic cell hybridization, we have assessed the capacity for A particle genome transfer from positive to negative cells. Since it has been previously shown that some chloramphenicol-resistant cell lines can transfer this resistance in the cytoplasm, we have used this characteristic as a marker for cytoplasmic fragments. Mouse cells containing A particles were mutagenized, and clones resistant to chloramphenicol were selected; by enucleating these cells and fusing the resultant cytoplasts to each of two recipient mouse cell lines negative for A particles, it is possible to identify clones of cells known to be the product of a fusion event between a cytoplast and a whole cell (cybrids). Under these conditions, intracisternal A particles appear in the cybrid clones as a phenotypic trait that has not been segregated over at least 60–80 cell generations.     

Tracking of single fluorescent particles in three dimensions: use of cylindrical optics to encode particle position.

We present a novel optical technique for three-dimensional tracking of single fluorescent particles using a modified epifluorescence microscope containing a weak cylindrical lens in the detection optics and a microstepper-controlled fine focus. Images of small, fluorescent particles were circular in focus but ellipsoidal above and below focus; the major axis of the ellipsoid shifted by 90 degrees in going through focus. Particle z position was determined from the image shape and orientation by applying a peak detection algorithm to image projections along the x and y axes; x, y position was determined from the centroid of the particle image. Typical spatial resolution was 12 nm along the optical axis and 5 nm in the image plane with a maximum sampling rate of 3-4 Hz. The method was applied to track fluorescent particles in artificial solutions and living cells. In a solution of viscosity 30 cP, the mean squared distance (MSD) traveled by a 264 nm diameter rhodamine-labeled bead was linear with time to 20 s. The measured diffusion coefficient, 0.0558 +/- 0.001 micron2/s (SE, n = 4), agreed with the theoretical value of 0.0556 micron2/s. Statistical variability of MSD curves for a freely diffusing bead was in quantitative agreement with Monte Carlo simulations of three-dimensional random walks. In a porous glass matrix, the MSD data was curvilinear and showed reduced bead diffusion. In cytoplasm of Swiss 3T3 fibroblasts, bead diffusion was restricted. The water permeability in individual Chinese Hamster Ovary cells was measured from the z movement of a fluorescent bead fixed at the cell surface in response osmotic gradients; water permeability was increased by > threefold in cells expressing CHIP28 water channels. The simplicity and precision of this tracking method may be useful to quantify the complex trajectories of fluorescent particles in living cells.