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The enormous species diversity of diatoms correlates with the remarkable range of cell sizes in this group. Nuclear DNA content relates fundamentally to cell volume in other eukaryotic cells. The relationship of cell volume to G1 DNA content was determined among selected members of the genus Thalassiosira, one of the most species-rich and well-studied centric diatom genera. Both minimum and maximum species-specific cell volume correlated positively with G1 DNA content. Phylogeny based on 5.8 S and ITS rDNA sequences indicated that multiple changes in G1 DNA content and cell volume occurred in Thalassiosira evolution, leading to a 1,000-fold range in both parameters in the group. Within the Thalassiosira weissflogii (Grunow) G. A. Fryxell et Grunow species complex, G1 DNA content varied 3-fold: differences related to geographic origin and time since isolation; doubling and tripling of G1 DNA content occurred since isolation in certain T. weissflogii isolates; and subcultures of T. weissflogii CCMP 1336 diverged in DNA content by 50% within 7 years of separation. Actin, β-tubulin, and Spo11/TopVIA genes were selected for quantitative PCR estimation of haploid genome size in subclones of selected T. weissflogii isolates because they occur only once in the T. pseudonana Hasle et Heimdal genome. Comparison of haploid genome size estimates with G1 DNA content suggested that the most recent T. weissflogii isolate was diploid, whereas other T. weissflogii isolates appeared to be polyploid and/or aneuploid. Aberrant meiotic and mitotic cell divisions were observed, which might relate to polyploidization. The structural flexibility of diatom genomes has important implications for their evolutionary diversification and stability during laboratory maintenance.

A sheathless flow cytometry system is disclosed wherein a fluid containing particles of interest, for example cells, flows through a sensing region, and is illuminated in the sensing region with one or more light source. Light resulting from the interaction of the particles with the illumination is received by an objective, and focused toward a field stop having an aperture comprising relatively large end portions and a relatively small center portion. Light deflectors, such as prisms, are disposed over the relatively large end portions of the aperture. The system is arranged such that light from particles in focus in the sensing region is focused on the relatively small center portion of the aperture. Peripheral detectors are positioned to receive light from the light deflectors, and a scatter detector is positioned to receive light passing through the center portion. The detector signals may be used to identify which of the detector signals correspond to particles in focus as they passed through the sensing region.

The enormous species diversity of diatoms correlates with the remarkable range of cell sizes in this group. Nuclear DNA content relates fundamentally to cell volume in other eukaryotic cells. The relationship of cell volume to G1 DNA content was determined among selected members of the genus Thalassiosira, one of the most species-rich and well-studied centric diatom genera. Both minimum and maximum species-specific cell volume correlated positively with G1 DNA content. Phylogeny based on 5.8 S and ITS rDNA sequences indicated that multiple changes in G1 DNA content and cell volume occurred in Thalassiosira evolution, leading to a 1,000-fold range in both parameters in the group. Within the Thalassiosira weissflogii (Grunow) G. A. Fryxell et Grunow species complex, G1 DNA content varied 3-fold: differences related to geographic origin and time since isolation; doubling and tripling of G1 DNA content occurred since isolation in certain T. weissflogii isolates; and subcultures of T. weissflogii CCMP 1336 diverged in DNA content by 50% within 7 years of separation. Actin, beta-tubulin, and Spo11/TopVIA genes were selected for quantitative PCR estimation of haploid genome size in subclones of selected T. weissflogii isolates because they occur only once in the T. pseudonana Hasle et Heimdal genome. Comparison of haploid genome size estimates with G1 DNA content suggested that the most recent T. weissflogii isolate was diploid, whereas other T. weissflogii isolates appeared to be polyploid and/or aneuploid. Aberrant meiotic and mitotic cell divisions were observed, which might relate to polyploidization. The structural flexibility of diatom genomes has important implications for their evolutionary diversification and stability during laboratory maintenance.

Dissolved organic matter (DOM) is a major carbon reservoir for the global carbon cycle, and its molecules play a key role in the biogeochemistry of the ocean. Colloidal DOM macromolecules assemble to form polymer hydrogels known as marine microgels. Marine microgels represent one of the most dynamic pools of organic carbon in the ocean. However, their optical characteristics and their contribution to ocean optical properties are largely unknown. In this work, we explore the optical and proteomic properties of spontaneously assembled DOM polymer microgels. Microgels from cultures and from Puget Sound seawater were sorted and counted using a dual-laser (365 nm/365 nm) high-speed cell sorter. This sorter has been adapted to interface with a scanning monochromator to measure the fluorescence emission spectrum of the microgels over the range from 300 to 850 nm. Surprisingly, the microgels show a broad fluorescence emission from 420 to 520 nm when excited with UV light. The microgels were classified according to their blue autofluorescence, and by three criteria that are used to define microgels: 1) staining with chlortetracycline 2) the ability to undergo phase transitions at low pH, and 3) dispersion following calcium chelation by EDTA. © 2007 Elsevier B.V. All rights reserved.

The side population (SP) phenotype has been reported as a method to identify hematopoietic stem cells in the bone marrow based upon differential staining with the fluorescent dye, Hoechst 33342. This technique has drawn great interest in the stem cell community, as it may provide a simple approach to the enrichment of progenitor cells from a variety of normal and malignant tissues. The frequency of these cells and their performance in functional assays has varied considerably within the literature. To investigate mechanisms that may contribute to the SP phenotype, we measured the fluorescence emission of Hoechst-stained bone marrow cells as a function of both time and dye concentration using a custom flow cytometer and data acquisition software. These measurements demonstrate that all nucleated cells within the bone marrow undergo an identical staining pattern at varying rates, even under conditions previously reported to abrogate the SP. Therefore, the SP phenotype is not unique to stem cells, but rather represents a transient feature of marrow cells exposed to Hoechst 33342 for varying amounts of time. We propose that heterogeneity of SP-defined populations may be a consequence of the rate at which differing cell populations accumulate Hoechst 33342. Further, we suggest that dye uptake kinetics will likely be an important factor for optimal use of Hoechst 33342 in isolating stem cells.

Transcriptional noise is known to be an important cause of cellular heterogeneity and phenotypic variation. The extent to which molecular interaction networks may have evolved to either filter or exploit transcriptional noise is a much debated question. The yeast genetic network regulating galactose metabolism involves two proteins, Gal3p and Gal80p, that feed back positively and negatively, respectively, on GAL gene expression. Using kinetic modeling and experimental validation, we demonstrate that these feedback interactions together are important for (i) controlling the cell-to-cell variability of GAL gene expression and (ii) ensuring that cells rapidly switch to an induced state for galactose uptake.

Genetic networks are often affected by stochastic noise, due to the low number of molecules taking part in certain reactions. In complex regulatory networks, noise in any one chemical species may induce noise in the rest of the system. In this paper, we analyse the sources of stochastic noise in the yeast galactose utilization pathway at the level of the complete system, by using both computer simulations, and experimental comparisons between wild-type yeast and a modified strain. A computer model was first used to determine the sources of network noise, as well as mechanisms by which noise is controlled. Results from an estimation tool, confirmed by detailed stochastic simulations, show that noise is caused primarily by fluctuations in mRNA concentrations due to their stochastic creation and decay. The noise is controlled by feedback loops which regulate transcription of certain genes. To test the effect of the feedback loops on the rest of the system, a modified strain of yeast was prepared in which regulation of two key genes is eliminated. As predicted by the model, the mutant strain is induced by galactose in a manner similar to that for the wild-type, but with a higher degree of stochastic noise.

Macrophages play a critical role in both innate and acquired immunity because of their unique ability to internalize, kill, and degrade bacterial pathogens through the process of phagocytosis. The adaptor protein, amphiphysin IIm, participates in phagocytosis and is transiently associated with early phagosomes. Certain pathogens, including Chlamydia pneumoniae, have evolved mechanisms to subvert macrophage phagosome maturation and, thus, are able to survive within these cells. We report here that, although amphiphysin IIm is usually only transiently associated with the phagosome, it is indefinitely retained on vacuoles containing C. pneumoniae. Under these wild-type conditions, C. pneumoniae do not elicit significant nitric oxide (NO) production and are not killed. Abrogation of amphiphysin IIm function results in C. pneumoniae–induced NO production and in the sterilization of the vacuole. The data suggest that C. pneumoniae retains amphiphysin IIm on the vacuole to survive within the macrophage.

High-speed jet-in-air cell sorters analyze and sort thousands of cells a second. The key to the accuracy and purity of their operation is the ability to maintain a stable location on the jet where the stream breaks into individual droplets. The physics of the instability of a capillary jet are well understood and date back to the 19th century. The time it takes for a jet of liquid to break into droplets depends exponentially on the amplitude of perturbations that develop on the surface of the jet. In this work, we describe a method to monitor the amplitude of these perturbations on the surface of a liquid jet using a novel detector. This detector may be combined with feedback mechanisms to actively stabilize the break off point in modern cell sorters. We also show that this detector can accurately monitor the phase of the perturbations with respect to the driving element, and this phase information, in turn, can be used to improve sort rates for high-throughput cell sorters.

The helical cell shape of Helicobacter pylori is highly conserved and contributes to its ability to swim through and colonize the viscous gastric mucus layer. A multi-faceted peptidoglycan (PG) modification programme involving four recently characterized peptidases and two accessory proteins is essential for maintaining H. pylori's helicity. To expedite identification of additional shape-determining genes, we employed flow cytometry with fluorescence-activated cell sorting (FACS) to enrich a transposon library for bacterial cells with altered light scattering profiles that correlate with perturbed cell morphology. After a single round of sorting, 15% of our clones exhibited a stable cell shape defect, reflecting 37-fold enrichment. Sorted clones with straight rod morphology contained insertions in known PG peptidases, as well as an insertion in csd6, which we demonstrated has ld-carboxypeptidase activity and cleaves monomeric tetrapeptides in the PG sacculus, yielding tripeptides. Other mutants had only slight changes in helicity due to insertions in genes encoding MviN/MurJ, a protein possibly involved in initiating PG synthesis, and the hypothetical protein HPG27_782. Our findings demonstrate FACS robustly detects perturbations of bacterial cell shape and identify additional PG peptide modifications associated with helical cell shape in H. pylori.

Background Identification and characterization of the prostate stem cell is important for understanding normal prostate development and carcinogenesis. The flow cytometry-based side population (SP) technique has been developed to isolate putative adult stem cells in several human tissue types including the prostate. This phenotype is mainly mediated by the ATP-binding cassette membrane transporter ABCG2. Methods Immunolocalization of ABCG2 was performed on normal prostate tissue obtained from radical prostatectomies. Normal human prostate SP cells and ABCG2+ cells were isolated and gene expression was determined with DNA array analysis and RT-PCR. Endothelial cells were removed by pre-sorting with CD31. Results ABCG2 positive cells were localized to the prostate basal epithelium and endothelium. ABCG2+ cells in the basal epithelium constituted less than 1% of the total basal cell population. SP cells constituted 0.5–3% of the total epithelial fraction. The SP transcriptome was essentially the same as ABCG2+ and both populations expressed genes indicative of a stem cell phenotype, however, the cells also expressed many genes in common with endothelial cells. Conclusion These results provide gene expression profiles for the prostate SP and ABCG2+ cells that will be critical for studying normal development and carcinogenesis, in particular as related to the cancer stem cell concept.

The innate immune system uses a wide variety of pattern recognition receptors including TLRs, scavenger receptors, and lectins to identify potential pathogens. A carefully regulated balance between activation and inhibition must be kept to avoid detrimental and inappropriate inflammatory responses. In this study, we identify murine-paired Ig-like receptor (PIR)-B, and its human orthologs Ig-like transcript 2 and Ig-like transcript 5 as novel receptors for Staphylococcus aureus. PIR-B contains four ITIM motifs and is thought to be an inhibitory receptor. Expression of these receptors enables NIH3T3 cells to bind S. aureus. In mouse bone marrow-derived macrophages, masking of PIR-B by anti-PIR mAb or genetic deletion of PIR-B shows significantly impaired recognition of S. aureus and enhanced TLR-mediated inflammatory responses to the bacteria. These data suggest a novel mechanism for innate immune regulation by paired Ig-like receptor family members.

Publisher Summary High-speed cell sorters are currently available from a handful of manufacturers with varying specifications, capabilities, facility requirements, and user interfaces. The purchase of any one particular instrument will depend on the desires of individual laboratories and should come after careful consideration of these differences. Successful high-speed cell sorting requires both careful preparation of the sample to be studied and maintenance of the instrument being used. In most cases, the upkeep of electronics, lasers, deflexion plates, software, and other hardware is left to service professionals, while alignment of optics and maintenance of fluidics and associated systems remain the purview of the user. Attaching an empty sample tube and sheath container to the sorter and pressurizing each for a brief period can accomplish air drying of the sample and sheath lines. The tubing and the sheath containers should be stored dry in a clean place. To separate the desired fraction of cells, an electrical charge may be applied to the droplets containing cells of interest as they separate from the main jet. In this manner, droplets with different cell types are directed toward collection vials by a static electrical field.

Background The fluorescence induced by polarized light sources, such as the lasers that are used in flow cytometry, is often polarized and anisotropic. In addition, most optical detector systems are sensitive to the direction of polarization. These two factors influence the accuracy of fluorescence intensity measurements. The intensity of two light sources can be compared only if all details of the direction and degree of polarization are known. In a previous study, we observed that fluorescence polarization might be modified by dye–dye interactions. This report further investigates the role of dye density in fluorescence polarization anisotropy. Methods We measured the polarization distribution of samples stained with commonly used DNA dyes. To determine the role of fluorophore proximity, we compared the monomeric and a dimeric form of the DNA dyes ethidium bromide (EB), thiazole orange (TO), and oxazole yellow (YO). Results In all dyes sampled, fluorescence polarization is less at high dye concentrations than at low concentrations. The monomeric dyes exhibit a higher degree of polarization than the dimeric dyes of the same species. Conclusions The polarization of fluorescence from DNA dyes is related to the density of incorporation into the DNA helix. Energy transfer between molecules that are in close proximity loosens the linkage between the excitation and emission dipoles, thereby reducing the degree of polarization of the emission. © 2004 Wiley-Liss, Inc.

Background Many methods in flow cytometry rely on staining DNA with a fluorescent dye to gauge DNA content. From the relative intensity of the fluorescence signature, one can then infer position in cell cycle, amount of DNA (i.e., for sperm selection), or, as in the case of flow karyotyping, to distinguish individual chromosomes. This work examines the staining of murine thymocytes with a common DNA dye, Hoechst 33342, to investigate nonlinearities in the florescence intensity as well as chromatic shifts. Methods Murine thymocytes were stained with Hoechst 33342 and measured in a flow cytometer at two fluorescence emission bands. In other measurements, cells were stained at different dye concentrations, and then centrifuged. The supernatant was then used for a second round of staining to test the amount of dye uptake. Finally, to test for resonant energy transfer, we measured fluorescence anisotropy at two different wavelengths. Results The fluorescence of cells stained with Hoechst 33342 is a nonlinear process that shows an overall decrease in intensity with increased dye uptake, and spectral shift to the red. Along with the spectral shift of the fluorescence to the longer wavelengths, we document decreases in the fluorescence anisotropy that may indicate resonant energy transfer. Conclusions At low concentrations, Hoechst 33342 binds to the minor groove of DNA and shows an increase in fluorescence and a blue shift upon binding. At higher concentrations, at which the dye molecules can no longer bind without overlapping, the blue fluorescence decreases and the red fluorescence increases until there is approximately one dye molecule per DNA base pair. The ratio of the blue fluorescence to the red fluorescence is an accurate indicator of the cellular dye concentration. © 2004 Wiley-Liss, Inc.

Background High-speed jet-in-air cytometric sorting requires knowledge of the time it takes a particle to travel from the laser to the point where the jet breaks into droplets. Variations in this breakoff time will result in poorer yields and poorer sort purities. Methods This work examined the physical mechanisms that lead to the break up of the jet into droplets and calculated the stability of the droplet breakoff time relative to physical parameters, which govern the behavior of the jet. Results We derived the variations in the breakoff time and found that small variations in the drive frequency, temperature, pressure, and drive amplitude can lead to correspondingly large changes in the breakoff time. We found explicitly that the time it takes the jet to break up is not necessarily correlated with the distance to the breakoff point. Conclusions Many high-speed cell sorters use active means to control the breakoff time. A common method to monitor the breakoff time is to visually monitor the breakoff point. This technique in fact may decrease the sorting purity and efficiency by inadvertently correcting for breakoff time variations. We show explicitly the breakoff time's dependence on a number of physical parameters that can be monitored to increase the stability of the breakoff time. Cytometry Part A 56A:63–70, 2003. © 2003 Wiley-Liss, Inc.

Dissolved organic matter is the largest reservoir of reduced carbon in the ocean and is primarily composed of small biopolymers. It is a critical substrate for the microbial community and plays a pivotal role in global carbon cycling.

A high speed flow cytometric cell sorter was modified to maintain a controlled anaerobic environment. This technology enabled coupling of the precise high-throughput analytical and cell separation capabilities of flow cytometry to the assessment of cell viability of evolved lineages of obligate anaerobic organisms from cocultures.