A dense ring of the trans-Neptunian object Quaoar outside its Roche limit.

Planetary rings are observed not only around giant planets1, but also around small bodies such as the Centaur Chariklo2 and the dwarf planet Haumea3. Up to now, all known dense rings were located close enough to their parent bodies, being inside the Roche limit, where tidal forces prevent material with reasonable densities from aggregating into a satellite. Here we report observations of an inhomogeneous ring around the trans-Neptunian body (50000) Quaoar. This trans-Neptunian object has an estimated radius4 of 555 km and possesses a roughly 80-km satellite5 (Weywot) that orbits at 24 Quaoar radii6,7. The detected ring orbits at 7.4 radii from the central body, which is well outside Quaoar's classical Roche limit, thus indicating that this limit does not always determine where ring material can survive. Our local collisional simulations show that elastic collisions, based on laboratory experiments8, can maintain a ring far away from the body. Moreover, Quaoar's ring orbits close to the 1/3 spin-orbit resonance9 with Quaoar, a property shared by Chariklo's2,10,11 and Haumea's3 rings, suggesting that this resonance plays a key role in ring confinement for small bodies.

The size, shape, density and ring of the dwarf planet Haumea from a stellar occultation.

Haumea-one of the four known trans-Neptunian dwarf planets-is a very elongated and rapidly rotating body. In contrast to other dwarf planets, its size, shape, albedo and density are not well constrained. The Centaur Chariklo was the first body other than a giant planet known to have a ring system, and the Centaur Chiron was later found to possess something similar to Chariklo's rings. Here we report observations from multiple Earth-based observatories of Haumea passing in front of a distant star (a multi-chord stellar occultation). Secondary events observed around the main body of Haumea are consistent with the presence of a ring with an opacity of 0.5, width of 70 kilometres and radius of about 2,287 kilometres. The ring is coplanar with both Haumea's equator and the orbit of its satellite Hi'iaka. The radius of the ring places it close to the 3:1 mean-motion resonance with Haumea's spin period-that is, Haumea rotates three times on its axis in the time that a ring particle completes one revolution. The occultation by the main body provides an instantaneous elliptical projected shape with axes of about 1,704 kilometres and 1,138 kilometres. Combined with rotational light curves, the occultation constrains the three-dimensional orientation of Haumea and its triaxial shape, which is inconsistent with a homogeneous body in hydrostatic equilibrium. Haumea's largest axis is at least 2,322 kilometres, larger than previously thought, implying an upper limit for its density of 1,885 kilograms per cubic metre and a geometric albedo of 0.51, both smaller than previous estimates. In addition, this estimate of the density of Haumea is closer to that of Pluto than are previous estimates, in line with expectations. No global nitrogen- or methane-dominated atmosphere was detected.

Charon's size and an upper limit on its atmosphere from a stellar occultation.

Pluto and its satellite, Charon (discovered in 1978; ref. 1), appear to form a double planet, rather than a hierarchical planet/satellite couple. Charon is about half Pluto's size and about one-eighth its mass. The precise radii of Pluto and Charon have remained uncertain, leading to large uncertainties on their densities. Although stellar occultations by Charon are in principle a powerful way of measuring its size, they are rare, as the satellite subtends less than 0.3 microradians (0.06 arcsec) on the sky. One occultation (in 1980) yielded a lower limit of 600 km for the satellite's radius, which was later refined to 601.5 km (ref. 4). Here we report observations from a multi-station stellar occultation by Charon, which we use to derive a radius, R(C) = 603.6 +/- 1.4 km (1sigma), and a density of rho = 1.71 +/- 0.08 g cm(-3). This occultation also provides upper limits of 110 and 15 (3sigma) nanobar for an atmosphere around Charon, assuming respectively a pure nitrogen or pure methane atmosphere.

Large changes in Pluto's atmosphere as revealed by recent stellar occultations.

Pluto's tenuous nitrogen atmosphere was first detected by the imprint left on the light curve of a star that was occulted by the planet in 1985 (ref. 1), and studied more extensively during a second occultation event in 1988 (refs 2-6). These events are, however, quite rare and Pluto's atmosphere remains poorly understood, as in particular the planet has not yet been visited by a spacecraft. Here we report data from the first occultations by Pluto since 1988. We find that, during the intervening 14 years, there seems to have been a doubling of the atmospheric pressure, a probable seasonal effect on Pluto.

Cell kinetic studies in mouse tongue mucosa by autoradiographic, immunohistochemical and flow cytometric techniques.

A number of techniques, including autoradiography after in vivo administration of tritiated thymidine ([3H]dT), immunohistochemistry after in vivo administration of bromodeoxyuridine (BrdUrd), and flow cytometry (FCM) with and without BrdUrd detection were compared in the epithelium of ventral mouse tongue. Investigation of the diurnal proliferative rhythm by immunohistochemical detection of incorporated BrdUrd with different primary antibodies in combination with the alkaline-phosphatase anti-alkaline-phosphatase technique, the peroxidase-anti-peroxidase method, and an indirect method with a polyclonal peroxidase-conjugated secondary antibody yielded results similar to standard autoradiography. Preparation of single cell suspensions for flow cytometry was not successful. A maximum yield of about 8.5% of the original cell number was achieved by ultrasound disintegration in combination with trypsin and dithioerythrol treatment, but neither a G0/G1 peak nor a G2 + M peak was observed in DNA histograms. A better yield of about 38% of the original nuclei number was obtained by preparation of suspensions of nuclei using citric acid and the detergent Tween 20 in combination with magnetic stirring. Both S-phase index and BrdUrd labelling index could be determined by FCM and showed the normal diurnal variations. However, the BrdUrd labelling index in suspensions of nuclei was significantly higher than the labelling index determined after immunohistochemistry. The FCM S-phase index at times of day with low DNA synthesizing activity was higher than the BrdUrd index, indicating a fraction of unlabelled S-phase cells. In conclusion, detection of incorporated BrdUrd in oral mucosa by immunohistochemical techniques or flow cytometry is feasible and provides a useful tool for fast measurements of proliferation.

Denaturation and condensation of intracellular nucleic acids monitored by fluorescence depolarization of intercalating dyes in individual cells.

The intercalating binding of planar aromatic dye molecules to nucleic acids can be analyzed using fluorescence depolarization measurements of the dye molecules excited by linearly polarized light. In this study, we investigated the conformational changes of the intracellular DNA-dye complex in single cells. Flow cytometry, combined with a newly developed double-beam autocompensation technique, permitted rapid high-precision fluorescence depolarization measurements on a large number of individual cells. The dyes ethidium bromide (EB), propidium iodide (PI), and acridine orange (AO) were used in this study. Depending on the dye-to-phosphate ratio of the nuclear acid-dye complex, as well as on the spatial dye structure itself, internal and external binding sites can be monitored by fluorescence depolarization analysis. Both energy transfer and rotation and vibration of the dye molecules cause depolarization of the fluorescence emission. Differences in the concentration-dependent dye fluorescence depolarization values between PI and EB on one side and AO on the other side can be interpreted as a denaturation and condensation of double-stranded DNA regions by AO. We further show that the fluorescence polarization measurement technique can be used in an alternative way to monitor thermal denaturation of cellular DNA.

Intraphagolysosomal pH in canine and rat alveolar macrophages: flow cytometric measurements.

Intracellular dissolution of inhaled inorganic particles is an important clearance mechanism of the lung and occurs in phagolysosomal vacuoles of phagocytes. Flow cytometric measurements of intraphagolysosomal pH in alveolar macrophages (AM) obtained from beagle dogs, Wistar rats, and from a baboon were made using fluorescein isothiocyanate-labeled amorphous silica particles (FSP). AM were obtained by bronchoalveolar lavage. FSP were phagocytized by AM in cell suspensions incubated in full media for 24 hr up to 6 days. Dual laser flow cytometry was performed and six-parameter list mode data were recorded from forward scatter, side scatter, and fluorescence intensities at 530 nm excited at 457 nm and 488 nm as well as logarithmic fluorescence intensity at wavelengths 630 nm excited at 488 nm. In this way it was possible to discriminate viable AM with phagocytized FSP from lysing AM with phagocytized FSP and from cells without FSP and from free FSP. Viable cells were distinguished from lysing cells by staining with propidium iodide immediately before the flow cytometric measurement. A calibration curve for the pH value was determined from FSP suspended in buffered media at pH values ranging from 3.5 to 7.5. First flow cytometrical results indicated that after an incubation time of 24 hr, the mean intraphagolysosomal pH of viable AM was 4.7 +/- 0.3 for dogs and 5.1 +/- 0.5 for rats. The intraphagolysosomal pH of the baboon AM was 4.5.

An automated flow cytometric micronucleus assay for human lymphocytes.

A new flow cytometric method is presented for scoring micronuclei (MN) in human lymphocytes after in vitro gamma-irradiation. Fifty to fifty-five hours after PHA-stimulation, the frequency of micronuclei per nucleus and the fraction of cells in the second cell cycle were measured using flow cytometry. All data were automatically analysed using our DAS-software package. Eight individual linear-quadratic dose response curves derived from five donors revealed inter- and intra-individual variabilities of all curve parameters. Since also an age dependence was found for spontaneous MN-frequencies and for the linear curve parameter, a combined linear-quadratic age-dose-effect model was used to fit the data. The 90% prediction intervals show that a reliable individual dose estimation for donors aged between 23 and 54 years cannot be achieved for exposures below 1 Gy.

Application of pulsed field gel electrophoresis to determine gamma-ray-induced double-strand breaks in yeast chromosomal molecules.

The frequency of DNA double-strand breaks (dsb) was determined in yeast cells exposed to gamma-rays under anoxic conditions. Genomic DNA of treated cells was separated by pulsed field gel electrophoresis, and two different approaches for the evaluation of the gels were employed: (1) The DNA mass distribution profile obtained by electrophoresis was compared to computed profiles, and the number of DSB per unit length was then derived in terms of a fitting procedure; (2) hybridization of selected chromosomes was performed, and a comparison of the hybridization signals in treated and untreated samples was then used to derive the frequency of dsb. The two assays gave similar results for the frequency of dsb ((1.07 +/- 0.06) x 10(-9) Gy-1 bp-1 and (0.93 +/- 0.09) x 10(-9) Gy-1 bp-1, respectively). The dsb frequency was found to be linearly dependent on dose.

Low mutagenic effects of mitomycin C in undifferentiated embryonic P19 cells are correlated with efficient cell cycle control.

Pluripotent undifferentiated embryonic carcinoma cells of line P19 and their differentiated progeny, epithelioid ectoderm-like EPI-7 cells, showed different responses to mitomycin C (MMC) with respect to induction of micronuclei, mutations at the HPRT-locus and cell cycle control. Cytotoxic effects of MMC after a 5-h treatment were lower in undifferentiated P19 cells than in differentiated EPI-7 cells with IC50 values of 1.3 and 0.25 microM for P19 and EPI-7 cells, respectively. MMC did not induce 6-thioguanine-resistant mutants in P19 cells but significantly increased the mutation frequency in EPI-7 cells with concentrations of 0.25, 0.5 and 1.0 microM MMC. Micronuclei determined by flow-cytometry were induced by MMC in both cell lines at equitoxic concentrations of 4.5 (P19) and 0.75 (EPI-7) microM, reducing the viability in both cell lines to 10%. Whereas the induction of micronuclei in P19 cells was maximal 28 h after treatment and declined thereafter, micronucleus induction peaked 48 h post treatment in EPI-7 cells and remained significantly increased even 67 h after the treatment. Flow-cytometric determination of the distribution of MMC-treated P19 and EPI-7 within the cell cycle revealed a distinct G2/M-block in P19 cells, whereas EPI-7 cells showed normal progression through S-phase and a negligible G2/M-block. Therefore, we conclude that the lower effectivity of MMC to induce gene mutations and micronuclei in P19 cells seemed to be correlated with a more efficient cell cycle control in undifferentiated compared to differentiated EPI-7 cells.

Immunophenotyping of canine bronchoalveolar and peripheral blood lymphocytes.

The immunophenotype of canine lymphocytes obtained by bronchoalveolar lavage (BAL) was investigated and compared with that of peripheral blood leukocytes (PBL). Indirect immunofluorescence, generated by monoclonal antibodies (mAb) specific for canine CD5, CD4, CD8, CD45pan, CD45RA, MHCII and THY-1, was detected by flow cytometry. In comparison with PBL, in BAL there are fewer lymphocytes positive for CD45RA (75.4 +/- 12.6% vs. 42.3 +/- 9.4%; P < 0.05) and MHCI I (97.0 +/- 2.9% vs. 74.0 +/- 7.6%; P < 0.01), while there are more cells positive for CD8 (19.0 +/- 3.6% vs. 29.5 +/- 12.0%; P < 0.05). Thus there is a lower CD4/CD8 ratio in the cell compartment accessible by BAL (2.2 +/- 0.3 vs. 1.3 +/- 0.6; P < 0.005). The immunophenotype may be stable over time, as indicated by reexamination of cells obtained from one dog at four times over 1 year. Investigating the phenotype of lymphocytes from three different locations of the right lung, the cranial lobe lymphocytes show a lower CD4/CD8 ratio in comparison with PBL (1.81 +/- 0.35 vs. 1.12 +/- 0.31, n = 5; P < 0.02). In general, less MHCII positive lymphocytes (P < 0.001) and greater immunophenotype variability of results were found in these separate samples compared with pooled samples from these locations.

Flow cytometric analysis of in vitro micronucleus induction in hepatocytes treated with carcinogens.

The micronucleus test is frequently used in established cell lines to detect genotoxic chemicals. In contrast, there is only very limited experience concerning the applicability of this test in primary cultures of hepatocytes. The induction of micronuclei (MN) by methyl methanesulphonate (2 mm) and by the indirect carcinogens cyclophosphamide (CP, 0.4-4 mm) and diethylnitrosamine (DEN, 1-10 mm) has therefore been studied in rat liver cells in vitro. Analysis and quantification of MN, as well as determination of the proliferative activity of the hepatocytes, was performed by flow cytometric techniques. All three chemicals increased the frequency of MN at incubation times of more than 48 hr. The relative increase in MN compared with that in untreated or solvent-treated cultures, however, was at the most only three-fold, since the frequency of MN increased markedly in the control cultures also. There was a marked decrease in proliferative activity of the hepatocytes, as shown by the decrease in frequency of cells in the second G1-phase at the highest concentration of CP and at all concentrations of DEN. In conclusion, flow cytometric analysis of MN enables a fast and reliable determination of cytogenetic effects in hepatocyte cultures treated with chemicals. However, the large number of MN in untreated hepatocytes, which is possibly a consequence of DNA damage induced by the isolation procedure, may limit the sensitivity of the method.

A new combined integral-light and slit-scan data analysis system (DAS) for flow cytometry.

Flow cytometry using list mode parameters such as fluorescence emission, light scatter and size on one hand and different slit-scan parameters on the other hand needs a fast, flexible, efficient and easy-to-use data analysis software. A new software package (data analysis system, DAS) has been developed that integrates data analysis for conventional (integral-light) flow cytometry and for slit-scan flow cytometry. The requirements, design and some examples are discussed and an implementation for IBM-compatible computers is presented. Special attention is directed to the handling of different data types from one-parameter histograms to multiparameter slit-scan data files. The package can be used as an interpreting programming language or as an interactive menu-driven command line interpreter with a large number of graphic, mathematical and statistical functions. DAS is not limited to use in flow cytometry only, but multidimensional data analysis, from astronomy to economics, can be done as well.

Principles of fluorescence polarization measurements.

Fluorescence polarization measurements using high-speed single-cell flow cytometry have found increasing use in cellular biology. In most flow systems, the detection axis generally is aligned orthogonally to the direction of flow and the excitation axis, and asymmetric apertures along the excitation and emission axes by cylindrical lenses and/or nonplanar transition between optical indices complicate the numerical correction of systematic depolarization effects. In addition, recent studies on fluorescence emission of structured particles have shown remarkable anisotropies of polarized fluorescence emission dependent on the direction of the detection axis.

Epi-illumination optical design for fluorescence polarization measurements in flow systems.

An epi-illumination design for fluorescence polarization measurements is introduced in flow cytometry with the optical axis orthogonally aligned to the cell stream. Various optical components and designs are discussed with respect to their influence on polarization measurements. Using the epi-configuration, paired measurements with the direction of polarization of the exciting light changed orthogonally are proposed for the compensation of system anisotropies and electronic mismatch. Large aperture corrections are employed for the excitation as well as for the emission pathway. Additional parameters such as fluorescence at 90 degrees, multiangle light scattering, and high precision cell-sizing by internally calibrated time of the flight measurements, as described previously, remain available with the design proposed here. Fluorescent latex microspheres, stained intracellular DNA, and algae have been used to test performance.

Double-beam autocompensation for fluorescence polarization measurements in flow cytometry.

The degree of depolarization of fluorescent light emitted from an organic dye, which is used as molecular probe, is a powerful tool in probing the microenvironment. By fluorescence depolarization the macromolecular structure can be investigated as well as the the mobility of the marker molecule itself or of the complex formed by the probe. Additional information such as energy transfer rates, donor-acceptor distances, and orientations are also measurable. These data are of particular interest if they can be measured from whole cells. Using flow cytometry, we can analyze a large number of cells with high statistical significance in a short period of time. We describe a newly developed double-beam epi-illumination arrangement for fluorescence polarization measurements that uses an autocompensation technique. This new technique permits the various depolarizing effects within the optical as well as the electronic components of the system to be continually compensated for on a cell by cell basis. Simultaneous measurements of other cell parameters for cell cycle analysis by total fluorescence intensity remains possible. The sensitivity of the system to measure polarization was determined as +/- 0.006 p (0 less than or equal to p less than or equal to 0.5 in isotropic media), which amounts to +/- 1.2% of the maximum p value. Polarization data for latex microspheres plotted in the histogram mode were measured with a standard deviation of 0.006, which proved the high resolution and the high performance of the system.

Measurement of the kinetics of DNA repair synthesis after uv irradiation using immunochemical staining of incorporated 5-bromo-2'-deoxyuridine and flow cytometry.

The kinetics of unscheduled DNA synthesis in normal human fibroblasts was characterized by flow cytometry utilizing the immunofluorescent detection of 5-bromo-2'-deoxyuridine (BrdUrd) incorporated into cellular DNA during the repair process. Quiescent normal human fibroblasts were irradiated with ultraviolet light and incubated in the presence of BrdUrd during a postirradiation repair period. The amount of unscheduled DNA synthesis was then quantified in the quiescent cells by immunofluorescence staining using monoclonal antibodies against BrdUrd incorporated into the DNA. Significant amounts of unscheduled DNA synthesis were measured after doses as low as 0.1 J/m2 and for time periods as short as 15 min. The initial repair rate was found to be linear with time at all doses tested until repair neared completion. Interestingly, the initial repair rate was constant for doses over the range of 5 to 40 J/m2, whereas the time to completion of repair was dose dependent. These results suggest that above 5 J/m2 in normal human fibroblasts, the repair process is saturated but continues to function until all available regions are repaired. Using this methodology for measuring unscheduled DNA synthesis in combination with second and third flow markers, it is now possible to measure unscheduled DNA synthesis in heterogeneous mixtures of cells.

Micronuclei induced by 2-chlorobenzylidene malonitrile contain single chromosomes as demonstrated by the combined use of flow cytometry and immunofluorescent staining with anti-kinetochore antibodies.

The effects of the tear gas 2-chlorobenzylidene malonitrile (CS) on micronucleus induction and cell cycle kinetics were studied in Chinese hamster and Ehrlich ascites tumour cells using flow cytometric analysis of micronuclei and nuclei in suspension, and indirect immunofluorescent staining of kinetochores in micronuclei. In both cell lines CS induced a concentration-dependent inhibition of the fraction of cells in mitosis as observed by simultaneous flow cytometric measurements of DNA content and side scatter intensities of cell nuclei. Micronucleus frequency increased during the delayed division of cells accumulated by CS in mitosis and reached a plateau when most of these cells have divided. The height of this plateau depended on the CS concentration. Results obtained by flow cytometric analysis of the frequency of CS-induced micronuclei did not agree quantitatively with results obtained by microscopic analysis due to cells showing CS-induced fragmented nuclei. Nearly all CS-induced micronuclei exhibited kinetochores, the majority of which (60-70%) showed one kinetochore per micronucleus implying the presence of a single metaphase chromosome in these micronuclei. DNA distributions of micronuclei measured by flow cytometry showed pronounced peaks corresponding to the DNA distribution of chromosomes measured by flow karyotyping. Even micronuclei containing two of the large chromosomes could be observed as distinct peaks in the distributions. The combined results of flow cytometric analysis of micronucleus distributions and immunofluorescence staining of kinetochores in micronuclei suggest that CS induces micronuclei mainly by damaging the spindle fibres of single chromosomes during mitosis, thus possibly leading to aneuploidy and polyploidy.

Optimizing fluorescent in situ hybridization with rRNA-targeted oligonucleotide probes for flow cytometric identification of microorganisms.

A combination of fluorescent rRNA-targeted oligonucleotide probes ("phylogenetic stains") and flow cytometry was used for a high resolution automated analysis of mixed microbial populations. Fixed cells of bacteria and yeasts were hybridized in suspension with fluorescein- or tetramethylrhodamine-labeled oligonucleotide probes complementary to group-specific regions of the 16S ribosomal RNA (rRNA) molecules. Quantifying probe-conferred cell fluorescence by flow cytometry, we could discriminate between target and nontarget cell populations. We critically examined changes of the hybridization conditions, kinetics of the hybridization, and posthybridization treatments. Intermediate probe concentrations, addition of detergent to the hybridization buffer, and a posthybridization washing step were found to increase the signal to noise ratio. We could demonstrate a linear correlation between growth rate and probe-conferred fluorescence of Escherichia coli and Pseudomonas cepacia cells. Oligonucleotides labeled with multiple fluorochromes showed elevated levels of nonspecific binding and therefore could not be used to lower the detection limits, which still restrict studies with fluorescing rRNA-targeted oligonucleotide probes to well-growing microbial cells. Two probes of different specificities--one labeled with fluorescein, the other with tetramethylrhodamine--could be applied simultaneously for dual color analysis.