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Sample records for monitoring single-cell activities

  1. A single-cell correlative nanoelectromechanosensing approach to detect cancerous transformation: monitoring the function of F-actin microfilaments in the modulation of the ion channel activity

    NASA Astrophysics Data System (ADS)

    AbdolahadThe Authors With Same Contributions., Mohammad; Saeidi, Ali; Janmaleki, Mohsen; Mashinchian, Omid; Taghinejad, Mohammad; Taghinejad, Hossein; Azimi, Soheil; Mahmoudi, Morteza; Mohajerzadeh, Shams

    2015-01-01

    Cancerous transformation may be dependent on correlation between electrical disruptions in the cell membrane and mechanical disruptions of cytoskeleton structures. Silicon nanotube (SiNT)-based electrical probes, as ultra-accurate signal recorders with subcellular resolution, may create many opportunities for fundamental biological research and biomedical applications. Here, we used this technology to electrically monitor cellular mechanosensing. The SiNT probe was combined with an electrically activated glass micropipette aspiration system to achieve a new cancer diagnostic technique that is based on real-time correlation between mechanical and electrical behaviour of single cells. Our studies demonstrated marked changes in the electrical response following increases in the mechanical aspiration force in healthy cells. In contrast, such responses were extremely weak for malignant cells. Confocal microscopy results showed the impact of actin microfilament remodelling on the reduction of the electrical response for aspirated cancer cells due to the significant role of actin in modulating the ion channel activity in the cell membrane.Cancerous transformation may be dependent on correlation between electrical disruptions in the cell membrane and mechanical disruptions of cytoskeleton structures. Silicon nanotube (SiNT)-based electrical probes, as ultra-accurate signal recorders with subcellular resolution, may create many opportunities for fundamental biological research and biomedical applications. Here, we used this technology to electrically monitor cellular mechanosensing. The SiNT probe was combined with an electrically activated glass micropipette aspiration system to achieve a new cancer diagnostic technique that is based on real-time correlation between mechanical and electrical behaviour of single cells. Our studies demonstrated marked changes in the electrical response following increases in the mechanical aspiration force in healthy cells. In contrast, such

  2. Bioluminescence Microscopy as a Method to Measure Single Cell Androgen Receptor Activity Heterogeneous Responses to Antiandrogens

    PubMed Central

    Jain, Pallavi; Neveu, Bertrand; Velot, Lauriane; Wu, Lily; Fradet, Yves; Pouliot, Frédéric

    2016-01-01

    Cancer cell heterogeneity is well-documented. Therefore, techniques to monitor single cell heterogeneous responses to treatment are needed. We developed a highly translational and quantitative bioluminescence microscopy method to measure single cell androgen receptor (AR) activity modulation by antiandrogens from fluid biopsies. We showed that this assay can detect heterogeneous cellular response to drug treatment and that the sum of single cell AR activity can mirror the response in the whole cell population. This method may thus be used to monitor heterogeneous dynamic treatment responses in cancer cells. PMID:27678181

  3. Optical twisting to monitor the rheology of single cells.

    PubMed

    de Saint Vincent, Matthieu Robert

    2016-05-26

    Biological cells exhibit complex mechanical properties which determine their responses to applied force. We developed an optical method to probe the temporal evolution of power-law rheology of single cells. The method consisted in applying optically a constant mechanical torque to a birefringent microparticle bound to the cell membrane, and observing dynamics of the particle's in-plane rotation. The deformation dynamics of the membrane followed a power law of time, which directly relates to cytoskeletal prestress as reported in the literature. The temporal evolution of this rheological behaviour, over time scales of several minutes, showed strong variations of the exponent on single adherent cells not subject to any specific treatment. The consistent observation of variations in the exponent suggests that, in their normal activity, living cells modulate their prestress by up to three orders of magnitude within minutes.

  4. Microwave-induced thermogenetic activation of single cells

    SciTech Connect

    Safronov, N. A.; Fedotov, I. V.; Ermakova, Yu. G.; Matlashov, M. E.; Belousov, V. V.; Sidorov-Biryukov, D. A.; Fedotov, A. B.; Zheltikov, A. M.

    2015-04-20

    Exposure to a microwave field is shown to enable thermogenetic activation of individual cells in a culture of cell expressing thermosensitive ion channels. Integration of a microwave transmission line with an optical fiber and a diamond quantum thermometer has been shown to allow thermogenetic single-cell activation to be combined with accurate local online temperature measurements based on an optical detection of electron spin resonance in nitrogen–vacancy centers in diamond.

  5. Monitoring single-cell bioenergetics via the coarsening of emulsion droplets.

    PubMed

    Boitard, L; Cottinet, D; Kleinschmitt, C; Bremond, N; Baudry, J; Yvert, G; Bibette, J

    2012-05-08

    Microorganisms are widely used to generate valuable products, and their efficiency is a major industrial focus. Bioreactors are typically composed of billions of cells, and available measurements only reflect the overall performance of the population. However, cells do not equally contribute, and process optimization would therefore benefit from monitoring this intrapopulation diversity. Such monitoring has so far remained difficult because of the inability to probe concentration changes at the single-cell level. Here, we unlock this limitation by taking advantage of the osmotically driven water flux between a droplet containing a living cell toward surrounding empty droplets, within a concentrated inverse emulsion. With proper formulation, excreted products are far more soluble within the continuous hydrophobic phase compared to initial nutrients (carbohydrates and salts). Fast diffusion of products induces an osmotic mismatch, which further relaxes due to slower diffusion of water through hydrophobic interfaces. By measuring droplet volume variations, we can deduce the metabolic activity down to isolated single cells. As a proof of concept, we present the first direct measurement of the maintenance energy of individual yeast cells. This method does not require any added probes and can in principle apply to any osmotically sensitive bioactivity, opening new routes for screening, and sorting large libraries of microorganisms and biomolecules.

  6. Monitoring single-cell bioenergetics via the coarsening of emulsion droplets

    PubMed Central

    Boitard, L.; Cottinet, D.; Kleinschmitt, C.; Bremond, N.; Baudry, J.; Yvert, G.; Bibette, J.

    2012-01-01

    Microorganisms are widely used to generate valuable products, and their efficiency is a major industrial focus. Bioreactors are typically composed of billions of cells, and available measurements only reflect the overall performance of the population. However, cells do not equally contribute, and process optimization would therefore benefit from monitoring this intrapopulation diversity. Such monitoring has so far remained difficult because of the inability to probe concentration changes at the single-cell level. Here, we unlock this limitation by taking advantage of the osmotically driven water flux between a droplet containing a living cell toward surrounding empty droplets, within a concentrated inverse emulsion. With proper formulation, excreted products are far more soluble within the continuous hydrophobic phase compared to initial nutrients (carbohydrates and salts). Fast diffusion of products induces an osmotic mismatch, which further relaxes due to slower diffusion of water through hydrophobic interfaces. By measuring droplet volume variations, we can deduce the metabolic activity down to isolated single cells. As a proof of concept, we present the first direct measurement of the maintenance energy of individual yeast cells. This method does not require any added probes and can in principle apply to any osmotically sensitive bioactivity, opening new routes for screening, and sorting large libraries of microorganisms and biomolecules. PMID:22538813

  7. Monitoring of dopamine release in single cell using ultrasensitive ITO microsensors modified with carbon nanotubes.

    PubMed

    Shi, Bao-Xian; Wang, Yu; Zhang, Kai; Lam, Tin-Lun; Chan, Helen Lai-Wa

    2011-02-15

    The study of single cell dynamics has been greatly adapted in biological and medical research and applications. In this work a novel microfluidic electrochemical sensor with carbon nanotubes (CNTs) modified indium tin oxide (ITO) microelectrode was developed for single cells release monitoring. The sensitivity of the electrochemical sensor after CNTs surface modification was improved by 2.5-3 orders of magnitude. The developed CNTs modified ITO sensor was successfully employed to monitor the dopamine release from single living rat pheochromocytoma (PC 12) cells. Its ultrahigh sensitivity, transparency and need for fewer agents enable this smart electrochemical sensor to become a powerful tool in recording dynamic release from various living tissues and organs optically and electrically.

  8. Quantitative telomerase enzyme activity determination using droplet digital PCR with single cell resolution

    PubMed Central

    Ludlow, Andrew T.; Robin, Jerome D.; Sayed, Mohammed; Litterst, Claudia M.; Shelton, Dawne N.; Shay, Jerry W.; Wright, Woodring E.

    2014-01-01

    The telomere repeat amplification protocol (TRAP) for the human reverse transcriptase, telomerase, is a PCR-based assay developed two decades ago and is still used for routine determination of telomerase activity. The TRAP assay can only reproducibly detect ∼2-fold differences and is only quantitative when compared to internal standards and reference cell lines. The method generally involves laborious radioactive gel electrophoresis and is not conducive to high-throughput analyzes. Recently droplet digital PCR (ddPCR) technologies have become available that allow for absolute quantification of input deoxyribonucleic acid molecules following PCR. We describe the reproducibility and provide several examples of a droplet digital TRAP (ddTRAP) assay for telomerase activity, including quantitation of telomerase activity in single cells, telomerase activity across several common telomerase positive cancer cells lines and in human primary peripheral blood mononuclear cells following mitogen stimulation. Adaptation of the TRAP assay to digital format allows accurate and reproducible quantification of the number of telomerase-extended products (i.e. telomerase activity; 57.8 ± 7.5) in a single HeLa cell. The tools developed in this study allow changes in telomerase enzyme activity to be monitored on a single cell basis and may have utility in designing novel therapeutic approaches that target telomerase. PMID:24861623

  9. A single-cell bioluminescence imaging system for monitoring cellular gene expression in a plant body.

    PubMed

    Muranaka, Tomoaki; Kubota, Saya; Oyama, Tokitaka

    2013-12-01

    Gene expression is a fundamental cellular process and expression dynamics are of great interest in life science. We succeeded in monitoring cellular gene expression in a duckweed plant, Lemna gibba, using bioluminescent reporters. Using particle bombardment, epidermal and mesophyll cells were transfected with the luciferase gene (luc+) under the control of a constitutive [Cauliflower mosaic virus 35S (CaMV35S)] and a rhythmic [Arabidopsis thaliana CIRCADIAN CLOCK ASSOCIATED 1 (AtCCA1)] promoter. Bioluminescence images were captured using an EM-CCD (electron multiply charged couple device) camera. Luminescent spots of the transfected cells in the plant body were quantitatively measured at the single-cell level. Luminescence intensities varied over a 1,000-fold range among CaMV35S::luc+-transfected cells in the same plant body and showed a log-normal-like frequency distribution. We monitored cellular gene expression under light-dark conditions by capturing bioluminescence images every hour. Luminescence traces of ≥50 individual cells in a frond were successfully obtained in each monitoring procedure. Rhythmic and constitutive luminescence behaviors were observed in cells transfected with AtCCA1::luc+ and CaMV35S::luc+, respectively. Diurnal rhythms were observed in every AtCCA1::luc+-introduced cell with traceable luminescence, and slight differences were detected in their rhythmic waveforms. Thus the single-cell bioluminescence monitoring system was useful for the characterization of cellular gene expression in a plant body.

  10. Electrochemiluminescence imaging for parallel single-cell analysis of active membrane cholesterol.

    PubMed

    Zhou, Junyu; Ma, Guangzhong; Chen, Yun; Fang, Danjun; Jiang, Dechen; Chen, Hong-Yuan

    2015-08-18

    Luminol electrochemiluminescence (ECL) imaging was developed for the parallel measurement of active membrane cholesterol at single living cells, thus establishing a novel electrochemical detection technique for single cells with high analysis throughput and low detection limit. In our strategy, the luminescence generated from luminol and hydrogen peroxide upon the potential was recorded in one image so that hydrogen peroxide at the surface of multiple cells could be simultaneously analyzed. Compared with the classic microelectrode array for the parallel single-cell analysis, the plat electrode only was needed in our ECL imaging, avoiding the complexity of electrode fabrication. The optimized ECL imaging system showed that hydrogen peroxide as low as 10 μM was visible and the efflux of hydrogen peroxide from cells could be determined. Coupled with the reaction between active membrane cholesterol and cholesterol oxidase to generate hydrogen peroxide, active membrane cholesterol at cells on the electrode was analyzed at single-cell level. The luminescence intensity was correlated with the amount of active membrane cholesterol, validating our system for single-cell cholesterol analysis. The relative high standard deviation on the luminescence suggested high cellular heterogeneities on hydrogen peroxide efflux and active membrane cholesterol, which exhibited the significance of single-cell analysis. This success in ECL imaging for single-cell analysis opens a new field in the parallel measurement of surface molecules at single cells.

  11. Single-cell bacteria growth monitoring by automated DEP-facilitated image analysis.

    PubMed

    Peitz, Ingmar; van Leeuwen, Rien

    2010-11-07

    Growth monitoring is the method of choice in many assays measuring the presence or properties of pathogens, e.g. in diagnostics and food quality. Established methods, relying on culturing large numbers of bacteria, are rather time-consuming, while in healthcare time often is crucial. Several new approaches have been published, mostly aiming at assaying growth or other properties of a small number of bacteria. However, no method so far readily achieves single-cell resolution with a convenient and easy to handle setup that offers the possibility for automation and high throughput. We demonstrate these benefits in this study by employing dielectrophoretic capturing of bacteria in microfluidic electrode structures, optical detection and automated bacteria identification and counting with image analysis algorithms. For a proof-of-principle experiment we chose an antibiotic susceptibility test with Escherichia coli and polymyxin B. Growth monitoring is demonstrated on single cells and the impact of the antibiotic on the growth rate is shown. The minimum inhibitory concentration as a standard diagnostic parameter is derived from a dose-response plot. This report is the basis for further integration of image analysis code into device control. Ultimately, an automated and parallelized setup may be created, using an optical microscanner and many of the electrode structures simultaneously. Sufficient data for a sound statistical evaluation and a confirmation of the initial findings can then be generated in a single experiment.

  12. Real-time characterization of cytotoxicity using single-cell impedance monitoring.

    PubMed

    Asphahani, Fareid; Thein, Myo; Wang, Kui; Wood, David; Wong, Sau Shun; Xu, Jian; Zhang, Miqin

    2012-07-07

    Cellular impedance sensors have attracted great attention as a powerful characterization tool for real-time, label-free detection of cytotoxic agents. However, impedance measurements with conventional cell-based sensors that host multiple cells on a single electrode neither provide optimal cell signal sensitivity nor are capable of recording individual cell responses. Here we use a single-cell based platform to monitor cellular impedance on planar microelectrodes to characterize cellular death. In this study, individual cells were selectively patterned on microelectrodes with each hosting one live cell through ligand-mediated natural cell adhesion. Changes in cellular morphology and cell-electrode adherence were monitored after the patterned cells were treated with varying concentrations of hydrogen peroxide, sodium arsenite, and disodium hydrogen arsenate, three potent toxicants related to neurotoxicity and oxidative stress. At low toxicant concentrations, impedance waveforms acquired from individual cells showed variable responses. A time- and concentration-dependent response was seen in the averaged single-cell impedance waveform for all three toxicants. The apoptosis and necrosis characterizations were performed to validate cell impedance results. Furthermore, time constants of apoptosis and necrosis in response to toxicant exposure were analytically established using an equivalent circuit model that characterized the mechanisms of cell death.

  13. Single cell activity reveals direct electron transfer in methanotrophic consortia

    NASA Astrophysics Data System (ADS)

    McGlynn, Shawn E.; Chadwick, Grayson L.; Kempes, Christopher P.; Orphan, Victoria J.

    2015-10-01

    Multicellular assemblages of microorganisms are ubiquitous in nature, and the proximity afforded by aggregation is thought to permit intercellular metabolic coupling that can accommodate otherwise unfavourable reactions. Consortia of methane-oxidizing archaea and sulphate-reducing bacteria are a well-known environmental example of microbial co-aggregation; however, the coupling mechanisms between these paired organisms is not well understood, despite the attention given them because of the global significance of anaerobic methane oxidation. Here we examined the influence of interspecies spatial positioning as it relates to biosynthetic activity within structurally diverse uncultured methane-oxidizing consortia by measuring stable isotope incorporation for individual archaeal and bacterial cells to constrain their potential metabolic interactions. In contrast to conventional models of syntrophy based on the passage of molecular intermediates, cellular activities were found to be independent of both species intermixing and distance between syntrophic partners within consortia. A generalized model of electric conductivity between co-associated archaea and bacteria best fit the empirical data. Combined with the detection of large multi-haem cytochromes in the genomes of methanotrophic archaea and the demonstration of redox-dependent staining of the matrix between cells in consortia, these results provide evidence for syntrophic coupling through direct electron transfer.

  14. Application of laser tweezers Raman spectroscopy techniques to the monitoring of single cell response to stimuli

    NASA Astrophysics Data System (ADS)

    Chan, James W.; Liu, Rui; Matthews, Dennis L.

    2012-06-01

    Laser tweezers Raman spectroscopy (LTRS) combines optical trapping with micro-Raman spectroscopy to enable label-free biochemical analysis of individual cells and small biological particles in suspension. The integration of the two technologies greatly simplifies the sample preparation and handling of suspension cells for spectroscopic analysis in physiologically meaningful conditions. In our group, LTRS has been used to study the effects of external perturbations, both chemical and mechanical, on the biochemistry of the cell. Single cell dynamics can be studied by performing longitudinal studies to continuously monitor the response of the cell as it interacts with its environment. The ability to carry out these measurements in-vitro makes LTRS an attractive tool for many biomedical applications. Here, we discuss the use of LTRS to study the response of cancer cells to chemotherapeutics and bacteria cells to antibiotics and show that the life cycle and apoptosis of the cells can be detected. These results show the promise of LTRS for drug discovery/screening, antibiotic susceptibility testing, and chemotherapy response monitoring applications. In separate experiments, we study the response of red blood cells to the mechanical forces imposed on the cell by the optical tweezers. A laser power dependent deoxygenation of the red blood cell in the single beam trap is reported. Normal, sickle cell, and fetal red blood cells have a different behavior that enables the discrimination of the cell types based on this mechanochemical response. These results show the potential utility of LTRS for diagnosing and studying red blood cell diseases.

  15. Direct observation of frequency modulated transcription in single cells using light activation

    PubMed Central

    Larson, Daniel R; Fritzsch, Christoph; Sun, Liang; Meng, Xiuhau; Lawrence, David S; Singer, Robert H

    2013-01-01

    Single-cell analysis has revealed that transcription is dynamic and stochastic, but tools are lacking that can determine the mechanism operating at a single gene. Here we utilize single-molecule observations of RNA in fixed and living cells to develop a single-cell model of steroid-receptor mediated gene activation. We determine that steroids drive mRNA synthesis by frequency modulation of transcription. This digital behavior in single cells gives rise to the well-known analog dose response across the population. To test this model, we developed a light-activation technology to turn on a single steroid-responsive gene and follow dynamic synthesis of RNA from the activated locus. DOI: http://dx.doi.org/10.7554/eLife.00750.001 PMID:24069527

  16. Microfluidic Device for Electric Field-Driven Single-Cell Captureand Activation

    SciTech Connect

    Toriello, Nicholas M.; Douglas, Erik S.; Mathies, Richard A.

    2005-09-20

    A microchip that performs directed capture and chemical activation of surface-modified single-cells has been developed. The cell-capture system is comprised of interdigitated gold electrodes microfabricated on a glass substrate within PDMS channels. The cell surface is labeled with thiol functional groups using endogenous RGD receptors and adhesion to exposed gold pads on the electrodes is directed by applying a driving electric potential. Multiple cell types can thus be sequentially and selectively captured on desired electrodes. Single-cell capture efficiency is optimized by varying the duration of field application. Maximum single-cell capture is attained for the 10 min trial, with 63+-9 percent (n=30) of the electrode pad rows having a single cell. In activation studies, single M1WT3 CHO cells loaded with the calcium-sensitive dye fluo-4 AM were captured; exposure to the muscarinic agonist carbachol increased the fluorescence to 220+-74percent (n=79) of the original intensity. These results demonstrate the ability to direct the adhesion of selected living single cells on electrodes in a microfluidic device and to analyze their response to chemical stimuli.

  17. Dynamic monitoring of single cell lysis in an impedance-based microfluidic device.

    PubMed

    Zhou, Ying; Basu, Srinjan; Laue, Ernest D; Seshia, Ashwin A

    2016-08-01

    A microfluidic device that is capable of trapping and sensing dynamic variations in the electrical properties of individual cells is demonstrated. The device is applied to the real-time recording of impedance measurements of mouse embryonic stem cells (mESCs) during the process of membrane lysis, with the resulting changes in the electrical properties of cells during this process being quantitatively tracked over time. It is observed that the impedance magnitude decreases dramatically after cell membrane lysis. A significant shift in the phase spectrum is also observed during the time course of this process. By fitting experimental data to physical models, the electrical parameters of cells can be extracted and parameter variations quantified during the process. In the cell lysis experiments, the equivalent conductivity of the cell membrane is found to increase significantly due to pore formation in the membrane during lysis. An increase in the specific capacitance of the membrane is also observed. On the other hand, the conductivity of the cytoplasm is observed to decrease, which may be explained the fact that excess water enters the cell through the gradual permeabilization of the membrane during lysis. Cells can be trapped in the device for periods up to several days, and their electrical response can be monitored by real-time impedance measurements in a label-free and non-invasive manner. Furthermore, due to the highly efficient single cell trapping capacity of the device, a number of cells can be trapped and held in separate wells for concurrent parallel experiments, allowing for the possibility of stepped parametric experiments and studying cell heterogeneity by combining measurements across the array.

  18. Detecting kinase activities from single cell lysate using concentration-enhanced mobility shift assay.

    PubMed

    Cheow, Lih Feng; Sarkar, Aniruddh; Kolitz, Sarah; Lauffenburger, Douglas; Han, Jongyoon

    2014-08-05

    Electrokinetic preconcentration coupled with mobility shift assays can give rise to very high detection sensitivities. We describe a microfluidic device that utilizes this principle to detect cellular kinase activities by simultaneously concentrating and separating substrate peptides with different phosphorylation states. This platform is capable of reliably measuring kinase activities of single adherent cells cultured in nanoliter volume microwells. We also describe a novel method utilizing spacer peptides that significantly increase separation resolution while maintaining high concentration factors in this device. Thus, multiplexed kinase measurements can be implemented with single cell sensitivity. Multiple kinase activity profiling from single cell lysate could potentially allow us to study heterogeneous activation of signaling pathways that can lead to multiple cell fates.

  19. [Impulse cytofluorometry of the redox activity of single cells using fluorescent formazan].

    PubMed

    Severin, E; Stellmach, J

    1984-01-01

    The first application of a newly developed fluorescent formazan in flow cytometry is described. The cell surface redox activity of isolated mouse hepatocytes after incubation with the tetrazolium salt forming the new fluorescent formazan and the DNA content after Hoechst staining have been measured simultaneously. 2 parametric distribution patterns have been obtained. This new and sensitive fluorometric technique can be used for automatic measurements of single cells correlating redox activity with other cell parameters.

  20. Automated single cell microbioreactor for monitoring intracellular dynamics and cell growth in free solution†

    PubMed Central

    Johnson-Chavarria, Eric M.; Agrawal, Utsav; Tanyeri, Melikhan; Kuhlman, Thomas E.

    2014-01-01

    We report an automated microfluidic-based platform for single cell analysis that allows for cell culture in free solution with the ability to control the cell growth environment. Using this approach, cells are confined by the sole action of gentle fluid flow, thereby enabling non-perturbative analysis of cell growth away from solid boundaries. In addition, the single cell microbioreactor allows for precise and time-dependent control over cell culture media, with the combined ability to observe the dynamics of non-adherent cells over long time scales. As a proof-of-principle demonstration, we used the platform to observe dynamic cell growth, gene expression, and intracellular diffusion of repressor proteins while precisely tuning the cell growth environment. Overall, this microfluidic approach enables the direct observation of cellular dynamics with exquisite control over environmental conditions, which will be useful for quantifying the behaviour of single cells in well-defined media. PMID:24836754

  1. Single-cell bioelectrical impedance platform for monitoring cellular response to drug treatment

    PubMed Central

    Asphahani, Fareid; Wang, Kui; Thein, Myo; Veiseh, Omid; Yung, Sandy; Xu, Jian; Zhang, Miqin

    2011-01-01

    The response of cells to a chemical or biological agent in terms of their impedance changes in real-time is a useful mechanism that can be utilized for a wide variety of biomedical and environmental applications. The use of a single-cell based analytical platform could be an effective approach to acquiring more sensitive cell impedance measurements, particularly in applications where only diminutive changes in impedance are expected. Here, we report the development of an on-chip cell impedance biosensor with two types of electrodes that hosts individual cells and cell populations, respectively, to study its efficacy in detecting cellular response. Human glioblastoma (U87MG) cells were patterned on single- and multi-cell electrodes through ligand-mediated natural cell adhesion. We comparatively investigated how these cancer cells on both types of electrodes respond to an ion channel inhibitor, chlorotoxin (CTX), in terms of their shape alternations and impedance changes to exploit the fine detectability of the single-cell based system. The detecting electrodes hosting single cells exhibited a significant reduction in the real impedance signal, while electrodes hosting confluent monolayer of cells showed little to no impedance change. When single-cell electrodes were treated with CTX of different doses, a dose-dependent impedance change was observed. This enables us to identify the effective dose needed for this particular treatment. Our study demonstrated that this single-cell impedance system may potentially serve as a useful analytical tool for biomedical applications such as environmental toxin detection and drug evaluation. PMID:21301069

  2. Femtosecond laser fabricated microfluorescence-activated cell sorter for single cell recovery

    NASA Astrophysics Data System (ADS)

    Bragheri, F.; Paiè, P.; Nava, G.; Yang, T.; Minzioni, P.; Martinez Vazquez, R.; Bellini, N.; Ramponi, R.; Cristiani, I.; Osellame, R.

    2014-03-01

    Manipulation, sorting and recovering of specific live cells from samples containing less than a few thousand cells is becoming a major hurdle in rare cell exploration such as stem cell research or cell based diagnostics. Moreover the possibility of recovering single specific cells for culturing and further analysis would be of great impact in many biological fields ranging from regenerative medicine to cancer therapy. In recent years considerable effort has been devoted to the development of integrated and low-cost optofluidic devices able to handle single cells, which usually rely on microfluidic circuits that guarantee a controlled flow of the cells. Among the different microfabrication technologies, femtosecond laser micromachining (FLM) is ideally suited for this purpose as it provides the integration of both microfluidic and optical functions on the same glass chip leading to monolithic, robust and portable devices. Here a new optofluidic device is presented, which is capable of sorting and recovering of single cells, through optical forces, on the basis of their fluorescence and. Both fluorescence detection and single cell sorting functions are integrated in the microfluidic chip by FLM. The device, which is specifically designed to operate with a limited amount of cells but with a very high selectivity, is fabricated by a two-step process that includes femtosecond laser irradiation followed by chemical etching. The capability of the device to act as a micro fluorescence-activated cell sorter has been tested on polystyrene beads and on tumor cells and the results on the single live cell recovery are reported.

  3. Single cell analysis of cancer cells using an improved RT-MLPA method has potential for cancer diagnosis and monitoring.

    PubMed

    Kvastad, L; Werne Solnestam, B; Johansson, E; Nygren, A O; Laddach, N; Sahlén, P; Vickovic, S; Bendigtsen, Schirmer C; Aaserud, M; Floer, L; Borgen, E; Schwind, C; Himmelreich, R; Latta, D; Lundeberg, J

    2015-11-12

    Single cell analysis techniques have great potential in the cancer genomics field. The detection and characterization of circulating tumour cells are important for identifying metastatic disease at an early stage and monitoring it. This protocol is based on transcript profiling using Reverse Transcriptase Multiplex Ligation-dependent Probe Amplification (RT-MLPA), which is a specific method for simultaneous detection of multiple mRNA transcripts. Because of the small amount of (circulating) tumour cells, a pre-amplification reaction is performed after reverse transcription to generate a sufficient number of target molecules for the MLPA reaction. We designed a highly sensitive method for detecting and quantifying a panel of seven genes whose expression patterns are associated with breast cancer, and optimized the method for single cell analysis. For detection we used a fluorescence-dependent semi-quantitative method involving hybridization of unique barcodes to an array. We evaluated the method using three human breast cancer cell lines and identified specific gene expression profiles for each line. Furthermore, we applied the method to single cells and confirmed the heterogeneity of a cell population. Successful gene detection from cancer cells in human blood from metastatic breast cancer patients supports the use of RT-MLPA as a diagnostic tool for cancer genomics.

  4. Living at the border: A community and single-cell assessment of lake bacterioneuston activity

    PubMed Central

    Hörtnagl, Paul; Pérez, María Teresa; Sommaruga, Ruben

    2010-01-01

    We assessed the physicochemical properties of the surface microlayer (SML: first 900 μm) and its underlying water (ULW: 0.2–0.5-m depth) and compared the composition and activity of their bacterial communities in six lakes located across an altitude gradient. Activity was assessed at both the community level, by measuring leucine bulk incorporation, and at the single-cell level, by using microautoradiography. Catalyzed reporter deposition fluorescence in situ hybridization was used to quantitatively assess the structure of the bacterial assemblage. Dissolved organic matter at the SML was significantly enriched in small-size molecules as compared to the ULW. Bacterial abundance in the SML ranged from 3.2 × 105 cells mL−1 to 3.2 × 106 cells mL−1 and was enriched in four out of six lakes when compared to the ULW. The SML and ULW showed lake-specific differences in bacterial community composition, although in most cases, both layers were dominated by Betaproteobacteria. This group also contributed the most to total activity in both layers in all lakes, followed by Actinobacteria. Despite large differences in environmental conditions among lakes, the fraction of active neustonic bacteria was very similar in most of them. Both bulk and single-cell activities are not necessarily lower in the SML than in the ULW, and well-adapted bacteria exist in the extreme conditions found in this habitat. PMID:20401318

  5. High-sensitivity measurements of multiple kinase activities in live single cells.

    PubMed

    Regot, Sergi; Hughey, Jacob J; Bajar, Bryce T; Carrasco, Silvia; Covert, Markus W

    2014-06-19

    Increasing evidence has shown that population dynamics are qualitatively different from single-cell behaviors. Reporters to probe dynamic, single-cell behaviors are desirable yet relatively scarce. Here, we describe an easy-to-implement and generalizable technology to generate reporters of kinase activity for individual cells. Our technology converts phosphorylation into a nucleocytoplasmic shuttling event that can be measured by epifluorescence microscopy. Our reporters reproduce kinase activity for multiple types of kinases and allow for calculation of active kinase concentrations via a mathematical model. Using this technology, we made several experimental observations that had previously been technicallyunfeasible, including stimulus-dependent patterns of c-Jun N-terminal kinase (JNK) and nuclear factor kappa B (NF-κB) activation. We also measured JNK, p38, and ERK activities simultaneously, finding that p38 regulates the peak number, but not the intensity, of ERK fluctuations. Our approach opens the possibility of analyzing a wide range of kinase-mediated processes in individual cells.

  6. Single cell active force generation under dynamic loading - Part I: AFM experiments.

    PubMed

    Weafer, P P; Reynolds, N H; Jarvis, S P; McGarry, J P

    2015-11-01

    A novel series of experiments are performed on single cells using a bespoke AFM system where the response of cells to dynamic loading at physiologically relevant frequencies is uncovered. Measured forces for the untreated cells are dramatically different to cytochalasin-D (cyto-D) treated cells, indicating that the contractile actin cytoskeleton plays a critical role in the response of cells to dynamic loading. Following a change in applied strain magnitude, while maintaining a constant applied strain rate, the compression force for contractile cells recovers to 88.9±7.8% of the steady state force. In contrast, cyto-D cell compression forces recover to only 38.0±6.7% of the steady state force. Additionally, untreated cells exhibit strongly negative (pulling) forces during unloading half-cycles when the probe is retracted. In comparison, negligible pulling forces are measured for cyto-D cells during probe retraction. The current study demonstrates that active contractile forces, generated by actin-myosin cross-bridge cycling, dominate the response of single cells to dynamic loading. Such active force generation is shown to be independent of applied strain magnitude. Passive forces generated by the applied deformation are shown to be of secondary importance, exhibiting a high dependence on applied strain magnitude, in contrast to the active forces in untreated cells. A novel series of experiments are performed on single cells using a bespoke AFM system where the response of cells to dynamic loading at physiologically relevant frequencies is uncovered. Contractile cells, which contain the active force generation machinery of the actin cytoskeleton, are shown to be insensitive to applied strain magnitude, exhibiting high resistance to dynamic compression and stretching. Such trends are not observed for cells in which the actin cytoskeleton has been chemically disrupted. These biomechanical insights have not been previously reported. This detailed characterisation of

  7. A Single-Cell Platform for Monitoring Viral Proteolytic Cleavage in Different Cellular Compartments

    PubMed Central

    Abbadessa, Darin; Smurthwaite, Cameron A.; Reed, Connor W.; Wolkowicz, Roland

    2015-01-01

    Infectious diseases affect human health despite advances in biomedical research and drug discovery. Among these, viruses are especially difficult to tackle due to the sudden transfer from animals to humans, high mutational rates, resistance to current treatments, and the intricacies of their molecular interactions with the host. As an example of these interactions, we describe a cell-based approach to monitor specific proteolytic events executed by either the viral-encoded protease or by host proteins on the virus. We then emphasize the significance of examining proteolysis within the subcellular compartment where cleavage occurs naturally. We show the power of stable expression, highlighting the usefulness of the cell-based multiplexed approach, which we have adapted to two independent assays previously developed to monitor (a) the activity of the HIV-1-encoded protease or (b) the cleavage of the HIV-1-encoded envelope protein by the host. Multiplexing was achieved by mixing cells each carrying a different assay or, alternatively, by engineering cells expressing two assays. Multiplexing relies on the robustness of the individual assays and their clear discrimination, further enhancing screening capabilities in an attempt to block proteolytic events required for viral infectivity and spread. PMID:27688710

  8. Monitoring and manipulation of the pH of single cells using infrared spectromicroscopy and a molecular switch.

    PubMed

    Carbone, Marilena; Zlateva, Theodora; Quaroni, Luca

    2013-04-01

    The pH of a biological system is a crucial determinant of the structures and reactivity of its components and cellular homeostasis of H(+) is critical for cell viability. Control and monitoring of cellular acidity are highly desirable for the purpose of studying biochemical processes in vivo. The effect of photolysis of a caged strong acid, the ester 1-(2-nitrophenyl)-ethylhexadecyl sulfonate (HDNS) is used to cause a controlled drop in pH in single cells. An isolated cell is selected under the IR microscope, irradiated with near-UV light and monitored by FTIR. We demonstrate the use of FTIR spectromicroscopy to monitor light-induced acidification of the cellular medium by measuring the increased concentration of CO2 and corresponding decrease of HCO3(-) in the cell and in the surrounding medium. We have demonstrated a method to control and accurately monitor the changes in pH of a cellular system by coupling a caged proton-releasing agent with FTIR spectromicroscopy detection. The overall implementation of photolysis and spectroscopic detection in a microscope optical configuration ensures single cell selectivity in both acidification and monitoring. We show the viability of monitoring of pH changes by FTIR spectromicroscopy with sensitivity comparable to that of glass electrodes, better than the existing methods for determining cell pH. Reporting the effect of small variations of cellular acidity provides a major improvement in the understanding of the interplay between molecular properties as assessed in vitro and cell physiology. Copyright © 2013 Elsevier B.V. All rights reserved.

  9. Metagenomics, metatranscriptomics and single cell genomics reveal functional response of active Oceanospirillales to Gulf oil spill

    SciTech Connect

    Mason, Olivia U.; Hazen, Terry C.; Borglin, Sharon; Chain, Patrick S. G.; Dubinsky, Eric A.; Fortney, Julian L.; Han, James; Holman, Hoi-Ying N.; Hultman, Jenni; Lamendella, Regina; Mackelprang, Rachel; Malfatti, Stephanie; Tom, Lauren M.; Tringe, Susannah G.; Woyke, Tanja; Zhou, Jizhong; Rubin, Edward M.; Jansson, Janet K.

    2012-06-12

    The Deepwater Horizon oil spill in the Gulf of Mexico resulted in a deep-sea hydrocarbon plume that caused a shift in the indigenous microbial community composition with unknown ecological consequences. Early in the spill history, a bloom of uncultured, thus uncharacterized, members of the Oceanospirillales was previously detected, but their role in oil disposition was unknown. Here our aim was to determine the functional role of the Oceanospirillales and other active members of the indigenous microbial community using deep sequencing of community DNA and RNA, as well as single-cell genomics. Shotgun metagenomic and metatranscriptomic sequencing revealed that genes for motility, chemotaxis and aliphatic hydrocarbon degradation were significantly enriched and expressed in the hydrocarbon plume samples compared with uncontaminated seawater collected from plume depth. In contrast, although genes coding for degradation of more recalcitrant compounds, such as benzene, toluene, ethylbenzene, total xylenes and polycyclic aromatic hydrocarbons, were identified in the metagenomes, they were expressed at low levels, or not at all based on analysis of the metatranscriptomes. Isolation and sequencing of two Oceanospirillales single cells revealed that both cells possessed genes coding for n-alkane and cycloalkane degradation. Specifically, the near-complete pathway for cyclohexane oxidation in the Oceanospirillales single cells was elucidated and supported by both metagenome and metatranscriptome data. The draft genome also included genes for chemotaxis, motility and nutrient acquisition strategies that were also identified in the metagenomes and metatranscriptomes. These data point towards a rapid response of members of the Oceanospirillales to aliphatic hydrocarbons in the deep sea.

  10. Integration of plasma-assisted surface chemical modification, soft lithography, and protein surface activation for single-cell patterning

    NASA Astrophysics Data System (ADS)

    Cheng, Q.; Komvopoulos, K.

    2010-07-01

    Surface patterning for single-cell culture was accomplished by combining plasma-assisted surface chemical modification, soft lithography, and protein-induced surface activation. Hydrophilic patterns were produced on Parylene C films deposited on glass substrates by oxygen plasma treatment through the windows of polydimethylsiloxane shadow masks. After incubation first with Pluronic F108 solution and then serum medium overnight, surface seeding with mesenchymal stem cells in serum medium resulted in single-cell patterning. The present method provides a means of surface patterning with direct implications in single-cell culture.

  11. Atmospheric CO2 monitoring with single-cell NDIR-based analyzers

    NASA Astrophysics Data System (ADS)

    Stephens, B. B.; Miles, N. L.; Richardson, S. J.; Watt, A. S.; Davis, K. J.

    2011-12-01

    We describe CO2 concentration measurement systems based on relatively inexpensive single-cell non-dispersive infrared CO2 sensors. The systems utilize signal averaging to obtain precision (1-σ in 100 s) of 0.1 parts per million dry air mole fraction (ppm), frequent calibrations and sample drying in order to achieve state-of-the-art compatibility, and can run autonomously for months at a time. Laboratory tests indicate compatibility among four to six systems to be ±0.1 ppm (1-σ), and field measurements of known reference-gases yield median errors of 0.01 to 0.17 ppm with 1-σ variance of ±0.1 to 0.2 ppm. From May to August 2007, a system co-located with a NOAA-ESRL dual-cell NDIR system at the WLEF tall tower in Wisconsin measured daytime-only daily averages of CO2 that differ by 0.26 ± 0.15 ppm (median ± 1 σ), and from August 2005 to April 2011 a system co-located with weekly NOAA-ESRL network flask collection at Niwot Ridge, Colorado measured coincident CO2 concentrations that differed by -0.06 ± 0.30 ppm (n = 585). Data from these systems are now supporting a wide range of analyses and this approach may be applicable in future studies where accuracy and initial cost of the sensors are priorities.

  12. Atmospheric CO2 monitoring with single-cell NDIR-based analyzers

    NASA Astrophysics Data System (ADS)

    Stephens, B. B.; Miles, N. L.; Richardson, S. J.; Watt, A. S.; Davis, K. J.

    2011-07-01

    We describe CO2 concentration measurement systems based on relatively inexpensive single-cell non-dispersive infrared CO2 sensors. The systems utilize signal averaging to obtain precision (1-σ in 100 s) of 0.1 parts per million dry air mole fraction (ppm), frequent calibrations and sample drying in order to achieve state-of-the-art compatibility, and can run autonomously for months at a time. Laboratory tests indicate compatibility among four to six systems to be ±0.1 ppm (1-σ), and field measurements of known reference-gases yield median errors of 0.01 to 0.17 ppm with 1-σ variance of ±0.1 to 0.2 ppm. From May to August 2007, a system co-located with a NOAA-ESRL dual-cell NDIR system at the WLEF tall tower in Wisconsin measured daytime-only daily averages of CO2 that differ by 0.26 ± 0.15 ppm (median ± 1 σ), and from August 2005 to April 2011 a system co-located with weekly NOAA-ESRL network flask collection at Niwot Ridge, Colorado measured coincident CO2 concentrations that differed by -0.06 ± 0.30 ppm (n = 585). Data from these systems are now supporting a wide range of analyses and this approach may be applicable in future studies where accuracy and initial cost of the sensors are priorities.

  13. Single-cell transcriptome analyses reveal signals to activate dormant neural stem cells.

    PubMed

    Luo, Yuping; Coskun, Volkan; Liang, Aibing; Yu, Juehua; Cheng, Liming; Ge, Weihong; Shi, Zhanping; Zhang, Kunshan; Li, Chun; Cui, Yaru; Lin, Haijun; Luo, Dandan; Wang, Junbang; Lin, Connie; Dai, Zachary; Zhu, Hongwen; Zhang, Jun; Liu, Jie; Liu, Hailiang; deVellis, Jean; Horvath, Steve; Sun, Yi Eve; Li, Siguang

    2015-05-21

    The scarcity of tissue-specific stem cells and the complexity of their surrounding environment have made molecular characterization of these cells particularly challenging. Through single-cell transcriptome and weighted gene co-expression network analysis (WGCNA), we uncovered molecular properties of CD133(+)/GFAP(-) ependymal (E) cells in the adult mouse forebrain neurogenic zone. Surprisingly, prominent hub genes of the gene network unique to ependymal CD133(+)/GFAP(-) quiescent cells were enriched for immune-responsive genes, as well as genes encoding receptors for angiogenic factors. Administration of vascular endothelial growth factor (VEGF) activated CD133(+) ependymal neural stem cells (NSCs), lining not only the lateral but also the fourth ventricles and, together with basic fibroblast growth factor (bFGF), elicited subsequent neural lineage differentiation and migration. This study revealed the existence of dormant ependymal NSCs throughout the ventricular surface of the CNS, as well as signals abundant after injury for their activation.

  14. Single Cell Assay for Molecular Diagnostics and Medicine: Monitoring Intracellular Concentrations of Macromolecules by Two-photon Fluorescence Lifetime Imaging.

    PubMed

    Pliss, Artem; Peng, Xiao; Liu, Lixin; Kuzmin, Andrey; Wang, Yan; Qu, Junle; Li, Yuee; Prasad, Paras N

    2015-01-01

    Molecular organization of a cell is dynamically transformed along the course of cellular physiological processes, pathologic developments or derived from interactions with drugs. The capability to measure and monitor concentrations of macromolecules in a single cell would greatly enhance studies of cellular processes in heterogeneous populations. In this communication, we introduce and experimentally validate a bio-analytical single-cell assay, wherein the overall concentration of macromolecules is estimated in specific subcellular domains, such as structure-function compartments of the cell nucleus as well as in nucleoplasm. We describe quantitative mapping of local biomolecular concentrations, either intrinsic relating to the functional and physiological state of a cell, or altered by a therapeutic drug action, using two-photon excited fluorescence lifetime imaging (FLIM). The proposed assay utilizes a correlation between the fluorescence lifetime of fluorophore and the refractive index of its microenvironment varying due to changes in the concentrations of macromolecules, mainly proteins. Two-photon excitation in Near-Infra Red biological transparency window reduced the photo-toxicity in live cells, as compared with a conventional single-photon approach. Using this new assay, we estimated average concentrations of proteins in the compartments of nuclear speckles and in the nucleoplasm at ~150 mg/ml, and in the nucleolus at ~284 mg/ml. Furthermore, we show a profound influence of pharmaceutical inhibitors of RNA synthesis on intracellular protein density. The approach proposed here will significantly advance theranostics, and studies of drug-cell interactions at the single-cell level, aiding development of personal molecular medicine.

  15. Alkaline single-cell gel (comet) assay and genotoxicity monitoring using two species of tadpoles.

    PubMed

    Ralph, S; Petras, M; Pandrangi, R; Vrzoc, M

    1996-01-01

    Small bodies of water (e.g., creeks, ponds, and drainage ditches) have received very little attention in genotoxicity studies, yet these areas are important because they are often the first to be affected by industrial effluents, sewage contaminants, accidental spills, internal combustion engine emissions, landfill runoffs, and pesticide uses. To address this deficiency, we examined erythrocytes in two species of tadpoles, Rana clamitans and Bufo americanus, using the alkaline single-cell gel (SCG) ("comet") assay. This approach involves detection, under alkaline conditions, of cell DNA fragments, which on electrophoresis migrate from the nuclear core, resulting in a "comet-with-tail" formation. Exposure of R. clamitans todpoles to a range of concentrations of methyl methanesulfonate (MMS) produced a linear increase in DNA length to DNA core width ratios. This is consistent with findings in a number of other species. Time-dose experiments using MMS suggest that the peak level of DNA damage in R. clamitans todpoles occurred 42 hr after exposure. B. americanus tadpoles exposed to 6.25 mg/l of MMS for 12 hours had a significant increase in DNA damage over that seen in the controls. Freshly caught R. clamitans tadpoles from Highgate and B. americanus tadpoles from Duart, both on the north shore of Lake Erie, gave ratios of 2.78 and 2.07, respectively. This region of Ontario is a prime agricultural area and pesticide use is extensive. Tadpoles from Highgate and Duart, maintained in the laboratory for 4 months and 6 weeks, respectively, gave ratios of 1.29 and 1.44. The results of the SCG procedure in tadpoles indicate that this assay is extremely sensitive and suitable for detecting genotoxicity in the environment.

  16. Single-cell activity of freshwater aerobic anoxygenic phototrophic bacteria and their contribution to biomass production.

    PubMed

    Garcia-Chaves, Maria C; Cottrell, Matthew T; Kirchman, David L; Ruiz-González, Clara; Del Giorgio, Paul A

    2016-07-01

    Aerobic anoxygenic phototrophic (AAP) bacteria are photoheterotrophs that despite their low abundances have been hypothesized to play an ecologically and biogeochemically important role in aquatic systems. Characterizing this role requires a better understanding of the in situ dynamics and activity of AAP bacteria. Here we provide the first assessment of the single-cell activity of freshwater AAP bacteria and their contribution to total bacterial production across lakes spanning a wide trophic gradient, and explore the role of light in regulating AAP activity. The proportion of cells that were active in leucine incorporation and the level of activity per cell were consistently higher for AAP than for bulk bacteria across lakes. As a result, AAP bacteria contributed disproportionately more to total bacterial production than to total bacterial abundance. Interestingly, although environmentally driven patterns in activity did not seem to differ largely between AAP and bulk bacteria, their response to light did, and exposure to light resulted in increases in the proportion of active AAP bacteria with no clear effect on their cell-specific activity. This suggests that light may play a role in the activation of AAP bacteria, enabling these photoheterotrophs to contribute more to the carbon cycle than suggested by their abundance.

  17. Single-cell western blotting

    PubMed Central

    Hughes, Alex J.; Spelke, Dawn P.; Xu, Zhuchen; Kang, Chi-Chih; Schaffer, David V.; Herr, Amy E.

    2014-01-01

    To measure cell-to-cell variation in protein-mediated functions — a hallmark of biological processes — we developed an approach to conduct ~103 concurrent single-cell western blots (scWesterns) in ~4 hours. A microscope slide supporting a 30 µm-thick photoactive polyacrylamide gel enables western blotting comprised of: settling of single cells into microwells, lysis in situ, gel electrophoresis, photoinitiated blotting to immobilize proteins, and antibody probing. We apply this scWestern to monitor single rat neural stem cell differentiation and responses to mitogen stimulation. The scWestern quantifies target proteins even with off-target antibody binding, multiplexes to 11 protein targets per single cell with detection thresholds of <30,000 molecules, and supports analyses of low starting cell numbers (~200) when integrated with fluorescence activated cell sorting. The scWestern thus overcomes limitations in single-cell protein analysis (i.e., antibody fidelity, sensitivity, and starting cell number) and constitutes a versatile tool for the study of complex cell populations at single-cell resolution. PMID:24880876

  18. Encapsulation of single cells into monodisperse droplets by fluorescence-activated droplet formation on a microfluidic chip.

    PubMed

    Hu, Rui; Liu, Pian; Chen, Pu; Wu, Liang; Wang, Yao; Feng, Xiaojun; Liu, Bi-Feng

    2016-06-01

    Random compartmentalization of cells by common droplet formation methods, i.e., T-junction and flow-focusing, results in low occupancy of droplets by single cells. To resolve this issue, a fluorescence-activated droplet formation method was developed for the on-command generation of droplets and encapsulation of single cells. In this method, droplets containing one cell were generated by switching on/off a two-phase hydrodynamic gating valve upon optical detection of single cells. To evaluate the developed method, flow visualization experiments were conducted with fluorescein. Results indicated that picoliter droplets of uniform sizes (RSD<4.9%) could be generated. Encapsulation of single fluorescent polystyrene beads demonstrated an average of 94.3% droplets contained one bead. Further application of the developed methods to the compartmentalization of individual HeLa cells indicated 82.5% occupancy of droplets by single cells, representing a 3 fold increase in comparison to random compartmentalization.

  19. 3D restoration microscopy improves quantification of enzyme-labeled fluorescence-based single-cell phosphatase activity in plankton.

    PubMed

    Diaz-de-Quijano, Daniel; Palacios, Pilar; Horňák, Karel; Felip, Marisol

    2014-10-01

    The ELF or fluorescence-labeled enzyme activity (FLEA) technique is a culture-independent single-cell tool for assessing plankton enzyme activity in close-to-in situ conditions. We demonstrate that single-cell FLEA quantifications based on two-dimensional (2D) image analysis were biased by up to one order of magnitude relative to deconvolved 3D. This was basically attributed to out-of-focus light, and partially to object size. Nevertheless, if sufficient cells were measured (25-40 cells), biases in individual 2D cell measurements were partially compensated, providing useful and comparable results to deconvolved 3D. We also discuss how much caution should be used when comparing the single-cell enzyme activities of different sized bacterio- and/or phytoplankton populations measured on 2D images. Finally, a novel method based on deconvolved 3D images (wide field restoration microscopy; WFR) was devised to improve the discrimination of similar single-cell enzyme activities, the comparison of enzyme activities between different size cells, the measurement of low fluorescence intensities, the quantification of less numerous species, and the combination of the FLEA technique with other single-cell methods. These improvements in cell enzyme activity measurements will provide a more precise picture of individual species' behavior in nature, which is essential to understand their functional role and evolutionary history.

  20. Concentric zones of active RhoA and Cdc42 around single cell wounds

    PubMed Central

    Benink, Hélène A.; Bement, William M.

    2005-01-01

    Rho GTPases control many cytoskeleton-dependent processes, but how they regulate spatially distinct features of cytoskeletal function within a single cell is poorly understood. Here, we studied active RhoA and Cdc42 in wounded Xenopus oocytes, which assemble and close a dynamic ring of actin filaments (F-actin) and myosin-2 around wound sites. RhoA and Cdc42 are rapidly activated around wound sites in a calcium-dependent manner and segregate into distinct, concentric zones around the wound, with active Cdc42 in the approximate middle of the F-actin array and active RhoA on the interior of the array. These zones form before F-actin accumulation, and then move in concert with the closing array. Microtubules and F-actin are required for normal zone organization and dynamics, as is crosstalk between RhoA and Cdc42. Each of the zones makes distinct contributions to the organization and function of the actomyosin wound array. We propose that similar rho activity zones control related processes such as cytokinesis. PMID:15684032

  1. Measuring activity in the ubiquitin-proteasome system: From large scale discoveries to single cells analysis

    PubMed Central

    Melvin, Adam T.; Woss, Gregery S.; Park, Jessica H.; Waters, Marcey L.; Allbritton, Nancy L.

    2013-01-01

    The ubiquitin proteasome system (UPS) is the primary pathway responsible for the recognition and degradation of misfolded, damaged, or tightly regulated proteins in addition to performing essential roles in DNA repair, cell cycle regulation, cell migration, and the immune response. While traditional biochemical techniques have proven useful in the identification of key proteins involved in this pathway, the implementation of novel reporters responsible for measuring enzymatic activity of the UPS have provided valuable insight into the effectiveness of therapeutics and role of the UPS in various human diseases such as multiple myeloma and Huntington’s disease. These reporters, usually consisting of a recognition sequences fused to an analytical handle, are designed to specifically evaluate enzymatic activity of certain members of the UPS including the proteasome, E3 ubiquitin ligases, and deubiquitinating enzymes (DUBs). This review highlights the more commonly used reporters employed in a variety of scenarios ranging from high-throughput screening of novel inhibitors to single cell microscopy techniques measuring E3 ligase or proteasome activity. Finally, recent work is presented highlighting the development of novel degron-based substrate designed to overcome the limitations of current reporting techniques in measuring E3 ligase and proteasome activity in patient samples. PMID:23686610

  2. Measuring activity in the ubiquitin-proteasome system: from large scale discoveries to single cells analysis.

    PubMed

    Melvin, Adam T; Woss, Gregery S; Park, Jessica H; Waters, Marcey L; Allbritton, Nancy L

    2013-09-01

    The ubiquitin-proteasome system (UPS) is the primary pathway responsible for the recognition and degradation of misfolded, damaged, or tightly regulated proteins in addition to performing essential roles in DNA repair, cell cycle regulation, cell migration, and the immune response. While traditional biochemical techniques have proven useful in the identification of key proteins involved in this pathway, the implementation of novel reporters responsible for measuring enzymatic activity of the UPS has provided valuable insight into the effectiveness of therapeutics and role of the UPS in various human diseases such as multiple myeloma and Huntington's disease. These reporters, usually consisting of a recognition sequence fused to an analytical handle, are designed to specifically evaluate enzymatic activity of certain members of the UPS including the proteasome, E3 ubiquitin ligases, and deubiquitinating enzymes. This review highlights the more commonly used reporters employed in a variety of scenarios ranging from high-throughput screening of novel inhibitors to single cell microscopy techniques measuring E3 ligase or proteasome activity. Finally, a recent study is presented highlighting the development of a novel degron-based substrate designed to overcome the limitations of current reporting techniques in measuring E3 ligase and proteasome activity in patient samples.

  3. Single Cell Analysis of Leukocyte Protease Activity Using Integrated Continuous-Flow Microfluidics.

    PubMed

    Jing, Tengyang; Lai, Zhangxing; Wu, Lidan; Han, Jongyoon; Lim, Chwee Teck; Chen, Chia-Hung

    2016-12-06

    Leukocytes are the essential cells of the immune system that protect the human body against bacteria, viruses, and other foreign invaders. Secretory products of individual leukocytes, such as matrix metalloproteinases (MMPs) and a disintegrin and metalloproteinase (ADAMs), are critical for regulating the inflammatory response and mediating host defense. Conventional single cell analytical methods, such as flow cytometry for cellular surface biomarker studies, are insufficient for performing functional assays of the protease activity of individual leukocytes. Here, an integrated continuous-flow microfluidic assay is developed to effectively detect secretory protease activity of individual viable leukocytes. Leukocytes in blood are first washed on-chip with defined buffer to remove background activity, followed by encapsulating individual leukocytes with protease sensors in water-in-oil droplets and incubating for 1 h to measure protease secretion. With this design, single leukocyte protease profiles under naive and phorbol 12-myristate 13-acetate (PMA)-stimulated conditions are reliably measured. It is found that PMA treatment not only elevates the average protease activity level but also reduces the cellular heterogeneity in protease secretion, which is important in understanding immune capability and the disease condition of individual patients.

  4. Capturing Single Cell Genomes of Active Polysaccharide Degraders: An Unexpected Contribution of Verrucomicrobia

    PubMed Central

    Martinez-Garcia, Manuel; Brazel, David M.; Swan, Brandon K.; Arnosti, Carol; Chain, Patrick S. G.; Reitenga, Krista G.; Xie, Gary; Poulton, Nicole J.; Gomez, Monica Lluesma; Masland, Dashiell E. D.; Thompson, Brian; Bellows, Wendy K.; Ziervogel, Kai; Lo, Chien-Chi; Ahmed, Sanaa; Gleasner, Cheryl D.; Detter, Chris J.; Stepanauskas, Ramunas

    2012-01-01

    Microbial hydrolysis of polysaccharides is critical to ecosystem functioning and is of great interest in diverse biotechnological applications, such as biofuel production and bioremediation. Here we demonstrate the use of a new, efficient approach to recover genomes of active polysaccharide degraders from natural, complex microbial assemblages, using a combination of fluorescently labeled substrates, fluorescence-activated cell sorting, and single cell genomics. We employed this approach to analyze freshwater and coastal bacterioplankton for degraders of laminarin and xylan, two of the most abundant storage and structural polysaccharides in nature. Our results suggest that a few phylotypes of Verrucomicrobia make a considerable contribution to polysaccharide degradation, although they constituted only a minor fraction of the total microbial community. Genomic sequencing of five cells, representing the most predominant, polysaccharide-active Verrucomicrobia phylotype, revealed significant enrichment in genes encoding a wide spectrum of glycoside hydrolases, sulfatases, peptidases, carbohydrate lyases and esterases, confirming that these organisms were well equipped for the hydrolysis of diverse polysaccharides. Remarkably, this enrichment was on average higher than in the sequenced representatives of Bacteroidetes, which are frequently regarded as highly efficient biopolymer degraders. These findings shed light on the ecological roles of uncultured Verrucomicrobia and suggest specific taxa as promising bioprospecting targets. The employed method offers a powerful tool to rapidly identify and recover discrete genomes of active players in polysaccharide degradation, without the need for cultivation. PMID:22536372

  5. Optical Recording of Suprathreshold Neural Activity with Single-cell and Single-spike Resolution

    PubMed Central

    Ranganathan, Gayathri Nattar; Koester, Helmut J.

    2012-01-01

    Signaling of information in the vertebrate central nervous system is often carried by populations of neurons rather than individual neurons. Also propagation of suprathreshold spiking activity involves populations of neurons. Empirical studies addressing cortical function directly thus require recordings from populations of neurons with high resolution. Here we describe an optical method and a deconvolution algorithm to record neural activity from up to 100 neurons with single-cell and single-spike resolution. This method relies on detection of the transient increases in intracellular somatic calcium concentration associated with suprathreshold electrical spikes (action potentials) in cortical neurons. High temporal resolution of the optical recordings is achieved by a fast random-access scanning technique using acousto-optical deflectors (AODs)1. Two-photon excitation of the calcium-sensitive dye results in high spatial resolution in opaque brain tissue2. Reconstruction of spikes from the fluorescence calcium recordings is achieved by a maximum-likelihood method. Simultaneous electrophysiological and optical recordings indicate that our method reliably detects spikes (>97% spike detection efficiency), has a low rate of false positive spike detection (< 0.003 spikes/sec), and a high temporal precision (about 3 msec) 3. This optical method of spike detection can be used to record neural activity in vitro and in anesthetized animals in vivo3,4. PMID:22972033

  6. Single cell monitoring of growth arrest and morphological changes induced by transfer of wild-type p53 alleles to glioblastoma cells.

    PubMed Central

    Van Meir, E G; Roemer, K; Diserens, A C; Kikuchi, T; Rempel, S A; Haas, M; Huang, H J; Friedmann, T; de Tribolet, N; Cavenee, W K

    1995-01-01

    Mutation of the p53 tumor suppressor gene is one of the earliest identified genetic lesions during malignant progression of human astrocytomas. To assess the functional significance of these mutations, wild-type (WT) p53 genes were introduced into glioblastoma cell lines having mutant, WT, or null endogenous p53 alleles. Populations of cells with mutant or null endogenous p53 alleles and exogenous WT p53 were spontaneously selected in culture for cells expressing only mutant p53 or no p53, which then displayed a growth and tumorigenic phenotype identical to the parental cells. To determine the phenotypic consequences of WT p53 expression before the occurrence of mutations, we developed a single cell assay to monitor WT p53-dependent transcription activity. Transfer and expression of exogenous WT p53 genes to cells with endogenous mutant or deleted, but not WT, p53 alleles caused growth arrest and morphological changes, including increased cell size and acquisition of multiple nuclei. This supports the hypothesis that genetic lesions of the p53 gene play an important role in the genesis of astrocytomas. Furthermore, the high sensitivity of the episomal single cell reporter strategy developed here has potential clinical applications in the rapid screening of patients for germ-line mutations of the p53 gene or any other gene with known targets for transcriptional transactivation. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:7862624

  7. Nanocoating of single cells: from maintenance of cell viability to manipulation of cellular activities.

    PubMed

    Park, Ji Hun; Yang, Sung Ho; Lee, Juno; Ko, Eun Hyea; Hong, Daewha; Choi, Insung S

    2014-04-02

    The chronological progresses in single-cell nanocoating are described. The historical developments in the field are divided into biotemplating, cytocompatible nanocoating, and cells in nano-nutshells, depending on the main research focuses. Each subfield is discussed in conjunction with the others, regarding how and why to manipulate living cells by nanocoating at the single-cell level. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Fluorescent protein-based FRET sensor for intracellular monitoring of redox status in bacteria at single cell level.

    PubMed

    Abraham, Bobin George; Santala, Ville; Tkachenko, Nikolai V; Karp, Matti

    2014-11-01

    Monitoring of intracellular redox status in a bacterial cell provides vital information about the physiological status of the cell, which can be exploited in several applications such as metabolic engineering and computational modeling. Fluorescent protein-based genetically encoded sensors can be used to monitor intracellular oxidation/reduction status. This study reports the development of a redox sensor for intracellular measurements using fluorescent protein pairs and the phenomenon of Förster resonance energy transfer (FRET). For the development of the sensor, fluorescent proteins Citrine and Cerulean were genetically modified to carry reactive cysteine residues on the protein surface close to the chromophore and a constructed FRET pair was fused using a biotinylation domain as a linker. In oxidized state, the FRET pairs are in close proximity by labile disulfide bond formation resulting in higher FRET efficiency. In reducing environment, the FRET is diminished due to the increased distance between FRET pairs providing large dynamic measurement range to the sensor. Intracellular studies in Escherichia coli mutants revealed the capability of the sensor in detecting real-time redox variations at single cell level. The results were validated by intensity based and time resolved measurements. The functional immobilization of the fluorescent protein-based FRET sensor at solid surfaces for in vitro applications was also demonstrated.

  9. A single-cell perspective on non-growing but metabolically active (NGMA) bacteria.

    PubMed

    Manina, Giulia; McKinney, John D

    2013-01-01

    A long-standing and fundamental problem in microbiology is the non-trivial discrimination between live and dead cells. The existence of physically intact and possibly viable bacterial cells that fail to replicate during a more or less protracted period of observation, despite environmental conditions that are ostensibly propitious for growth, has been extensively documented in many different organisms. In clinical settings, non-culturable cells may contribute to non-apparent infections capable of reactivating after months or years of clinical latency, a phenomenon that has been well documented in the specific case of Mycobacterium tuberculosis. The prevalence of these silent but potentially problematic bacterial reservoirs has been highlighted by classical approaches such as limiting culture dilution till extinction of growing cells, followed by resuscitation of apparently "viable but non-culturable" (VBNC) subpopulations. Although these assays are useful to demonstrate the presence of VBNC cells in a population, they are effectively retrospective and are not well suited to the analysis of non-replicating cells per se. Here, we argue that research on a closely related problem, which we shall refer to as the "non-growing but metabolically active" state, is poised to advance rapidly thanks to the recent development of novel technologies and methods for real-time single-cell analysis. In particular, the combination of fluorescent reporter dyes and strains, microfluidic and microelectromechanical systems, and time-lapse fluorescence microscopy offers tremendous and largely untapped potential for future exploration of the physiology of non-replicating cells.

  10. Novel methods for the guidance and monitoring of single cells and simple networks in culture.

    PubMed

    Dow, J A; Clark, P; Connolly, P; Curtis, A S; Wilkinson, C D

    1987-01-01

    The effects of the topography, adhesiveness and chemistry of surfaces in modulating the behaviour of cells in vivo and in vitro have been extensively researched. However, few natural systems are simple enough to allow straightforward conclusions to be drawn, as many different cues are likely to be present at one time. Microelectronic fabrication, normally employed in making integrated circuits, can produce substrates patterned on scales highly relevant to studies of cell behaviour. In this paper, we describe progress in fabricating simple artificial substrata both at the micrometre and sub-micrometre scales. The former can be considered as models for contact guidance along other cells or axonal processes: the latter, models for guidance along aligned collagen matrices. We have systematically studied the reactions of different cell types to simple cues (steps and grooves). Additionally, it may be possible to produce fine-resolution patterns with differential adhesiveness, or with other cell-specific surface-chemical properties, such as the differential deposition of proteins, e.g. cell adhesion molecules. We also describe early results in using topographic and other cues to guide cells onto patterned metal electrodes, forming simple electrically active networks of controlled design, from which long-term recordings can conveniently be made.

  11. Using single cell cultivation system for on-chip monitoring of the interdivision timer in Chlamydomonas reinhardtii cell cycle

    PubMed Central

    2010-01-01

    Regulation of cell cycle progression in changing environments is vital for cell survival and maintenance, and different regulation mechanisms based on cell size and cell cycle time have been proposed. To determine the mechanism of cell cycle regulation in the unicellular green algae Chlamydomonas reinhardtii, we developed an on-chip single-cell cultivation system that allows for the strict control of the extracellular environment. We divided the Chlamydomonas cell cycle into interdivision and division phases on the basis of changes in cell size and found that, regardless of the amount of photosynthetically active radiation (PAR) and the extent of illumination, the length of the interdivision phase was inversely proportional to the rate of increase of cell volume. Their product remains constant indicating the existence of an 'interdivision timer'. The length of the division phase, in contrast, remained nearly constant. Cells cultivated under light-dark-light conditions did not divide unless they had grown to twice their initial volume during the first light period. This indicates the existence of a 'commitment sizer'. The ratio of the cell volume at the beginning of the division phase to the initial cell volume determined the number of daughter cells, indicating the existence of a 'mitotic sizer'. PMID:20868509

  12. Using single cell cultivation system for on-chip monitoring of the interdivision timer in Chlamydomonas reinhardtii cell cycle.

    PubMed

    Matsumura, Kazunori; Yagi, Toshiki; Hattori, Akihiro; Soloviev, Mikhail; Yasuda, Kenji

    2010-09-25

    Regulation of cell cycle progression in changing environments is vital for cell survival and maintenance, and different regulation mechanisms based on cell size and cell cycle time have been proposed. To determine the mechanism of cell cycle regulation in the unicellular green algae Chlamydomonas reinhardtii, we developed an on-chip single-cell cultivation system that allows for the strict control of the extracellular environment. We divided the Chlamydomonas cell cycle into interdivision and division phases on the basis of changes in cell size and found that, regardless of the amount of photosynthetically active radiation (PAR) and the extent of illumination, the length of the interdivision phase was inversely proportional to the rate of increase of cell volume. Their product remains constant indicating the existence of an 'interdivision timer'. The length of the division phase, in contrast, remained nearly constant. Cells cultivated under light-dark-light conditions did not divide unless they had grown to twice their initial volume during the first light period. This indicates the existence of a 'commitment sizer'. The ratio of the cell volume at the beginning of the division phase to the initial cell volume determined the number of daughter cells, indicating the existence of a 'mitotic sizer'.

  13. Ultra-high throughput detection of single cell β-galactosidase activity in droplets using micro-optical lens array

    NASA Astrophysics Data System (ADS)

    Lim, Jiseok; Vrignon, Jérémy; Gruner, Philipp; Karamitros, Christos S.; Konrad, Manfred; Baret, Jean-Christophe

    2013-11-01

    We demonstrate the use of a hybrid microfluidic-micro-optical system for the screening of enzymatic activity at the single cell level. Escherichia coli β-galactosidase activity is revealed by a fluorogenic assay in 100 pl droplets. Individual droplets containing cells are screened by measuring their fluorescence signal using a high-speed camera. The measurement is parallelized over 100 channels equipped with microlenses and analyzed by image processing. A reinjection rate of 1 ml of emulsion per minute was reached corresponding to more than 105 droplets per second, an analytical throughput larger than those obtained using flow cytometry.

  14. Single-cell nanosurgery.

    PubMed

    Zeigler, Maxwell B; Chiu, Daniel T

    2013-01-01

    This chapter explains the steps necessary to perform laser surgery upon single adherent mammalian cells, where individual organelles are extracted from the cells by optical tweezers and the cells are monitored post-surgery to check their viability. Single-cell laser nanosurgery is used in an increasing range of methodologies because it offers great flexibility. Its main advantages are (a) there is not any physical contact with the cells so they remain in a sterile environment, (b) high spatial selectivity so that single organelles can be extracted from specific areas of individual cells, (c) the method can be conducted in the cell's native media, and (d) in comparison to other techniques that target single cells, such as micromanipulators, laser nanosurgery has a comparatively high throughput.

  15. Measurement of enzyme activity in single cells by voltammetry using a microcell with a positionable dual electrode.

    PubMed

    Gao, Ning; Zhao, Minghui; Zhang, Xiaoli; Jin, Wenrui

    2006-01-01

    The electrochemical single-cell analysis for enzyme activity was developed using microcells on a microcell array coupled with a positionable dual microelectrode. The microcell array with the nanoliter-scale microcells was constructed using simple chemical etching without photolithographic techniques. The positionable dual microelectrodes consisted of the nanometer-to-micrometer-radius Au disk working electrode and a approximately 80-microm-radius Ag/AgCl reference electrode. Peroxidase was chosen as the model enzyme. Factors that concern electrochemical single-cell analysis in microcells such as solution evaporation, interference of soluble oxygen, electrode size, solution volume, and electrode fouling were investigated and discussed. The 20 or 100 nL of detection volume was found to be suitable for peroxidase determination in single neutrophils or single acute promyelocytic leukemia cells without interference from intracellular macromolecules and electrode fouling, when the dual electrode with a 10-microm-radius Au disk working electrode was used. Cells were perforated with digitonin before transferring them into the microcells, to lyse cells easily. The perforated cells were transferred into the microcells by pushing a microscope slide on a drop of the cell suspension on the microcell array. After a single cell in the microcell was lysed using a freeze-thawing technique and allowed to dry, physiological buffer saline containing 2.0 x 10(-3) mol/L hydroquinone and 2.0 x 10(-3) mol/L H2O2 as the substrates of the enzyme-catalyzed reaction was added. The microcell array was positioned in a constant-humidity chamber to prevent evaporation. Then the dual electrode was inserted into the microcell by means of a scanning electrochemical microscope and the product benzoquinone of the enzyme-catalyzed reaction was voltammetrically detected. Peroxidase activity could be quantified using the steady-state current on the voltammogram after subtracting the blank and using the

  16. Parallel single-cell analysis of active caspase-3/7 in apoptotic and non-apoptotic cells.

    PubMed

    Ledvina, Vojtěch; Janečková, Eva; Matalová, Eva; Klepárník, Karel

    2017-01-01

    Analysing the chemical content of individual cells has already been proven to reveal unique information on various biological processes. Single-cell analysis provides more accurate and reliable results for biology and medicine than analyses of extracts from cell populations, where a natural heterogeneity is averaged. To meet the requirements in the research of important biologically active molecules, such as caspases, we have developed a miniaturized device for simultaneous analyses of individual cells. A stainless steel body with a carousel holder enables high-sensitivity parallel detections in eight microvials. The holder is mounted in front of a photomultiplier tube with cooled photocathode working in photon counting mode. The detection of active caspase-3/7, central effector caspases in apoptosis, in single cells is based on the bioluminescence chemistry commercially available as Caspase-Glo(®) 3/7 reagent developed by Promega. Individual cells were captured from a culture medium under microscope and transferred by micromanipulator into detection microvial filled with the reagent. As a result of testing, the limits of detection and quantification were determined to be 0.27/0.86 of active caspase-3/7 content in an average apoptotic cell and 0.46/2.92 for non-apoptotic cells. Application potential of this technology in laboratory diagnostics and related medical research is discussed. Graphical abstract Miniaturized device for simultaneous analyses of individual cells.

  17. The 40-year history of modeling active dendrites in cerebellar Purkinje cells: emergence of the first single cell "community model".

    PubMed

    Bower, James M

    2015-01-01

    The subject of the effects of the active properties of the Purkinje cell dendrite on neuronal function has been an active subject of study for more than 40 years. Somewhat unusually, some of these investigations, from the outset have involved an interacting combination of experimental and model-based techniques. This article recounts that 40-year history, and the view of the functional significance of the active properties of the Purkinje cell dendrite that has emerged. It specifically considers the emergence from these efforts of what is arguably the first single cell "community" model in neuroscience. The article also considers the implications of the development of this model for future studies of the complex properties of neuronal dendrites.

  18. Metabolic-Activity-Based Assessment of Antimicrobial Effects by D2O-Labeled Single-Cell Raman Microspectroscopy.

    PubMed

    Tao, Yifan; Wang, Yun; Huang, Shi; Zhu, Pengfei; Huang, Wei E; Ling, Junqi; Xu, Jian

    2017-04-04

    To combat the spread of antibiotic resistance, methods that quantitatively assess the metabolism-inhibiting effects of drugs in a rapid and culture-independent manner are urgently needed. Here using four oral bacteria as models, we show that heavy water (D2O)-based single-cell Raman microspectroscopy (D2O-Raman) can probe bacterial response to different drugs using the Raman shift at the C-D (carbon-deuterium vibration) band in 2040 to 2300 cm(-1) as a universal biomarker for metabolic activity at single-bacterial-cell resolution. The "minimum inhibitory concentration based on metabolic activity" (MIC-MA), defined as the minimal dose under which the median ΔC-D-ratio at 8 h of drug exposure is ≤0 and the standard deviation (SD) of the ΔC-D ratio among individual cells is ≤0.005, was proposed to evaluate the metabolism-inhibiting efficacy of drugs. In addition, heterogeneity index of MIC-MA (MIC-MA-HI), defined as SD of C-D ratio among individual cells, quantitatively assesses the among-cell heterogeneity of metabolic activity after drug regimens. When exposed to 1× MIC of sodium fluoride (NaF), 1× MIC of chlorhexidine (CHX), or 60× MIC of ampicillin, the cariogenic oral pathogen Streptococcus mutans UA159 ceased propagation yet remained metabolically highly active. This underscores the advantage of MIC-MA over the growth-based MIC in being able to detect the "nongrowing but metabolically active" (NGMA) cells that underlie many latent or recurring infections. Moreover, antibiotic susceptible and resistant S. mutans strains can be readily discriminated at as early as 0.5 h. Thus, D2O-Raman can serve as a universal method for rapid and quantitative assessment of antimicrobial effects based on general metabolic activity at single-cell resolution.

  19. Single-cell telomere-length quantification couples telomere length to meristem activity and stem cell development in Arabidopsis.

    PubMed

    González-García, Mary-Paz; Pavelescu, Irina; Canela, Andrés; Sevillano, Xavier; Leehy, Katherine A; Nelson, Andrew D L; Ibañes, Marta; Shippen, Dorothy E; Blasco, Maria A; Caño-Delgado, Ana I

    2015-05-12

    Telomeres are specialized nucleoprotein caps that protect chromosome ends assuring cell division. Single-cell telomere quantification in animals established a critical role for telomerase in stem cells, yet, in plants, telomere-length quantification has been reported only at the organ level. Here, a quantitative analysis of telomere length of single cells in Arabidopsis root apex uncovered a heterogeneous telomere-length distribution of different cell lineages showing the longest telomeres at the stem cells. The defects in meristem and stem cell renewal observed in tert mutants demonstrate that telomere lengthening by TERT sets a replicative limit in the root meristem. Conversely, the long telomeres of the columella cells and the premature stem cell differentiation plt1,2 mutants suggest that differentiation can prevent telomere erosion. Overall, our results indicate that telomere dynamics are coupled to meristem activity and continuous growth, disclosing a critical association between telomere length, stem cell function, and the extended lifespan of plants. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  20. Diverse activities of viral cis-acting RNA regulatory elements revealed using multicolor, long-term, single-cell imaging.

    PubMed

    Pocock, Ginger M; Zimdars, Laraine L; Yuan, Ming; Eliceiri, Kevin W; Ahlquist, Paul; Sherer, Nathan M

    2017-02-01

    Cis-acting RNA structural elements govern crucial aspects of viral gene expression. How these structures and other posttranscriptional signals affect RNA trafficking and translation in the context of single cells is poorly understood. Herein we describe a multicolor, long-term (>24 h) imaging strategy for measuring integrated aspects of viral RNA regulatory control in individual cells. We apply this strategy to demonstrate differential mRNA trafficking behaviors governed by RNA elements derived from three retroviruses (HIV-1, murine leukemia virus, and Mason-Pfizer monkey virus), two hepadnaviruses (hepatitis B virus and woodchuck hepatitis virus), and an intron-retaining transcript encoded by the cellular NXF1 gene. Striking behaviors include "burst" RNA nuclear export dynamics regulated by HIV-1's Rev response element and the viral Rev protein; transient aggregations of RNAs into discrete foci at or near the nuclear membrane triggered by multiple elements; and a novel, pulsiform RNA export activity regulated by the hepadnaviral posttranscriptional regulatory element. We incorporate single-cell tracking and a data-mining algorithm into our approach to obtain RNA element-specific, high-resolution gene expression signatures. Together these imaging assays constitute a tractable, systems-based platform for studying otherwise difficult to access spatiotemporal features of viral and cellular gene regulation.

  1. Spontaneous Neuronal Activity in Developing Neocortical Networks: From Single Cells to Large-Scale Interactions

    PubMed Central

    Luhmann, Heiko J.; Sinning, Anne; Yang, Jenq-Wei; Reyes-Puerta, Vicente; Stüttgen, Maik C.; Kirischuk, Sergei; Kilb, Werner

    2016-01-01

    Neuronal activity has been shown to be essential for the proper formation of neuronal circuits, affecting developmental processes like neurogenesis, migration, programmed cell death, cellular differentiation, formation of local and long-range axonal connections, synaptic plasticity or myelination. Accordingly, neocortical areas reveal distinct spontaneous and sensory-driven neuronal activity patterns already at early phases of development. At embryonic stages, when immature neurons start to develop voltage-dependent channels, spontaneous activity is highly synchronized within small neuronal networks and governed by electrical synaptic transmission. Subsequently, spontaneous activity patterns become more complex, involve larger networks and propagate over several neocortical areas. The developmental shift from local to large-scale network activity is accompanied by a gradual shift from electrical to chemical synaptic transmission with an initial excitatory action of chloride-gated channels activated by GABA, glycine and taurine. Transient neuronal populations in the subplate (SP) support temporary circuits that play an important role in tuning early neocortical activity and the formation of mature neuronal networks. Thus, early spontaneous activity patterns control the formation of developing networks in sensory cortices, and disturbances of these activity patterns may lead to long-lasting neuronal deficits. PMID:27252626

  2. Monitoring human leukocyte antigen class I molecules by micro-Raman spectroscopy at single-cell level

    NASA Astrophysics Data System (ADS)

    Das, Gobind; La Rocca, Rosanna; Lakshmikanth, Tadepally; Gentile, Francesco; Tallerico, Rossana; Zambetti, Lia P.; Devitt, J.; Candeloro, Patrizio; de Angelis, Francesco; Carbone, Ennio; di Fabrizio, Enzo

    2010-03-01

    Human leukocyte antigen (HLA) class I molecules are formed by three immunoglobulin-like domains (α1, α2, and α3) once folded by peptide and β2-microglobulin show the presence of two α-helix streams and one β-sheet limiting the pocket for the antigenic peptide. The loss of HLA class I expression in tumors and virus-infected cells, on one hand, prevents T cell recognition, while on the other hand, it leads to natural killer (NK) cell mediated cytotoxicity. We propose the possibility of using Raman spectroscopy to measure the relative expression of HLA class I molecules at the single-cell level. Raman spectra are recorded for three cell lines (K562, T2, and T3) and monomers (HLA class I folded, unfolded and peptide+β2-microlobulin refolded) using 830 nm laser line. Our data are consistent with the hypothesis that in the Raman spectra, ranging from 1600 to 1800 cm-1, the intensity variation of cells associated with HLA class I molecules could be measured.

  3. Single Cell Electrical Characterization Techniques.

    PubMed

    Mansor, Muhammad Asraf; Ahmad, Mohd Ridzuan

    2015-06-04

    Electrical properties of living cells have been proven to play significant roles in understanding of various biological activities including disease progression both at the cellular and molecular levels. Since two decades ago, many researchers have developed tools to analyze the cell's electrical states especially in single cell analysis (SCA). In depth analysis and more fully described activities of cell differentiation and cancer can only be accomplished with single cell analysis. This growing interest was supported by the emergence of various microfluidic techniques to fulfill high precisions screening, reduced equipment cost and low analysis time for characterization of the single cell's electrical properties, as compared to classical bulky technique. This paper presents a historical review of single cell electrical properties analysis development from classical techniques to recent advances in microfluidic techniques. Technical details of the different microfluidic techniques are highlighted, and the advantages and limitations of various microfluidic devices are discussed.

  4. Investigating Microbial Activity in Diazotrophic Methane Seep Sediment via Transcript Analysis and Single-Cell FISH-NanoSIMS

    NASA Astrophysics Data System (ADS)

    Dekas, A. E.; Connon, S. A.; Chadwick, G.; Orphan, V. J.

    2012-12-01

    Methane seep microbial ecosystems are phylogenetically diverse and physiologically complex, and require culture-independent techniques to accurately investigate metabolic activity. In the present study we combine an RNA analysis of four key microbial genes with FISH-NanoSIMS analysis of single cells to determine the diversity of nitrogen fixing microorganisms (diazotrophs) present at a deep-sea methane-seeping site, as well as investigate the methane-dependency of a variety of community members. Recently, methane-dependent nitrogen fixation was observed in Mound 12 Costa Rica sediments, and was spatially correlated with the abundance of aggregates of anaerobic methanotrophic archaea (ANME) and sulfate reducing bacterial symbionts (SRB). Combined with the detection of 15N uptake from 15N2 in these aggregates, this suggested that the ANME-SRB aggregates are the primary diazotrophs in seep sediment. However, the diversity of dinitrogenase reductase (nifH) sequences recovered from several deep-sea locales, including Mound 12, suggests a greater diversity of diazotrophs in marine sediment. To investigate the activity of these potential diazotrophs in Mound 12 sediment, we investigated a suite of RNA transcripts in 15N2 incubations in both the presence and absence of methane: nifH, bacterial 16S rRNA, methyl coenzyme M reductase A (mcrA), and adenosine-5'-phosposulfate reductase alpha subunit (aprA). No nifH transcripts were recovered in incubations without methane, consistent with previous measurements lacking 15N2 uptake in the same sediments. The activity of the bacterial community in general, assessed by variable transcription, was also greatly affected by the presence or absence of methane. Single-cell fluorescence in situ hybridization coupled to nanoscale secondary ion mass spectrometry (FISH-NanoSIMS) was employed to confirm diazotrophic activity (15N2 uptake) and protein synthesis (15NH4+ uptake) of particular species implicated as ecologically important by the

  5. Excitability in the p53 network mediates robust signaling with tunable activation thresholds in single cells

    PubMed Central

    Mönke, Gregor; Cristiano, Elena; Finzel, Ana; Friedrich, Dhana; Herzel, Hanspeter; Falcke, Martin; Loewer, Alexander

    2017-01-01

    Cellular signaling systems precisely transmit information in the presence of molecular noise while retaining flexibility to accommodate the needs of individual cells. To understand design principles underlying such versatile signaling, we analyzed the response of the tumor suppressor p53 to varying levels of DNA damage in hundreds of individual cells and observed a switch between distinct signaling modes characterized by isolated pulses and sustained oscillations of p53 accumulation. Guided by dynamic systems theory we show that this requires an excitable network structure comprising positive feedback and provide experimental evidence for its molecular identity. The resulting data-driven model reproduced all features of measured signaling responses and is sufficient to explain their heterogeneity in individual cells. We present evidence that heterogeneity in the levels of the feedback regulator Wip1 sets cell-specific thresholds for p53 activation, providing means to modulate its response through interacting signaling pathways. Our results demonstrate how excitable signaling networks can provide high specificity, sensitivity and robustness while retaining unique possibilities to adjust their function to the physiology of individual cells. PMID:28417973

  6. Stochastic expression of a multiple antibiotic resistance activator confers transient resistance in single cells.

    PubMed

    El Meouche, Imane; Siu, Yik; Dunlop, Mary J

    2016-01-13

    Transient resistance can allow microorganisms to temporarily survive lethal concentrations of antibiotics. This can be accomplished through stochastic mechanisms, where individual cells within a population display diverse phenotypes to hedge against the appearance of an antibiotic. To date, research on transient stochastic resistance has focused primarily on mechanisms where a subpopulation of cells enters a dormant, drug-tolerant state. However, a fundamental question is whether stochastic gene expression can also generate variable resistance levels among growing cells in a population. We hypothesized that stochastic expression of antibiotic-inducible resistance mechanisms might play such a role. To investigate this, we focused on a prototypical example of such a system: the multiple antibiotic resistance activator MarA. Previous studies have shown that induction of MarA can lead to a multidrug resistant phenotype at the population level. We asked whether MarA expression also has a stochastic component, even when uninduced. Time lapse microscopy showed that isogenic cells express heterogeneous, dynamic levels of MarA, which were correlated with transient antibiotic survival. This finding has important clinical implications, as stochastic expression of resistance genes may be widespread, allowing populations to hedge against the sudden appearance of an antibiotic.

  7. MCO Monitoring activity description

    SciTech Connect

    SEXTON, R.A.

    1998-11-09

    Spent Nuclear Fuel remaining from Hanford's N-Reactor operations in the 1970s has been stored under water in the K-Reactor Basins. This fuel will be repackaged, dried and stored in a new facility in the 200E Area. The safety basis for this process of retrieval, drying, and interim storage of the spent fuel has been established. The monitoring of MCOS in dry storage is a currently identified issue in the SNF Project. This plan outlines the key elements of the proposed monitoring activity. Other fuel stored in the K-Reactor Basins, including SPR fuel, will have other monitoring considerations and is not addressed by this activity description.

  8. Direct methods for dynamic monitoring of secretions from single cells by capillary electrophoresis and microscopy with laser-induced native fluorescence detection

    SciTech Connect

    Tong, Wei

    1997-10-08

    Microscale separation and detection methods for real-time monitoring of dynamic cellular processes (e.g., secretion) by capillary electrophoresis (CE) and microscopic imaging were developed. Ultraviolet laser-induced native fluorescence (LINF) provides simple, sensitive and direct detection of neurotransmitters and proteins without any derivatization. An on-column CE-LINF protocol for quantification of the release from single cell was demonstrated. Quantitative measurements of both the amount of insulin released from and the amount remaining in the cell (βTC3) were achieved simultaneously. Secretion of catecholamines (norepinephrine (NE) and epinephrine (E)) from individual bovine adrenal chromaffin cells was determined using the on-column CE-LINF. Direct visualization of the secretion process of individual bovine adrenal chromaffin cells was achieved by LINF imaging microscopy with high temporal and spatial resolution. The secretion of serotonin from individual leech Retzius neurons was directly characterized by LINF microscopy with high spatial resolution.

  9. Single cell wound repair

    PubMed Central

    Abreu-Blanco, Maria Teresa; Verboon, Jeffrey M

    2011-01-01

    Cell wounding is a common event in the life of many cell types, and the capacity of the cell to repair day-to-day wear-and-tear injuries, as well as traumatic ones, is fundamental for maintaining tissue integrity. Cell wounding is most frequent in tissues exposed to high levels of stress. Survival of such plasma membrane disruptions requires rapid resealing to prevent the loss of cytosolic components, to block Ca2+ influx and to avoid cell death. In addition to patching the torn membrane, plasma membrane and cortical cytoskeleton remodeling are required to restore cell function. Although a general understanding of the cell wound repair process is in place, the underlying mechanisms of each step of this response are not yet known. We have developed a model to study single cell wound repair using the early Drosophila embryo. Our system combines genetics and live imaging tools, allowing us to dissect in vivo the dynamics of the single cell wound response. We have shown that cell wound repair in Drosophila requires the coordinated activities of plasma membrane and cytoskeleton components. Furthermore, we identified an unexpected role for E-cadherin as a link between the contractile actomyosin ring and the newly formed plasma membrane plug. PMID:21922041

  10. Single Cell Physiology

    NASA Astrophysics Data System (ADS)

    Neveu, Pierre; Sinha, Deepak Kumar; Kettunen, Petronella; Vriz, Sophie; Jullien, Ludovic; Bensimon, David

    The possibility to control at specific times and specific places the activity of biomolecules (enzymes, transcription factors, RNA, hormones, etc.) is opening up new opportunities in the study of physiological processes at the single cell level in a live organism. Most existing gene expression systems allow for tissue specific induction upon feeding the organism with exogenous inducers (e.g., tetracycline). Local genetic control has earlier been achieved by micro-injection of the relevant inducer/repressor molecule, but this is an invasive and possibly traumatic technique. In this chapter, we present the requirements for a noninvasive optical control of the activity of biomolecules and review the recent advances in this new field of research.

  11. Intracellular pH of Mycobacterium avium subsp. paratuberculosis following exposure to antimicrobial compounds monitored at the single cell level.

    PubMed

    Gaggìa, Francesca; Nielsen, Dennis Sandris; Biavati, Bruno; Siegumfeldt, Henrik

    2010-07-31

    Mycobacterium avium subsp. paratuberculosis (MAP) is the etiologic agent of Johne's disease; moreover, it seems to be implicated in the development of Crohn's disease in humans. In the present study, fluorescence ratio imaging microscopy (FRIM) was used to assess changes in intracellular pH (pH(i)) of one strain of MAP after exposure to nisin and neutralized cell-free supernatants (NCSs) from five bacteriocin-producing lactic acid bacteria (LAB) with known probiotic properties. The evaluation of pH(i) by FRIM provides information about the physiological state of bacterial cells, bypassing the long and problematic incubations needed for methods relying upon growth of MAP such as determination of colony forming units. The FRIM results showed that both nisin and the cell-free supernatant from Lactobacillus plantarum PCA 236 affected the pH(i) of MAP within a few hours. However, monitoring the population for 24h revealed the presence of a subpopulation of cells probably resistant to the antimicrobial compounds tested. Use of nisin and bacteriocin-producing LAB strains could lead to new intervention strategies for the control of MAP based on in vivo application of probiotic cultures as feed additives at farm level.

  12. Fast methods for analysis of neurotransmitters from single cell and monitoring their releases in central nervous system by capillary electrophoresis, fluorescence microscopy and luminescence imaging

    SciTech Connect

    Wang, Ziqiang

    1999-12-10

    Fast methods for separation and detection of important neurotransmitters and the releases in central nervous system (CNS) were developed. Enzyme based immunoassay combined with capillary electrophoresis was used to analyze the contents of amino acid neurotransmitters from single neuron cells. The release of amino acid neurotransmitters from neuron cultures was monitored by laser induced fluorescence imaging method. The release and signal transduction of adenosine triphosphate (ATP) in CNS was studied with sensitive luminescence imaging method. A new dual-enzyme on-column reaction method combined with capillary electrophoresis has been developed for determining the glutamate content in single cells. Detection was based on monitoring the laser-induced fluorescence of the reaction product NADH, and the measured fluorescence intensity was related to the concentration of glutamate in each cell. The detection limit of glutamate is down to 10-8 M level, which is 1 order of magnitude lower than the previously reported detection limit based on similar detection methods. The mass detection limit of a few attomoles is far superior to that of any other reports. Selectivity for glutamate is excellent over most of amino acids. The glutamate content in single human erythrocyte and baby rat brain neurons were determined with this method and results agreed well with literature values.

  13. Identifying contact-mediated, localized toxic effects of MWCNT aggregates on epithelial monolayers: a single-cell monitoring toxicity assay.

    PubMed

    Rotoli, Bianca M; Gatti, Rita; Movia, Dania; Bianchi, Massimiliano G; Di Cristo, Luisana; Fenoglio, Ivana; Sonvico, Fabio; Bergamaschi, Enrico; Prina-Mello, Adriele; Bussolati, Ovidio

    2015-03-01

    Aggregates of multiwalled carbon nanotubes (MWCNT) impair the barrier properties of human airway cell monolayers. To resolve the mechanism of the barrier alteration, monolayers of Calu-3 human airway epithelial cells were exposed to aggregated MWCNT. At the cell-population level, trans-epithelial electrical resistance (TEER) was used as an indicator of barrier competence, caspase activity was assessed with standard biochemical assays, and cell viability was investigated by biochemical techniques and high-throughput screening (HTS) technique based on automated epifluorescence microscopy. At cell level, the response to MWCNT was investigated with confocal microscopy, by evaluating cell death (calcein/propidium iodide (PI)), proliferation (Ki-67), and apoptosis (caspase activity). At the cell-population level, exposure to aggregated MWCNT caused a decrease in TEER, which was not associated with a decrease in cell viability or onset of apoptosis even after an 8-d exposure. In contrast, confocal imaging demonstrated contact with MWCNT aggregates triggered cell death after 24 h of exposure. In the presence of a natural surfactant, both TEER decrease and contact-mediated toxicity were mitigated. With confocal imaging, increased proliferation and apoptosis were detected in Calu-3 cells next to the aggregates. Contact-mediated cytotoxicity was recorded in two additional cell lines (BEAS-2B and A549) derived from human airways. Similar results were confirmed by adopting two additional MWCNT preparations with different physico-chemical features. This indicates MWCNT caused localized damage to airway epithelial monolayers in vitro and altered the apoptotic and proliferative rate of epithelial cells in close proximity to the aggregates. These findings provide evidence on the pathway by which MWCNT aggregates impair airway barrier function, and support the use of imaging techniques as a possible regulatory-decision supporting tool to identify effects of aggregated nanomaterials

  14. Single Cell Electrical Characterization Techniques

    PubMed Central

    Mansor, Muhammad Asraf; Ahmad, Mohd Ridzuan

    2015-01-01

    Electrical properties of living cells have been proven to play significant roles in understanding of various biological activities including disease progression both at the cellular and molecular levels. Since two decades ago, many researchers have developed tools to analyze the cell’s electrical states especially in single cell analysis (SCA). In depth analysis and more fully described activities of cell differentiation and cancer can only be accomplished with single cell analysis. This growing interest was supported by the emergence of various microfluidic techniques to fulfill high precisions screening, reduced equipment cost and low analysis time for characterization of the single cell’s electrical properties, as compared to classical bulky technique. This paper presents a historical review of single cell electrical properties analysis development from classical techniques to recent advances in microfluidic techniques. Technical details of the different microfluidic techniques are highlighted, and the advantages and limitations of various microfluidic devices are discussed. PMID:26053399

  15. Single cell genomics reveals activation signatures of endogenous SCAR's networks in aneuploid human embryos and clinically intractable malignant tumors.

    PubMed

    Glinsky, Gennadi V

    2016-10-10

    Somatic mutations and chromosome instability are hallmarks of genomic aberrations in cancer cells. Aneuploidies represent common manifestations of chromosome instability, which is frequently observed in human embryos and malignant solid tumors. Activation of human endogenous retroviruses (HERV)-derived loci is documented in preimplantation human embryos, hESC, and multiple types of human malignancies. It remains unknown whether the HERV activation may highlight a common molecular pathway contributing to the frequent occurrence of chromosome instability in the early stages of human embryonic development and the emergence of genomic aberrations in cancer. Single cell RNA sequencing analysis of human preimplantation embryos reveals activation of specific LTR7/HERVH loci during the transition from the oocytes to zygotes and identifies HERVH network signatures associated with the aneuploidy in human embryos. The correlation patterns' analysis links transcriptome signatures of the HERVH network activation of the in vivo matured human oocytes with gene expression profiles of clinical samples of prostate tumors supporting the existence of a cancer progression pathway from putative precursor lesions (prostatic intraepithelial neoplasia) to localized and metastatic prostate cancers. Tracking signatures of HERVH networks' activation in tumor samples from cancer patients with known long-term therapy outcomes enabled patients' stratification into sub-groups with markedly distinct likelihoods of therapy failure and death from cancer. Genome-wide analyses of human-specific genetic elements of stem cell-associated retroviruses (SCARs)-regulated networks in 12,093 clinical tumor samples across 29 cancer types revealed pan-cancer genomic signatures of clinically-lethal therapy resistant disease defined by the presence of somatic non-silent mutations (SNMs), gene-level copy number changes, and transcripts and proteins' expression of SCARs-regulated host genes. More than 73% of all

  16. Small Active Radiation Monitor

    NASA Technical Reports Server (NTRS)

    Badhwar, Gautam D.

    2004-01-01

    A device, named small active radiation monitor, allows on-orbit evaluations during periods of increased radiation, after extravehicular activities, or at predesignated times for crews on such long-duration space missions as on the International Space Station. It also permits direct evaluation of biological doses, a task now performed using a combination of measurements and potentially inaccurate simulations. Indeed the new monitor can measure a full array of radiation levels, from soft x-rays to hard galactic cosmic-ray particles. With refinement, it will benefit commercial (nuclear power-plant workers, airline pilots, medical technicians, physicians/dentists, and others) and military personnel as well as the astronauts for whom thermoluminescent dosimeters are inadequate. Civilian and military personnel have long since graduated from film badges to thermoluminescent dosimeters. Once used, most dosimeters must be returned to a central facility for processing, a step that can take days or even weeks. While this suffices for radiation workers for whom exposure levels are typically very low and of brief duration, it does not work for astronauts. Even in emergencies and using express mail, the results can often be delayed by as much as 24 hours. Electronic dosimeters, which are the size of electronic oral thermometers, and tattlers, small electronic dosimeters that sound an alarm when the dose/dose rate exceeds preset values, are also used but suffer disadvantages similar to those of thermoluminescent dosimeters. None of these devices fully answers the need of rapid monitoring during the space missions. Instead, radiation is monitored by passive detectors, which are read out after the missions. Unfortunately, these detectors measure only the absorbed dose and not the biologically relevant dose equivalent. The new monitor provides a real-time readout, a time history of radiation exposures (both absorbed dose and biologically relevant dose equivalent), and a count of the

  17. Genome-Wide Single-Cell Analysis of Recombination Activity and de novo Mutation Rates in Human Sperm

    PubMed Central

    Wang, Jianbin; Fan, H. Christina; Behr, Barry; Quake, Stephen R.

    2012-01-01

    SUMMARY Meiotic recombination and de novo mutation are the two main contributions towards gamete genome diversity, and many questions remain about how an individual human’s genome is edited by these two processes. Here, we describe a high-throughput method for single-cell whole-genome analysis which was used to measure the genomic diversity in one individual’s gamete genomes. A microfluidic system was used for highly parallel sample processing and to minimize non-specific amplification. High-density genotyping results from 91 single cells were used to create a personal recombination map, which was consistent with population-wide data at low resolution but revealed significant differences from pedigree data at higher resolution. We used the data to test for meiotic drive and found evidence for gene conversion. High throughput sequencing on 31 single cells was used to measure the frequency of large-scale genome instability, and deeper sequencing of eight single cells revealed de novo mutation rates with distinct characteristics. PMID:22817899

  18. Visualization of RelB expression and activation at the single-cell level during dendritic cell maturation in Relb-Venus knock-in mice.

    PubMed

    Seki, Takao; Yamamoto, Mami; Taguchi, Yuu; Miyauchi, Maki; Akiyama, Nobuko; Yamaguchi, Noritaka; Gohda, Jin; Akiyama, Taishin; Inoue, Jun-ichiro

    2015-12-01

    RelB is activated by the non-canonical NF-κB pathway, which is crucial for immunity by establishing lymphoid organogenesis and B-cell and dendritic cell (DC) maturation. To elucidate the mechanism of the RelB-mediated immune cell maturation, a precise understanding of the relationship between cell maturation and RelB expression and activation at the single-cell level is required. Therefore, we generated knock-in mice expressing a fusion protein between RelB and fluorescent protein (RelB-Venus) from the Relb locus. The Relb(Venus/Venus) mice developed without any abnormalities observed in the Relb(-/-) mice, allowing us to monitor RelB-Venus expression and nuclear localization as RelB expression and activation. Relb(Venus/Venus) DC analyses revealed that DCs consist of RelB(-), RelB(low) and RelB(high) populations. The RelB(high) population, which included mature DCs with projections, displayed RelB nuclear localization, whereas RelB in the RelB(low) population was in the cytoplasm. Although both the RelB(low) and RelB(-) populations barely showed projections, MHC II and co-stimulatory molecule expression were higher in the RelB(low) than in the RelB(-) splenic conventional DCs. Taken together, our results identify the RelB(low) population as a possible novel intermediate maturation stage of cDCs and the Relb(Venus/Venus) mice as a useful tool to analyse the dynamic regulation of the non-canonical NF-κB pathway.

  19. Single cell analysis of low-power laser irradiation-induced activation of signaling pathway in cell proliferation

    NASA Astrophysics Data System (ADS)

    Xing, Da; Gao, Xuejuan

    2007-02-01

    Low-power laser irradiation (LPLI) has been shown to promote cell proliferation in various cell types, yet the mechanism of which has not been fully clarified. Investigating the signaling pathways involved in the laser irradiation is important for understanding these processes. The small G protein Ras works as a binary switch in many important intracellular signaling pathways and, therefore, has been one of the focal targets of signal-transduction investigations and drug development. The Ras/Raf/MEK/ERK (extracellular-signal-regulated kinase) signaling pathway is a network that governs proliferation, differentiation and cell survival. Recent studies suggest that Ras/Raf signaling pathway is involved in the LPLI-induced cell proliferation. On the other hand, Protein kinase Cs (PKCs), the Ca 2+ activated, phospholipid-dependent serine/threonine protein kinases, have been recently presumed to be involved in the regulation of cell proliferation induced by LPLI. In this report, to monitor the direct activations of Ras and PKCs after LPLI treatment in living cells in real time, Raichu-Ras reporter and C kinase activity reporter (CKAR) were utilized, both of which were constructed based on fluorescence resonance energy transfer (FRET) technique. The direct activation of Ras is predominantly initiated from the different microdomains of the plasma membrane. The results are monitored during cell proliferation induced by LPLI (0.8 J/cm2) in serum-starved COS-7 cells expressing Raichu-Ras reporter using FRET imaging on laser scanning confocal microscope. Furthermore, the increasing activation of PKCs is also monitored during cell proliferation induced by LPLI (0.8 J/cm2) in serum-starved human lung adenocarcinoma cells (ASTC-a-1) expressing CKAR reporter using the similar way. Taken together, the dynamic increases of H-Ras and PKCs activities are observed during the processes of cell proliferation induced by LPLI.

  20. Organic Cyanide Decorated SERS Active Nanopipettes for Quantitative Detection of Hemeproteins and Fe(3+) in Single Cells.

    PubMed

    Hanif, Sumaira; Liu, Hailing; Chen, Ming; Muhammad, Pir; Zhou, Yue; Cao, Jiao; Ahmed, Saud Asif; Xu, Jingjuan; Xia, Xinghua; Chen, Hongyuan; Wang, Kang

    2017-02-21

    It is challenging to develop a robust nanoprobe for real-time operational and accurate detection of heavy metals in single cells. Fe-CN coordination chemistry has been well studied to determine the structural characteristics of hemeproteins by different techniques. However, the frequently used cyanide ligands are inorganic molecules that release cyanide anion under particular conditions and cause cyanide poisoning. In the present study, organic cyanide (4-mercaptobenzonitrile, MBN) was utilized for the first time in developing a facile nanoprobe based on surface-enhanced Raman scattering (SERS) for quantitative detection of hemeproteins (oxy-Hb) and trivalent iron (Fe(3+)) ions. The nanoprobe prepared by coating the glass capillary tip (100 nm) with a thin gold film, which enables highly localized study in living cell system. The cyanide stretching vibration in MBN was highly sensitive and selective to Fe(3+) and oxy-Hb with excellent binding affinity (Kd 0.4 pM and 0.1 nM, respectively). The high sensitivity of the nanoprobe to analyte (Fe(3+)) was attributed to the two adsorption conformations (-SH and -CN) of MBN to the gold surface. Therefore, MBN showed an exceptional dual-peak (2126 and 2225 cm(-1)) behavior. Furthermore, the special Raman peaks of cyanide in 2100-2300 cm(-1) (silent region of SERS spectra) are distinguishable from other biomolecules characteristic peaks. The selective detection of Fe(3+) in both free and protein-bound states in aqueous solution is achieved with 0.1 pM and 0.08 μM levels of detection limits, respectively. Furthermore, practical applicability of fabricated nanoprobe was validated by detection of free Fe(3+) in pretreated living HeLa cells by direct insertion of a SERS active nanoprobe. Regarding the appropriate precision, good reproducibility (relative standard deviation, RSD 7.2-7.6%), and recyclability (retain good Raman intensity even after three renewing cycles) of the method, the developed sensing strategy on a

  1. Rare Earth Ion Mediated Fluorescence Accumulation on a Single Microbead: An Ultrasensitive Strategy for the Detection of Protein Kinase Activity at the Single-Cell Level.

    PubMed

    Zhang, Xiaobo; Liu, Chenghui; Wang, Honghong; Wang, Hui; Li, Zhengping

    2015-12-07

    A single microbead-based fluorescence imaging (SBFI) strategy that enables detection of protein kinase activity from single cell lysates is reported. We systematically investigated the ability of various rare earth (RE) ions, immobilized on the microbead, for specific capturing of kinase-induced phosphopeptides, and Dy(3+) was found to be the most prominent one. Through the efficient concentration of kinase-induced fluorescent phosphopeptides on a Dy(3+) -functionalized single microbead, kinase activity can be detected and quantified by reading the fluorescence on the microbead with a confocal fluorescence microscope. Owing to the extremely specific recognition of Dy(3+) towards phosphopeptides and the highly-concentrated fluorescence accumulation on only one microbead, ultrahigh sensitivity has been achieved for the SBFI strategy which allows direct kinase analysis at the single-cell level. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. Monitoring international nuclear activity

    SciTech Connect

    Firestone, R.B.

    2006-05-19

    The LBNL Table of Isotopes website provides primary nuclearinformation to>150,000 different users annually. We have developedthe covert technology to identify users by IP address and country todetermine the kinds of nuclear information they are retrieving. Wepropose to develop pattern recognition software to provide an earlywarning system to identify Unusual nuclear activity by country or regionSpecific nuclear/radioactive material interests We have monitored nuclearinformation for over two years and provide this information to the FBIand LLNL. Intelligence is gleaned from the website log files. Thisproposal would expand our reporting capabilities.

  3. Progress toward single cell metabolomics

    PubMed Central

    Rubakhin, Stanislav S.; Lanni, Eric J.; Sweedler, Jonathan V.

    2012-01-01

    The metabolome refers to the entire set of small molecules, or metabolites, within a biological sample. These molecules are involved in many fundamental intracellular functions and reflect the cell’s physiological condition. The ability to detect and identify metabolites and determine and monitor their amounts at the single cell level enables an exciting range of studies of biological variation and functional heterogeneity between cells, even within a presumably homogenous cell population. Significant progress has been made in the development and application of bioanalytical tools for single cell metabolomics based on mass spectrometry, microfluidics, and capillary separations. Remarkable improvements in the sensitivity, specificity, and throughput of these approaches enable investigation of multiple metabolites simultaneously in a range of individual cell samples. PMID:23246232

  4. Microfluidics for single-cell genetic analysis.

    PubMed

    Thompson, A M; Paguirigan, A L; Kreutz, J E; Radich, J P; Chiu, D T

    2014-09-07

    The ability to correlate single-cell genetic information to cellular phenotypes will provide the kind of detailed insight into human physiology and disease pathways that is not possible to infer from bulk cell analysis. Microfluidic technologies are attractive for single-cell manipulation due to precise handling and low risk of contamination. Additionally, microfluidic single-cell techniques can allow for high-throughput and detailed genetic analyses that increase accuracy and decrease reagent cost compared to bulk techniques. Incorporating these microfluidic platforms into research and clinical laboratory workflows can fill an unmet need in biology, delivering the highly accurate, highly informative data necessary to develop new therapies and monitor patient outcomes. In this perspective, we describe the current and potential future uses of microfluidics at all stages of single-cell genetic analysis, including cell enrichment and capture, single-cell compartmentalization and manipulation, and detection and analyses.

  5. Single-Cell Metabolomics.

    PubMed

    Emara, Samy; Amer, Sara; Ali, Ahmed; Abouleila, Yasmine; Oga, April; Masujima, Tsutomu

    2017-01-01

    The dynamics of a cell is always changing. Cells move, divide, communicate, adapt, and are always reacting to their surroundings non-synchronously. Currently, single-cell metabolomics has become the leading field in understanding the phenotypical variations between them, but sample volumes, low analyte concentrations, and validating gentle sample techniques have proven great barriers toward achieving accurate and complete metabolomics profiling. Certainly, advanced technologies such as nanodevices and microfluidic arrays are making great progress, and analytical techniques, such as matrix-assisted laser desorption ionization (MALDI), are gaining popularity with high-throughput methodology. Nevertheless, live single-cell mass spectrometry (LCSMS) values the sample quality and precision, turning once theoretical speculation into present-day applications in a variety of fields, including those of medicine, pharmaceutical, and agricultural industries. While there is still room for much improvement, it is clear that the metabolomics field is progressing toward analysis and discoveries at the single-cell level.

  6. Single Cell Oncogenesis

    NASA Astrophysics Data System (ADS)

    Lu, Xin

    It is believed that cancer originates from a single cell that has gone through generations of evolution of genetic and epigenetic changes that associate with the hallmarks of cancer. In some cancers such as various types of leukemia, cancer is clonal. Yet in other cancers like glioblastoma (GBM), there is tremendous tumor heterogeneity that is likely to be caused by simultaneous evolution of multiple subclones within the same tissue. It is obvious that understanding how a single cell develops into a clonal tumor upon genetic alterations, at molecular and cellular levels, holds the key to the real appreciation of tumor etiology and ultimate solution for therapeutics. Surprisingly very little is known about the process of spontaneous tumorigenesis from single cells in human or vertebrate animal models. The main reason is the lack of technology to track the natural process of single cell changes from a homeostatic state to a progressively cancerous state. Recently, we developed a patented compound, photoactivatable (''caged'') tamoxifen analogue 4-OHC and associated technique called optochemogenetic switch (OCG switch), which we believe opens the opportunity to address this urgent biological as well as clinical question about cancer. We propose to combine OCG switch with genetically engineered mouse models of head and neck squamous cell carcinoma and high grade astrocytoma (including GBM) to study how single cells, when transformed through acute loss of tumor suppressor genes PTEN and TP53 and gain of oncogenic KRAS, can develop into tumor colonies with cellular and molecular heterogeneity in these tissues. The abstract is for my invited talk in session ``Beyond Darwin: Evolution in Single Cells'' 3/18/2016 11:15 AM.

  7. Spatiotemporally controlled single cell sonoporation

    PubMed Central

    Fan, Zhenzhen; Liu, Haiyan; Mayer, Michael; Deng, Cheri X.

    2012-01-01

    This paper presents unique approaches to enable control and quantification of ultrasound-mediated cell membrane disruption, or sonoporation, at the single-cell level. Ultrasound excitation of microbubbles that were targeted to the plasma membrane of HEK-293 cells generated spatially and temporally controlled membrane disruption with high repeatability. Using whole-cell patch clamp recording combined with fluorescence microscopy, we obtained time-resolved measurements of single-cell sonoporation and quantified the size and resealing rate of pores. We measured the intracellular diffusion coefficient of cytoplasmic RNA/DNA from sonoporation-induced transport of an intercalating fluorescent dye into and within single cells. We achieved spatiotemporally controlled delivery with subcellular precision and calcium signaling in targeted cells by selective excitation of microbubbles. Finally, we utilized sonoporation to deliver calcein, a membrane-impermeant substrate of multidrug resistance protein-1 (MRP1), into HEK-MRP1 cells, which overexpress MRP1, and monitored the calcein efflux by MRP1. This approach made it possible to measure the efflux rate in individual cells and to compare it directly to the efflux rate in parental control cells that do not express MRP1. PMID:23012425

  8. Single-cell analysis of HIV-1 transcriptional activity reveals expression of proviruses in expanded clones during ART.

    PubMed

    Wiegand, Ann; Spindler, Jonathan; Hong, Feiyu F; Shao, Wei; Cyktor, Joshua C; Cillo, Anthony R; Halvas, Elias K; Coffin, John M; Mellors, John W; Kearney, Mary F

    2017-05-02

    Little is known about the fraction of human immunodeficiency virus type 1 (HIV-1) proviruses that express unspliced viral RNA in vivo or about the levels of HIV RNA expression within single infected cells. We developed a sensitive cell-associated HIV RNA and DNA single-genome sequencing (CARD-SGS) method to investigate fractional proviral expression of HIV RNA (1.3-kb fragment of p6, protease, and reverse transcriptase) and the levels of HIV RNA in single HIV-infected cells from blood samples obtained from individuals with viremia or individuals on long-term suppressive antiretroviral therapy (ART). Spiking experiments show that the CARD-SGS method can detect a single cell expressing HIV RNA. Applying CARD-SGS to blood mononuclear cells in six samples from four HIV-infected donors (one with viremia and not on ART and three with viremia suppressed on ART) revealed that an average of 7% of proviruses (range: 2-18%) expressed HIV RNA. Levels of expression varied from one to 62 HIV RNA molecules per cell (median of 1). CARD-SGS also revealed the frequent expression of identical HIV RNA sequences across multiple single cells and across multiple time points in donors on suppressive ART consistent with constitutive expression of HIV RNA in infected cell clones. Defective proviruses were found to express HIV RNA at levels similar to those proviruses that had no obvious defects. CARD-SGS is a useful tool to characterize fractional proviral expression in single infected cells that persist despite ART and to assess the impact of experimental interventions on proviral populations and their expression.

  9. Magnetic levitation of single cells

    PubMed Central

    Durmus, Naside Gozde; Tekin, H. Cumhur; Guven, Sinan; Sridhar, Kaushik; Arslan Yildiz, Ahu; Calibasi, Gizem; Davis, Ronald W.; Steinmetz, Lars M.; Demirci, Utkan

    2015-01-01

    Several cellular events cause permanent or transient changes in inherent magnetic and density properties of cells. Characterizing these changes in cell populations is crucial to understand cellular heterogeneity in cancer, immune response, infectious diseases, drug resistance, and evolution. Although magnetic levitation has previously been used for macroscale objects, its use in life sciences has been hindered by the inability to levitate microscale objects and by the toxicity of metal salts previously applied for levitation. Here, we use magnetic levitation principles for biological characterization and monitoring of cells and cellular events. We demonstrate that each cell type (i.e., cancer, blood, bacteria, and yeast) has a characteristic levitation profile, which we distinguish at an unprecedented resolution of 1 × 10−4 g⋅mL−1. We have identified unique differences in levitation and density blueprints between breast, esophageal, colorectal, and nonsmall cell lung cancer cell lines, as well as heterogeneity within these seemingly homogenous cell populations. Furthermore, we demonstrate that changes in cellular density and levitation profiles can be monitored in real time at single-cell resolution, allowing quantification of heterogeneous temporal responses of each cell to environmental stressors. These data establish density as a powerful biomarker for investigating living systems and their responses. Thereby, our method enables rapid, density-based imaging and profiling of single cells with intriguing applications, such as label-free identification and monitoring of heterogeneous biological changes under various physiological conditions, including antibiotic or cancer treatment in personalized medicine. PMID:26124131

  10. Magnetic levitation of single cells.

    PubMed

    Durmus, Naside Gozde; Tekin, H Cumhur; Guven, Sinan; Sridhar, Kaushik; Arslan Yildiz, Ahu; Calibasi, Gizem; Ghiran, Ionita; Davis, Ronald W; Steinmetz, Lars M; Demirci, Utkan

    2015-07-14

    Several cellular events cause permanent or transient changes in inherent magnetic and density properties of cells. Characterizing these changes in cell populations is crucial to understand cellular heterogeneity in cancer, immune response, infectious diseases, drug resistance, and evolution. Although magnetic levitation has previously been used for macroscale objects, its use in life sciences has been hindered by the inability to levitate microscale objects and by the toxicity of metal salts previously applied for levitation. Here, we use magnetic levitation principles for biological characterization and monitoring of cells and cellular events. We demonstrate that each cell type (i.e., cancer, blood, bacteria, and yeast) has a characteristic levitation profile, which we distinguish at an unprecedented resolution of 1 × 10(-4) g ⋅ mL(-1). We have identified unique differences in levitation and density blueprints between breast, esophageal, colorectal, and nonsmall cell lung cancer cell lines, as well as heterogeneity within these seemingly homogenous cell populations. Furthermore, we demonstrate that changes in cellular density and levitation profiles can be monitored in real time at single-cell resolution, allowing quantification of heterogeneous temporal responses of each cell to environmental stressors. These data establish density as a powerful biomarker for investigating living systems and their responses. Thereby, our method enables rapid, density-based imaging and profiling of single cells with intriguing applications, such as label-free identification and monitoring of heterogeneous biological changes under various physiological conditions, including antibiotic or cancer treatment in personalized medicine.

  11. SERS assay of telomerase activity at single-cell level and colon cancer tissues via quadratic signal amplification.

    PubMed

    Shi, Muling; Zheng, Jing; Liu, Changhui; Tan, Guixiang; Qing, Zhihe; Yang, Sheng; Yang, Jinfeng; Tan, Yongjun; Yang, Ronghua

    2016-03-15

    As an important biomarker and therapeutic target, telomerase has attracted extensive attention concerning its detection and monitoring. Recently, enzyme-assisted amplification approaches have provided useful platforms for the telomerase activity detection, however, further improvement in sensitivity is still hindered by the single-step signal amplification. Herein, we develop a quadratic signal amplification strategy for ultrasensitive surface-enhanced Raman scattering (SERS) detection of telomerase activity. The central idea of our design is using telomerase-induced silver nanoparticles (AgNPs) assembly and silver ions (Ag(+))-mediated cascade amplification. In our approach, each telomerase-aided DNA sequence extension could trigger the formation of a long double-stranded DNA (dsDNA), making numerous AgNPs assembling along with this long strand through specific Ag-S bond, to form a primary amplification element. For secondary amplification, each conjugated AgNP was dissolved into Ag(+), which can effectively induce the 4-aminobenzenethiol (4-ABT) modified gold nanoparticles (AuNPs@4-ABT) to undergo aggregation to form numerous "hot-spots". Through quadratic amplifications, a limit of detection down to single HeLa cell was achieved. More importantly, this method demonstrated good performance when applied to tissues from colon cancer patients, which exhibits great potential in the practical application of telomerase-based cancer diagnosis in early stages. To demonstrate the potential in screening the telomerase inhibitors and telomerase-targeted drugs, the proposed design is successfully employed to measure the inhibition of telomerase activity by 3'-azido-3'-deoxythymidine. Copyright © 2015 Elsevier B.V. All rights reserved.

  12. Culture-independent method for identification of microbial enzyme-encoding genes by activity-based single-cell sequencing using a water-in-oil microdroplet platform.

    PubMed

    Nakamura, Kazuki; Iizuka, Ryo; Nishi, Shinro; Yoshida, Takao; Hatada, Yuji; Takaki, Yoshihiro; Iguchi, Ayaka; Yoon, Dong Hyun; Sekiguchi, Tetsushi; Shoji, Shuichi; Funatsu, Takashi

    2016-02-26

    Environmental microbes are a great source of industrially valuable enzymes with potent and unique catalytic activities. Unfortunately, the majority of microbes remain unculturable and thus are not accessible by culture-based methods. Recently, culture-independent metagenomic approaches have been successfully applied, opening access to untapped genetic resources. Here we present a methodological approach for the identification of genes that encode metabolically active enzymes in environmental microbes in a culture-independent manner. Our method is based on activity-based single-cell sequencing, which focuses on microbial cells showing specific enzymatic activities. First, at the single-cell level, environmental microbes were encapsulated in water-in-oil microdroplets with a fluorogenic substrate for the target enzyme to screen for microdroplets that contain microbially active cells. Second, the microbial cells were recovered and subjected to whole genome amplification. Finally, the amplified genomes were sequenced to identify the genes encoding target enzymes. Employing this method, we successfully identified 14 novel β-glucosidase genes from uncultured bacterial cells in marine samples. Our method contributes to the screening and identification of genes encoding industrially valuable enzymes.

  13. Culture-independent method for identification of microbial enzyme-encoding genes by activity-based single-cell sequencing using a water-in-oil microdroplet platform

    PubMed Central

    Nakamura, Kazuki; Iizuka, Ryo; Nishi, Shinro; Yoshida, Takao; Hatada, Yuji; Takaki, Yoshihiro; Iguchi, Ayaka; Yoon, Dong Hyun; Sekiguchi, Tetsushi; Shoji, Shuichi; Funatsu, Takashi

    2016-01-01

    Environmental microbes are a great source of industrially valuable enzymes with potent and unique catalytic activities. Unfortunately, the majority of microbes remain unculturable and thus are not accessible by culture-based methods. Recently, culture-independent metagenomic approaches have been successfully applied, opening access to untapped genetic resources. Here we present a methodological approach for the identification of genes that encode metabolically active enzymes in environmental microbes in a culture-independent manner. Our method is based on activity-based single-cell sequencing, which focuses on microbial cells showing specific enzymatic activities. First, at the single-cell level, environmental microbes were encapsulated in water-in-oil microdroplets with a fluorogenic substrate for the target enzyme to screen for microdroplets that contain microbially active cells. Second, the microbial cells were recovered and subjected to whole genome amplification. Finally, the amplified genomes were sequenced to identify the genes encoding target enzymes. Employing this method, we successfully identified 14 novel β-glucosidase genes from uncultured bacterial cells in marine samples. Our method contributes to the screening and identification of genes encoding industrially valuable enzymes. PMID:26915788

  14. The 40-year history of modeling active dendrites in cerebellar Purkinje cells: emergence of the first single cell “community model”

    PubMed Central

    Bower, James M.

    2015-01-01

    The subject of the effects of the active properties of the Purkinje cell dendrite on neuronal function has been an active subject of study for more than 40 years. Somewhat unusually, some of these investigations, from the outset have involved an interacting combination of experimental and model-based techniques. This article recounts that 40-year history, and the view of the functional significance of the active properties of the Purkinje cell dendrite that has emerged. It specifically considers the emergence from these efforts of what is arguably the first single cell “community” model in neuroscience. The article also considers the implications of the development of this model for future studies of the complex properties of neuronal dendrites. PMID:26539104

  15. Tumor Heterogeneity, Single-Cell Sequencing, and Drug Resistance

    PubMed Central

    Schmidt, Felix; Efferth, Thomas

    2016-01-01

    Tumor heterogeneity has been compared with Darwinian evolution and survival of the fittest. The evolutionary ecosystem of tumors consisting of heterogeneous tumor cell populations represents a considerable challenge to tumor therapy, since all genetically and phenotypically different subpopulations have to be efficiently killed by therapy. Otherwise, even small surviving subpopulations may cause repopulation and refractory tumors. Single-cell sequencing allows for a better understanding of the genomic principles of tumor heterogeneity and represents the basis for more successful tumor treatments. The isolation and sequencing of single tumor cells still represents a considerable technical challenge and consists of three major steps: (1) single cell isolation (e.g., by laser-capture microdissection), fluorescence-activated cell sorting, micromanipulation, whole genome amplification (e.g., with the help of Phi29 DNA polymerase), and transcriptome-wide next generation sequencing technologies (e.g., 454 pyrosequencing, Illumina sequencing, and other systems). Data demonstrating the feasibility of single-cell sequencing for monitoring the emergence of drug-resistant cell clones in patient samples are discussed herein. It is envisioned that single-cell sequencing will be a valuable asset to assist the design of regimens for personalized tumor therapies based on tumor subpopulation-specific genetic alterations in individual patients. PMID:27322289

  16. Tumor Heterogeneity, Single-Cell Sequencing, and Drug Resistance.

    PubMed

    Schmidt, Felix; Efferth, Thomas

    2016-06-16

    Tumor heterogeneity has been compared with Darwinian evolution and survival of the fittest. The evolutionary ecosystem of tumors consisting of heterogeneous tumor cell populations represents a considerable challenge to tumor therapy, since all genetically and phenotypically different subpopulations have to be efficiently killed by therapy. Otherwise, even small surviving subpopulations may cause repopulation and refractory tumors. Single-cell sequencing allows for a better understanding of the genomic principles of tumor heterogeneity and represents the basis for more successful tumor treatments. The isolation and sequencing of single tumor cells still represents a considerable technical challenge and consists of three major steps: (1) single cell isolation (e.g., by laser-capture microdissection), fluorescence-activated cell sorting, micromanipulation, whole genome amplification (e.g., with the help of Phi29 DNA polymerase), and transcriptome-wide next generation sequencing technologies (e.g., 454 pyrosequencing, Illumina sequencing, and other systems). Data demonstrating the feasibility of single-cell sequencing for monitoring the emergence of drug-resistant cell clones in patient samples are discussed herein. It is envisioned that single-cell sequencing will be a valuable asset to assist the design of regimens for personalized tumor therapies based on tumor subpopulation-specific genetic alterations in individual patients.

  17. Single cell profiling of surface carbohydrates on Bacillus cereus

    PubMed Central

    Wang, Congzhou; Ehrhardt, Christopher J.; Yadavalli, Vamsi K.

    2015-01-01

    Cell surface carbohydrates are important to various bacterial activities and functions. It is well known that different types of Bacillus display heterogeneity of surface carbohydrate compositions, but detection of their presence, quantitation and estimation of variation at the single cell level have not been previously solved. Here, using atomic force microscopy (AFM)-based recognition force mapping coupled with lectin probes, the specific carbohydrate distributions of N-acetylglucosamine and mannose/glucose were detected, mapped and quantified on single B. cereus surfaces at the nanoscale across the entire cell. Further, the changes of the surface carbohydrate compositions from the vegetative cell to spore were shown. These results demonstrate AFM-based ‘recognition force mapping’ as a versatile platform to quantitatively detect and spatially map key bacterial surface biomarkers (such as carbohydrate compositions), and monitor in situ changes in surface biochemical properties during intracellular activities at the single cell level. PMID:25505137

  18. Fluidic Logic Used in a Systems Approach to Enable Integrated Single-Cell Functional Analysis

    PubMed Central

    Ramalingam, Naveen; Fowler, Brian; Szpankowski, Lukasz; Leyrat, Anne A.; Hukari, Kyle; Maung, Myo Thu; Yorza, Wiganda; Norris, Michael; Cesar, Chris; Shuga, Joe; Gonzales, Michael L.; Sanada, Chad D.; Wang, Xiaohui; Yeung, Rudy; Hwang, Win; Axsom, Justin; Devaraju, Naga Sai Gopi Krishna; Angeles, Ninez Delos; Greene, Cassandra; Zhou, Ming-Fang; Ong, Eng-Seng; Poh, Chang-Chee; Lam, Marcos; Choi, Henry; Htoo, Zaw; Lee, Leo; Chin, Chee-Sing; Shen, Zhong-Wei; Lu, Chong T.; Holcomb, Ilona; Ooi, Aik; Stolarczyk, Craig; Shuga, Tony; Livak, Kenneth J.; Larsen, Cate; Unger, Marc; West, Jay A. A.

    2016-01-01

    The study of single cells has evolved over the past several years to include expression and genomic analysis of an increasing number of single cells. Several studies have demonstrated wide spread variation and heterogeneity within cell populations of similar phenotype. While the characterization of these populations will likely set the foundation for our understanding of genomic- and expression-based diversity, it will not be able to link the functional differences of a single cell to its underlying genomic structure and activity. Currently, it is difficult to perturb single cells in a controlled environment, monitor and measure the response due to perturbation, and link these response measurements to downstream genomic and transcriptomic analysis. In order to address this challenge, we developed a platform to integrate and miniaturize many of the experimental steps required to study single-cell function. The heart of this platform is an elastomer-based integrated fluidic circuit that uses fluidic logic to select and sequester specific single cells based on a phenotypic trait for downstream experimentation. Experiments with sequestered cells that have been performed include on-chip culture, exposure to various stimulants, and post-exposure image-based response analysis, followed by preparation of the mRNA transcriptome for massively parallel sequencing analysis. The flexible system embodies experimental design and execution that enable routine functional studies of single cells. PMID:27709111

  19. Monitoring of Single-Cell Responses in the Optic Tectum of Adult Zebrafish with Dextran-Coupled Calcium Dyes Delivered via Local Electroporation

    PubMed Central

    Kassing, Vanessa

    2013-01-01

    The zebrafish (Danio rerio) has become one of the major animal models for in vivo examination of sensory and neuronal computation. Similar to Xenopus tadpoles neural activity in the optic tectum, the major region controlling visually guided behavior, can be examined in zebrafish larvae by optical imaging. Prerequisites of these approaches are usually the transparency of larvae up to a certain age and the use of two-photon microscopy. This principle of fluorescence excitation was necessary to suppress crosstalk between signals from individual neurons, which is a critical issue when using membrane-permeant dyes. This makes the equipment to study neuronal processing costly and limits the approach to the study of larvae. Thus there is lack of knowledge about the properties of neurons in the optic tectum of adult animals. We established a procedure to circumvent these problems, enabling in vivo calcium imaging in the optic tectum of adult zebrafish. Following local application of dextran-coupled dyes single-neuron activity of adult zebrafish can be monitored with conventional widefield microscopy, because dye labeling remains restricted to tens of neurons or less. Among the neurons characterized with our technique we found neurons that were selective for a certain pattern orientation as well as neurons that responded in a direction-selective way to visual motion. These findings are consistent with previous studies and indicate that the functional integrity of neuronal circuits in the optic tectum of adult zebrafish is preserved with our staining technique. Overall, our protocol for in vivo calcium imaging provides a useful approach to monitor visual responses of individual neurons in the optic tectum of adult zebrafish even when only widefield microscopy is available. This approach will help to obtain valuable insight into the principles of visual computation in adult vertebrates and thus complement previous work on developing visual circuits. PMID:23667529

  20. Thermomicrocapillaries as temperature biosensors in single cells

    NASA Astrophysics Data System (ADS)

    Herth, Simone; Giesguth, Miriam; Wedel, Waldemar; Reiss, Günther; Dietz, Karl-Josef

    2013-03-01

    Temperature is an important physical parameter in biology and its deviation from optimum can cause damage in biosystems. Thermocouples based on the Seebeck effect can be structured on glass microcapillaries to obtain thermomicrocapillaries (TMCs) usable in a micromanipulation setup. The suitability of the setup was proven by monitoring the temperature increase upon illumination of leaves and single cells following insertion of the TMC. The increase was 1.5 K in green tissue and 0.75 K in white leaf sections due to lower absorption. In single cells of trichomes, the increase was 0.5 K due to heat dissipation to the surrounding air.

  1. Autoradiography and Immunofluorescence Combined for Autecological Study of Single Cell Activity with Nitrobacter as a Model System1

    PubMed Central

    Fliermans, C. B.; Schmidt, E. L.

    1975-01-01

    Specific detection of a particular bacterium by immunofluorescence was combined with estimation of its metabolic activity by autoradiography. The nitrifying bacteria Nitrobacter agilis and N. winogradskyi were used as a model system. Nitrobacter were incubated with NaH14CO3 and 14CO2 prior to study. The same preparations made for autoradiograms were stained with fluorescent antibodies specific for the Nitrobacter species. Examination by epifluorescence and transmitted dark-field microscopy revealed Nitrobacter cells with and without associated silver grains. Direct detection and simultaneous evaluation of metabolic activity of Nitrobacter was demonstrated in pure cultures, in a simple mixed culture, and in a natural soil. Images PMID:1103733

  2. Voltage clamp measurements of the hyperpolarization-activated inward current I(f) in single cells from rabbit sino-atrial node.

    PubMed Central

    van Ginneken, A C; Giles, W

    1991-01-01

    1. The kinetics and ion transfer characteristics of the hyperpolarization-activated inward current, I(f), have been studied in single cells obtained by enzymatic dispersion from the rabbit sino-atrial (S-A) node. These experiments were done to assess the role of I(f) in the generation of the pacemaker depolarization in the S-A node. 2. The activation and the deactivation of I(f) in these single cells are accompanied by significant conductance increases and decreases respectively, confirming earlier findings from multicellular man-made strips of rabbit S-A node, and from mammalian Purkinje fibres. 3. The steady-state activation of I(f) lies between -40 and -120 mV, and its voltage dependence can be described by a Boltzmann relation with the half-activation point at approximately -70 mV. 4. The delay or sigmoidicity in both the onset of I(f) and the deactivation of the tail currents can be accounted for semi-quantitatively by using a second-order Hodgkin-Huxley kinetic scheme. 5. The reversal potential for I(f) is -24 +/- 2 mV (mean +/- S.E.M., n = 6). It does not change significantly as a function of the amount of I(f) which is activated, indicating that ion accumulation or depletion phenomena are not important variables controlling the time course of I(f), or its selectivity. 6. The fully-activated current-voltage relationship for I(f) is approximately linear with a slope conductance of 12.0 +/- 0.88 nS per cell (mean +/- S.E.M., n = 6). 7. A simple mathematical model based on the measured values of maximum conductance, reversal potential, and kinetics of I(f) has been developed to simulate the size and time course of I(f) during typical spontaneous pacemaker activity in rabbit sino-atrial node cells. The calculations show that I(f) can change significantly during pacing and suggest that this current change is, at least in part, responsible for the pacemaker depolarization. Images Fig. 1 PMID:1708824

  3. Rational Design of a Dephosphorylation-Resistant Reporter Enables Single-Cell Measurement of Tyrosine Kinase Activity.

    PubMed

    Turner, Abigail H; Lebhar, Michael S; Proctor, Angela; Wang, Qunzhao; Lawrence, David S; Allbritton, Nancy L

    2016-02-19

    Although peptide-based reporters of protein tyrosine kinase (PTK) activity have been used to study PTK enzymology in vitro, the application of these reporters to intracellular conditions is compromised by their dephosphorylation, preventing PTK activity measurements. Nonproteinogenic amino acids may be utilized to rationally design selective peptidic ligands by accessing greater chemical and structural diversity than is available using the native amino acids. We describe a peptidic reporter that, upon phosphorylation by the epidermal growth factor receptor (EGFR), is resistant to dephosphorylation both in vitro and in cellulo. The reporter contains a conformationally constrained phosphorylatable moiety (7-(S)-hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) in the place of L-tyrosine and is efficiently phosphorylated in A431 epidermoid carcinoma cells. Dephosphorylation of the reporter occurs 3 orders of magnitude more slowly compared with that of the conventional tyrosine-containing reporter.

  4. Monitoring active volcanoes

    USGS Publications Warehouse

    Tilling, Robert I.

    1987-01-01

    One of the most spectacular, awesomely beautiful, and at times destructive displays of natural energy is an erupting volcano, belching fume and ash thousands of meters into the atmosphere and pouring out red-hot molten lava in fountains and streams. Countless eruptions in the geologic past have produced volcanic rocks that form much of the Earth's present surface. The gradual disintegration and weathering of these rocks have yielded some of the richest farmlands in the world, and these fertile soils play a significant role in sustaining our large and growing population. Were it not for volcanic activity, the Hawaiian Islands with their sugar cane and pineapple fields and magnificent landscapes and seascapes would not exist to support their residents and to charm their visitors. Yet, the actual eruptive processes are catastrophic and can claim life and property.

  5. Abundance and single-cell activity of heterotrophic bacterial groups in the western Arctic Ocean in summer and winter.

    PubMed

    Nikrad, Mrinalini P; Cottrell, M T; Kirchman, D L

    2012-04-01

    Environmental conditions in the western Arctic Ocean range from constant light and nutrient depletion in summer to complete darkness and sea ice cover in winter. This seasonal environmental variation is likely to have an effect on the use of dissolved organic matter (DOM) by heterotrophic bacteria in surface water. However, this effect is not well studied and we know little about the activity of specific bacterial clades in the surface oceans. The use of DOM by three bacterial subgroups in both winter and summer was examined by microautoradiography combined with fluorescence in situ hybridization. We found selective use of substrates by these groups, although the abundances of Ant4D3 (Antarctic Gammaproteobacteria), Polaribacter (Bacteroidetes), and SAR11 (Alphaproteobacteria) were not different between summer and winter in the Beaufort and Chukchi Seas. The number of cells taking up glucose within all three bacterial groups decreased significantly from summer to winter, while the percentage of cells using leucine did not show a clear pattern between seasons. The uptake of the amino acid mix increased substantially from summer to winter by the Ant4D3 group, although such a large increase in uptake was not seen for the other two groups. Use of glucose by bacteria, but not use of leucine or the amino acid mix, related strongly to inorganic nutrients, chlorophyll a, and other environmental factors. Our results suggest a switch in use of dissolved organic substrates from summer to winter and that the three phylogenetic subgroups examined fill different niches in DOM use in the two seasons.

  6. Human T cells monitored by impedance spectrometry using field-effect transistor arrays: a novel tool for single-cell adhesion and migration studies.

    PubMed

    Law, Jessica Ka Yan; Susloparova, Anna; Vu, Xuan Thang; Zhou, Xiao; Hempel, Felix; Qu, Bin; Hoth, Markus; Ingebrandt, Sven

    2015-05-15

    Cytotoxic T lymphocytes (CTLs) play an important role in the immune system by recognizing and eliminating pathogen-infected and tumorigenic cells. In order to achieve their function, T cells have to migrate throughout the whole body and identify the respective targets. In conventional immunology studies, interactions between CTLs and targets are usually investigated using tedious and time-consuming immunofluorescence imaging. However, there is currently no straightforward measurement tool available to examine the interaction strengths. In the present study, adhesion strengths and migration of single human CD8(+) T cells on pre-coated field-effect transistor (FET) devices (i.e. fibronectin, anti-CD3 antibody, and anti-LFA-1 antibody) were measured using impedance spectroscopy. Adhesion strengths to different protein and antibody coatings were compared. By fitting the data to an electronically equivalent circuit model, cell-related parameters (cell membrane capacitance referring to cell morphology and seal resistance referring to adhesion strength) were obtained. This electronically-assessed adhesion strength provides a novel, fast, and important index describing the interaction efficiency. Furthermore, the size of our detection transistor gates as well as their sensitivity reaches down to single cell resolution. Real-time motions of individually migrating T cells can be traced using our FET devices. The in-house fabricated FETs used in the present study are providing a novel and very efficient insight to individual cell interactions.

  7. Core-shell self-assembly triggered via a thiol-disulfide exchange reaction for reduced glutathione detection and single cells monitoring

    PubMed Central

    Zhang, Zhen; Jiao, Yuting; Wang, Yuanyuan; Zhang, Shusheng

    2016-01-01

    A novel core-shell DNA self-assembly catalyzed by thiol-disulfide exchange reactions was proposed, which could realize GSH-initiated hybridization chain reaction (HCR) for signal amplification and molecules gathering. Significantly, these self-assembled products via electrostatic interaction could accumulate into prominent and clustered fluorescence-bright spots in single cancer cells for reduced glutathione monitoring, which will effectively drive cell monitoring into a new era. PMID:27412605

  8. Single-cell RNA sequencing highlights transcription activity of autophagy-related genes during hematopoietic stem cell formation in mouse embryos.

    PubMed

    Hu, Yongfei; Huang, Yan; Yi, Ying; Wang, Hongwei; Liu, Bing; Yu, Jia; Wang, Dong

    2017-04-03

    Accumulating evidence has demonstrated that macroautophagy/autophagy plays an essential role in self-renewal and differentiation in embryonic hematopoiesis. Here, according to the RNA sequencing data sets of 5 population cells related to hematopoietic stem cell (HSC) formation during mouse embryogenesis (endothelial cells, PTPRC/CD45(-) and PTPRC/CD45(+) pre-HSCs in the E11 aorta-gonad-mesonephros (AGM) region, mature HSCs in E12 and E14 fetal liver), we explored the dynamic expression of mouse autophagy-related genes in this course at the single-cell level. Our results revealed that the transcription activity of autophagy-related genes had a substantial increase when endothelial cells (ECs) specified into pre-HSCs, and the upregulation of autophagy-essential genes correlated with reduced NOTCH signaling in pre-HSCs, suggesting the autophagy activity may be greatly enhanced during pre-HSC specification from endothelial precursors. In summary, our results presented strong evidence that autophagy plays a critical role in HSC emergence during mouse midgestation.

  9. A New Generation of FRET Sensors for Robust Measurement of Gαi1, Gαi2 and Gαi3 Activation Kinetics in Single Cells

    PubMed Central

    van Unen, Jakobus; Stumpf, Anette D.; Schmid, Benedikt; Reinhard, Nathalie R.; Hordijk, Peter L.; Hoffmann, Carsten; Gadella, Theodorus W. J.; Goedhart, Joachim

    2016-01-01

    G-protein coupled receptors (GPCRs) can activate a heterotrimeric G-protein complex with subsecond kinetics. Genetically encoded biosensors based on Förster resonance energy transfer (FRET) are ideally suited for the study of such fast signaling events in single living cells. Here we report on the construction and characterization of three FRET biosensors for the measurement of Gαi1, Gαi2 and Gαi3 activation. To enable quantitative long-term imaging of FRET biosensors with high dynamic range, fluorescent proteins with enhanced photophysical properties are required. Therefore, we use the currently brightest and most photostable CFP variant, mTurquoise2, as donor fused to Gαi subunit, and cp173Venus fused to the Gγ2 subunit as acceptor. The Gαi FRET biosensors constructs are expressed together with Gβ1 from a single plasmid, providing preferred relative expression levels with reduced variation in mammalian cells. The Gαi FRET sensors showed a robust response to activation of endogenous or over-expressed alpha-2A-adrenergic receptors, which was inhibited by pertussis toxin. Moreover, we observed activation of the Gαi FRET sensor in single cells upon stimulation of several GPCRs, including the LPA2, M3 and BK2 receptor. Furthermore, we show that the sensors are well suited to extract kinetic parameters from fast measurements in the millisecond time range. This new generation of FRET biosensors for Gαi1, Gαi2 and Gαi3 activation will be valuable for live-cell measurements that probe Gαi activation. PMID:26799488

  10. Low-coverage single-cell mRNA sequencing reveals cellular heterogeneity and activated signaling pathways in developing cerebral cortex

    PubMed Central

    Pollen, Alex A; Nowakowski, Tomasz J; Shuga, Joe; Wang, Xiaohui; Leyrat, Anne A; Lui, Jan H; Li, Nianzhen; Szpankowski, Lukasz; Fowler, Brian; Chen, Peilin; Ramalingam, Naveen; Sun, Gang; Thu, Myo; Norris, Michael; Lebofsky, Ronald; Toppani, Dominique; Kemp, Darnell; Wong, Michael; Clerkson, Barry; Jones, Brittnee N; Wu, Shiquan; Knutsson, Lawrence; Alvarado, Beatriz; Wang, Jing; Weaver, Lesley S; May, Andrew P; Jones, Robert C; Unger, Marc A; Kriegstein, Arnold R; West, Jay AA

    2014-01-01

    Large-scale surveys of single-cell gene expression have the potential to reveal rare cell populations and lineage relationships, but require efficient methods for cell capture and mRNA sequencing1–4. Although cellular barcoding strategies allow parallel sequencing of single cells at ultra-low depths5, the limitations of shallow sequencing have not been directly investigated. By capturing 301 single cells from 11 populations using microfluidics and analyzing single-cell transcriptomes across downsampled sequencing depths, we demonstrate that shallow single-cell mRNA sequencing (~50,000 reads per cell) is sufficient for unbiased cell-type classification and biomarker identification. In developing cortex we identify diverse cell types including multiple progenitor and neuronal subtypes, and we identify EGR1 and FOS as previously unreported candidate targets of Notch signaling in human but not mouse radial glia. Our strategy establishes an efficient method for unbiased analysis and comparison of cell populations from heterogeneous tissue by microfluidic single-cell capture and low-coverage sequencing of many cells. PMID:25086649

  11. Active Job Monitoring in Pilots

    NASA Astrophysics Data System (ADS)

    Kuehn, Eileen; Fischer, Max; Giffels, Manuel; Jung, Christopher; Petzold, Andreas

    2015-12-01

    Recent developments in high energy physics (HEP) including multi-core jobs and multi-core pilots require data centres to gain a deep understanding of the system to monitor, design, and upgrade computing clusters. Networking is a critical component. Especially the increased usage of data federations, for example in diskless computing centres or as a fallback solution, relies on WAN connectivity and availability. The specific demands of different experiments and communities, but also the need for identification of misbehaving batch jobs, requires an active monitoring. Existing monitoring tools are not capable of measuring fine-grained information at batch job level. This complicates network-aware scheduling and optimisations. In addition, pilots add another layer of abstraction. They behave like batch systems themselves by managing and executing payloads of jobs internally. The number of real jobs being executed is unknown, as the original batch system has no access to internal information about the scheduling process inside the pilots. Therefore, the comparability of jobs and pilots for predicting run-time behaviour or network performance cannot be ensured. Hence, identifying the actual payload is important. At the GridKa Tier 1 centre a specific tool is in use that allows the monitoring of network traffic information at batch job level. This contribution presents the current monitoring approach and discusses recent efforts and importance to identify pilots and their substructures inside the batch system. It will also show how to determine monitoring data of specific jobs from identified pilots. Finally, the approach is evaluated.

  12. Technologies for Single-Cell Isolation

    PubMed Central

    Gross, Andre; Schoendube, Jonas; Zimmermann, Stefan; Steeb, Maximilian; Zengerle, Roland; Koltay, Peter

    2015-01-01

    The handling of single cells is of great importance in applications such as cell line development or single-cell analysis, e.g., for cancer research or for emerging diagnostic methods. This review provides an overview of technologies that are currently used or in development to isolate single cells for subsequent single-cell analysis. Data from a dedicated online market survey conducted to identify the most relevant technologies, presented here for the first time, shows that FACS (fluorescence activated cell sorting) respectively Flow cytometry (33% usage), laser microdissection (17%), manual cell picking (17%), random seeding/dilution (15%), and microfluidics/lab-on-a-chip devices (12%) are currently the most frequently used technologies. These most prominent technologies are described in detail and key performance factors are discussed. The survey data indicates a further increasing interest in single-cell isolation tools for the coming years. Additionally, a worldwide patent search was performed to screen for emerging technologies that might become relevant in the future. In total 179 patents were found, out of which 25 were evaluated by screening the title and abstract to be relevant to the field. PMID:26213926

  13. Single-cell transcriptomics for microbial eukaryotes.

    PubMed

    Kolisko, Martin; Boscaro, Vittorio; Burki, Fabien; Lynn, Denis H; Keeling, Patrick J

    2014-11-17

    One of the greatest hindrances to a comprehensive understanding of microbial genomics, cell biology, ecology, and evolution is that most microbial life is not in culture. Solutions to this problem have mainly focused on whole-community surveys like metagenomics, but these analyses inevitably loose information and present particular challenges for eukaryotes, which are relatively rare and possess large, gene-sparse genomes. Single-cell analyses present an alternative solution that allows for specific species to be targeted, while retaining information on cellular identity, morphology, and partitioning of activities within microbial communities. Single-cell transcriptomics, pioneered in medical research, offers particular potential advantages for uncultivated eukaryotes, but the efficiency and biases have not been tested. Here we describe a simple and reproducible method for single-cell transcriptomics using manually isolated cells from five model ciliate species; we examine impacts of amplification bias and contamination, and compare the efficacy of gene discovery to traditional culture-based transcriptomics. Gene discovery using single-cell transcriptomes was found to be comparable to mass-culture methods, suggesting single-cell transcriptomics is an efficient entry point into genomic data from the vast majority of eukaryotic biodiversity.

  14. Technologies for Single-Cell Isolation.

    PubMed

    Gross, Andre; Schoendube, Jonas; Zimmermann, Stefan; Steeb, Maximilian; Zengerle, Roland; Koltay, Peter

    2015-07-24

    The handling of single cells is of great importance in applications such as cell line development or single-cell analysis, e.g., for cancer research or for emerging diagnostic methods. This review provides an overview of technologies that are currently used or in development to isolate single cells for subsequent single-cell analysis. Data from a dedicated online market survey conducted to identify the most relevant technologies, presented here for the first time, shows that FACS (fluorescence activated cell sorting) respectively Flow cytometry (33% usage), laser microdissection (17%), manual cell picking (17%), random seeding/dilution (15%), and microfluidics/lab-on-a-chip devices (12%) are currently the most frequently used technologies. These most prominent technologies are described in detail and key performance factors are discussed. The survey data indicates a further increasing interest in single-cell isolation tools for the coming years. Additionally, a worldwide patent search was performed to screen for emerging technologies that might become relevant in the future. In total 179 patents were found, out of which 25 were evaluated by screening the title and abstract to be relevant to the field.

  15. PCR-free and label-free fluorescent detection of telomerase activity at single-cell level based on triple amplification.

    PubMed

    Gao, Yanfang; Xu, Jing; Li, Baoxin; Jin, Yan

    2016-07-15

    As a universal biomarker for cancer diagnostics and cancer therapeutics, telomerase has attracted extensive attention concerning its detection and discovery of its inhibitors. Herein, we developed a PCR-free and label-free fluorescent strategy for facile, reliable and highly sensitive assay of human telomerase activity from crude cancer cell extracts. A G-quadruplex-selective fluorescent dye, N-methyl mesoporphyrin IX (NMM), was utilized as signal probe. Two hairpin probes with hidden G-quadruplex strand in their stem were designed as assembly components of strand displacement reaction (SDR). In this strategy, one telomerase elongation product contains several hexamer repeats which can hybridize with numerous assistant DNA to release a lot of trigger DNA (T-DNA) of SDR for achieving first step amplification. Then, strand displacement reaction led to the formation of G-quadruplex at the both end of two hairpin DNA probes for realizing second step amplification. Finally, the re-released T-DNA initiated another cycle of SDR, resulting in a significant increase in the fluorescence intensity of NMM. By taking advantage of triple signal amplification, the telomerase activity in the HeLa extracts equivalent to 1-3000 cells was detected in homogeneous solution. Telomerase activities of different cell lines, including cancer cells and normal cell, were also successfully evaluated. Meanwhile, the inhibition effect of 3'-azido-3'-deoxythymidine (AZT) was also investigated. Therefore, it offers a simple and reliable method for detecting telomerase activity at single-cell level without complex pre-modification of probe and enzyme auxiliary signal amplification, which has the merits of simplicity, rapid response, low cost and high reliability.

  16. Assessment of nutritional value of single-cell protein from waste-activated sludge as a protein supplement in poultry feed.

    PubMed

    Nkhalambayausi-Chirwa, Evans M; Lebitso, Moses T

    2012-12-01

    The amount of protein wasted through sludge in Gauteng, South Africa, amounts to 95 000 metric tonne/yr, with the order of magnitude of the national protein requirement of approximately 145 000 metric tonne/yr. Waste-activated sludge (WAS) from wastewater treatment plants (WWTPs) that treat domestic wastewater contains protein in a ratio of 2:1 against fishmeal. This protein source has not been utilized because of the high content of toxic heavy metals and other potential carcinogenic pollutants in the sludge. In this study, a pretreatment method of modified aqua regia dilute acid wash was used to lower the metal content by approximately 60%. However, this resulted in a 33% loss of amino acids in the acid-washed WAS. A feed substitution test in poultry with different fishmeal-sludge ratios (0%, 25%, 50%, 75%, and 100% WAS as percent substitution of fishmeal) showed no impact of sludge single-cell protein (SCP) on mortality rate. However, sludge substitution in the feed yielded weight gains and cost savings up to 46%.

  17. Clonal Expansions of Cd8+ T Cells Dominate the T Cell Infiltrate in Active Multiple Sclerosis Lesions as Shown by Micromanipulation and Single Cell Polymerase Chain Reaction

    PubMed Central

    Babbe, Holger; Roers, Axel; Waisman, Ari; Lassmann, Hans; Goebels, Norbert; Hohlfeld, Reinhard; Friese, Michael; Schröder, Roland; Deckert, Martina; Schmidt, Stephan; Ravid, Rivka; Rajewsky, Klaus

    2000-01-01

    Clonal composition and T cell receptor (TCR) repertoire of CD4+ and CD8+ T cells infiltrating actively demyelinating multiple sclerosis (MS) lesions were determined with unprecedented resolution at the level of single cells. Individual CD4+ or CD8+ T cells were isolated from frozen sections of lesional tissue by micromanipulation and subjected to single target amplification of TCR-β gene rearrangements. This strategy allows the assignment of a TCR variable region (V region) sequence to the particular T cell from which it was amplified. Sequence analysis revealed that in both cases investigated, the majority of CD8+ T cells belonged to few clones. One of these clones accounted for 35% of CD8+ T cells in case 1. V region sequence comparison revealed signs of selection for common peptide specificities for some of the CD8+ T cells in case 1. In both cases, the CD4+ T cell population was more heterogeneous. Most CD4+ and CD8+ clones were represented in perivascular infiltrates as well as among parenchymal T cells. In case 2, two of the CD8+ clones identified in brain tissue were also detected in peripheral blood. Investigation of the antigenic specificities of expanded clones may help to elucidate their functional properties. PMID:10934227

  18. Single Cell Analysis of a Bacterial Sender-Receiver System

    PubMed Central

    Mückl, Andrea; Kapsner, Korbinian; Gerland, Ulrich; Simmel, Friedrich C.

    2016-01-01

    Monitoring gene expression dynamics on the single cell level provides important information on cellular heterogeneity and stochasticity, and potentially allows for more accurate quantitation of gene expression processes. We here study bacterial senders and receivers genetically engineered with components of the quorum sensing system derived from Aliivibrio fischeri on the single cell level using microfluidics-based bacterial chemostats and fluorescence video microscopy. We track large numbers of bacteria over extended periods of time, which allows us to determine bacterial lineages and filter out subpopulations within a heterogeneous population. We quantitatively determine the dynamic gene expression response of receiver bacteria to varying amounts of the quorum sensing inducer N-3-oxo-C6-homoserine lactone (AHL). From this we construct AHL response curves and characterize gene expression dynamics of whole bacterial populations by investigating the statistical distribution of gene expression activity over time. The bacteria are found to display heterogeneous induction behavior within the population. We therefore also characterize gene expression in a homogeneous bacterial subpopulation by focusing on single cell trajectories derived only from bacteria with similar induction behavior. The response at the single cell level is found to be more cooperative than that obtained for the heterogeneous total population. For the analysis of systems containing both AHL senders and receiver cells, we utilize the receiver cells as ‘bacterial sensors’ for AHL. Based on a simple gene expression model and the response curves obtained in receiver-only experiments, the effective AHL concentration established by the senders and their ‘sending power’ is determined. PMID:26808777

  19. Transcriptome analyses of inner cell mass and trophectoderm cells isolated by magnetic-activated cell sorting from bovine blastocysts using single cell RNA-seq.

    PubMed

    Zhao, X-M; Cui, L-S; Hao, H-S; Wang, H-Y; Zhao, S-J; Du, W-H; Wang, D; Liu, Y; Zhu, H-B

    2016-10-01

    Research on bovine embryonic stem cells (bESCs) has been hampered because bESCs are cultured in conditions that are based on information obtained from culturing mouse and human inner cell mass (ICM) cells. The aim of this study was to compare gene expression in ICM and trophectoderm (TE) cell lineages of bovine embryos and to discuss the findings relative to information available for mice and humans. We separated a high-purity (>90%) ICM and TE from bovine blastocysts by magnetic-activated cell sorting and analysed their transcriptomes by single cell RNA-seq. Differentially expressed genes (DEGs) were assessed using Gene Ontology (GO) and Kyoto Encyclopaedia of Genes and Genomes (KEGG) databases. Finally, qRT-PCR was performed to validate the RNA-seq results. From 207 DEGs identified (adjusted p ≤ .05; fold change ≥2), 159 and 48 had greater expression in the ICM and TE cells respectively. We validated 27 genes using qRT-PCR and found their expression patterns were mostly similar to those of RNA-seq, including 12 novel ICM-dominant (HNF4A, CCL24, FGFR4, IFITM3, PTCHD2, GJB5, FN1, KLK7, PRDM14, GRP, FGF19 and GCM1) and two novel TE-dominant (SLC10A1 and WNT4) genes. Bioinformatics analysis showed that these DEGs are involved in many important pathways, such as MAPK and cancer cell pathways, and these pathways have been shown to play essential roles in mouse and human ESCs in the self-renewal and pluripotent maintenance. As a conclusion, there were sufficient differences to allow us to conclude that the control of pluripotency in bovine ICM cells is species-specific.

  20. Single cell elemental analysis using nuclear microscopy

    NASA Astrophysics Data System (ADS)

    Ren, M. Q.; Thong, P. S. P.; Kara, U.; Watt, F.

    1999-04-01

    The use of Particle Induced X-ray Emission (PIXE), Rutherford Backscattering Spectrometry (RBS) and Scanning Transmission Ion Microscopy (STIM) to provide quantitative elemental analysis of single cells is an area which has high potential, particularly when the trace elements such as Ca, Fe, Zn and Cu can be monitored. We describe the methodology of sample preparation for two cell types, the procedures of cell imaging using STIM, and the quantitative elemental analysis of single cells using RBS and PIXE. Recent work on single cells at the Nuclear Microscopy Research Centre,National University of Singapore has centred around two research areas: (a) Apoptosis (programmed cell death), which has been recently implicated in a wide range of pathological conditions such as cancer, Parkinson's disease etc, and (b) Malaria (infection of red blood cells by the malaria parasite). Firstly we present results on the elemental analysis of human Chang liver cells (ATTCC CCL 13) where vanadium ions were used to trigger apoptosis, and demonstrate that nuclear microscopy has the capability of monitoring vanadium loading within individual cells. Secondly we present the results of elemental changes taking place in individual mouse red blood cells which have been infected with the malaria parasite and treated with the anti-malaria drug Qinghaosu (QHS).

  1. Cytometry-based single-cell analysis of intact epithelial signaling reveals MAPK activation divergent from TNF-α-induced apoptosis in vivo

    PubMed Central

    Simmons, Alan J; Banerjee, Amrita; McKinley, Eliot T; Scurrah, Cherie' R; Herring, Charles A; Gewin, Leslie S; Masuzaki, Ryota; Karp, Seth J; Franklin, Jeffrey L; Gerdes, Michael J; Irish, Jonathan M; Coffey, Robert J; Lau, Ken S

    2015-01-01

    Understanding heterogeneous cellular behaviors in a complex tissue requires the evaluation of signaling networks at single-cell resolution. However, probing signaling in epithelial tissues using cytometry-based single-cell analysis has been confounded by the necessity of single-cell dissociation, where disrupting cell-to-cell connections inherently perturbs native cell signaling states. Here, we demonstrate a novel strategy (Disaggregation for Intracellular Signaling in Single Epithelial Cells from Tissue—DISSECT) that preserves native signaling for Cytometry Time-of-Flight (CyTOF) and fluorescent flow cytometry applications. A 21-plex CyTOF analysis encompassing core signaling and cell-identity markers was performed on the small intestinal epithelium after systemic tumor necrosis factor-alpha (TNF-α) stimulation. Unsupervised and supervised analyses robustly selected signaling features that identify a unique subset of epithelial cells that are sensitized to TNF-α-induced apoptosis in the seemingly homogeneous enterocyte population. Specifically, p-ERK and apoptosis are divergently regulated in neighboring enterocytes within the epithelium, suggesting a mechanism of contact-dependent survival. Our novel single-cell approach can broadly be applied, using both CyTOF and multi-parameter flow cytometry, for investigating normal and diseased cell states in a wide range of epithelial tissues. PMID:26519361

  2. Quantification of Circadian Rhythms in Single Cells

    PubMed Central

    Westermark, Pål O.; Welsh, David K.; Okamura, Hitoshi; Herzel, Hanspeter

    2009-01-01

    Bioluminescence techniques allow accurate monitoring of the circadian clock in single cells. We have analyzed bioluminescence data of Per gene expression in mouse SCN neurons and fibroblasts. From these data, we extracted parameters such as damping rate and noise intensity using two simple mathematical models, one describing a damped oscillator driven by noise, and one describing a self-sustained noisy oscillator. Both models describe the data well and enabled us to quantitatively characterize both wild-type cells and several mutants. It has been suggested that the circadian clock is self-sustained at the single cell level, but we conclude that present data are not sufficient to determine whether the circadian clock of single SCN neurons and fibroblasts is a damped or a self-sustained oscillator. We show how to settle this question, however, by testing the models' predictions of different phases and amplitudes in response to a periodic entrainment signal (zeitgeber). PMID:19956762

  3. Single Cell Oxygen Mapping (SCOM) by Scanning Electrochemical Microscopy Uncovers Heterogeneous Intracellular Oxygen Consumption.

    PubMed

    Santos, Carla Santana; Kowaltowski, Alicia J; Bertotti, Mauro

    2017-09-12

    We developed a highly sensitive oxygen consumption scanning microscopy system using platinized platinum disc microelectrodes. The system is capable of reliably detecting single-cell respiration, responding to classical regulators of mitochondrial oxygen consumption activity as expected. Comparisons with commercial multi-cell oxygen detection systems show that the system has comparable errors (if not smaller), with the advantage of being able to monitor inter and intra-cell heterogeneity in oxygen consumption characteristics. Our results uncover heterogeneous oxygen consumption characteristics between cells and within the same cell´s microenvironments. Single Cell Oxygen Mapping (SCOM) is thus capable of reliably studying mitochondrial oxygen consumption characteristics and heterogeneity at a single-cell level.

  4. Plant single-cell and single-cell-type metabolomics.

    PubMed

    Misra, Biswapriya B; Assmann, Sarah M; Chen, Sixue

    2014-10-01

    In conjunction with genomics, transcriptomics, and proteomics, plant metabolomics is providing large data sets that are paving the way towards a comprehensive and holistic understanding of plant growth, development, defense, and productivity. However, dilution effects from organ- and tissue-based sampling of metabolomes have limited our understanding of the intricate regulation of metabolic pathways and networks at the cellular level. Recent advances in metabolomics methodologies, along with the post-genomic expansion of bioinformatics knowledge and functional genomics tools, have allowed the gathering of enriched information on individual cells and single cell types. Here we review progress, current status, opportunities, and challenges presented by single cell-based metabolomics research in plants.

  5. Anatahan Activity and Monitoring, 2005

    NASA Astrophysics Data System (ADS)

    Lockhart, A.; White, R.; Koyanagi, S.; Trusdell, F.; Kauahikaua, J.; Marso, J.; Ewert, J.

    2005-12-01

    Anatahan volcano began erupting in 2003 and continued with a second eruptive phase in 2004. In January 2005 the volcano began a sequence of eruptions and unrest that continues as of September 2005. The activity has been characterized by punctuated episodes of very steamy strombolian activity and vigorous ash emission. Some of the ash emissions have reached 50,000-foot elevations, with VOG and ash occasionally reaching the Philippines and southernmost Japan, over 1000 miles away. Vigorous ash emission has been almost continuous since June 2005. A M4.8 long-period earthquake (LP) occurred in mid-August, one of the largest LPs recorded on the planet in the last quarter-century. Real-time monitoring consisting of a few telemetered short-period seismometers and acoustic sensors has been severely hampered by ashfall on the small island. Monitoring efforts have been focused on the aircraft/ash hazard, with the goal of providing the FAA and airline industry with rapid notice of seismic signatures that may indicate ash columns rising to the altitude of airline traffic, or nominally above 20,000-30,000 ft.

  6. Introduction: why analyze single cells?

    PubMed

    Di Carlo, Dino; Tse, Henry Tat Kwong; Gossett, Daniel R

    2012-01-01

    Powerful methods in molecular biology are abundant; however, in many fields including hematology, stem cell biology, tissue engineering, and cancer biology, data from tools and assays that analyze the average signals from many cells may not yield the desired result because the cells of interest may be in the minority-their behavior masked by the majority-or because the dynamics of the populations of interest are offset in time. Accurate characterization of samples with high cellular heterogeneity may only be achieved by analyzing single cells. In this chapter, we discuss the rationale for performing analyses on individual cells in more depth, cover the fields of study in which single-cell behavior is yielding new insights into biological and clinical questions, and speculate on how single-cell analysis will be critical in the future.

  7. Single Cell Isolation and Analysis

    PubMed Central

    Hu, Ping; Zhang, Wenhua; Xin, Hongbo; Deng, Glenn

    2016-01-01

    Individual cell heterogeneity within a population can be critical to its peculiar function and fate. Subpopulations studies with mixed mutants and wild types may not be as informative regarding which cell responds to which drugs or clinical treatments. Cell to cell differences in RNA transcripts and protein expression can be key to answering questions in cancer, neurobiology, stem cell biology, immunology, and developmental biology. Conventional cell-based assays mainly analyze the average responses from a population of cells, without regarding individual cell phenotypes. To better understand the variations from cell to cell, scientists need to use single cell analyses to provide more detailed information for therapeutic decision making in precision medicine. In this review, we focus on the recent developments in single cell isolation and analysis, which include technologies, analyses and main applications. Here, we summarize the historical background, limitations, applications, and potential of single cell isolation technologies. PMID:27826548

  8. Continuous cultivation of fission yeast: analysis of single-cell protein synthesis kinetics

    SciTech Connect

    Agar, D.W.; Bailey, J.E.

    1981-01-01

    A fundamental problem in microbial reactor analysis is identification of the relation between environment and individual cell metabolic activity. Population balance equations provide a link between experimental measurements of composition frequency functions in microbial populations on the one hand and macromolecule synthesis kinetics and cell division control parameters for single cells on the other. Flow microfluorometry measurements of frequency functions for single-cell protein content in Schizosaccharomyces pombe in balanced exponential growth were analyzed by 2 different methods. One approach utilizes the integrated form of the population balance equation known as the Collins-Richmond equation, and the other method involves optimization of parameters in assumed kinetic and cell division functional forms to fit measured frequency functions with corresponding model solutions. Both data interpretation techniques indicate that rates of protein synthesis increase most in low-protein-content cells as the population specific growth rate increases, leading to parabolic single-cell protein synthesis kinetics at large specific growth rates. Utilization of frequency function data for an asynchronous population is in this case a far more sensitive method for determination of single-cell kinetics than is monitoring the metabolic dynamics of a single cell or, equivalently, synchronous culture analyses.

  9. Single Cell Proteolytic Assays to Investigate Cancer Clonal Heterogeneity and Cell Dynamics Using an Efficient Cell Loading Scheme

    NASA Astrophysics Data System (ADS)

    Chen, Yu-Chih; Cheng, Yu-Heng; Ingram, Patrick; Yoon, Euisik

    2016-06-01

    Proteolytic degradation of the extracellular matrix (ECM) is critical in cancer invasion, and recent work suggests that heterogeneous cancer populations cooperate in this process. Despite the importance of cell heterogeneity, conventional proteolytic assays measure average activity, requiring thousands of cells and providing limited information about heterogeneity and dynamics. Here, we developed a microfluidic platform that provides high-efficiency cell loading and simple valveless isolation, so the proteolytic activity of a small sample (10–100 cells) can be easily characterized. Combined with a single cell derived (clonal) sphere formation platform, we have successfully demonstrated the importance of microenvironmental cues for proteolytic activity and also investigated the difference between clones. Furthermore, the platform allows monitoring single cells at multiple time points, unveiling different cancer cell line dynamics in proteolytic activity. The presented tool facilitates single cell proteolytic analysis using small samples, and our findings illuminate the heterogeneous and dynamic nature of proteolytic activity.

  10. Single Cell Proteolytic Assays to Investigate Cancer Clonal Heterogeneity and Cell Dynamics Using an Efficient Cell Loading Scheme

    PubMed Central

    Chen, Yu-Chih; Cheng, Yu-Heng; Ingram, Patrick; Yoon, Euisik

    2016-01-01

    Proteolytic degradation of the extracellular matrix (ECM) is critical in cancer invasion, and recent work suggests that heterogeneous cancer populations cooperate in this process. Despite the importance of cell heterogeneity, conventional proteolytic assays measure average activity, requiring thousands of cells and providing limited information about heterogeneity and dynamics. Here, we developed a microfluidic platform that provides high-efficiency cell loading and simple valveless isolation, so the proteolytic activity of a small sample (10–100 cells) can be easily characterized. Combined with a single cell derived (clonal) sphere formation platform, we have successfully demonstrated the importance of microenvironmental cues for proteolytic activity and also investigated the difference between clones. Furthermore, the platform allows monitoring single cells at multiple time points, unveiling different cancer cell line dynamics in proteolytic activity. The presented tool facilitates single cell proteolytic analysis using small samples, and our findings illuminate the heterogeneous and dynamic nature of proteolytic activity. PMID:27283981

  11. Detection of cAMP and of PKA activity in Saccharomyces cerevisiae single cells using Fluorescence Resonance Energy Transfer (FRET) probes.

    PubMed

    Colombo, Sonia; Broggi, Serena; Collini, Maddalena; D'Alfonso, Laura; Chirico, Giuseppe; Martegani, Enzo

    2017-06-03

    In Saccharomyces cerevisiae the second messenger cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) play a central role in metabolism regulation, stress resistance and cell cycle progression. To monitor cAMP levels and PKA activity in vivo in single S. cerevisiae cells, we expressed an Epac-based FRET probe and a FRET-based A-kinase activity reporter, which were proven to be useful live-cell biosensors for cAMP levels and PKA activity in mammalian cells. Regarding detection of cAMP in single yeast cells, we show that in wild type strains the CFP/YFP fluorescence ratio increased immediately after glucose addition to derepressed cells, while no changes were observed when glucose was added to a strain that is not able to produce cAMP. In addition, we had evidence for damped oscillations in cAMP levels at least in SP1 strain. Regarding detection of PKA activity, we show that in wild type strains the FRET increased after glucose addition to derepressed cells, while no changes were observed when glucose was added to either a strain that is not able to produce cAMP or to a strain with absent PKA activity. Taken together these probes are useful to follow activation of the cAMP/PKA pathway in single yeast cells and for long times (up to one hour). Copyright © 2017 Elsevier Inc. All rights reserved.

  12. Digital microfluidic immunocytochemistry in single cells

    PubMed Central

    Ng, Alphonsus H. C.; Chamberlain, M. Dean; Situ, Haozhong; Lee, Victor; Wheeler, Aaron R.

    2015-01-01

    We report a new technique called Digital microfluidic Immunocytochemistry in Single Cells (DISC). DISC automates protocols for cell culture, stimulation and immunocytochemistry, enabling the interrogation of protein phosphorylation on pulsing with stimulus for as little as 3 s. DISC was used to probe the phosphorylation states of platelet-derived growth factor receptor (PDGFR) and the downstream signalling protein, Akt, to evaluate concentration- and time-dependent effects of stimulation. The high time resolution of the technique allowed for surprising new observations—for example, a 10 s pulse stimulus of a low concentration of PDGF is sufficient to cause >30% of adherent fibroblasts to commit to Akt activation. With the ability to quantitatively probe signalling events with high time resolution at the single-cell level, we propose that DISC may be an important new technique for a wide range of applications, especially for screening signalling responses of a heterogeneous cell population. PMID:26104298

  13. Nanokit for single-cell electrochemical analyses

    PubMed Central

    Pan, Rongrong; Xu, Mingchen; Jiang, Dechen; Burgess, Jame D.; Chen, Hong-Yuan

    2016-01-01

    The development of more intricate devices for the analysis of small molecules and protein activity in single cells would advance our knowledge of cellular heterogeneity and signaling cascades. Therefore, in this study, a nanokit was produced by filling a nanometer-sized capillary with a ring electrode at the tip with components from traditional kits, which could be egressed outside the capillary by electrochemical pumping. At the tip, femtoliter amounts of the kit components were reacted with the analyte to generate hydrogen peroxide for the electrochemical measurement by the ring electrode. Taking advantage of the nanotip and small volume injection, the nanokit was easily inserted into a single cell to determine the intracellular glucose levels and sphingomyelinase (SMase) activity, which had rarely been achieved. High cellular heterogeneities of these two molecules were observed, showing the significance of the nanokit. Compared with the current methods that use a complicated structural design or surface functionalization for the recognition of the analytes, the nanokit has adapted features of the well-established kits and integrated the kit components and detector in one nanometer-sized capillary, which provides a specific device to characterize the reactivity and concentrations of cellular compounds in single cells. PMID:27671654

  14. UV Decontamination of MDA Reagents for Single Cell Genomics

    SciTech Connect

    Lee, Janey; Tighe, Damon; Sczyrba, Alexander; Malmatrom, Rex; Clingenpeel, Scott; Malfatti, Stephanie; Rinke, Christian; Wang, Zhong; Stepanauskas, Ramunas; Cheng, Jan-Fang; Woyke, Tanja

    2011-03-18

    Single cell genomics, the amplification and sequencing of genomes from single cells, can provide a glimpse into the genetic make-up and thus life style of the vast majority of uncultured microbial cells, making it an immensely powerful and increasingly popular tool. This is accomplished by use of multiple displacement amplification (MDA), which can generate billions of copies of a single bacterial genome producing microgram-range DNA required for shotgun sequencing. Here, we address a key challenge inherent to this approach and propose a solution for the improved recovery of single cell genomes. While DNA-free reagents for the amplification of a single cell genome are a prerequisite for successful single cell sequencing and analysis, DNA contamination has been detected in various reagents, which poses a considerable challenge. Our study demonstrates the effect of UV irradiation in efficient elimination of exogenous contaminant DNA found in MDA reagents, while maintaining Phi29 activity. Consequently, we also find that increased UV exposure to Phi29 does not adversely affect genome coverage of MDA amplified single cells. While additional challenges in single cell genomics remain to be resolved, the proposed methodology is relatively quick and simple and we believe that its application will be of high value for future single cell sequencing projects.

  15. Superposition of Individual Activities: Urea-Mediated Suppression of Nitrate Uptake in the Dinoflagellate Prorocentrum minimum Revealed at the Population and Single-Cell Levels

    PubMed Central

    Matantseva, Olga; Skarlato, Sergei; Vogts, Angela; Pozdnyakov, Ilya; Liskow, Iris; Schubert, Hendrik; Voss, Maren

    2016-01-01

    Dinoflagellates readily use diverse inorganic and organic compounds as nitrogen sources, which is advantageous in eutrophied coastal areas exposed to high loads of anthropogenic nutrients, e.g., urea, one of the most abundant organic nitrogen substrates in seawater. Cell-to-cell variability in nutritional physiology can further enhance the diversity of metabolic strategies among dinoflagellates of the same species, but it has not been studied in free-living microalgae. We applied stable isotope tracers, isotope ratio mass spectrometry and nanoscale secondary ion mass spectrometry (NanoSIMS) to investigate the response of cultured nitrate-acclimated dinoflagellates Prorocentrum minimum to a sudden input of urea and the effect of urea on the concurrent nitrate uptake at the population and single-cell levels. We demonstrate that inputs of urea lead to suppression of nitrate uptake by P. minimum, and urea uptake exceeds the concurrent uptake of nitrate. Individual dinoflagellate cells within a population display significant heterogeneity in the rates of nutrient uptake and extent of the urea-mediated inhibition of the nitrate uptake, thus forming several groups characterized by different modes of nutrition. We conclude that urea originating from sporadic sources is rapidly utilized by dinoflagellates and can be used in biosynthesis or stored intracellularly depending on the nutrient status; therefore, sudden urea inputs can represent one of the factors triggering or supporting harmful algal blooms. Significant physiological heterogeneity revealed at the single-cell level is likely to play a role in alleviation of intra-population competition for resources and can affect the dynamics of phytoplankton populations and their maintenance in natural environments. PMID:27610101

  16. Measuring and Modeling Apoptosis in Single Cells

    PubMed Central

    Spencer, Sabrina L.; Sorger, Peter K.

    2011-01-01

    Cell death plays an essential role in the development of tissues and organisms, the etiology of disease, and the responses of cells to therapeutic drugs. Here we review progress made over the last decade in using mathematical models and quantitative, often single-cell, data to study apoptosis. We discuss the delay that follows exposure of cells to prodeath stimuli, control of mitochondrial outer membrane permeabilization, switch-like activation of effector caspases, and variability in the timing and probability of death from one cell to the next. Finally, we discuss challenges facing the fields of biochemical modeling and systems pharmacology. PMID:21414484

  17. Measuring single-cell density

    PubMed Central

    Grover, William H.; Bryan, Andrea K.; Diez-Silva, Monica; Suresh, Subra; Higgins, John M.; Manalis, Scott R.

    2011-01-01

    We have used a microfluidic mass sensor to measure the density of single living cells. By weighing each cell in two fluids of different densities, our technique measures the single-cell mass, volume, and density of approximately 500 cells per hour with a density precision of 0.001 g mL-1. We observe that the intrinsic cell-to-cell variation in density is nearly 100-fold smaller than the mass or volume variation. As a result, we can measure changes in cell density indicative of cellular processes that would be otherwise undetectable by mass or volume measurements. Here, we demonstrate this with four examples: identifying Plasmodium falciparum malaria-infected erythrocytes in a culture, distinguishing transfused blood cells from a patient’s own blood, identifying irreversibly sickled cells in a sickle cell patient, and identifying leukemia cells in the early stages of responding to a drug treatment. These demonstrations suggest that the ability to measure single-cell density will provide valuable insights into cell state for a wide range of biological processes. PMID:21690360

  18. Multiplex single-cell quantification of rare RNA transcripts from protoplasts in a model plant system.

    PubMed

    Kadam, Ulhas S; Schulz, Burkhard; Irudayaraj, Joseph M K

    2017-06-01

    Here we demonstrate multiplex and simultaneous detection of four different rare RNA species from plant, Arabidopsis thaliana, using surface-enhanced Raman spectroscopy (SERS) and gold nanoprobes at single-cell resolution. We show the applicability of nanoparticle-based Raman spectroscopic sensor to study intracellular RNA copies. First, we demonstrate that gold-nanoparticles decorated with Raman probes and carrying specific nucleic acid probe sequences can be uptaken by the protoplasts. We confirm the internalization of gold nanoprobes by transmission electron microscopy, inductively-coupled plasma-mass spectrometry and fluorescence imaging. Second, we show the utility of a SERS platform to monitor individual alternatively spliced (AS) variants and miRNA copies within single cells. Finally, the distinctive spectral features of Raman-active dyes were exploited for multiplex analysis of AtPTB2, AtDCL2, miR156a and miR172a. Furthermore, single-cell studies were validated by in vitro quantification and evaluation of nanotoxicity of gold probes. Raman tag functionalized gold nanosensors yielded an approach for the tracking of rare RNAs within the protoplasts. The SERS-based approach for quantification of RNAs has the capability to be a highly sensitive, accurate and discerning method for single-cell studies including AS variants quantification and rare miRNA detection in specific plant species. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

  19. Single-Cell Genomics for Virology.

    PubMed

    Ciuffi, Angela; Rato, Sylvie; Telenti, Amalio

    2016-05-04

    Single-cell sequencing technologies, i.e., single cell analysis followed by deep sequencing investigate cellular heterogeneity in many biological settings. It was only in the past year that single-cell sequencing analyses has been applied in the field of virology, providing new ways to explore viral diversity and cell response to viral infection, which are summarized in the present review.

  20. Single-cell RNA-seq reveals activation of unique gene groups as a consequence of stem cell-parenchymal cell fusion.

    PubMed

    Freeman, Brian T; Jung, Jangwook P; Ogle, Brenda M

    2016-03-21

    Fusion of donor mesenchymal stem cells with parenchymal cells of the recipient can occur in the brain, liver, intestine and heart following transplantation. The therapeutic benefit or detriment of resultant hybrids is unknown. Here we sought a global view of phenotypic diversification of mesenchymal stem cell-cardiomyocyte hybrids and associated time course. Using single-cell RNA-seq, we found hybrids consistently increase ribosome components and decrease genes associated with the cell cycle suggesting an increase in protein production and decrease in proliferation to accommodate the fused state. But in the case of most other gene groups, hybrids were individually distinct. In fact, though hybrids can express a transcriptome similar to individual fusion partners, approximately one-third acquired distinct expression profiles in a single day. Some hybrids underwent reprogramming, expressing pluripotency and cardiac precursor genes latent in parental cells and associated with developmental and morphogenic gene groups. Other hybrids expressed genes associated with ontologic cancer sets and two hybrids of separate experimental replicates clustered with breast cancer cells, expressing critical oncogenes and lacking tumor suppressor genes. Rapid transcriptional diversification of this type garners consideration in the context of cellular transplantation to damaged tissues, those with viral infection or other microenvironmental conditions that might promote fusion.

  1. Lhcb transcription is coordinated with cell size and chlorophyll accumulation. Studies on fluorescence-activated, cell-sorter-purified single cells from wild-type and immutans Arabidopsis thaliana

    SciTech Connect

    Meehan, L.; Harkins, K.; Rodermel, S.

    1996-11-01

    To study the mechanisms that integrate pigment and chlorophyll a/b-binding apoprotein biosynthesis during light-harvesting complex II assembly, we have examined {beta}-glucuronidase (GUS) enzyme activities, cell-sorting-separated single cells sizes in fluorescence activated, cell-sorting-separated single cells from transgenic Arabidopsis thaliana wild-type and immutans variegation mutant plants that express an Lhcb (photosystem II chlorophyll a/b-binding polypeptide gene)/GUS promoter fusion. We found that GUS activities are positively correlated with chlorophyll content and cell size in green cells from the control and immutans plants, indicating that Lhcb gene transcription is coordinated with cell size in this species. Compared with the control plants, however, chlorophyll production is enhanced in the green cells of immutans; this may represent part of a strategy to maximize photosynthesis in the white sectors of the mutant. Lhcb transcription is significantly higher in pure-white cells of the transgenic immutans plants than in pure-white cells from norflurazon-treated, photooxidized A. thaliana leaves. This suggests that immutans partially uncouples Lhcb transcription from its normal dependence on chlorophyll accumulation and chloroplast development. We conclude that immutans may play a role in regulating Lhcb transcription, and may be a key component in the signal transduction pathways that control chloroplast biogenesis. 58 refs., 5 figs., 2 tabs.

  2. Single-Cell Imaging Mass Spectrometry

    PubMed Central

    Passarelli, Melissa K.; Ewing, Andrew G.

    2013-01-01

    Single-cell imaging mass spectrometry (IMS) is a powerful technique used to map the distributions of endogenous biomolecules with sub-cellular resolution. Currently, secondary ion mass spectrometry is the predominant technique for single-cell IMS, thanks to its sub-micron lateral resolution and surface sensitivity. However, recent methodological and technological developments aimed at improving the spatial resolution of matrix assisted laser desorption ionization (MALDI) have made this technique a potential platform of single-cell IMS. MALDI opens the field of single-cell IMS to new possibilities, including single cell proteomic imaging and atmospheric pressure analyses; however, sensitivity is a challenge. In this report, we estimate the availability of proteins and lipids in a single cell and discuss strategies employed to improve sensitivity at the single-cell level. PMID:23948695

  3. Monitoring by Control Technique - Activated Carbon Adsorber

    EPA Pesticide Factsheets

    Stationary source emissions monitoring is required to demonstrate that a source is meeting the requirements in Federal or state rules. This page is about Activated Carbon Adsorber control techniques used to reduce pollutant emissions.

  4. Monitoring ion activities in and around cells using ion-selective liquid-membrane microelectrodes.

    PubMed

    Lee, Seong-Ki; Boron, Walter F; Parker, Mark D

    2013-01-15

    Determining the effective concentration (i.e., activity) of ions in and around living cells is important to our understanding of the contribution of those ions to cellular function. Moreover, monitoring changes in ion activities in and around cells is informative about the actions of the transporters and/or channels operating in the cell membrane. The activity of an ion can be measured using a glass microelectrode that includes in its tip a liquid-membrane doped with an ion-selective ionophore. Because these electrodes can be fabricated with tip diameters that are less than 1 μm, they can be used to impale single cells in order to monitor the activities of intracellular ions. This review summarizes the history, theory, and practice of ion-selective microelectrode use and brings together a number of classic and recent examples of their usefulness in the realm of physiological study.

  5. Monitoring Ion Activities In and Around Cells Using Ion-Selective Liquid-Membrane Microelectrodes

    PubMed Central

    Lee, Seong-Ki; Boron, Walter F.; Parker, Mark D.

    2013-01-01

    Determining the effective concentration (i.e., activity) of ions in and around living cells is important to our understanding of the contribution of those ions to cellular function. Moreover, monitoring changes in ion activities in and around cells is informative about the actions of the transporters and/or channels operating in the cell membrane. The activity of an ion can be measured using a glass microelectrode that includes in its tip a liquid-membrane doped with an ion-selective ionophore. Because these electrodes can be fabricated with tip diameters that are less than 1 μm, they can be used to impale single cells in order to monitor the activities of intracellular ions. This review summarizes the history, theory, and practice of ion-selective microelectrode use and brings together a number of classic and recent examples of their usefulness in the realm of physiological study. PMID:23322102

  6. A microfluidic approach to parallelized transcriptional profiling of single cells.

    PubMed

    Sun, Hao; Olsen, Timothy; Zhu, Jing; Tao, Jianguo; Ponnaiya, Brian; Amundson, Sally A; Brenner, David J; Lin, Qiao

    2015-12-01

    The ability to correlate single-cell genetic information with cellular phenotypes is of great importance to biology and medicine, as it holds the potential to gain insight into disease pathways that is unavailable from ensemble measurements. We present a microfluidic approach to parallelized, rapid, quantitative analysis of messenger RNA from single cells via RT-qPCR. The approach leverages an array of single-cell RT-qPCR analysis units formed by a set of parallel microchannels concurrently controlled by elastomeric pneumatic valves, thereby enabling parallelized handling and processing of single cells in a drastically simplified operation procedure using a relatively small number of microvalves. All steps for single-cell RT-qPCR, including cell isolation and immobilization, cell lysis, mRNA purification, reverse transcription and qPCR, are integrated on a single chip, eliminating the need for off-chip manual cell and reagent transfer and qPCR amplification as commonly used in existing approaches. Additionally, the approach incorporates optically transparent microfluidic components to allow monitoring of single-cell trapping without the need for molecular labeling that can potentially alter the targeted gene expression and utilizes a polycarbonate film as a barrier against evaporation to minimize the loss of reagents at elevated temperatures during the analysis. We demonstrate the utility of the approach by the transcriptional profiling for the induction of the cyclin-dependent kinase inhibitor 1a and the glyceraldehyde 3-phosphate dehydrogenase in single cells from the MCF-7 breast cancer cell line. Furthermore, the methyl methanesulfonate is employed to allow measurement of the expression of the genes in individual cells responding to a genotoxic stress.

  7. High-Throughput Single-Cell Manipulation in Brain Tissue

    PubMed Central

    Steinmeyer, Joseph D.; Yanik, Mehmet Fatih

    2012-01-01

    The complexity of neurons and neuronal circuits in brain tissue requires the genetic manipulation, labeling, and tracking of single cells. However, current methods for manipulating cells in brain tissue are limited to either bulk techniques, lacking single-cell accuracy, or manual methods that provide single-cell accuracy but at significantly lower throughputs and repeatability. Here, we demonstrate high-throughput, efficient, reliable, and combinatorial delivery of multiple genetic vectors and reagents into targeted cells within the same tissue sample with single-cell accuracy. Our system automatically loads nanoliter-scale volumes of reagents into a micropipette from multiwell plates, targets and transfects single cells in brain tissues using a robust electroporation technique, and finally preps the micropipette by automated cleaning for repeating the transfection cycle. We demonstrate multi-colored labeling of adjacent cells, both in organotypic and acute slices, and transfection of plasmids encoding different protein isoforms into neurons within the same brain tissue for analysis of their effects on linear dendritic spine density. Our platform could also be used to rapidly deliver, both ex vivo and in vivo, a variety of genetic vectors, including optogenetic and cell-type specific agents, as well as fast-acting reagents such as labeling dyes, calcium sensors, and voltage sensors to manipulate and track neuronal circuit activity at single-cell resolution. PMID:22536416

  8. High-throughput single-cell manipulation in brain tissue.

    PubMed

    Steinmeyer, Joseph D; Yanik, Mehmet Fatih

    2012-01-01

    The complexity of neurons and neuronal circuits in brain tissue requires the genetic manipulation, labeling, and tracking of single cells. However, current methods for manipulating cells in brain tissue are limited to either bulk techniques, lacking single-cell accuracy, or manual methods that provide single-cell accuracy but at significantly lower throughputs and repeatability. Here, we demonstrate high-throughput, efficient, reliable, and combinatorial delivery of multiple genetic vectors and reagents into targeted cells within the same tissue sample with single-cell accuracy. Our system automatically loads nanoliter-scale volumes of reagents into a micropipette from multiwell plates, targets and transfects single cells in brain tissues using a robust electroporation technique, and finally preps the micropipette by automated cleaning for repeating the transfection cycle. We demonstrate multi-colored labeling of adjacent cells, both in organotypic and acute slices, and transfection of plasmids encoding different protein isoforms into neurons within the same brain tissue for analysis of their effects on linear dendritic spine density. Our platform could also be used to rapidly deliver, both ex vivo and in vivo, a variety of genetic vectors, including optogenetic and cell-type specific agents, as well as fast-acting reagents such as labeling dyes, calcium sensors, and voltage sensors to manipulate and track neuronal circuit activity at single-cell resolution.

  9. Inside Single Cells: Quantitative Analysis with Advanced Optics and Nanomaterials

    PubMed Central

    Cui, Yi; Irudayaraj, Joseph

    2014-01-01

    Single cell explorations offer a unique window to inspect molecules and events relevant to mechanisms and heterogeneity constituting the central dogma of biology. A large number of nucleic acids, proteins, metabolites and small molecules are involved in determining and fine-tuning the state and function of a single cell at a given time point. Advanced optical platforms and nanotools provide tremendous opportunities to probe intracellular components with single-molecule accuracy, as well as promising tools to adjust single cell activity. In order to obtain quantitative information (e.g. molecular quantity, kinetics and stoichiometry) within an intact cell, achieving the observation with comparable spatiotemporal resolution is a challenge. For single cell studies both the method of detection and the biocompatibility are critical factors as they determine the feasibility, especially when considering live cell analysis. Although a considerable proportion of single cell methodologies depend on specialized expertise and expensive instruments, it is our expectation that the information content and implication will outweigh the costs given the impact on life science enabled by single cell analysis. PMID:25430077

  10. Highly multiplexed quantitation of gene expression on single cells

    PubMed Central

    Dominguez, Maria H.; Chattopadhyay, Pratip K.; Ma, Steven; Lamoreaux, Laurie; McDavid, Andrew; Finak, Greg; Gottardo, Raphael; Koup, Richard A.; Roederer, Mario

    2013-01-01

    Highly multiplexed, single-cell technologies reveal important heterogeneity within cell populations. Recently, technologies to simultaneously measure expression of 96 (or more) genes from a single cell have been developed for immunologic monitoring. Here, we report a rigorous, optimized, quantitative methodology for using this technology. Specifically: we describe a unique primer/probe qualification method necessary for quantitative results; we show that primers do not compete in highly multiplexed amplifications; we define the limit of detection for this assay as a single mRNA transcript; and, we show that the technical reproducibility of the system is very high. We illustrate two disparate applications of the platform: a “bulk” approach that measures expression patterns from 100 cells at a time in high throughput to define gene signatures, and a single-cell approach to define the coordinate expression patterns of multiple genes and reveal unique subsets of cells. PMID:23500781

  11. Future medical applications of single-cell sequencing in cancer

    PubMed Central

    2011-01-01

    Advances in whole genome amplification and next-generation sequencing methods have enabled genomic analyses of single cells, and these techniques are now beginning to be used to detect genomic lesions in individual cancer cells. Previous approaches have been unable to resolve genomic differences in complex mixtures of cells, such as heterogeneous tumors, despite the importance of characterizing such tumors for cancer treatment. Sequencing of single cells is likely to improve several aspects of medicine, including the early detection of rare tumor cells, monitoring of circulating tumor cells (CTCs), measuring intratumor heterogeneity, and guiding chemotherapy. In this review we discuss the challenges and technical aspects of single-cell sequencing, with a strong focus on genomic copy number, and discuss how this information can be used to diagnose and treat cancer patients. PMID:21631906

  12. Microfluidic whole genome amplification device for single cell sequencing.

    PubMed

    Yu, Zhilong; Lu, Sijia; Huang, Yanyi

    2014-10-07

    We developed a microfluidic device to perform multiplex single-cell whole-genome amplification (WGA) using multiple annealing and looping-based amplification cycles (MALBAC). This device, made of polydimethylsiloxane (PDMS), allows us to monitor the whole process of cell loading and single-cell WGA for sequencing. We show that the genome coverage of MALBAC amplifications is reproducible between chambers on a single chip and between different chips, which enables data normalization using standard samples to accurately identify copy number variations (CNVs). This device provides an easy-to-operate approach to perform single cell sequencing library preparation with minimum hands-on time. It reduces the requirement of manual expertise as well as the risk of contamination, which is essential in future applications especially the medical diagnosis.

  13. Future medical applications of single-cell sequencing in cancer.

    PubMed

    Navin, Nicholas; Hicks, James

    2011-05-31

    Advances in whole genome amplification and next-generation sequencing methods have enabled genomic analyses of single cells, and these techniques are now beginning to be used to detect genomic lesions in individual cancer cells. Previous approaches have been unable to resolve genomic differences in complex mixtures of cells, such as heterogeneous tumors, despite the importance of characterizing such tumors for cancer treatment. Sequencing of single cells is likely to improve several aspects of medicine, including the early detection of rare tumor cells, monitoring of circulating tumor cells (CTCs), measuring intratumor heterogeneity, and guiding chemotherapy. In this review we discuss the challenges and technical aspects of single-cell sequencing, with a strong focus on genomic copy number, and discuss how this information can be used to diagnose and treat cancer patients.

  14. Assessing physical activity using wearable monitors: measures of physical activity.

    PubMed

    Butte, Nancy F; Ekelund, Ulf; Westerterp, Klaas R

    2012-01-01

    Physical activity may be defined broadly as "all bodily actions produced by the contraction of skeletal muscle that increase energy expenditure above basal level." Physical activity is a complex construct that can be classified into major categories qualitatively, quantitatively, or contextually. The quantitative assessment of physical activity using wearable monitors is grounded in the measurement of energy expenditure. Six main categories of wearable monitors are currently available to investigators: pedometers, load transducers/foot-contact monitors, accelerometers, HR monitors, combined accelerometer and HR monitors, and multiple sensor systems. Currently available monitors are capable of measuring total physical activity as well as components of physical activity that play important roles in human health. The selection of wearable monitors for measuring physical activity will depend on the physical activity component of interest, study objectives, characteristics of the target population, and study feasibility in terms of cost and logistics. Future development of sensors and analytical techniques for assessing physical activity should focus on the dynamic ranges of sensors, comparability for sensor output across manufacturers, and the application of advanced modeling techniques to predict energy expenditure and classify physical activities. New approaches for qualitatively classifying physical activity should be validated using direct observation or recording. New sensors and methods for quantitatively assessing physical activity should be validated in laboratory and free-living populations using criterion methods of calorimetry or doubly labeled water.

  15. An automated programmable platform enabling multiplex dynamic stimuli delivery and cellular response monitoring for high-throughput suspension single-cell signaling studies† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c4lc01070a Click here for additional data file.

    PubMed Central

    He, Luye; Kniss, Ariel; San-Miguel, Adriana; Rouse, Tel; Kemp, Melissa L.

    2015-01-01

    Cell signaling events are orchestrated by dynamic external biochemical cues. By rapidly perturbing cells with dynamic inputs and examining the output from these systems, one could study the structure and dynamic properties of a cellular signaling network. Conventional experimental techniques limit the implementation of these systematic approaches due to the lack of sophistication in manipulating individual cells and the fluid microenvironment around them; existing microfluidic technologies thus far are mainly targeting adherent cells. In this paper we present an automated platform to interrogate suspension cells with dynamic stimuli while simultaneously monitoring cellular responses in a high-throughput manner at single-cell resolution. We demonstrate the use of this platform in an experiment to measure Jurkat T cells in response to distinct dynamic patterns of stimuli; we find cells exhibit highly heterogeneous responses under each stimulation condition. More interestingly, these cells act as low-pass filters, only entrained to the low frequency stimulus signals. We also demonstrate that this platform can be easily programmed to actively generate arbitrary dynamic signals. We envision our platform to be useful in other contexts to study cellular signaling dynamics, which may be difficult using conventional experimental methods. PMID:25609410

  16. Scaling and automation of a high-throughput single-cell-derived tumor sphere assay chip.

    PubMed

    Cheng, Yu-Heng; Chen, Yu-Chih; Brien, Riley; Yoon, Euisik

    2016-10-07

    Recent research suggests that cancer stem-like cells (CSCs) are the key subpopulation for tumor relapse and metastasis. Due to cancer plasticity in surface antigen and enzymatic activity markers, functional tumorsphere assays are promising alternatives for CSC identification. To reliably quantify rare CSCs (1-5%), thousands of single-cell suspension cultures are required. While microfluidics is a powerful tool in handling single cells, previous works provide limited throughput and lack automatic data analysis capability required for high-throughput studies. In this study, we present the scaling and automation of high-throughput single-cell-derived tumor sphere assay chips, facilitating the tracking of up to ∼10 000 cells on a chip with ∼76.5% capture rate. The presented cell capture scheme guarantees sampling a representative population from the bulk cells. To analyze thousands of single-cells with a variety of fluorescent intensities, a highly adaptable analysis program was developed for cell/sphere counting and size measurement. Using a Pluronic® F108 (poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol)) coating on polydimethylsiloxane (PDMS), a suspension culture environment was created to test a controversial hypothesis: whether larger or smaller cells are more stem-like defined by the capability to form single-cell-derived spheres. Different cell lines showed different correlations between sphere formation rate and initial cell size, suggesting heterogeneity in pathway regulation among breast cancer cell lines. More interestingly, by monitoring hundreds of spheres, we identified heterogeneity in sphere growth dynamics, indicating the cellular heterogeneity even within CSCs. These preliminary results highlight the power of unprecedented high-throughput and automation in CSC studies.

  17. Monitoring Biological Activity at Geothermal Power Plants

    SciTech Connect

    Peter Pryfogle

    2005-09-01

    The economic impact of microbial growth in geothermal power plants has been estimated to be as high as $500,000 annually for a 100 MWe plant. Many methods are available to monitor biological activity at these facilities; however, very few plants have any on-line monitoring program in place. Metal coupon, selective culturing (MPN), total organic carbon (TOC), adenosine triphosphate (ATP), respirometry, phospholipid fatty acid (PLFA), and denaturing gradient gel electrophoresis (DGGE) characterizations have been conducted using water samples collected from geothermal plants located in California and Utah. In addition, the on-line performance of a commercial electrochemical monitor, the BIoGEORGE?, has been evaluated during extended deployments at geothermal facilities. This report provides a review of these techniques, presents data on their application from laboratory and field studies, and discusses their value in characterizing and monitoring biological activities at geothermal power plants.

  18. Functional activity monitoring from wearable sensor data.

    PubMed

    Nawab, S Hamid; Roy, Serge H; De Luca, Carlo J

    2004-01-01

    A novel approach is presented for the interpretation and use of EMG and accelerometer data to monitor, identify, and categorize functional motor activities in individuals whose movements are unscripted, unrestrained, and take place in the "real world". Our proposed solution provides a novel and practical way of conceptualizing physical activities that facilitates the deployment of modern signal processing and interpretation techniques to carry out activity monitoring. A hierarchical approach is adopted that is based upon: 1) blackboard and rule-based technology from artificial intelligence to support a process in which coarse-grained activity partitioning forms the context for finer-grained activity partitioning; 2) neural network technology to support initial activity classification; and 3) integrated processing and understanding of signals (IPUS) technology for revising the initial classifications to account for the high degrees of anticipated signal variability and overlap during freeform activity.

  19. Active personal radiation monitor for lunar EVA

    NASA Astrophysics Data System (ADS)

    Straume, Tore; Borak, Tom; Braby, L. A.; Lusby, Terry; Semones, Edward J.; Vazquez, Marcelo E.

    As astronauts return to the Moon-and this time, work for extended periods-there will be a critical need for crew personnel radiation monitoring as they operate lunar rovers or otherwise perform a myriad of extravehicular activities (EVAs). Our focus is on development of a small personal radiation monitor for lunar EVA that responds to the complex radiation quality and changing dose rates on the Moon. Of particular concern are active monitoring capabilities that provide both early warning and radiation dosimetry information during solar particle events (SPEs). To accomplish this, we are developing small detectors integrated with modern high speed, low power microelectronics to measure dose-rate and dose-mean lineal energy in real time. The monitor is designed to perform over the range of dose rates and LETs expected from both GCR and SPE radiations during lunar EVA missions. The monitor design provides simultaneous measurement of dose-equivalent rates at two tissue-equivalent depths simulating skin and marrow. The compact personal monitor is estimated to be the size of a cell phone and would fit on an EVA spacesuit (e.g., in backpack) or in a toolbox. The four-year development effort (which began December 2007) will result in a prototype radiation monitor field tested and characterized for the major radiations expected on the surface of the Moon. We acknowledge support from NSBRI through grants to NASA Ames Research Center (T. Straume, PI) and Colorado State University (T. Borak, PI).

  20. Nanowire-based single-cell endoscopy

    NASA Astrophysics Data System (ADS)

    Yan, Ruoxue; Park, Ji-Ho; Choi, Yeonho; Heo, Chul-Joon; Yang, Seung-Man; Lee, Luke P.; Yang, Peidong

    2012-03-01

    One-dimensional smart probes based on nanowires and nanotubes that can safely penetrate the plasma membrane and enter biological cells are potentially useful in high-resolution and high-throughput gene and drug delivery, biosensing and single-cell electrophysiology. However, using such probes for optical communication across the cellular membrane at the subwavelength level remains limited. Here, we show that a nanowire waveguide attached to the tapered tip of an optical fibre can guide visible light into intracellular compartments of a living mammalian cell, and can also detect optical signals from subcellular regions with high spatial resolution. Furthermore, we show that through light-activated mechanisms the endoscope can deliver payloads into cells with spatial and temporal specificity. Moreover, insertion of the endoscope into cells and illumination of the guided laser did not induce any significant toxicity in the cells.

  1. Stochastic and Differential Activation of σB and PrfA in Listeria monocytogenes at the Single Cell Level under Different Environmental Stress Conditions

    PubMed Central

    Guldimann, Claudia; Guariglia-Oropeza, Veronica; Harrand, Sophia; Kent, David; Boor, Kathryn J.; Wiedmann, Martin

    2017-01-01

    During host infection, the foodborne pathogen Listeria monocytogenes must sense and respond to rapidly changing environmental conditions. Two transcriptional regulators, the alternative sigma factor B (σB) and the Positive Regulatory Factor A (PrfA), are key contributors to the transcriptomic responses that enable bacterial survival in the host gastrointestinal tract and invasion of host duodenal cells. Increases in temperature and osmolarity induce activity of these proteins; such conditions may be encountered in food matrices as well as within the host gastrointestinal tract. Differences in PrfA and σB activity between individual cells might affect the fate of a cell during host invasion, therefore, we hypothesized that PrfA and σB activities differ among individual cells under heat and salt stress. We used fluorescent reporter fusions to determine the relative proportions of cells with active σB or PrfA following exposure to 45°C heat or 4% NaCl. Activities of both PrfA and σB were induced stochastically, with fluorescence levels ranging from below detection to high among individual cells. The proportion of cells with active PrfA was significantly higher than the proportion with active σB under all tested conditions; under some conditions, nearly all cells had active PrfA. Our findings further support the growing body of evidence illustrating the stochastic nature of bacterial gene expression under conditions that are relevant for host invasion via food-borne, oral infection. PMID:28352251

  2. Stochastic and Differential Activation of σ(B) and PrfA in Listeria monocytogenes at the Single Cell Level under Different Environmental Stress Conditions.

    PubMed

    Guldimann, Claudia; Guariglia-Oropeza, Veronica; Harrand, Sophia; Kent, David; Boor, Kathryn J; Wiedmann, Martin

    2017-01-01

    During host infection, the foodborne pathogen Listeria monocytogenes must sense and respond to rapidly changing environmental conditions. Two transcriptional regulators, the alternative sigma factor B (σ(B)) and the Positive Regulatory Factor A (PrfA), are key contributors to the transcriptomic responses that enable bacterial survival in the host gastrointestinal tract and invasion of host duodenal cells. Increases in temperature and osmolarity induce activity of these proteins; such conditions may be encountered in food matrices as well as within the host gastrointestinal tract. Differences in PrfA and σ(B) activity between individual cells might affect the fate of a cell during host invasion, therefore, we hypothesized that PrfA and σ(B) activities differ among individual cells under heat and salt stress. We used fluorescent reporter fusions to determine the relative proportions of cells with active σ(B) or PrfA following exposure to 45°C heat or 4% NaCl. Activities of both PrfA and σ(B) were induced stochastically, with fluorescence levels ranging from below detection to high among individual cells. The proportion of cells with active PrfA was significantly higher than the proportion with active σ(B) under all tested conditions; under some conditions, nearly all cells had active PrfA. Our findings further support the growing body of evidence illustrating the stochastic nature of bacterial gene expression under conditions that are relevant for host invasion via food-borne, oral infection.

  3. Single-cell regulome data analysis by SCRAT.

    PubMed

    Ji, Zhicheng; Zhou, Weiqiang; Ji, Hongkai

    2017-09-15

    Emerging single-cell technologies (e.g. single-cell ATAC-seq, DNase-seq or ChIP-seq) have made it possible to assay regulome of individual cells. Single-cell regulome data are highly sparse and discrete. Analyzing such data is challenging. User-friendly software tools are still lacking. We present SCRAT, a Single-Cell Regulome Analysis Toolbox with a graphical user interface, for studying cell heterogeneity using single-cell regulome data. SCRAT can be used to conveniently summarize regulatory activities according to different features (e.g. gene sets, transcription factor binding motif sites, etc.). Using these features, users can identify cell subpopulations in a heterogeneous biological sample, infer cell identities of each subpopulation, and discover distinguishing features such as gene sets and transcription factors that show different activities among subpopulations. SCRAT is freely available at https://zhiji.shinyapps.io/scrat as an online web service and at https://github.com/zji90/SCRAT as an R package. hji@jhu.edu. Supplementary data are available at Bioinformatics online.

  4. Active Sites Environmental Monitoring Program: Program plan

    SciTech Connect

    Ashwood, T.L.; Wickliff, D.S.; Morrissey, C.M.

    1990-10-01

    DOE Order 5820.2A requires that low-level waste (LLW) disposal sites active on or after September 1988 and all transuranic (TRU) waste storage sites be monitored periodically to assure that radioactive contamination does not escape from the waste sites and pose a threat to the public or to the environment. This plan describes such a monitoring program for the active LLW disposal sites in SWSA 6 and the TRU waste storage sites in SWSA 5 North. 14 refs., 8 figs.

  5. Thermogenetic neurostimulation with single-cell resolution

    PubMed Central

    Ermakova, Yulia G.; Lanin, Aleksandr A.; Fedotov, Ilya V.; Roshchin, Matvey; Kelmanson, Ilya V.; Kulik, Dmitry; Bogdanova, Yulia A.; Shokhina, Arina G.; Bilan, Dmitry S.; Staroverov, Dmitry B.; Balaban, Pavel M.; Fedotov, Andrei B.; Sidorov-Biryukov, Dmitry A.; Nikitin, Evgeny S.; Zheltikov, Aleksei M.; Belousov, Vsevolod V.

    2017-01-01

    Thermogenetics is a promising innovative neurostimulation technique, which enables robust activation of neurons using thermosensitive transient receptor potential (TRP) cation channels. Broader application of this approach in neuroscience is, however, hindered by a limited variety of suitable ion channels, and by low spatial and temporal resolution of neuronal activation when TRP channels are activated by ambient temperature variations or chemical agonists. Here, we demonstrate rapid, robust and reproducible repeated activation of snake TRPA1 channels heterologously expressed in non-neuronal cells, mouse neurons and zebrafish neurons in vivo by infrared (IR) laser radiation. A fibre-optic probe that integrates a nitrogen−vacancy (NV) diamond quantum sensor with optical and microwave waveguide delivery enables thermometry with single-cell resolution, allowing neurons to be activated by exceptionally mild heating, thus preventing the damaging effects of excessive heat. The neuronal responses to the activation by IR laser radiation are fully characterized using Ca2+ imaging and electrophysiology, providing, for the first time, a complete framework for a thermogenetic manipulation of individual neurons using IR light. PMID:28530239

  6. Integrated Electrowetting Nanoinjector for Single Cell Transfection

    PubMed Central

    Shekaramiz, Elaheh; Varadarajalu, Ganeshkumar; Day, Philip J.; Wickramasinghe, H. Kumar

    2016-01-01

    Single cell transfection techniques are essential to understand the heterogeneity between cells. We have developed an integrated electrowetting nanoinjector (INENI) to transfect single cells. The high transfection efficiency, controlled dosage delivery and ease of INENI fabrication promote the widespread application of the INENI in cell transfection assays. PMID:27374766

  7. Novel approaches in function-driven single-cell genomics

    DOE PAGES

    Doud, Devin F. R.; Woyke, Tanja

    2017-06-07

    Deeper sequencing and improved bioinformatics in conjunction with single-cell and metagenomic approaches continue to illuminate undercharacterized environmental microbial communities. This has propelled the 'who is there, and what might they be doing' paradigm to the uncultivated and has already radically changed the topology of the tree of life and provided key insights into the microbial contribution to biogeochemistry. While characterization of 'who' based on marker genes can describe a large fraction of the community, answering 'what are they doing' remains the elusive pinnacle for microbiology. Function-driven single-cell genomics provides a solution by using a function-based screen to subsample complex microbialmore » communities in a targeted manner for the isolation and genome sequencing of single cells. This enables single-cell sequencing to be focused on cells with specific phenotypic or metabolic characteristics of interest. Recovered genomes are conclusively implicated for both encoding and exhibiting the feature of interest, improving downstream annotation and revealing activity levels within that environment. This emerging approach has already improved our understanding of microbial community functioning and facilitated the experimental analysis of uncharacterized gene product space. Here we provide a comprehensive review of strategies that have been applied for function-driven single-cell genomics and the future directions we envision.« less

  8. 34 CFR 300.120 - Monitoring activities.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 34 Education 2 2013-07-01 2013-07-01 false Monitoring activities. 300.120 Section 300.120 Education Regulations of the Offices of the Department of Education (Continued) OFFICE OF SPECIAL EDUCATION AND REHABILITATIVE SERVICES, DEPARTMENT OF EDUCATION ASSISTANCE TO STATES FOR THE EDUCATION...

  9. 34 CFR 300.120 - Monitoring activities.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 34 Education 2 2014-07-01 2013-07-01 true Monitoring activities. 300.120 Section 300.120 Education Regulations of the Offices of the Department of Education (Continued) OFFICE OF SPECIAL EDUCATION AND REHABILITATIVE SERVICES, DEPARTMENT OF EDUCATION ASSISTANCE TO STATES FOR THE EDUCATION OF CHILDREN...

  10. 34 CFR 300.120 - Monitoring activities.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 34 Education 2 2010-07-01 2010-07-01 false Monitoring activities. 300.120 Section 300.120 Education Regulations of the Offices of the Department of Education (Continued) OFFICE OF SPECIAL EDUCATION AND REHABILITATIVE SERVICES, DEPARTMENT OF EDUCATION ASSISTANCE TO STATES FOR THE EDUCATION...

  11. 34 CFR 300.120 - Monitoring activities.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 34 Education 2 2012-07-01 2012-07-01 false Monitoring activities. 300.120 Section 300.120 Education Regulations of the Offices of the Department of Education (Continued) OFFICE OF SPECIAL EDUCATION AND REHABILITATIVE SERVICES, DEPARTMENT OF EDUCATION ASSISTANCE TO STATES FOR THE EDUCATION...

  12. 34 CFR 300.120 - Monitoring activities.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 34 Education 2 2011-07-01 2010-07-01 true Monitoring activities. 300.120 Section 300.120 Education Regulations of the Offices of the Department of Education (Continued) OFFICE OF SPECIAL EDUCATION AND REHABILITATIVE SERVICES, DEPARTMENT OF EDUCATION ASSISTANCE TO STATES FOR THE EDUCATION OF CHILDREN...

  13. Single-cell microfluidics: opportunity for bioprocess development.

    PubMed

    Grünberger, Alexander; Wiechert, Wolfgang; Kohlheyer, Dietrich

    2014-10-01

    Cell-to-cell heterogeneity in microbial biotechnological processes caused by biological (intrinsic) and environmental (extrinsic) fluctuations can have a severe impact on productivity. However, as yet little is known about the complex interplay between environmental reactor dynamics and cellular activity. A few years ago, innovative microfluidic systems were introduced facilitating the spatiotemporal analysis of single cells under well-defined environmental conditions allowing so far unachievable insights into population heterogeneity and bioreactor inhomogeneity. Examples of microfabricated systems include microfluidic cavities harbouring micropopulations of several thousand cells down to femtolitre-size structures entrapping individual bacteria. In well-defined perfusion experiments, central questions in biotechnology regarding, for example, growth, productivity, and heterogeneity on the single-cell level have been addressed for the first time. Microfluidics will take its place as a single-cell analytical technique in biotechnological process and strain characterization. Copyright © 2014 Elsevier Ltd. All rights reserved.

  14. GHRP-6-induced changes in electrical activity of single cells in the arcuate, ventromedial and periventricular nucleus neurones [correction of nuclei] of a hypothalamic slice preparation in vitro.

    PubMed

    Hewson, A K; Viltart, O; McKenzie, D N; Dyball, R E; Dickson, S L

    1999-12-01

    Previously, we demonstrated that systemic injection of the growth hormone secretagogue, growth hormone-releasing peptide (GHRP)-6, selectively activated cells in the hypothalamic arcuate nucleus, as reflected by increased electrical activity and induction of the immediate early gene c-fos. The growth hormone secretagogue receptor distribution is not confined to the arcuate nucleus, suggesting that additional sites of action may exist. In the present study we characterized the electrophysiological responses of cells in the arcuate nucleus, ventromedial nucleus and periventricular nucleus in an in-vitro hypothalamic slice preparation, following bath application of GHRP-6. Additionally, since central somatostatin administration has been shown to attenuate the induction of the c-fos gene by GHRP-6, we sought to determine whether the arcuate cells activated by GHRP-6 are also somatostatin-sensitive. Male Wistar rats (100-150 g body weight (BW)) were anaesthetized (urethane; 1.2 g/kg BW) and the brains removed. Coronal sections (400 microm thickness) were cut through a block of hypothalamus and were transferred to a slice chamber perfused with artificial cerebrospinal fluid. Forty-one arcuate nucleus cells were tested with bath application of 15 microm GHRP-6 for 10 min, 16 of which were tested subsequently (>30 min later) with application of 10 microM somatostatin. Following GHRP-6 administration, 19 cells (46. 3%) showed a significant increase in firing rate during the 15-min period after GHRP-6 application (P<0.001), 17 cells (41.5%) did not respond and the remaining five cells (12.2%) were significantly inhibited. Six of the eight arcuate nucleus cells that were excited by GHRP-6 were significantly inhibited by somatostatin. By contrast, five of the six arcuate nucleus cells that were unresponsive to GHRP-6 were also unresponsive to somatostatin. In the ventromedial nucleus, of 19 cells tested, eight cells (42.1%) were excited by GHRP-6, eight cells (42.1%) were

  15. Apoptosis induction-related cytosolic calcium responses revealed by the dual FRET imaging of calcium signals and caspase-3 activation in a single cell.

    PubMed

    Miyamoto, Akitoshi; Miyauchi, Hiroshi; Kogure, Takako; Miyawaki, Atsushi; Michikawa, Takayuki; Mikoshiba, Katsuhiko

    2015-04-24

    Stimulus-induced changes in the intracellular Ca(2+) concentration control cell fate decision, including apoptosis. However, the precise patterns of the cytosolic Ca(2+) signals that are associated with apoptotic induction remain unknown. We have developed a novel genetically encoded sensor of activated caspase-3 that can be applied in combination with a genetically encoded sensor of the Ca(2+) concentration and have established a dual imaging system that enables the imaging of both cytosolic Ca(2+) signals and caspase-3 activation, which is an indicator of apoptosis, in the same cell. Using this system, we identified differences in the cytosolic Ca(2+) signals of apoptotic and surviving DT40 B lymphocytes after B cell receptor (BCR) stimulation. In surviving cells, BCR stimulation evoked larger initial Ca(2+) spikes followed by a larger sustained elevation of the Ca(2+) concentration than those in apoptotic cells; BCR stimulation also resulted in repetitive transient Ca(2+) spikes, which were mediated by the influx of Ca(2+) from the extracellular space. Our results indicate that the observation of both Ca(2+) signals and cells fate in same cell is crucial to gain an accurate understanding of the function of intracellular Ca(2+) signals in apoptotic induction.

  16. Monitoring Malware Activity on the LAN Network

    NASA Astrophysics Data System (ADS)

    Skrzewski, Mirosław

    Many security related organizations periodically publish current network and systems security information, with the lists of top malware programs. These lists raises the question how these threats spreads out, if the worms (the only threat with own communication abilities) are low or missing on these lists. The paper discuss the research on malware network activity, aimed to deliver the answer to the question, what is the main infection channel of modern malware, done with the usage of virtual honeypot systems on dedicated, unprotected network. Systems setup, network and systems monitoring solutions, results of over three months of network traffic and malware monitoring are presented, along with the proposed answer to our research question.

  17. Active Monitoring Complex-Envelope Processing

    NASA Astrophysics Data System (ADS)

    Unger, R.

    2011-12-01

    Pseudo-random 3-D signal transmission followed by quadrature signal processing significantly enhances seismic Active Monitoring (AM). The algorithm produces complex-envelope seismograms consisting of the dual time series of instantaneous amplitude and phase. Minimum-standard-deviation signal arrival detection and timing from coherent instantaneous-phase seismogram ensembles yields signal time errors inversely proportional to frequency and signal-to-noise ratio. In particular, application to piezo-electric acoustic cross well monitoring as in SAFOD experiments should result in nano-second multiple-signal detection and timing precision. The method is applicable to all types (seismic, EM, acoustic) of AM.

  18. Reporters to monitor cellular MMP12 activity

    NASA Astrophysics Data System (ADS)

    Cobos-Correa, Amanda; Mall, Marcus A.; Schultz, Carsten

    2010-02-01

    Macrophage elastase, also called MMP12, belongs to a family of proteolytic enzymes whose best known physiological function is the remodeling of the extracellular matrix. Under certain pathological conditions, including inflammation, chronic overexpression of MMP12 has been observed and its elevated proteolytic activity has been suggested to be the cause of pulmonary emphysema. However, it was until recently impossible to monitor the activity of MMP12 under disease conditions, mainly due to a lack of detection methods. Recent development of new reporters for monitoring MMP12 activity in living cells, such as LaRee1, provided novel insights into the pathobiology of MMP12 in pulmonary inflammation.1 In the future, these reporters might contribute to improved diagnosis and in finding better treatments for chronic inflammatory lung diseases and emphysema. Our approach for visualizing MMP12 activity is based on peptidic, membrane-targeted FRET (Foerster Resonance Energy Transfer) reporters. Here we describe a set of new reporters containing different fluorophore pairs as well as modifications in the membrane-targeting lipid moiety. We studied the influence of these modifications on reporter performance and the reporter mobility on live cell membranes by FRAP (fluorescence recovery after photobleaching). Finally, we generated several new fluorescently labeled MMP inhibitors based on the peptidic reporter structures as prototypes for future tools to inhibit and monitor MMP activity at the same time.

  19. Increased activity of 6-phosphogluconate dehydrogenase and glucose-6-phosphate dehydrogenase in purified cell suspensions and single cells from the uterine cervix in cervical intraepithelial neoplasia.

    PubMed Central

    Jonas, S. K.; Benedetto, C.; Flatman, A.; Hammond, R. H.; Micheletti, L.; Riley, C.; Riley, P. A.; Spargo, D. J.; Zonca, M.; Slater, T. F.

    1992-01-01

    The activities of 6-phosphogluconate dehydrogenase and glucose-6-phosphate dehydrogenase have been measured in squamous epithelial cells of the uterine cervix from normal patients and cases of cervical intraepithelial neoplasia (CIN). A biochemical cycling method, which uses only simple equipment and is suited to routine use and to automation, was applied to cells separated by gradient centrifugation. In addition, cells were examined cytochemically, and the intensity of staining in the cytoplasm of single whole cells was measured using computerised microcytospectrophotometry. Twenty per cent of cells in samples from normal patients (n=61) showed staining intensities above an extinction of 0.15 at 540 nm, compared to 71% of cases of CIN 1 (n=14), 91% of cases of CIN 2 (n=11) and 67% of cases of CIN 3 (n=15). The cytochemical data do not allow definitive distinctions to be made between different grades of CIN whereas the biochemical assay applied to cell lysates shows convincing differences between normal samples and cases of CIN. There are no false negatives for CIN 3 (n=14) and CIN 2 (n=10) and 11% false negatives for CIN 1 (n=9) and 14% of false positives for normal cases (n=21). The results of this preliminary study with reference to automation are discussed [corrected]. Images Figure 1 PMID:1637668

  20. Multiwavelength Monitoring of Active Galactic Nuclei

    NASA Technical Reports Server (NTRS)

    Peterson, Bradley M.

    2001-01-01

    By intensive monitoring of AGN variability over a large range in wavelength, we can probe the structure and physics of active galactic nuclei on microarcsecond angular scales. For example, multi-wavelength variability data allow us (a) to establish causal relationships between variations in different wavebands, and thus determine which physical processes are primary and which spectral changes are induced by variations at other wavelengths, and (b) through reverberation mapping of the UV/optical emission lines, to determine the structure and kinematics of the line-emitting region, and thus accurately determine the central masses in AGNs. Multiwavelength monitoring is resource-intensive, and is difficult to implement with general-purpose facilities. As a result, virtually all programs undertaken to date have been either sparsely sampled, or short in duration, or both. The potentially high return on this type of investigation, however, argues for dedicated facilities for multiwavelength monitoring programs.

  1. Single cell sequencing: a distinct new field.

    PubMed

    Wang, Jian; Song, Yuanlin

    2017-12-01

    Single cell sequencing (SCS) has become a new approach to study biological heterogeneity. The advancement in technologies for single cell isolation, amplification of genome/transcriptome and next-generation sequencing enables SCS to reveal the inherent properties of a single cell from the large scale of the genome, transcriptome or epigenome at high resolution. Recently, SCS has been widely applied in various clinical and research fields, such as cancer biology and oncology, immunology, microbiology, neurobiology and prenatal diagnosis. In this review, we will discuss the development of SCS methods and focus on the latest clinical and research applications of SCS.

  2. Automated Single Cell Data Decontamination Pipeline

    SciTech Connect

    Tennessen, Kristin; Pati, Amrita

    2014-03-21

    Recent technological advancements in single-cell genomics have encouraged the classification and functional assessment of microorganisms from a wide span of the biospheres phylogeny.1,2 Environmental processes of interest to the DOE, such as bioremediation and carbon cycling, can be elucidated through the genomic lens of these unculturable microbes. However, contamination can occur at various stages of the single-cell sequencing process. Contaminated data can lead to wasted time and effort on meaningless analyses, inaccurate or erroneous conclusions, and pollution of public databases. A fully automated decontamination tool is necessary to prevent these instances and increase the throughput of the single-cell sequencing process

  3. Lhcb Transcription Is Coordinated with Cell Size and Chlorophyll Accumulation (Studies on Fluorescence-Activated, Cell-Sorter-Purified Single Cells from Wild-Type and immutans Arabidopsis thaliana).

    PubMed Central

    Meehan, L.; Harkins, K.; Chory, J.; Rodermel, S.

    1996-01-01

    To study the mechanisms that integrate pigment and chlorophyll a/b-binding apoprotein biosynthesis during light-harvesting complex II assembly, we have examined [beta]-glucuronidase (GUS) enzyme activities, chlorophyll contents, and cell sizes in fluorescence-activated, cell-sorting-separated single cells from transgenic Arabidopsis thaliana wild-type and immutans variegation mutant plants that express an Lhcb (photosystem II chlorophyll a/b-binding polypeptide gene)/GUS promoter fusion. We found that GUS activities are positively correlated with chlorophyll content and cell size in green cells from the control and immutans plants, indicating that Lhcb gene transcription is coordinated with cell size in this species. Compared with the control plants, however, chlorophyll production is enhanced in the green cells of immutans; this may represent part of a strategy to maximize photosynthesis in the green sectors to compensate for a lack of photosynthesis in the white sectors of the mutant. Lhcb transcription is significantly higher in pure-white cells of the transgenic immutans plants than in pure-white cells from norflurazon-treated, photooxidized A. thaliana leaves. This suggests that immutans partially uncouples Lhcb transcription from its normal dependence on chlorophyll accumulation and chloroplast development. We conclude that immutans may play a role in regulating Lhcb transcription, and may be a key component in the signal transduction pathways that control chloroplast biogenesis. PMID:12226428

  4. Active Sites Environmental Monitoring Program: Program plan

    SciTech Connect

    Ashwood, T.L.; Wickliff, D.S.; Morrissey, C.M.

    1992-02-01

    The Active Sites Environmental Monitoring Program (ASEMP), initiated in 1989, provides early detection and performance monitoring of transuranic (TRU) waste and active low-level waste (LLW) facilities at Oak Ridge National Laboratory (ORNL) in accordance with US Department of Energy (DOE) Order 5820.2A. Active LLW facilities in Solid Waste Storage Area (SWSA) 6 include Tumulus I and Tumulus II, the Interim Waste Management Facility (IWMF), LLW silos, high-range wells, asbestos silos, and fissile wells. The tumulus pads and IWMF are aboveground, high-strength concrete pads on which concrete vaults containing metal boxes of LLW are placed; the void space between the boxes and vaults is filled with grout. Eventually, these pads and vaults will be covered by an engineered multilayered cap. All other LLW facilities in SWSA 6 are below ground. In addition, this plan includes monitoring of the Hillcut Disposal Test Facility (HDTF) in SWSA 6, even though this facility was completed prior to the data of the DOE order. In SWSA 5 North, the TRU facilities include below-grade engineered caves, high-range wells, and unlined trenches. All samples from SWSA 6 are screened for alpha and beta activity, counted for gamma-emitting isotopes, and analyzed for tritium. In addition to these analytes, samples from SWSA 5 North are analyzed for specific transuranic elements.

  5. Epigenetics reloaded: the single-cell revolution.

    PubMed

    Bheda, Poonam; Schneider, Robert

    2014-11-01

    Mechanistically, how epigenetic states are inherited through cellular divisions remains an important open question in the chromatin field and beyond. Defining the heritability of epigenetic states and the underlying chromatin-based mechanisms within a population of cells is complicated due to cell heterogeneity combined with varying levels of stability of these states; thus, efforts must be focused toward single-cell analyses. The approaches presented here constitute the forefront of epigenetics research at the single-cell level using classic and innovative methods to dissect epigenetics mechanisms from the limited material available in a single cell. This review further outlines exciting future avenues of research to address the significance of epigenetic heterogeneity and the contributions of microfluidics technologies to single-cell isolation and analysis. Copyright © 2014 Elsevier Ltd. All rights reserved.

  6. Efficient Synergistic Single-Cell Genome Assembly.

    PubMed

    Movahedi, Narjes S; Embree, Mallory; Nagarajan, Harish; Zengler, Karsten; Chitsaz, Hamidreza

    2016-01-01

    As the vast majority of all microbes are unculturable, single-cell sequencing has become a significant method to gain insight into microbial physiology. Single-cell sequencing methods, currently powered by multiple displacement genome amplification (MDA), have passed important milestones such as finishing and closing the genome of a prokaryote. However, the quality and reliability of genome assemblies from single cells are still unsatisfactory due to uneven coverage depth and the absence of scattered chunks of the genome in the final collection of reads caused by MDA bias. In this work, our new algorithm Hybrid De novo Assembler (HyDA) demonstrates the power of coassembly of multiple single-cell genomic data sets through significant improvement of the assembly quality in terms of predicted functional elements and length statistics. Coassemblies contain significantly more base pairs and protein coding genes, cover more subsystems, and consist of longer contigs compared to individual assemblies by the same algorithm as well as state-of-the-art single-cell assemblers SPAdes and IDBA-UD. Hybrid De novo Assembler (HyDA) is also able to avoid chimeric assemblies by detecting and separating shared and exclusive pieces of sequence for input data sets. By replacing one deep single-cell sequencing experiment with a few single-cell sequencing experiments of lower depth, the coassembly method can hedge against the risk of failure and loss of the sample, without significantly increasing sequencing cost. Application of the single-cell coassembler HyDA to the study of three uncultured members of an alkane-degrading methanogenic community validated the usefulness of the coassembly concept. HyDA is open source and publicly available at http://chitsazlab.org/software.html, and the raw reads are available at http://chitsazlab.org/research.html.

  7. Regenerable activated bauxite adsorbent alkali monitor probe

    DOEpatents

    Lee, S.H.D.

    1992-12-22

    A regenerable activated bauxite adsorber alkali monitor probe for field applications to provide reliable measurement of alkali-vapor concentration in combustion gas with special emphasis on pressurized fluidized-bed combustion (PFBC) off-gas. More particularly, the invention relates to the development of a easily regenerable bauxite adsorbent for use in a method to accurately determine the alkali-vapor content of PFBC exhaust gases. 6 figs.

  8. Regenerable activated bauxite adsorbent alkali monitor probe

    SciTech Connect

    Lee, S.H.D.

    1991-01-22

    This invention relates to a regenerable activated bauxite adsorber alkali monitor probe for field applications to provide reliable measurement of alkali-vapor 5 concentration in combustion gas with special emphasis on pressurized fluidized-bed combustion (PFBC) off-gas. More particularly, the invention relates to the development of a easily regenerable bauxite adsorbent for use in a method to accurately determine the alkali-vapor content of PFBC 10 exhaust gases.

  9. [A monitor of the biomechanical cardiac activity].

    PubMed

    Masloboev, Iu P; Okhritskiĭ, A A; Prilutskiĭ, D A; Selishchev, S V

    2004-01-01

    A monitor of the biomechanical cardiac activity is described, which was elaborated on the basis of the accelerometer sensor and sigma-delta ADC for the purpose of registering the ballistocardiograms and seismocardiograms. The device ensures a non-stop signal recording for as long as 8 hours with the data being preserved in an inbuilt memory. Data are fed to the computer through the USB port. An algorithm is suggested for recordings processing by using the neuron-net technologies.

  10. Regenerable activated bauxite adsorbent alkali monitor probe

    DOEpatents

    Lee, Sheldon H. D.

    1992-01-01

    A regenerable activated bauxite adsorber alkali monitor probe for field applications to provide reliable measurement of alkali-vapor concentration in combustion gas with special emphasis on pressurized fluidized-bed combustion (PFBC) off-gas. More particularly, the invention relates to the development of a easily regenerable bauxite adsorbent for use in a method to accurately determine the alkali-vapor content of PFBC exhaust gases.

  11. Activity monitor accuracy in persons using canes.

    PubMed

    Wendland, Deborah Michael; Sprigle, Stephen H

    2012-01-01

    The StepWatch activity monitor has not been validated on multiple indoor and outdoor surfaces in a population using ambulation aids. The aims of this technical report are to report on strategies to configure the StepWatch activity monitor on subjects using a cane and to report the accuracy of both leg-mounted and cane-mounted StepWatch devices on people ambulating over different surfaces while using a cane. Sixteen subjects aged 67 to 85 yr (mean 75.6) who regularly use a cane for ambulation participated. StepWatch calibration was performed by adjusting sensitivity and cadence. Following calibration optimization, accuracy was tested on both the leg-mounted and cane-mounted devices on different surfaces, including linoleum, sidewalk, grass, ramp, and stairs. The leg-mounted device had an accuracy of 93.4% across all surfaces, while the cane-mounted device had an aggregate accuracy of 84.7% across all surfaces. Accuracy of the StepWatch on the stairs was significantly less accurate (p < 0.001) when comparing surfaces using repeated measures analysis of variance. When monitoring community mobility, placement of a StepWatch on a person and his/her ambulation aid can accurately document both activity and device use.

  12. PHASE I SINGLE CELL ELECTROLYZER TEST RESULTS

    SciTech Connect

    Steimke, J; Timothy Steeper, T

    2008-08-05

    This document reports the results of Phase I Single Cell testing of an SO{sub 2}-Depolarized Water Electrolyzer. Testing was performed primarily during the first quarter of FY 2008 at the Savannah River National Laboratory (SRNL) using an electrolyzer cell designed and built at SRNL. Other facility hardware were also designed and built at SRNL. This test further advances this technology for which work began at SRNL in 2005. This research is valuable in achieving the ultimate goal of an economical hydrogen production process based on the Hybrid Sulfur (HyS) Cycle. The focus of this work was to conduct single cell electrolyzer tests to further develop the technology of SO{sub 2}-depolarized electrolysis as part of the HyS Cycle. The HyS Cycle is a hybrid thermochemical cycle that may be used in conjunction with advanced nuclear reactors or centralized solar receivers to produce hydrogen by water-splitting. Like all other sulfur-based cycles, HyS utilizes the high temperature thermal decomposition of sulfuric acid to produce oxygen and regenerate sulfur dioxide. The unique aspect of HyS is the generation of hydrogen in a water electrolyzer that is operated under conditions where dissolved sulfur dioxide depolarizes the anodic reaction, resulting in substantial voltage reduction. Low cell voltage is essential for both thermodynamic efficiency and hydrogen cost. Sulfur dioxide is oxidized at the anode, producing sulfuric acid that is sent to the high temperature acid decomposition portion of the cycle. The electrolyzer cell uses the membrane electrode assembly (MEA) concept. The anode and cathode are formed by spraying platinum containing catalyst on both sides of a Proton Exchange Membrane (PEM). In most testing the material of the PEM was NafionR. The electrolyzer cell active area can be as large as 54.8 cm{sup 2}. Feed to the anode of the electrolyzer is a sulfuric acid solution containing sulfur dioxide. The partial pressure of sulfur dioxide could be varied in the

  13. Research highlights: microfluidic-enabled single-cell epigenetics.

    PubMed

    Dhar, Manjima; Khojah, Reem; Tay, Andy; Di Carlo, Dino

    2015-11-07

    Individual cells are the fundamental unit of life with diverse functions from metabolism to motility. In multicellular organisms, a single genome can give rise to tremendous variability across tissues at the single-cell level due to epigenetic differences in the genes that are expressed. Signals from the local environment or a history of signals can drive these variations, and tissues have many cell types that play separate roles. This epigenetic heterogeneity is of biological importance in normal functions such as tissue morphogenesis and can contribute to development or resistance of cancer, or other disease states. Therefore, an improved understanding of variations at the single cell level are fundamental to understanding biology and developing new approaches to combating disease. Traditional approaches to characterize epigenetic modifications of chromatin or the transcriptome of cells have often focused on blended responses of many cells in a tissue; however, such bulk measures lose spatial and temporal differences that occur from cell to cell, and cannot uncover novel or rare populations of cells. Here we highlight a flurry of recent activity to identify the mRNA profiles from thousands of single-cells as well as chromatin accessibility and histone marks on single to few hundreds of cells. Microfluidics and microfabrication have played a central role in the range of new techniques, and will likely continue to impact their further development towards routine single-cell epigenetic analysis.

  14. Quantification noise in single cell experiments

    PubMed Central

    Reiter, M.; Kirchner, B.; Müller, H.; Holzhauer, C.; Mann, W.; Pfaffl, M. W.

    2011-01-01

    In quantitative single-cell studies, the critical part is the low amount of nucleic acids present and the resulting experimental variations. In addition biological data obtained from heterogeneous tissue are not reflecting the expression behaviour of every single-cell. These variations can be derived from natural biological variance or can be introduced externally. Both have negative effects on the quantification result. The aim of this study is to make quantitative single-cell studies more transparent and reliable in order to fulfil the MIQE guidelines at the single-cell level. The technical variability introduced by RT, pre-amplification, evaporation, biological material and qPCR itself was evaluated by using RNA or DNA standards. Secondly, the biological expression variances of GAPDH, TNFα, IL-1β, TLR4 were measured by mRNA profiling experiment in single lymphocytes. The used quantification setup was sensitive enough to detect single standard copies and transcripts out of one solitary cell. Most variability was introduced by RT, followed by evaporation, and pre-amplification. The qPCR analysis and the biological matrix introduced only minor variability. Both conducted studies impressively demonstrate the heterogeneity of expression patterns in individual cells and showed clearly today's limitation in quantitative single-cell expression analysis. PMID:21745823

  15. Phenotype classification of single cells using SRS microscopy, RNA sequencing, and microfluidics (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Streets, Aaron M.; Cao, Chen; Zhang, Xiannian; Huang, Yanyi

    2016-03-01

    Phenotype classification of single cells reveals biological variation that is masked in ensemble measurement. This heterogeneity is found in gene and protein expression as well as in cell morphology. Many techniques are available to probe phenotypic heterogeneity at the single cell level, for example quantitative imaging and single-cell RNA sequencing, but it is difficult to perform multiple assays on the same single cell. In order to directly track correlation between morphology and gene expression at the single cell level, we developed a microfluidic platform for quantitative coherent Raman imaging and immediate RNA sequencing (RNA-Seq) of single cells. With this device we actively sort and trap cells for analysis with stimulated Raman scattering microscopy (SRS). The cells are then processed in parallel pipelines for lysis, and preparation of cDNA for high-throughput transcriptome sequencing. SRS microscopy offers three-dimensional imaging with chemical specificity for quantitative analysis of protein and lipid distribution in single cells. Meanwhile, the microfluidic platform facilitates single-cell manipulation, minimizes contamination, and furthermore, provides improved RNA-Seq detection sensitivity and measurement precision, which is necessary for differentiating biological variability from technical noise. By combining coherent Raman microscopy with RNA sequencing, we can better understand the relationship between cellular morphology and gene expression at the single-cell level.

  16. System and method for monitoring cellular activity

    NASA Technical Reports Server (NTRS)

    Bearman, Gregory H. (Inventor); Fraser, Scott E. (Inventor); Lansford, Russell D. (Inventor)

    2004-01-01

    A system and method for monitoring cellular activity in a cellular specimen. According to one embodiment, a plurality of excitable markers are applied to the specimen. A multi-photon laser microscope is provided to excite a region of the specimen and cause fluorescence to be radiated from the region. The radiating fluorescence is processed by a spectral analyzer to separate the fluorescence into respective wavelength bands. The respective bands of fluorescence are then collected by an array of detectors, with each detector receiving a corresponding one of the wavelength bands.

  17. System and method for monitoring cellular activity

    NASA Technical Reports Server (NTRS)

    Bearman, Gregory H. (Inventor); Fraser, Scott E. (Inventor); Lansford, Russell D. (Inventor)

    2002-01-01

    A system and method for monitoring cellular activity in a cellular specimen. According to one embodiment, a plurality of excitable markers are applied to the specimen. A multi-photon laser microscope is provided to excite a region of the specimen and cause fluorescence to be radiated from the region. The radiating fluorescence is processed by a spectral analyzer to separate the fluorescence into respective wavelength bands. The respective bands of fluorescence are then collected by an array of detectors, with each detector receiving a corresponding one of the wavelength bands.

  18. Single-cell transcriptome analysis of endometrial tissue.

    PubMed

    Krjutškov, K; Katayama, S; Saare, M; Vera-Rodriguez, M; Lubenets, D; Samuel, K; Laisk-Podar, T; Teder, H; Einarsdottir, E; Salumets, A; Kere, J

    2016-04-01

    How can we study the full transcriptome of endometrial stromal and epithelial cells at the single-cell level? By compiling and developing novel analytical tools for biopsy, tissue cryopreservation and disaggregation, single-cell sorting, library preparation, RNA sequencing (RNA-seq) and statistical data analysis. Although single-cell transcriptome analyses from various biopsied tissues have been published recently, corresponding protocols for human endometrium have not been described. The frozen-thawed endometrial biopsies were fluorescence-activated cell sorted (FACS) to distinguish CD13-positive stromal and CD9-positive epithelial cells and single-cell transcriptome analysis performed from biopsied tissues without culturing the cells. We studied gene transcription, applying a modern and efficient RNA-seq protocol. In parallel, endometrial stromal cells were cultured and global expression profiles were compared with uncultured cells. For method validation, we used two endometrial biopsies, one from mid-secretory phase (Day 21, LH+8) and another from late-secretory phase (Day 25). The samples underwent single-cell FACS sorting, single-cell RNA-seq library preparation and Illumina sequencing. Here we present a complete pipeline for single-cell gene-expression studies, from clinical sampling to statistical data analysis. Tissue manipulation, starting from disaggregation and cell-type-specific labelling and ending with single-cell automated sorting, is managed within 90 min at low temperature to minimize changes in the gene expression profile. The single living stromal and epithelial cells were sorted using CD13- and CD9-specific antibodies, respectively. Of the 8622 detected genes, 2661 were more active in cultured stromal cells than in biopsy cells. In the comparison of biopsy versus cultured cells, 5603 commonly expressed genes were detected, with 241 significantly differentially expressed genes. Of these, 231 genes were up- and 10 down-regulated in cultured cells

  19. Single-cell transcriptome analysis of endometrial tissue

    PubMed Central

    Krjutškov, K.; Katayama, S.; Saare, M.; Vera-Rodriguez, M.; Lubenets, D.; Samuel, K.; Laisk-Podar, T.; Teder, H.; Einarsdottir, E.; Salumets, A.; Kere, J.

    2016-01-01

    STUDY QUESTION How can we study the full transcriptome of endometrial stromal and epithelial cells at the single-cell level? SUMMARY ANSWER By compiling and developing novel analytical tools for biopsy, tissue cryopreservation and disaggregation, single-cell sorting, library preparation, RNA sequencing (RNA-seq) and statistical data analysis. WHAT IS KNOWN ALREADY Although single-cell transcriptome analyses from various biopsied tissues have been published recently, corresponding protocols for human endometrium have not been described. STUDY DESIGN, SIZE, DURATION The frozen-thawed endometrial biopsies were fluorescence-activated cell sorted (FACS) to distinguish CD13-positive stromal and CD9-positive epithelial cells and single-cell transcriptome analysis performed from biopsied tissues without culturing the cells. We studied gene transcription, applying a modern and efficient RNA-seq protocol. In parallel, endometrial stromal cells were cultured and global expression profiles were compared with uncultured cells. PARTICIPANTS/MATERIALS, SETTING, METHODS For method validation, we used two endometrial biopsies, one from mid-secretory phase (Day 21, LH+8) and another from late-secretory phase (Day 25). The samples underwent single-cell FACS sorting, single-cell RNA-seq library preparation and Illumina sequencing. MAIN RESULTS AND THE ROLE OF CHANCE Here we present a complete pipeline for single-cell gene-expression studies, from clinical sampling to statistical data analysis. Tissue manipulation, starting from disaggregation and cell-type-specific labelling and ending with single-cell automated sorting, is managed within 90 min at low temperature to minimize changes in the gene expression profile. The single living stromal and epithelial cells were sorted using CD13- and CD9-specific antibodies, respectively. Of the 8622 detected genes, 2661 were more active in cultured stromal cells than in biopsy cells. In the comparison of biopsy versus cultured cells, 5603

  20. MicroBioRobots for single cell manipulation

    NASA Astrophysics Data System (ADS)

    Sakar, Mahmut Selman

    One of the great challenges in nano and micro scale science and engineering is the independent manipulation of biological cells and small man-made objects with active sensing. For such biomedical applications as single cell manipulation, telemetry, and localized targeted delivery of chemicals, it is important to fabricate microstructures that can be powered and controlled without a tether in fluidic environments. These microstructures can be used to develop microrobots that have the potential to make existing therapeutic and diagnostic procedures less invasive. Actuation can be realized using various different organic and inorganic methods. Previous studies explored different forms of actuation and control with microorganisms. Bacteria, in particular, offer several advantages as controllable microactuators: they draw chemical energy directly from their environment, they are genetically modifiable, and they are scalable and configurable in the sense that any number of bacteria can be selectively patterned. Additionally, the study of bacteria inspires inorganic schemes of actuation and control. For these reasons, we chose to employ bacteria while controlling their motility using optical and electrical stimuli. In the first part of the thesis, we demonstrate a biointegrated approach by introducing MicroBioRobots (MBRs). MBRs are negative photosensitive epoxy (SU8) microfabricated structures with typical feature sizes ranging from 1-100 mum coated with a monolayer of the swarming Serratia marcescens . The adherent bacterial cells naturally coordinate to propel the microstructures in fluidic environments which we call Self-Actuation. First, we demonstrate the control of MBRs using self-actuation, DC electric fields and ultra-violet radiation and develop an experimentally-validated mathematical model for the MBRs. This model allows us to to steer the MBR to any position and orientation in a planar micro channel using visual feedback and an inverted microscope. Examples

  1. Live single-cell mass spectrometry.

    PubMed

    Masujima, Tsutomu

    2009-08-01

    The history from bio-imaging to live single-cell mass spectrometry (MS) is herein reviewed. The limitation of the current bio-imaging method is probing only known molecules, and a method for finding new molecules is needed for cells which, however, show individual behaviors even in the same incubation dish. Single-cell MALDI-TOF/MS has been developed, but it can detect only molecules that can be easily ionized, and not be exhaustive. Recently, the contents of a single cell have been sucked out by a nano-electro spray tip, and directly introduced into MS by nano-spray ionization. Thousands of molecular peaks have been successfully and exhaustively detected, and an extraction method for key molecules was also developed. This new method is now being widely applied to explore site- or state-specific molecules in various aspects of cell dynamisms.

  2. Current techniques for single-cell lysis.

    PubMed

    Brown, Robert B; Audet, Julie

    2008-10-06

    Owing to the small quantities of analytes and small volumes involved in single-cell analysis techniques, manipulation strategies must be chosen carefully. The lysis of single cells for downstream chemical analysis in capillaries and lab-on-a-chip devices can be achieved by optical, acoustic, mechanical, electrical or chemical means, each having their respective strengths and weaknesses. Selection of the most appropriate lysis method will depend on the particulars of the downstream cell lysate processing. Ultrafast lysis techniques such as the use of highly focused laser pulses or pulses of high voltage are suitable for applications requiring high temporal resolution. Other factors, such as whether the cells are adherent or in suspension and whether the proteins to be collected are desired to be native or denatured, will determine the suitability of detergent-based lysis methods. Therefore, careful selection of the proper lysis technique is essential for gathering accurate data from single cells.

  3. Single-cell sequencing in cancer research.

    PubMed

    Mato Prado, Mireia; Frampton, Adam E; Stebbing, Justin; Krell, Jonathan

    2016-01-01

    Genome-wide single-cell sequencing investigations have the potential to classify individual cells within a tumor mass. In recent years, various single-cell DNA and RNA quantification techniques have facilitated significant advances in our ability to classify subpopulations of cells within a heterogeneous population. These approaches provide the possibility of unraveling the complex variability in genetic, epigenetic and transcriptional interactions that occur within identical cells in a tumor. This should enhance our knowledge of the underlying biological phenotypes and could have a huge impact in designing more precise anticancer treatments in order to improve outcomes and avoid tumor resistance. In addition, single-cell sequencing analysis has the potential to allow the development of better diagnostic and prognostic biomarkers, and thus aid the delivery of more personalized targeted cancer therapy. Nevertheless, further research is still required to overcome technical, biological and computational problems before clinical application.

  4. Sampling techniques for single-cell electrophoresis.

    PubMed

    Cecala, Christine; Sweedler, Jonathan V

    2012-07-07

    Cells are extraordinarily complex, containing thousands of different analytes with concentrations spanning at least nine orders of magnitude. Analyzing single cells instead of tissue homogenates provides unique insights into cell-to-cell heterogeneity and aids in distinguishing normal cells from pathological ones. The high sensitivity and low sample consumption of capillary and on-chip electrophoresis, when integrated with fluorescence, electrochemical, and mass spectrometric detection methods, offer an ideal toolset for examining single cells and even subcellular organelles; however, the isolation and loading of such small samples into these devices is challenging. Recent advances have addressed this issue by interfacing a variety of enhanced mechanical, microfluidic, and optical sampling techniques to capillary and on-chip electrophoresis instruments for single-cell analyses.

  5. Single-cell analysis and isolation for microbiology and biotechnology: methods and applications.

    PubMed

    Ishii, Satoshi; Tago, Kanako; Senoo, Keishi

    2010-05-01

    Various single-cell isolation techniques, including dilution, micromanipulation, flow cytometry, microfluidics, and compartmentalization, have been developed. These techniques can be used to cultivate previously uncultured microbes, to assess and monitor cell physiology and function, and to screen for novel microbiological products. Various other techniques, such as viable staining, in situ hybridization, and those using autofluorescence proteins, are frequently combined with these single-cell isolation techniques depending on the purpose of the study. In this review article, we summarize currently available single-cell isolation techniques and their applications, when used in combination with other techniques, in microbiological and biotechnological studies.

  6. Defining cell types and states with single-cell genomics

    PubMed Central

    Trapnell, Cole

    2015-01-01

    A revolution in cellular measurement technology is under way: For the first time, we have the ability to monitor global gene regulation in thousands of individual cells in a single experiment. Such experiments will allow us to discover new cell types and states and trace their developmental origins. They overcome fundamental limitations inherent in measurements of bulk cell population that have frustrated efforts to resolve cellular states. Single-cell genomics and proteomics enable not only precise characterization of cell state, but also provide a stunningly high-resolution view of transitions between states. These measurements may finally make explicit the metaphor that C.H. Waddington posed nearly 60 years ago to explain cellular plasticity: Cells are residents of a vast “landscape” of possible states, over which they travel during development and in disease. Single-cell technology helps not only locate cells on this landscape, but illuminates the molecular mechanisms that shape the landscape itself. However, single-cell genomics is a field in its infancy, with many experimental and computational advances needed to fully realize its full potential. PMID:26430159

  7. Single-Cell-Precision Microplasma-Induced Cancer Cell Apoptosis

    PubMed Central

    Lu, Xinpei; He, Guangyuan; Ostrikov, Kostya

    2014-01-01

    The issue of single-cell control has recently attracted enormous interest. However, in spite of the presently achievable intracellular-level physiological probing through bio-photonics, nano-probe-based, and some other techniques, the issue of inducing selective, single-cell-precision apoptosis, without affecting neighbouring cells remains essentially open. Here we resolve this issue and report on the effective single-cell-precision cancer cell treatment using the reactive chemistry of the localized corona-type plasma discharge around a needle-like electrode with the spot size ∼1 µm. When the electrode is positioned with the micrometer precision against a selected cell, a focused and highly-localized micro-plasma discharge induces apoptosis in the selected individual HepG2 and HeLa cancer cells only, without affecting any surrounding cells, even in small cell clusters. This is confirmed by the real-time monitoring of the morphological and structural changes at the cellular and cell nucleus levels after the plasma exposure. PMID:24971517

  8. Single Cell Genomics: Advances and Future Perspectives

    PubMed Central

    Macaulay, Iain C.; Voet, Thierry

    2014-01-01

    Advances in whole-genome and whole-transcriptome amplification have permitted the sequencing of the minute amounts of DNA and RNA present in a single cell, offering a window into the extent and nature of genomic and transcriptomic heterogeneity which occurs in both normal development and disease. Single-cell approaches stand poised to revolutionise our capacity to understand the scale of genomic, epigenomic, and transcriptomic diversity that occurs during the lifetime of an individual organism. Here, we review the major technological and biological breakthroughs achieved, describe the remaining challenges to overcome, and provide a glimpse into the promise of recent and future developments. PMID:24497842

  9. Dynamics of single-cell gene expression

    PubMed Central

    Longo, Diane; Hasty, Jeff

    2006-01-01

    Cellular behavior has traditionally been investigated by utilizing bulk-scale methods that measure average values for a population of cells. Such population-wide studies mask the behavior of individual cells and are often insufficient for characterizing biological processes in which cellular heterogeneity plays a key role. A unifying theme of many recent studies has been a focus on the development and utilization of single-cell experimental techniques that are capable of probing key biological phenomena in individual living cells. Recently, novel information about gene expression dynamics has been obtained from single-cell experiments that draw upon the unique capabilities of fluorescent reporter proteins. PMID:17130866

  10. New technology for single-cell protein

    SciTech Connect

    Not Available

    1983-08-31

    New technology used by three companies for the production of single cell protein is described. Phillips petroleum is reported to be ready to license a new process that uses methanol or ethanol as feedstock yielding a product called Provesteen which contains 60% protein. Envirocon (Vancouver) uses pulp-mill sludge for protein production while Imperial Chemical Industries uses methanol. ICI targets its Pruteen, which contains 72% protein, as a substitute for fish meal and milk in animal feed, while Phillips is putting special stress as premium markets. Both Phillips and ICI are examining single cell protein as a human food source.

  11. Linking Microbial Phylogeny to Metabolic Activity at the Single-Cell Level by Using Enhanced Element Labeling-Catalyzed Reporter Deposition Fluorescence In Situ Hybridization (EL-FISH) and NanoSIMS▿ †

    PubMed Central

    Behrens, Sebastian; Lösekann, Tina; Pett-Ridge, Jennifer; Weber, Peter K.; Ng, Wing-On; Stevenson, Bradley S.; Hutcheon, Ian D.; Relman, David A.; Spormann, Alfred M.

    2008-01-01

    To examine phylogenetic identity and metabolic activity of individual cells in complex microbial communities, we developed a method which combines rRNA-based in situ hybridization with stable isotope imaging based on nanometer-scale secondary-ion mass spectrometry (NanoSIMS). Fluorine or bromine atoms were introduced into cells via 16S rRNA-targeted probes, which enabled phylogenetic identification of individual cells by NanoSIMS imaging. To overcome the natural fluorine and bromine backgrounds, we modified the current catalyzed reporter deposition fluorescence in situ hybridization (FISH) technique by using halogen-containing fluorescently labeled tyramides as substrates for the enzymatic tyramide deposition. Thereby, we obtained an enhanced element labeling of microbial cells by FISH (EL-FISH). The relative cellular abundance of fluorine or bromine after EL-FISH exceeded natural background concentrations by up to 180-fold and allowed us to distinguish target from non-target cells in NanoSIMS fluorine or bromine images. The method was optimized on single cells of axenic Escherichia coli and Vibrio cholerae cultures. EL-FISH/NanoSIMS was then applied to study interrelationships in a dual-species consortium consisting of a filamentous cyanobacterium and a heterotrophic alphaproteobacterium. We also evaluated the method on complex microbial aggregates obtained from human oral biofilms. In both samples, we found evidence for metabolic interactions by visualizing the fate of substrates labeled with 13C-carbon and 15N-nitrogen, while individual cells were identified simultaneously by halogen labeling via EL-FISH. Our novel approach will facilitate further studies of the ecophysiology of known and uncultured microorganisms in complex environments and communities. PMID:18359832

  12. Multiwavelength monitoring of active galactic nuclei

    NASA Technical Reports Server (NTRS)

    Urry, C. M.

    1993-01-01

    Recent multiwavelength monitoring of active galactic nuclei (AGN), particularly with the IUE satellite, has produced extraordinay advances in our understanding of the energy-generation mechanism(s) in the central engine and of the structure of the surrounding material. Examples discussed here include both ordinary AGN and blazars (the collective name for highly variable, radio-loud AGN like BL Lac objects and Optically Violently Variable quasars). In the last decade, efforts to obtain single-epoch multiwavelength spectra led to fundamentally new models for the structure of AGN, involving accretion disks for AGN and relativistic jets for blazars. Recent extensions of multiwavelength spectroscopy into the temporal domain have shown that while these general pictures may be correct, the details were probably wrong. Campaigns to monitor Seyfert 1 galaxies like NGC 4151, NGC 5548 and Fairall 9 at infrared, optical, ultraviolet and X-ray wavelengths indicate that broad-emission line regions are stratified by ionization, density, and velocity; argue against a standard thin accretion disk model; and suggest that X-rays represent primary rather than reprocessed radiation. For blazars, years of radio monitoring indicated emission from an inhomogeneous synchrotron-emitting plasma, which could also produce at least some of the shorter-wavelength emission. The recent month-long campaign to observe the BL Lac object PKS 2155-304 has revealed remarkably rapid variability that extends from the infrared through the X-ray with similar amplitude and little or no discernible lag. This lends strong support to relativistic jet models and rules out the proposed accretion disk model for the ultraviolet-X-ray continuum.

  13. Quality Assurance Project Plan for Facility Effluent Monitoring Plan activities

    SciTech Connect

    Frazier, T.P.

    1994-10-20

    This Quality Assurance Project Plan addresses the quality assurance requirements for the activities associated with the Facility Effluent Monitoring Plans, which are part of the overall Hanford Site Environmental Protection Plan. This plan specifically applies to the sampling and analysis activities and continuous monitoring performed for all Facility Effluent Monitoring Plan activities conducted by Westinghouse Hanford Company. It is generic in approach and will be implemented in conjunction with the specific requirements of the individual Facility Effluent Monitoring Plans.

  14. Capillary Electrophoretic Technologies for Single Cell Metabolomics

    ERIC Educational Resources Information Center

    Lapainis, Theodore E.

    2009-01-01

    Understanding the functioning of the brain is hindered by a lack of knowledge of the full complement of neurotransmitters and neuromodulatory compounds. Single cell measurements aid in the discovery of neurotransmitters used by small subsets of neurons that would be diluted below detection limits or masked by ubiquitous compounds when working with…

  15. Capillary Electrophoretic Technologies for Single Cell Metabolomics

    ERIC Educational Resources Information Center

    Lapainis, Theodore E.

    2009-01-01

    Understanding the functioning of the brain is hindered by a lack of knowledge of the full complement of neurotransmitters and neuromodulatory compounds. Single cell measurements aid in the discovery of neurotransmitters used by small subsets of neurons that would be diluted below detection limits or masked by ubiquitous compounds when working with…

  16. [Single-cell sequencing and tumour heterogeneity].

    PubMed

    Jordan, Bertrand

    2014-12-01

    The heterogeneity of tumours is now beginning to be documented precisely by single-cell new-generation sequencing. Recently published results on breast tumours show that each of the cells analysed displays a unique pattern of point mutations. This extensive genetic diversity is present before any treatment, and is likely to cause resistance to initially successful targeted therapies.

  17. Laser tweezers Raman spectroscopy of single cells

    NASA Astrophysics Data System (ADS)

    Chen, De

    Raman scattering is an inelastic collision between the vibrating molecules inside the sample and the incident photons. During this process, energy exchange takes place between the photon and the scattering molecule. By measuring the energy change of the photon, the molecular vibration mode can be probed. The vibrational spectrum contains valuable information about the disposition of atomic nuclei and chemical bonds within a molecule, the chemical compositions and the interactions between the molecule and its surroundings. In this dissertation, laser tweezers Raman spectroscopy (LTRS) technique is applied for the analysis of biological cells and human cells at single cell level. In LTRS, an individual cell is trapped in aqueous medium with laser tweezers, and Raman scattering spectra from the trapped cell are recorded in real-time. The Raman spectra of these cells can be used to reveal the dynamical processes of cell growth, cell response to environment changes, and can be used as the finger print for the identification of a bacterial cell species. Several biophysical experiments were carried out using LTRS: (1) the dynamic germination process of individual spores of Bacillus thuringiensis was detected via Ca-DPA, a spore-specific biomarker molecule; (2) inactivation and killing of Bacillus subtilis spores by microwave irradiation and wet heat were studied at single cell level; (3) the heat shock activation process of single B. subtilis spores were analyzed, in which the reversible transition from glass-like state at low temperature to liquid-like state at high temperature in spore was revealed at the molecular level; (4) the kinetic processes of bacterial cell lysis of E. coli by lysozyme and by temperature induction of lambda phage were detected real-time; (5) the fixation and rehydration of human platelets were quantitatively evaluated and characterized with Raman spectroscopy method, which provided a rapid way to quantify the quality of freeze-dried therapeutic

  18. Monitoring of Crew Activity with FAMOS

    NASA Astrophysics Data System (ADS)

    Wolf, L.; Cajochen, C.; Bromundt, V.

    2007-10-01

    The success of long duration space missions, such as manned missions to Mars, depends on high and sustained levels of vigilance and performance of astronauts and operators working in the technology rich environment of a spacecraft. Experiment 'Monitoring of Crew Activity with FAMOS' was set up to obtain operational experience with complimentary methods / technologies to assess the alertness / sleepiness status of selected AustroMars crewmembers on a daily basis. We applied a neurobehavioral test battery consisting of 1) Karolinska Sleepiness Scale KSS, 2) Karolinska Drowsiness Test KDT, 3) Psychomotor Vigilance Task PVT, combined with 4) left eye video recordings with an early prototype of the FAMOS Fatigue Monitoring System headset currently being developed by Sowoon Technologies (CH), and 5) Actiwatches that were worn continuously. A test battery required approximately 15 minutes and was repeated up to 4 times daily by 2 to 4 subjects. Here we present the data analysis of methods 1, 2, 3, and 5, while data analysis of method 4 is still in progress.

  19. Active chaotic excitation for bolted joint monitoring

    NASA Astrophysics Data System (ADS)

    Fasel, Timothy R.; Todd, Michael D.; Park, Gyuhae

    2006-03-01

    Recent research has shown that high frequency chaotic excitation and state space reconstruction may be used to identify incipient damage (loss of preload) in a bolted joint. In this study, a new experiment is undertaken with updated test equipment, including a piezostack actuator that allows for precise control of bolt preload. The excitation waveform is applied to a macro-fiber composite (MFC) patch that is bonded to the test structure and is sensed in an active manner using a second MFC patch. A novel prediction error algorithm, based on comparing filtered properties of the guided chaotic waves, is used to determine the damage state of a frame structure and is shown to be highly sensitive to small levels of bolt preload loss. The performance of the prediction error method is compared with standard structural health monitoring damage features that are based on time series analysis using auto-regressive (AR) models.

  20. WFSD fault monitoring using active seismic source

    NASA Astrophysics Data System (ADS)

    Yang, W.; Ge, H.; Wang, B.; Yuan, S.; Song, L.

    2010-12-01

    The Wenchuan Fault Scientific Drilling(WFSD)is a rapid response drilling project to the great Wenchuan earthquake. It focuses on the fault structure, earthquake physical mechanism, fluid and in-situ stress, energy budget and so on. Temporal variation of stress and physical property in the fault zone is important information for understanding earthquake physics, especially when the fault is still under the post-seismic recovery or stress modification. Seismic velocity is a good indicator of the medium mechanics, stress state within the fault zone. After the great Wenchuan Ms 8.0 earthquake, May 12, 2008, we built up a fault dynamic monitoring system using active seismic source cross the WFSD fault. It consists of a 10 ton accurately controlled eccentric mass source and eight receivers to continuously monitor the seismic velocity cross the fault zone. Combining the aftershock data, we try to monitor the fault recovery and some aftershock physical process. The observatory is located at the middle of the Longmenshan range-front fault, Mianzhu, Sichuan Province. The No.3 hole of WFSD is on the survey line near the No.4 receiver. The source and receiver site were carefully treated. All instruments were well installed to ensure the system's repeatability. Seismic velocity across the fault zone was monitored with continuous observation. The recording system consists of Guralp-40T short period seismometer and RefTek-130B recorder which was continuously GPS timed up to 20us. The active source ran since June 20, 2009. It was operated routinely at night and working continuously from 21:00 to 02:00 of the next day. So far, we have gotten almost one year recording. The seismic velocity variation may be caused by changes of the fault zone medium mechanical property, fault stress, fluid, and earth tide, barometric pressure and rainfall. Deconvolution, stacking and cross-correlation analysis were used for the velocity analysis. Results show that the relationship between seismic

  1. Single cell induced optical confinement in biological lasers

    NASA Astrophysics Data System (ADS)

    Karl, M.; Dietrich, C. P.; Schubert, M.; Samuel, I. D. W.; Turnbull, G. A.; Gather, M. C.

    2017-03-01

    Biological single cell lasers have shown great potential for fundamental research and next generation sensing applications. In this study, the potential of fluorescent biological cells as refractive index landscapes and active optical elements is investigated using a combined Fourier- and hyperspectral imaging technique. We show that the refractive index contrast between cell and surrounding leads to 3D confinement of photons inside living cells. The Fourier- and real-space emission characteristics of these biological lasers are closely related and can be predicted from one another. Investigations of the lasing threshold for different energy and momentum position in Fourier-space give insight into the fundamental creation of longitudinal and transverse lasing modes within the cell. These findings corroborate the potential of living biological materials for precision engineering of photonic structures and may pave the way towards low threshold polariton lasing from single cells.

  2. Exploring Metabolic Configurations of Single Cells within Complex Tissue Microenvironments.

    PubMed

    Miller, Anne; Nagy, Csörsz; Knapp, Bernhard; Laengle, Johannes; Ponweiser, Elisabeth; Groeger, Marion; Starkl, Philipp; Bergmann, Michael; Wagner, Oswald; Haschemi, Arvand

    2017-09-05

    Over the past years, plenty of evidence has emerged illustrating how metabolism supports many aspects of cellular function and how metabolic reprogramming can drive cell differentiation and fate. Here, we present a method to assess the metabolic configuration of single cells within their native tissue microenvironment via the visualization and quantification of multiple enzymatic activities measured at saturating substrate conditions combined with subsequent cell type identification. After careful validation of the approach and to demonstrate its potential, we assessed the intracellular metabolic configuration of different human immune cell populations in healthy and tumor colon tissue. Additionally, we analyzed the intercellular metabolic relationship between cancer cells and cancer-associated fibroblasts in a breast cancer tissue array. This study demonstrates that the determination of metabolic configurations in single cells could be a powerful complementary tool for every researcher interested to study metabolic networks in situ. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. Studying bacterial quorum-sensing at the single cell level

    NASA Astrophysics Data System (ADS)

    Delfino Perez, Pablo; Pelakh, Leslie; Young, Jonathan; Johnson, Elaine; Hagen, Stephen

    2010-03-01

    Like many bacterial species, Vibrio fischeri can detect its own population density through a quorum sensing (QS) mechanism. The bacterium releases a signal molecule (AI, autoinducer), which accumulates at high population density and triggers a genetic switch. In V.fischeri this leads to bioluminescence. Little is known about how stochastic gene expression affects QS at the level of single cells. We are imaging the luminescence of individual V.fischeri cells in a flow chamber and directly measuring the intercell variability in AI activation of the QS circuit. Our single-cell luminescence experiments allow us to track cells over time and characterize variations in their response to AI levels. We find heterogeneous response to the external signal: at a given AI concentration some cells may be strongly luminescent while others are virtually dark. The analysis of noise in the individual cell response can eventually lead to a better understanding of how cells use QS to gather information about their environment.

  4. Single-cell approaches for molecular classification of endocrine tumors

    PubMed Central

    Koh, James; Allbritton, Nancy L.; Sosa, Julie A.

    2015-01-01

    Purpose of review In this review, we summarize recent developments in single-cell technologies that can be employed for the functional and molecular classification of endocrine cells in normal and neoplastic tissue. Recent findings The emergence of new platforms for the isolation, analysis, and dynamic assessment of individual cell identity and reactive behavior enables experimental deconstruction of intratumoral heterogeneity and other contexts, where variability in cell signaling and biochemical responsiveness inform biological function and clinical presentation. These tools are particularly appropriate for examining and classifying endocrine neoplasias, as the clinical sequelae of these tumors are often driven by disrupted hormonal responsiveness secondary to compromised cell signaling. Single-cell methods allow for multidimensional experimental designs incorporating both spatial and temporal parameters with the capacity to probe dynamic cell signaling behaviors and kinetic response patterns dependent upon sequential agonist challenge. Summary Intratumoral heterogeneity in the provenance, composition, and biological activity of different forms of endocrine neoplasia presents a significant challenge for prognostic assessment. Single-cell technologies provide an array of powerful new approaches uniquely well suited for dissecting complex endocrine tumors. Studies examining the relationship between clinical behavior and tumor compositional variations in cellular activity are now possible, providing new opportunities to deconstruct the underlying mechanisms of endocrine neoplasia. PMID:26632769

  5. Single-Cell Lineage Analysis of Oogenesis in Mice.

    PubMed

    Lei, Lei; Spradling, Allan C

    2017-01-01

    Lineage analysis is widely used because it provides a very powerful tool for characterizing the developmental behavior of the cells in vivo. In this chapter, we describe a particularly informative variant of lineage analysis that we term "single-cell lineage analysis". As in traditional lineage analysis, the method employs a Tamoxifen (Tmx)-inducible CAGCreER mouse line, which is crossed to an R26R reporter line that can be activated by Cre-mediated DNA recombination. However, instead of driving CreER at a high level within a subset of cells defined by a particular promoter, CreER is driven with a generic promoter that is active in essentially all cells throughout the lifespan of the mouse. Specificity comes from using only a very low dose of Tmx so that just a few random, widely separated individual cells undergo recombination and become labeled. The growth and behavior of most such initially marked cells can subsequently be followed over time because each one forms a growing clone of marked cells that does not overlap with other clones due to their rarity. Following individual cell growth patterns provides much more information than can be derived from traditional lineage analysis, which relies on promoter specificity and uses high doses of Tmx that cannot resolve the behavior of single cells. We illustrate the value of single-cell lineage analysis using a recent study of fetal germ cell development and a recent search for female germ-line stem cells in adult mouse ovaries.

  6. Monitoring Photosynthetic Activity in Microalgal Cells by Raman Spectroscopy with Deuterium Oxide as a Tracking Probe.

    PubMed

    Yonamine, Yusuke; Suzuki, Yuta; Ito, Takuro; Miura, Yoshiko; Goda, Keisuke; Ozeki, Yasuyuki; Hoshino, Yu

    2017-08-14

    Microalgae offer great potential for the production of biofuel, but high photosynthetic activity is demanded for the practical realisation of microalgal biofuels. To this end, it is essential to evaluate the photosynthetic activity of single microalgal cells in a heterogeneous population. In this study, we present a method to monitor the photosynthetic activity of microalgae (in particular Euglena gracilis, a microalgal species of unicellular, photosynthetic, flagellate protists as our model organism) at single-cell resolution by Raman spectroscopy with deuterium from deuterium oxide (D2 O) as a tracking probe. Specifically, we replaced H2 O in culture media with D2 O up to a concentration of 20 % without disturbing the growth rate of E. gracilis cells and evaluated C-D bond formation as a consequence of photosynthetic reactions by Raman spectroscopy. We used the probe to monitor the kinetics of the C-D bond formation in E. gracilis cells by incubating them in D2 O media under light irradiation. Furthermore, we demonstrated Raman microscopy imaging of each single E. gracilis cell to discriminate deuterated cells from normal cells. Our results hold great promise for Raman-based screening of E. gracilis and potentially other microalgae with high photosynthetic activity by using D2 O as a tracking probe. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. Single-Cell Analysis of [18F]Fluorodeoxyglucose Uptake by Droplet Radiofluidics

    PubMed Central

    Türkcan, Silvan; Nguyen, Julia; Vilalta, Marta; Shen, Bin; Chin, Frederick T.; Pratx, Guillem; Abbyad, Paul

    2015-01-01

    Radiolabels can be used to detect small biomolecules with high sensitivity and specificity, and without interfering with the biochemical activity of the labeled molecule. For instance, the radiolabeled glucose analogue, [18F]fluorodeoxyglucose (FDG), is routinely used in positron emission tomography (PET) scans for cancer diagnosis, staging and monitoring. However, despite their widespread usage, conventional radionuclide techniques are unable to measure the variability and modulation of FDG uptake in single cells. We present here a novel microfluidic technique, dubbed droplet radiofluidics, that can measure radiotracer uptake for single cells encapsulated into an array of microdroplets. The advantages of this approach are multiple. First, droplets can be quickly and easily positioned in a predetermined pattern for optimal imaging throughput. Second, droplet encapsulation reduces cell efflux as a confounding factor, because any effluxed radionuclide is trapped in the droplet. Last, multiplexed measurements can be performed using fluorescent labels. In this new approach, intracellular radiotracers are imaged on a conventional fluorescence microscope by capturing individual flashes of visible light that are produced as individual positrons, emitted during radioactive decay, traverse a scintillator plate placed below the cells. This method is used to measure the cell-to-cell heterogeneity in the uptake of tracers such as FDG in cell lines and cultured primary cells. The capacity of the platform to perform multiplexed measurements was demonstrated by measuring differential FDG uptake in single cells subjected to different incubation conditions and expressing different types of glucose transporters. This method opens many new avenues of research in basic cell biology and human disease by capturing the full range of stochastic variations in highly heterogeneous cell populations in a repeatable and high-throughput manner. PMID:26035453

  8. Single-Cell Analysis of Mast Cell Degranulation Induced by Airway Smooth Muscle-Secreted Chemokines

    PubMed Central

    Manning, Benjamin M.; Meyer, Audrey F.; Gruba, Sarah M.; Haynes, Christy L.

    2015-01-01

    Background Asthma is a chronic inflammatory disease characterized by narrowed airways, bronchial hyper-responsiveness, mucus hyper-secretion, and airway remodeling. Mast cell (MC) infiltration into airway smooth muscle (ASM) is a defining feature of asthma, and ASM regulates the inflammatory response by secreting chemokines, including CXCL10 and CCL5. Single cell analysis offers a unique approach to study specific cellular signaling interactions within large and complex signaling networks such as the inflammatory microenvironment in asthma. Methods Carbon fiber microelectrode amperometry was used to study the effects of ASM–secreted chemokines on mouse peritoneal MC degranulation. Results MC degranulation in response to CXCL10 and CCL5 was monitored at the single cell level. Relative to IgE-mediated degranulation, CXCL10- and CCL5-stimulated MCs released a decreased amount of serotonin per granule with fewer release events per cell. Decreased serotonin released per granule was correlated with increased spike half-width and rise-time values. Conclusions MCs are directly activated with ASM-associated chemokines. CXCL10 and CCL5 induce less robust MC degranulation compared to IgE- and A23187-stimulation. The kinetics of MC degranulation are signaling pathway-dependent, suggesting a biophysical mechanism of regulated degranulation that incorporates control over granule trafficking, transport, and docking machinery. General Significance The biophysical mechanisms, including variations in number of exocytotic release events, serotonin released per granule, and the membrane kinetics of exocytosis that underlie MC degranulation in response to CXCL10 and CCL5 were characterized at the single cell level. These findings clarify the function of ASM-derived chemokines as instigators of MC degranulation relative to classical mechanisms of MC stimulation. PMID:25986989

  9. An Automatic Tremor Activity Monitoring System (TAMS)

    NASA Astrophysics Data System (ADS)

    Kao, H.; Thompson, P. J.; Rogers, G.; Dragert, H.; Spence, G.

    2006-12-01

    We have developed an algorithm that quantitatively characterizes the level of seismic tremors from recorded seismic waveforms. For each hour of waveform at a given station, the process begins with the calculation of scintillation index and moving average with various time lengths. The scintillation index (essentially the `normalized variance of intensity of the signal') is adapted from the studies of pulses in radio waves and is an efficient tool to identify the energy bursts of tremor signals. Both scintillation index and moving average values are fed into a series of logic gates to determine if tremor activity exists. This algorithm is implemented in the Tremor Activity Monitoring System (TAMS) to provide automatic early alerts for episodic tremor and slip (ETS) events in the northern Cascadia margin. Currently, TAMS retrieves the digital waveforms recorded during the previous day from the Canadian National Seismographic Network (CNSN) archive server at 1 AM every morning. The detecting process is repeated for all stations and hours to determine the level of tremor activity of the previous day. If a sufficient number of stations within a radius of 100 km are determined to have tremor patterns and coherent tremor arrivals can be found at more than 3 stations, TAMS automatically sends out alert emails to a list of subscribers with a figure summarizing the hours and locations of coherent tremors. TAMS outputs are very consistent with the work done by visual inspection, especially for major ETS events. It is straightforward to configure TAMS into a near-real-time system that can send out hourly (or shorter) reports if necessary.

  10. Single cell microfluidics for systems oncology

    NASA Astrophysics Data System (ADS)

    Fan, Rong

    2012-02-01

    The singular term ``cancer'' is never one kind of disease, but deceivingly encompasses a large number of heterogeneous disease states, which makes it impossible to completely treat cancer using a generic approach. Rather systems approaches are urgently required to assess cancer heterogeneity, stratify patients and enable the most effective, individualized treatment. The heterogeneity of tumors at the single cell level is reflected by the hierarchical complexity of the tumor microenvironment. To identify all the cellular components, including both tumor and infiltrating immune cells, and to delineate the associated cell-to-cell signaling network that dictates tumor initiation, progression and metastasis, we developed a single cell microfluidics chip that can analyze a panel of proteins that are potentially associated inter-cellular signaling network in tumor microenvironment from hundreds of single cells in parallel. This platform integrates two advanced technologies -- microfluidic single cell handling and ultra-high density protein array. This device was first tested for highly multiplexed profiling of secreted proteins including tumor-immune signaling molecules from monocytic leukemia cells. We observed profound cellular heterogeneity with all functional phenotypes quantitatively identified. Correlation analysis further indicated the existence of an intercellular cytokine network in which TNFα-induced secondary signaling cascades further increased functional cellular diversity. It was also exploited to evaluate polyfunctionality of tumor antigen-specific T cells from melanoma patients being treated with adoptive T cell transfer immunotherapy. This platform could be further extended to analyze both solid tumor cells (e.g. human lung carcinoma cells) and infiltrating immune cells (e.g. macrophages) so as to enable systems analysis of the complex tumor microenvironment from small amounts of clinical specimens, e.g. skinny needle biopsies. Thus, it could potentially

  11. Parallel single-cell analysis microfluidic platform.

    PubMed

    van den Brink, Floris T G; Gool, Elmar; Frimat, Jean-Philippe; Bomer, Johan; van den Berg, Albert; Le Gac, Séverine

    2011-11-01

    We report a PDMS microfluidic platform for parallel single-cell analysis (PaSCAl) as a powerful tool to decipher the heterogeneity found in cell populations. Cells are trapped individually in dedicated pockets, and thereafter, a number of invasive or non-invasive analysis schemes are performed. First, we report single-cell trapping in a fast (2-5  min) and reproducible manner with a single-cell capture yield of 85% using two cell lines (P3x63Ag8 and MCF-7), employing a protocol which is scalable and easily amenable to automation. Following this, a mixed population of P3x63Ag8 and MCF-7 cells is stained in situ using the nucleic acid probe (Hoechst) and a phycoerythrin-labeled monoclonal antibody directed at EpCAM present on the surface of the breast cancer cells MCF-7 and absent on the myeloma cells P3x63Ag8 to illustrate the potential of the device to analyze cell population heterogeneity. Next, cells are porated in situ using chemicals in a reversible (digitonin) or irreversible way (lithium dodecyl sulfate). This is visualized by the transportation of fluorescent dyes through the membrane (propidium iodide and calcein). Finally, an electrical protocol is developed for combined cell permeabilization and electroosmotic flow (EOF)-based extraction of the cell content. It is validated here using calcein-loaded cells and visualized through the progressive recovery of calcein in the side channels, indicating successful retrieval of individual cell content.

  12. Analysis of mitochondria isolated from single cells.

    PubMed

    Johnson, Ryan D; Navratil, Marian; Poe, Bobby G; Xiong, Guohua; Olson, Karen J; Ahmadzadeh, Hossein; Andreyev, Dmitry; Duffy, Ciarán F; Arriaga, Edgar A

    2007-01-01

    Bulk studies are not suitable to describe and study cell-to-cell variation, which is of high importance in biological processes such as embryogenesis, tissue differentiation, and disease. Previously, capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) was used to measure the properties of organelles isolated from millions of cells. As such, these bulk measurements reported average properties for the organelles of cell populations. Similar measurements for organelles released from single cells would be highly relevant to describe the subcellular variations among cells. Toward this goal, here we introduce an approach to analyze the mitochondria released from single mammalian cells. Osteosarcoma 143B cells are labeled with either the fluorescent mitochondrion-specific 10-N-nonyl acridine orange (NAO) or via expression of the fluorescent protein DsRed2. Subsequently, a single cell is introduced into the CE-LIF capillary where the organelles are released by a combined treatment of digitonin and trypsin. After this treatment, an electric field is applied and the released organelles electromigrate toward the LIF detector. From an electropherogram, the number of detected events per cell, their individual electrophoretic mobilities, and their individual fluorescence intensities are calculated. The results obtained from DsRed2 labeling, which is retained in intact mitochondria, and NAO labeling, which labels all mitochondria, are the basis for discussion of the strengths and limitations of this single-cell approach.

  13. Monitoring channel activities of proteoliposomes with SecA and Cx26 gap junction in single oocytes.

    PubMed

    Hsieh, Ying-Hsin; Zou, Juan; Jin, Jin-Shan; Yang, Hsiuchin; Chen, Yanyi; Jiang, Chun; Yang, Jenny; Tai, Phang C

    2015-07-01

    Establishing recordable channels in membranes of oocytes formed by expressing exogenous complementary DNA (cDNA) or messenger RNA (mRNA) has contributed greatly to understanding the molecular mechanisms of channel functions. Here, we report the extension of this semi-physiological system for monitoring the channel activity of preassembled membrane proteins in single cell oocytes by injecting reconstituted proteoliposomes along with substrates or regulatory molecules. We build on the observation that SecA from various bacteria forms active protein-conducting channels with injection of proteoliposomes, protein precursors, and ATP-Mg(2+). Such activity was enhanced by reconstituted SecYEG-SecDF•YajC liposome complexes that could be monitored easily and efficiently, providing correlation of in vitro and intact cell functionality. In addition, inserting reconstituted gap junction Cx26 liposomes into the oocytes allowed the demonstration of intracellular/extracellular Ca(2+)-regulated hemi-channel activities. The channel activities can be detected rapidly after injection, can be monitored for various effectors, and are dependent on specific exogenous lipid compositions. This simple and effective functional system with low endogenous channel activity should have broad applications for monitoring the specific channel activities of complex interactions of purified membrane proteins with their effectors and regulatory molecules. Copyright © 2015 Elsevier Inc. All rights reserved.

  14. ATP Consumption of Eukaryotic Flagella Measured at a Single-Cell Level

    NASA Astrophysics Data System (ADS)

    Chen, Daniel T. N.; Heymann, Michael; Fraden, Seth; Nicastro, Daniela; Dogic, Zvonimir

    2015-12-01

    The motility of cilia and flagella is driven by thousands of dynein motors that hydrolyze adenosine triphosphate (ATP). Despite decades of genetic, biochemical, structural and biophysical studies, some aspects of ciliary motility remain elusive, such as the regulation of beating patterns and the energetic efficiency of these nanomachines. Here, we introduce an experimental method to measure ATP consumption of actively beating axonemes on a single-cell level. We encapsulated individual sea urchin sperm with demembranated flagellum inside water-in-oil emulsion droplets and measured the axonemes ATP consumption by monitoring fluorescence intensity of a fluorophore-coupled reporter system for ATP turnover in the droplet. Concomitant phase contrast imaging allowed us to extract a linear dependence between the ATP consumption rate and the flagellar beating frequency, with ~2.3e5 ATP molecules consumed per beat of a demembranated flagellum. Increasing the viscosity of the aqueous medium led to modified beating waveforms of the axonemes and to higher energy consumption per beat cycle. Our single-cell experimental platform provides both new insights into the beating mechanism of flagella and a powerful tool for future studies.

  15. ATP Consumption of Eukaryotic Flagella Measured at a Single-Cell Level.

    PubMed

    Chen, Daniel T N; Heymann, Michael; Fraden, Seth; Nicastro, Daniela; Dogic, Zvonimir

    2015-12-15

    The motility of cilia and flagella is driven by thousands of dynein motors that hydrolyze adenosine triphosphate (ATP). Despite decades of genetic, biochemical, structural, and biophysical studies, some aspects of ciliary motility remain elusive, such as the regulation of beating patterns and the energetic efficiency of these nanomachines. In this study, we introduce an experimental method to measure ATP consumption of actively beating axonemes on a single-cell level. We encapsulated individual sea urchin sperm with demembranated flagellum inside water-in-oil emulsion droplets and measured the axoneme's ATP consumption by monitoring fluorescence intensity of a fluorophore-coupled reporter system for ATP turnover in the droplet. Concomitant phase contrast imaging allowed us to extract a linear dependence between the ATP consumption rate and the flagellar beating frequency, with ∼2.3 × 10(5) ATP molecules consumed per beat of a demembranated flagellum. Increasing the viscosity of the aqueous medium led to modified beating waveforms of the axonemes and to higher energy consumption per beat cycle. Our single-cell experimental platform provides both new insights, to our knowledge, into the beating mechanism of flagella and a powerful tool for future studies.

  16. ATP Consumption of Eukaryotic Flagella Measured at a Single-Cell Level

    PubMed Central

    Chen, Daniel T.N.; Heymann, Michael; Fraden, Seth; Nicastro, Daniela; Dogic, Zvonimir

    2015-01-01

    The motility of cilia and flagella is driven by thousands of dynein motors that hydrolyze adenosine triphosphate (ATP). Despite decades of genetic, biochemical, structural, and biophysical studies, some aspects of ciliary motility remain elusive, such as the regulation of beating patterns and the energetic efficiency of these nanomachines. In this study, we introduce an experimental method to measure ATP consumption of actively beating axonemes on a single-cell level. We encapsulated individual sea urchin sperm with demembranated flagellum inside water-in-oil emulsion droplets and measured the axoneme’s ATP consumption by monitoring fluorescence intensity of a fluorophore-coupled reporter system for ATP turnover in the droplet. Concomitant phase contrast imaging allowed us to extract a linear dependence between the ATP consumption rate and the flagellar beating frequency, with ∼2.3 × 105 ATP molecules consumed per beat of a demembranated flagellum. Increasing the viscosity of the aqueous medium led to modified beating waveforms of the axonemes and to higher energy consumption per beat cycle. Our single-cell experimental platform provides both new insights, to our knowledge, into the beating mechanism of flagella and a powerful tool for future studies. PMID:26682814

  17. How consumer physical activity monitors could transform human physiology research.

    PubMed

    Wright, Stephen P; Hall Brown, Tyish S; Collier, Scott R; Sandberg, Kathryn

    2017-03-01

    A sedentary lifestyle and lack of physical activity are well-established risk factors for chronic disease and adverse health outcomes. Thus, there is enormous interest in measuring physical activity in biomedical research. Many consumer physical activity monitors, including Basis Health Tracker, BodyMedia Fit, DirectLife, Fitbit Flex, Fitbit One, Fitbit Zip, Garmin Vivofit, Jawbone UP, MisFit Shine, Nike FuelBand, Polar Loop, Withings Pulse O2, and others have accuracies similar to that of research-grade physical activity monitors for measuring steps. This review focuses on the unprecedented opportunities that consumer physical activity monitors offer for human physiology and pathophysiology research because of their ability to measure activity continuously under real-life conditions and because they are already widely used by consumers. We examine current and potential uses of consumer physical activity monitors as a measuring or monitoring device, or as an intervention in strategies to change behavior and predict health outcomes. The accuracy, reliability, reproducibility, and validity of consumer physical activity monitors are reviewed, as are limitations and challenges associated with using these devices in research. Other topics covered include how smartphone apps and platforms, such as the Apple ResearchKit, can be used in conjunction with consumer physical activity monitors for research. Lastly, the future of consumer physical activity monitors and related technology is considered: pattern recognition, integration of sleep monitors, and other biosensors in combination with new forms of information processing.

  18. A System for Monitoring Posture and Physical Activity Using Accelerometers

    DTIC Science & Technology

    2007-11-02

    Abstract- Accelerometers can be used to monitor physical activity in the home over prolonged periods. We describe a novel system for...processing schema in which these parameters are extracted is described. Keywords - physical activity , accelerometers, congestive heart failure, chronic...When monitoring the condition of patients with neurodegenerative or chronic diseases, a knowledge of their body movement and physical activity

  19. High-Throughput Single-Cell Derived Sphere Formation for Cancer Stem-Like Cell Identification and Analysis

    PubMed Central

    Chen, Yu-Chih; Ingram, Patrick N.; Fouladdel, Shamileh; McDermott, Sean P.; Azizi, Ebrahim; Wicha, Max S.; Yoon, Euisik

    2016-01-01

    Considerable evidence suggests that many malignancies are driven by a cellular compartment that displays stem cell properties. Cancer stem-like cells (CSCs) can be identified by expression of cell surface markers or enzymatic activity, but these methods are limited by phenotypic heterogeneity and plasticity of CSCs. An alternative phenotypic methodology based on in-vitro sphere formation has been developed, but it is typically labor-intensive and low-throughput. In this work, we present a 1,024-microchamber microfluidic platform for single-cell derived sphere formation. Utilizing a hydrodynamic capturing scheme, more than 70% of the microchambers capture only one cell, allowing for monitoring of sphere formation from heterogeneous cancer cell populations for identification of CSCs. Single-cell derived spheres can be retrieved and dissociated for single-cell analysis using a custom 96-gene panel to probe heterogeneity within the clonal CSC spheres. This microfluidic platform provides reliable and high-throughput sphere formation for CSC identification and downstream clonal analysis. PMID:27292795

  20. High-Throughput Single-Cell Derived Sphere Formation for Cancer Stem-Like Cell Identification and Analysis

    NASA Astrophysics Data System (ADS)

    Chen, Yu-Chih; Ingram, Patrick N.; Fouladdel, Shamileh; McDermott, Sean P.; Azizi, Ebrahim; Wicha, Max S.; Yoon, Euisik

    2016-06-01

    Considerable evidence suggests that many malignancies are driven by a cellular compartment that displays stem cell properties. Cancer stem-like cells (CSCs) can be identified by expression of cell surface markers or enzymatic activity, but these methods are limited by phenotypic heterogeneity and plasticity of CSCs. An alternative phenotypic methodology based on in-vitro sphere formation has been developed, but it is typically labor-intensive and low-throughput. In this work, we present a 1,024-microchamber microfluidic platform for single-cell derived sphere formation. Utilizing a hydrodynamic capturing scheme, more than 70% of the microchambers capture only one cell, allowing for monitoring of sphere formation from heterogeneous cancer cell populations for identification of CSCs. Single-cell derived spheres can be retrieved and dissociated for single-cell analysis using a custom 96-gene panel to probe heterogeneity within the clonal CSC spheres. This microfluidic platform provides reliable and high-throughput sphere formation for CSC identification and downstream clonal analysis.

  1. Parallel measurement of dynamic changes in translation rates in single cells.

    PubMed

    Han, Kyuho; Jaimovich, Ariel; Dey, Gautam; Ruggero, Davide; Meyuhas, Oded; Sonenberg, Nahum; Meyer, Tobias

    2014-01-01

    Protein concentrations are often regulated by dynamic changes in translation rates. Nevertheless, it has been challenging to directly monitor changes in translation in living cells. We have developed a reporter system to measure real-time changes of translation rates in human or mouse individual cells by conjugating translation regulatory motifs to sequences encoding a nuclear targeted fluorescent protein and a controllable destabilization domain. Application of the method showed that individual cells undergo marked fluctuations in the translation rate of mRNAs whose 5' terminal oligopyrimidine (5' TOP) motif regulates the synthesis of ribosomal proteins. Furthermore, we show that small reductions in amino acid levels signal through different mTOR-dependent pathways to control TOP mRNA translation, whereas larger reductions in amino acid levels control translation through eIF2A. Our study demonstrates that dynamic measurements of single-cell activities of translation regulatory motifs can be used to identify and investigate fundamental principles of translation.

  2. Three-dimensional reconstruction of single-cell chromosome structure using recurrence plots

    NASA Astrophysics Data System (ADS)

    Hirata, Yoshito; Oda, Arisa; Ohta, Kunihiro; Aihara, Kazuyuki

    2016-10-01

    Single-cell analysis of the three-dimensional (3D) chromosome structure can reveal cell-to-cell variability in genome activities. Here, we propose to apply recurrence plots, a mathematical method of nonlinear time series analysis, to reconstruct the 3D chromosome structure of a single cell based on information of chromosomal contacts from genome-wide chromosome conformation capture (Hi-C) data. This recurrence plot-based reconstruction (RPR) method enables rapid reconstruction of a unique structure in single cells, even from incomplete Hi-C information.

  3. Three-dimensional reconstruction of single-cell chromosome structure using recurrence plots

    PubMed Central

    Hirata, Yoshito; Oda, Arisa; Ohta, Kunihiro; Aihara, Kazuyuki

    2016-01-01

    Single-cell analysis of the three-dimensional (3D) chromosome structure can reveal cell-to-cell variability in genome activities. Here, we propose to apply recurrence plots, a mathematical method of nonlinear time series analysis, to reconstruct the 3D chromosome structure of a single cell based on information of chromosomal contacts from genome-wide chromosome conformation capture (Hi-C) data. This recurrence plot-based reconstruction (RPR) method enables rapid reconstruction of a unique structure in single cells, even from incomplete Hi-C information. PMID:27725694

  4. Deformation Monitoring of AN Active Fault

    NASA Astrophysics Data System (ADS)

    Ostapchuk, A.

    2015-12-01

    The discovery of low frequency earthquakes, slow slip events and other deformation phenomena, new for geophysics, change our understanding of how the energy accumulated in the Earth's crust do release. The new geophysical data make one revise the underlying mechanism of geomechanical processes taking place in fault zones. Conditions for generating different slip modes are still unclear. The most vital question is whether a certain slip mode is intrinsic for a fault or may be controlled by external factors. This work presents the results of two and a half year deformation monitoring of a discontinuity in the zone of the Main Sayanskiy Fault. Main Sayanskiy Fault is right-lateral strike-slip fault. Observations were performed in the tunnel of Talaya seismic station (TLY), Irkutsk region, Russia. Measurements were carried out 70 m away from the entrance of the tunnel, the thickness of overlying rock was about 30 m. Inductive sensors of displacement were mounted at the both sides of a discontinuity, which recorded three components of relative fault side displacement with the accuracy of 0.2 mcm. Temperature variation inside the tunnel didn't exceed 0.5oC during the all period of observations. Important information about deformation properties of an active fault was obtained. A pronounced seasonality of deformation characteristics of discontinuity is observed in the investigated segment of rock. A great number of slow slip events with durations from several hours to several weeks were registered. Besides that alterations of fault deformation characteristics before the megathrust earthquake M9.0 Tohoku Oki 11 March 2011 and reaction to the event itself were detected. The work was supported by the Russian Science Foundation (grant no. 14-17-00719).

  5. Get to Understand More from Single-Cells: Current Studies of Microfluidic-Based Techniques for Single-Cell Analysis

    PubMed Central

    Lo, Shih-Jie; Yao, Da-Jeng

    2015-01-01

    This review describes the microfluidic techniques developed for the analysis of a single cell. The characteristics of microfluidic (e.g., little sample amount required, high-throughput performance) make this tool suitable to answer and to solve biological questions of interest about a single cell. This review aims to introduce microfluidic related techniques for the isolation, trapping and manipulation of a single cell. The major approaches for detection in single-cell analysis are introduced; the applications of single-cell analysis are then summarized. The review concludes with discussions of the future directions and opportunities of microfluidic systems applied in analysis of a single cell. PMID:26213918

  6. Quantitative assessments of glycolysis from single cells.

    PubMed

    Shin, Young Shik; Kim, Jungwoo; Johnson, Dazy; Dooraghi, Alex A; Mai, Wilson X; Ta, Lisa; Chatziioannou, Arion F; Phelps, Michael E; Nathanson, David A; Heath, James R

    2015-06-01

    The most common positron emission tomography (PET) radio-labeled probe for molecular diagnostics in patient care and research is the glucose analog, 2-deoxy-2-[F-18]fluoro-D-glucose ((18)F-FDG). We report on an integrated microfluidics-chip/beta particle imaging system for in vitro(18)F-FDG radioassays of glycolysis with single cell resolution. We investigated the kinetic responses of single glioblastoma cancer cells to targeted inhibitors of receptor tyrosine kinase signaling. Further, we find a weak positive correlation between cell size and rate of glycolysis.

  7. Quantitative assessments of glycolysis from single cells

    PubMed Central

    Shin, Young Shik; Kim, Jungwoo; Johnson, Dazy; Dooraghi, Alex A.; Mai, Wilson X.; Ta, Lisa; Chatziioannou, Arion F.; Phelps, Michael E.; Nathanson, David A.; Heath, James R.

    2015-01-01

    The most common positron emission tomography (PET) radio-labeled probe for molecular diagnostics in patient care and research is the glucose analog, 2-deoxy-2-[F-18]fluoro-D-glucose (18F-FDG). We report on an integrated microfluidics-chip/beta particle imaging system for in vitro 18F-FDG radioassays of glycolysis with single cell resolution. We investigated the kinetic responses of single glioblastoma cancer cells to targeted inhibitors of receptor tyrosine kinase signaling. Further, we find a weak positive correlation between cell size and rate of glycolysis. PMID:26835505

  8. Systems nanobiology: from quantitative single molecule biophysics to microfluidic-based single cell analysis.

    PubMed

    Martini, Joerg; Hellmich, Wibke; Greif, Dominik; Becker, Anke; Merkle, Thomas; Ros, Robert; Ros, Alexandra; Toensing, Katja; Anselmetti, Dario

    2007-01-01

    Detailed and quantitative information about structure-function relation, concentrations and interaction kinetics of biological molecules and subcellular components is a key prerequisite to understand and model cellular organisation and temporal dynamics. In systems nanobi-ology, cellular processes are quantitatively investigated at the sensitivity level of single molecules and cells. This approach provides direct access to biomolecular information without being statistically ensemble-averaged, their associated distribution functions, and possible subpopulations. Moreover at the single cell level, the interplay of regulated genomic information and proteomic variabilities can be investigated and attributed to functional peculiarities. These requirements necessitate the development of novel and ultrasensitive methods and instruments for single molecule detection, microscopy and spectroscopy for analysis without the need of amplification and preconcentration. In this chapter, we present three methodological applications that demonstrate how quantitative informations can be accessed that are representative for cellular processes or single cell analysis like gene expression regulation, intracellular protein translocation dynamics, and single cell protein fingerprinting. First, the interaction kinetics of transcriptionally regulated DNA-protein interaction can be quantitatively investigated with single molecule force spectroscopy allowing a molecular affinity ranking. Second, intracellular protein dynamics for a transcription regulator migrating form the nucleus to the cytoplasm can be quantitatively monitored by photoactivable GFP and two-photon laser scanning microscopy. And third, a microfluidic-based method for label-free single cell proteomics and fingerprinting and first label-free single cell electropherograms are presented which include the manipulation and steering of single cells in a microfluidic device.

  9. Single cell genomic quantification by non-fluorescence nonlinear microscopy

    NASA Astrophysics Data System (ADS)

    Kota, Divya; Liu, Jing

    2017-02-01

    Human epidermal growth receptor 2 (Her2) is a gene which plays a major role in breast cancer development. The quantification of Her2 expression in single cells is limited by several drawbacks in existing fluorescence-based single molecule techniques, such as low signal-to-noise ratio (SNR), strong autofluorescence and background signals from biological components. For rigorous genomic quantification, a robust method of orthogonal detection is highly desirable and we demonstrated it by two non-fluorescent imaging techniques -transient absorption microscopy (TAM) and second harmonic generation (SHG). In TAM, gold nanoparticles (AuNPs) are chosen as an orthogonal probes for detection of single molecules which gives background-free quantifications of single mRNA transcript. In SHG, emission from barium titanium oxide (BTO) nanoprobes was demonstrated which allows stable signal beyond the autofluorescence window. Her2 mRNA was specifically labeled with nanoprobes which are conjugated with antibodies or oligonucleotides and quantified at single copy sensitivity in the cancer cells and tissues. Furthermore, a non-fluorescent super-resolution concept, named as second harmonic super-resolution microscopy (SHaSM), was proposed to quantify individual Her2 transcripts in cancer cells beyond the diffraction limit. These non-fluorescent imaging modalities will provide new dimensions in biomarker quantification at single molecule sensitivity in turbid biological samples, offering a strong cross-platform strategy for clinical monitoring at single cell resolution.

  10. In vivo lipidomics using single-cell Raman spectroscopy

    PubMed Central

    Wu, Huawen; Volponi, Joanne V.; Oliver, Ann E.; Parikh, Atul N.; Simmons, Blake A.; Singh, Seema

    2011-01-01

    We describe a method for direct, quantitative, in vivo lipid profiling of oil-producing microalgae using single-cell laser-trapping Raman spectroscopy. This approach is demonstrated in the quantitative determination of the degree of unsaturation and transition temperatures of constituent lipids within microalgae. These properties are important markers for determining engine compatibility and performance metrics of algal biodiesel. We show that these factors can be directly measured from a single living microalgal cell held in place with an optical trap while simultaneously collecting Raman data. Cellular response to different growth conditions is monitored in real time. Our approach circumvents the need for lipid extraction and analysis that is both slow and invasive. Furthermore, this technique yields real-time chemical information in a label-free manner, thus eliminating the limitations of impermeability, toxicity, and specificity of the fluorescent probes common in currently used protocols. Although the single-cell Raman spectroscopy demonstrated here is focused on the study of the microalgal lipids with biofuel applications, the analytical capability and quantitation algorithms demonstrated are applicable to many different organisms and should prove useful for a diverse range of applications in lipidomics. PMID:21310969

  11. In vivo lipidomics using single-cell Raman spectroscopy.

    PubMed

    Wu, Huawen; Volponi, Joanne V; Oliver, Ann E; Parikh, Atul N; Simmons, Blake A; Singh, Seema

    2011-03-01

    We describe a method for direct, quantitative, in vivo lipid profiling of oil-producing microalgae using single-cell laser-trapping Raman spectroscopy. This approach is demonstrated in the quantitative determination of the degree of unsaturation and transition temperatures of constituent lipids within microalgae. These properties are important markers for determining engine compatibility and performance metrics of algal biodiesel. We show that these factors can be directly measured from a single living microalgal cell held in place with an optical trap while simultaneously collecting Raman data. Cellular response to different growth conditions is monitored in real time. Our approach circumvents the need for lipid extraction and analysis that is both slow and invasive. Furthermore, this technique yields real-time chemical information in a label-free manner, thus eliminating the limitations of impermeability, toxicity, and specificity of the fluorescent probes common in currently used protocols. Although the single-cell Raman spectroscopy demonstrated here is focused on the study of the microalgal lipids with biofuel applications, the analytical capability and quantitation algorithms demonstrated are applicable to many different organisms and should prove useful for a diverse range of applications in lipidomics.

  12. Single-cell ATAC-seq: strength in numbers.

    PubMed

    Pott, Sebastian; Lieb, Jason D

    2015-08-21

    Single-cell ATAC-seq detects open chromatin in individual cells. Currently data are sparse, but combining information from many single cells can identify determinants of cell-to-cell chromatin variation.

  13. Geophysical Mapping and Monitoring of Active Planets (GMAP)

    NASA Astrophysics Data System (ADS)

    McGovern, P. J.; Goossens, S. J.; Lemoine, F. G.

    2017-02-01

    Recent findings require a strongly upward revision of volcano-tectonic activity rate estimates for Venus and Mars. We propose a program of Geophysical Mapping and Monitoring of Active Planets (GMAP) including seismology, gravimetry, InSAR, and GPS.

  14. Instructional physical activity monitor video in english and spanish

    USDA-ARS?s Scientific Manuscript database

    The ActiGraph activity monitor is a widely used method for assessing physical activity. Compliance with study procedures in critical. A common procedure is for the research team to meet with participants and demonstrate how and when to attach and remove the monitor and convey how many wear-days are ...

  15. Quality Assurance Project Plan for Facility Effluent Monitoring Plan activities

    SciTech Connect

    Nickels, J.M.

    1991-06-01

    This Quality Assurance Project Plan addresses the quality assurance requirements for the Facility Monitoring Plans of the overall site-wide environmental monitoring plan. This plan specifically applies to the sampling and analysis activities and continuous monitoring performed for all Facility Effluent Monitoring Plan activities conducted by Westinghouse Hanford Company. It is generic in approach and will be implemented in conjunction with the specific requirements of individual Facility Effluent Monitoring Plans. This document is intended to be a basic road map to the Facility Effluent Monitoring Plan documents (i.e., the guidance document for preparing Facility Effluent Monitoring Plans, Facility Effluent Monitoring Plan determinations, management plan, and Facility Effluent Monitoring Plans). The implementing procedures, plans, and instructions are appropriate for the control of effluent monitoring plans requiring compliance with US Department of Energy, US Environmental Protection Agency, state, and local requirements. This Quality Assurance Project Plan contains a matrix of organizational responsibilities, procedural resources from facility or site manuals used in the Facility Effluent Monitoring Plans, and a list of the analytes of interest and analytical methods for each facility preparing a Facility Effluent Monitoring Plan. 44 refs., 1 figs., 2 tabs.

  16. SINCERITIES: Inferring gene regulatory networks from time-stamped single cell transcriptional expression profiles.

    PubMed

    Papili Gao, Nan; Ud-Dean, S M Minhaz; Gandrillon, Olivier; Gunawan, Rudiyanto

    2017-09-14

    Single cell transcriptional profiling opens up a new avenue in studying the functional role of cell-to-cell variability in physiological processes. The analysis of single cell expression profiles creates new challenges due to the distributive nature of the data and the stochastic dynamics of gene transcription process. The reconstruction of gene regulatory networks (GRNs) using single cell transcriptional profiles is particularly challenging, especially when directed gene-gene relationships are desired. We developed SINCERITIES (SINgle CEll Regularized Inference using TIme-stamped Expression profileS) for the inference of GRNs from single cell transcriptional profiles. We focused on time-stamped cross-sectional expression data, commonly generated from transcriptional profiling of single cells collected at multiple time points after cell stimulation. SINCERITIES recovers directed regulatory relationships among genes by employing regularized linear regression (ridge regression), using temporal changes in the distributions of gene expressions. Meanwhile, the modes of the gene regulations (activation and repression) come from partial correlation analyses between pairs of genes. We demonstrated the efficacy of SINCERITIES in inferring GRNs using in silico time-stamped single cell expression data and single cell transcriptional profiles of THP-1 monocytic human leukemia cells. The case studies showed that SINCERITIES could provide accurate GRN predictions, significantly better than other GRN inference algorithms such as TSNI, GENIE3 and JUMP3. Moreover, SINCERITIES has a low computational complexity and is amenable to problems of extremely large dimensionality. Finally, an application of SINCERITIES to single cell expression data of T2EC chicken erythrocytes pointed to BATF as a candidate novel regulator of erythroid development. The MATLAB and R version of SINCERITIES is freely available from the following websites: http://www.cabsel.ethz.ch/tools/sincerities.html and

  17. Pseudotime estimation: deconfounding single cell time series

    PubMed Central

    Reid, John E.; Wernisch, Lorenz

    2016-01-01

    Motivation: Repeated cross-sectional time series single cell data confound several sources of variation, with contributions from measurement noise, stochastic cell-to-cell variation and cell progression at different rates. Time series from single cell assays are particularly susceptible to confounding as the measurements are not averaged over populations of cells. When several genes are assayed in parallel these effects can be estimated and corrected for under certain smoothness assumptions on cell progression. Results: We present a principled probabilistic model with a Bayesian inference scheme to analyse such data. We demonstrate our method’s utility on public microarray, nCounter and RNA-seq datasets from three organisms. Our method almost perfectly recovers withheld capture times in an Arabidopsis dataset, it accurately estimates cell cycle peak times in a human prostate cancer cell line and it correctly identifies two precocious cells in a study of paracrine signalling in mouse dendritic cells. Furthermore, our method compares favourably with Monocle, a state-of-the-art technique. We also show using held-out data that uncertainty in the temporal dimension is a common confounder and should be accounted for in analyses of repeated cross-sectional time series. Availability and Implementation: Our method is available on CRAN in the DeLorean package. Contact: john.reid@mrc-bsu.cam.ac.uk Supplementary information: Supplementary data are available at Bioinformatics online. PMID:27318198

  18. Continuous differential impedance spectroscopy of single cells

    PubMed Central

    Nevill, J. Tanner; Lee, Luke P.; Morgan, Hywel

    2009-01-01

    A device for continuous differential impedance analysis of single cells held by a hydrodynamic cell trapping is presented. Measurements are accomplished by recording the current from two closely-situated electrode pairs, one empty (reference) and one containing a cell. We demonstrate time-dependent measurement of single cell impedance produced in response to dynamic chemical perturbations. First, the system is used to assay the response of HeLa cells to the effects of the surfactant Tween, which reduces the impedance of the trapped cells in a concentration dependent way and is interpreted as gradual lysis of the cell membrane. Second, the effects of the bacterial pore-forming toxin, Streptolysin-O are measured: a transient exponential decay in the impedance is recorded as the cell membrane becomes increasingly permeable. The decay time constant is inversely proportional to toxin concentration (482, 150, and 30 s for 0.1, 1, and 10 kU/ml, respectively). Electronic supplementary material The online version of this article (doi:10.1007/s10404-009-0534-2) contains supplementary material, which is available to authorized users. PMID:20927185

  19. Single molecule and single cell epigenomics.

    PubMed

    Hyun, Byung-Ryool; McElwee, John L; Soloway, Paul D

    2015-01-15

    Dynamically regulated changes in chromatin states are vital for normal development and can produce disease when they go awry. Accordingly, much effort has been devoted to characterizing these states under normal and pathological conditions. Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is the most widely used method to characterize where in the genome transcription factors, modified histones, modified nucleotides and chromatin binding proteins are found; bisulfite sequencing (BS-seq) and its variants are commonly used to characterize the locations of DNA modifications. Though very powerful, these methods are not without limitations. Notably, they are best at characterizing one chromatin feature at a time, yet chromatin features arise and function in combination. Investigators commonly superimpose separate ChIP-seq or BS-seq datasets, and then infer where chromatin features are found together. While these inferences might be correct, they can be misleading when the chromatin source has distinct cell types, or when a given cell type exhibits any cell to cell variation in chromatin state. These ambiguities can be eliminated by robust methods that directly characterize the existence and genomic locations of combinations of chromatin features in very small inputs of cells or ideally, single cells. Here we review single molecule epigenomic methods under development to overcome these limitations, the technical challenges associated with single molecule methods and their potential application to single cells. Copyright © 2014 Elsevier Inc. All rights reserved.

  20. Single Molecule and Single Cell Epigenomics

    PubMed Central

    Hyun, Byung-Ryool; McElwee, John L.; Soloway, Paul D.

    2014-01-01

    Dynamically regulated changes in chromatin states are vital for normal development and can produce disease when they go awry. Accordingly, much effort has been devoted to characterizing these states under normal and pathological conditions. Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is the most widely used method to characterize where in the genome transcription factors, modified histones, modified nucleotides and chromatin binding proteins are found; bisulfite sequencing (BS-seq) and its variants are commonly used to characterize the locations of DNA modifications. Though very powerful, these methods are not without limitations. Notably, they are best at characterizing one chromatin feature at a time, yet chromatin features arise and function in combination. Investigators commonly superimpose separate ChIP-seq or BS-seq datasets, and then infer where chromatin features are found together. While these inferences might be correct, they can be misleading when the chromatin source has distinct cell types, or when a given cell type exhibits any cell to cell variation in chromatin state. These ambiguities can be eliminated by robust methods that directly characterize the existence and genomic locations of combinations of chromatin features in very small inputs of cells or ideally, single cells. Here we review single molecule epigenomic methods under development to overcome these limitations, the technical challenges associated with single molecule methods and their potential application to single cells. PMID:25204781

  1. Pseudotime estimation: deconfounding single cell time series.

    PubMed

    Reid, John E; Wernisch, Lorenz

    2016-10-01

    Repeated cross-sectional time series single cell data confound several sources of variation, with contributions from measurement noise, stochastic cell-to-cell variation and cell progression at different rates. Time series from single cell assays are particularly susceptible to confounding as the measurements are not averaged over populations of cells. When several genes are assayed in parallel these effects can be estimated and corrected for under certain smoothness assumptions on cell progression. We present a principled probabilistic model with a Bayesian inference scheme to analyse such data. We demonstrate our method's utility on public microarray, nCounter and RNA-seq datasets from three organisms. Our method almost perfectly recovers withheld capture times in an Arabidopsis dataset, it accurately estimates cell cycle peak times in a human prostate cancer cell line and it correctly identifies two precocious cells in a study of paracrine signalling in mouse dendritic cells. Furthermore, our method compares favourably with Monocle, a state-of-the-art technique. We also show using held-out data that uncertainty in the temporal dimension is a common confounder and should be accounted for in analyses of repeated cross-sectional time series. Our method is available on CRAN in the DeLorean package. john.reid@mrc-bsu.cam.ac.uk Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press.

  2. Single Cell Chromatography, LDRD Feasibility Study

    SciTech Connect

    Knize, M G; Bailey, C G

    2007-02-22

    A limitation in the mass spectrometry of biological materials is the reduced ion formation caused by sample complexity. We proposed to develop an enabling technology, single cell planar chromatography, which will greatly increase the amount of chemical information that can be obtained from single biological cells when using imaging mass spectrometry or other surface analysis methods. The sample preparation methods were developed for the time-of-flight secondary mass spectrometer (ToF-SIMS) at LLNL. This instrument has a measured zeptomole (10{sup -21} mole, 600 atoms) limit-of-detection for a molecule with a mass to charge ratio of 225[1]. Our goal was to use planar chromatographic separation to approach similar low limits of detection even with the chemically complex contents of a single cell. The process was proposed to reduce ion suppression and at the same time expose more of the cell contents to the ion beam. The method of work was to deposit biological cells on a silicon chip with suitable chromatographic and electrical properties, dissolve the cell with a droplet of solvent, allow the solvent to evaporate, and then allow the movement of cell contents laterally by immersing an edge of the chip in to a chromatographic solvent, that then moves through the chromatographic matrix allowing the components to interact with, and be separated by, the chromatographic substrate. This process is a miniaturized version of thin layer chromatography with detection by surface mass spectrometry.

  3. Microfabricated devices for single cell analysis

    NASA Astrophysics Data System (ADS)

    Gao, Yuanfang

    BioMEMS or lab-on-a-chip technology is promising technology and enables the possibility of microchip devices with higher throughput or better performance for single cell analysis. We have designed and fabricated microdevices for single cell analysis, with impedance based device for fast cell screening and microchannel based flow systems for high throughput, high time resolution quantal exocytosis measurement with automatic cell positioning and reusability. The automatic cell positioning is realized by differential forces of fluidic dynamics. Microelectrodes are patterned at automatic trap positions for electrochemical detection quantal release of hormones like catecholamines secreted by cells. We also developed diamond-like carbon (DLC) microelectrodes onto chip device for low noise exocytosis measurement. The DLC microelectrodes were deposited by magnetron sputtering process with nitrogen doping and a bottom ITO conductive layer. Test results show the developed DLC can detect exocytosis with low noise and a stable background current which are comparable to that of carbon-fiber electrodes. They are batch producible at low cost and can realize high-throughput on-chip measurement of quantal exocytosis. The technology developed in this research can have wide ranging applications in fields such as electrophysiology, cell based sensors, high throughput screening of new drug development.

  4. Remote Physical Activity Monitoring in Neurological Disease: A Systematic Review

    PubMed Central

    Block, Valerie A. J.; Pitsch, Erica; Tahir, Peggy; Cree, Bruce A. C.; Allen, Diane D.; Gelfand, Jeffrey M.

    2016-01-01

    Objective To perform a systematic review of studies using remote physical activity monitoring in neurological diseases, highlighting advances and determining gaps. Methods Studies were systematically identified in PubMed/MEDLINE, CINAHL and SCOPUS from January 2004 to December 2014 that monitored physical activity for ≥24 hours in adults with neurological diseases. Studies that measured only involuntary motor activity (tremor, seizures), energy expenditure or sleep were excluded. Feasibility, findings, and protocols were examined. Results 137 studies met inclusion criteria in multiple sclerosis (MS) (61 studies); stroke (41); Parkinson's Disease (PD) (20); dementia (11); traumatic brain injury (2) and ataxia (1). Physical activity levels measured by remote monitoring are consistently low in people with MS, stroke and dementia, and patterns of physical activity are altered in PD. In MS, decreased ambulatory activity assessed via remote monitoring is associated with greater disability and lower quality of life. In stroke, remote measures of upper limb function and ambulation are associated with functional recovery following rehabilitation and goal-directed interventions. In PD, remote monitoring may help to predict falls. In dementia, remote physical activity measures correlate with disease severity and can detect wandering. Conclusions These studies show that remote physical activity monitoring is feasible in neurological diseases, including in people with moderate to severe neurological disability. Remote monitoring can be a psychometrically sound and responsive way to assess physical activity in neurological disease. Further research is needed to ensure these tools provide meaningful information in the context of specific neurological disorders and patterns of neurological disability. PMID:27124611

  5. Investigating intratumour heterogeneity by single-cell sequencing

    PubMed Central

    Ren, Shan-Cheng; Qu, Min; Sun, Ying-Hao

    2013-01-01

    Intratumour heterogeneity is a longstanding field of focus for both researchers and clinicians. It refers to the diversity amongst cells within the same tumour. Two major hypotheses have attempted to explain the existence of intratumour heterogeneity: (i) the clonal evolution (CE) theory and (ii) the cancer stem cell (CSC) model. CE theory emphasizes the evolutionary biological characteristics of the tumour, underscoring the initiation and progression of the disease. In contrast, the CSC model focuses on stem cell differentiation into distinct functions in order to stabilize the tumour microenvironment. Here we consider single-cell sequencing (SCS) as a newly developed technique for application to the investigation of intratumour heterogeneity and assess its relevance within research and clinical environments. Early detection of rare tumour cells, monitoring of circulating tumour cells (CTCs) and control of the occurrence of drug resistance are important goals in early diagnosis, prognosis prediction and individualized medicine. PMID:24141534

  6. Investigating intratumour heterogeneity by single-cell sequencing.

    PubMed

    Ren, Shan-Cheng; Qu, Min; Sun, Ying-Hao

    2013-11-01

    Intratumour heterogeneity is a longstanding field of focus for both researchers and clinicians. It refers to the diversity amongst cells within the same tumour. Two major hypotheses have attempted to explain the existence of intratumour heterogeneity: (i) the clonal evolution (CE) theory and (ii) the cancer stem cell (CSC) model. CE theory emphasizes the evolutionary biological characteristics of the tumour, underscoring the initiation and progression of the disease. In contrast, the CSC model focuses on stem cell differentiation into distinct functions in order to stabilize the tumour microenvironment. Here we consider single-cell sequencing (SCS) as a newly developed technique for application to the investigation of intratumour heterogeneity and assess its relevance within research and clinical environments. Early detection of rare tumour cells, monitoring of circulating tumour cells (CTCs) and control of the occurrence of drug resistance are important goals in early diagnosis, prognosis prediction and individualized medicine.

  7. Magnetic domain wall conduits for single cell applications.

    PubMed

    Donolato, M; Torti, A; Kostesha, N; Deryabina, M; Sogne, E; Vavassori, P; Hansen, M F; Bertacco, R

    2011-09-07

    The ability to trap, manipulate and release single cells on a surface is important both for fundamental studies of cellular processes and for the development of novel lab-on-chip miniaturized tools for biological and medical applications. In this paper we demonstrate how magnetic domain walls generated in micro- and nano-structures fabricated on a chip surface can be used to handle single yeast cells labeled with magnetic beads. In detail, first we show that the proposed approach maintains the microorganism viable, as proven by monitoring the division of labeled yeast cells trapped by domain walls over 16 hours. Moreover, we demonstrate the controlled transport and release of individual yeast cells via displacement and annihilation of individual domain walls in micro- and nano-sized magnetic structures. These results pave the way to the implementation of magnetic devices based on domain walls technology in lab-on-chip systems devoted to accurate individual cell trapping and manipulation.

  8. Monitoring metal concentrations in tissues and single cells using ultramicrosensors.

    PubMed Central

    Malinski, T; Grunfeld, S; Taha, Z; Tomboulian, P

    1994-01-01

    Intercellular and extracellular metal concentrations were measured using carbon fiber ultramicrosensors plated with mercury or with polymeric porphyrinic p-type semiconductors. Concentrations of unbound nickel and lead ions were studied within individual BC3H-1 myocytes, and H4-11-C3 rat hepatoma cells. Unbound ions are predominantly solvated inorganic ions not coordinated to biological cellular components. Fabrication of ultramicrosensors appropriate for the cells under investigation is described, including procedures for sharpening and waxing the microsensors in order to control the shape, area, and dimensions of the electroactive surface. Metal ion movement through cell membranes and intracellular ion diffusion in aorta tissue were studied. Images Figure 2. PMID:7843090

  9. Dissecting the multicellular ecosystem of metastatic melanoma by single-cell RNA-seq

    PubMed Central

    Tirosh, Itay; Izar, Benjamin; Prakadan, Sanjay M.; Wadsworth, Marc H.; Treacy, Daniel; Trombetta, John J.; Rotem, Asaf; Rodman, Christopher; Lian, Christine; Murphy, George; Fallahi-Sichani, Mohammad; Dutton-Regester, Ken; Lin, Jia-Ren; Cohen, Ofir; Shah, Parin; Lu, Diana; Genshaft, Alex S.; Hughes, Travis K.; Ziegler, Carly G. K.; Kazer, Samuel W.; Gaillard, Aleth; Kolb, Kellie E.; Villani, Alexandra-Chloé; Johannessen, Cory M.; Andreev, Aleksandr Y.; Van Allen, Eliezer M.; Bertagnolli, Monica; Sorger, Peter K.; Sullivan, Ryan J.; Flaherty, Keith T.; Frederick, Dennie T.; Jané-Valbuena, Judit; Yoon, Charles H.; Rozenblatt-Rosen, Orit; Shalek, Alex K.; Regev, Aviv; Garraway, Levi A.

    2016-01-01

    To explore the distinct genotypic and phenotypic states of melanoma tumors we applied single-cell RNA-seq to 4,645 single cells isolated from 19 patients, profiling malignant, immune, stromal and endothelial cells. Malignant cells within the same tumor displayed transcriptional heterogeneity associated with the cell cycle, spatial context, and a drug resistance program. In particular, all tumors harbored malignant cells from two distinct transcriptional cell states, such that “MITF-high” tumors also contained “AXL-high” tumor cells. Single-cell analyses suggested distinct tumor micro-environmental patterns, including cell-to-cell interactions. Analysis of tumor-infiltrating T cells revealed exhaustion programs, their connection to T cell activation and to clonal expansion, and their variability across patients. Overall, we begin to unravel the cellular ecosystem of tumors and how single cell genomics offers insights with implications for both targeted and immune therapies. PMID:27124452

  10. A fast solution switching system with temperature control for single cell measurements

    PubMed Central

    Koh, Duk-Su; Chen, Liangyi; Ufret-Vincenty, Carmen A.; Jung, Seung-Ryoung

    2011-01-01

    This article describes a perfusion system for biophysical single cell experiments at the physiological temperature. Our system regulates temperature of test solutions using a small heat exchanger that includes several capillaries. Water circulating inside the heat exchanger warms or cools test solutions flowing inside the capillaries. Temperature-controlled solutions are delivered directly to a single cell(s) through a multibarreled manifold that switches solutions bathing a cell in less than 1 s. This solution exchange is optimal for patch clamp, single-cell microamperometry, and microfluorometry experiments. Using this system, we demonstrate that exocytosis from pancreatic β cells and activation of TRPV1 channels are temperature sensitive. We also discuss how to measure local temperature near a single cell under investigation. PMID:21536068

  11. Dissecting the multicellular ecosystem of metastatic melanoma by single-cell RNA-seq.

    PubMed

    Tirosh, Itay; Izar, Benjamin; Prakadan, Sanjay M; Wadsworth, Marc H; Treacy, Daniel; Trombetta, John J; Rotem, Asaf; Rodman, Christopher; Lian, Christine; Murphy, George; Fallahi-Sichani, Mohammad; Dutton-Regester, Ken; Lin, Jia-Ren; Cohen, Ofir; Shah, Parin; Lu, Diana; Genshaft, Alex S; Hughes, Travis K; Ziegler, Carly G K; Kazer, Samuel W; Gaillard, Aleth; Kolb, Kellie E; Villani, Alexandra-Chloé; Johannessen, Cory M; Andreev, Aleksandr Y; Van Allen, Eliezer M; Bertagnolli, Monica; Sorger, Peter K; Sullivan, Ryan J; Flaherty, Keith T; Frederick, Dennie T; Jané-Valbuena, Judit; Yoon, Charles H; Rozenblatt-Rosen, Orit; Shalek, Alex K; Regev, Aviv; Garraway, Levi A

    2016-04-08

    To explore the distinct genotypic and phenotypic states of melanoma tumors, we applied single-cell RNA sequencing (RNA-seq) to 4645 single cells isolated from 19 patients, profiling malignant, immune, stromal, and endothelial cells. Malignant cells within the same tumor displayed transcriptional heterogeneity associated with the cell cycle, spatial context, and a drug-resistance program. In particular, all tumors harbored malignant cells from two distinct transcriptional cell states, such that tumors characterized by high levels of the MITF transcription factor also contained cells with low MITF and elevated levels of the AXL kinase. Single-cell analyses suggested distinct tumor microenvironmental patterns, including cell-to-cell interactions. Analysis of tumor-infiltrating T cells revealed exhaustion programs, their connection to T cell activation and clonal expansion, and their variability across patients. Overall, we begin to unravel the cellular ecosystem of tumors and how single-cell genomics offers insights with implications for both targeted and immune therapies.

  12. Design and Analysis of Single-Cell Sequencing Experiments.

    PubMed

    Grün, Dominic; van Oudenaarden, Alexander

    2015-11-05

    Recent advances in single-cell sequencing hold great potential for exploring biological systems with unprecedented resolution. Sequencing the genome of individual cells can reveal somatic mutations and allows the investigation of clonal dynamics. Single-cell transcriptome sequencing can elucidate the cell type composition of a sample. However, single-cell sequencing comes with major technical challenges and yields complex data output. In this Primer, we provide an overview of available methods and discuss experimental design and single-cell data analysis. We hope that these guidelines will enable a growing number of researchers to leverage the power of single-cell sequencing.

  13. Surviving apoptosis: life-death signaling in single cells

    PubMed Central

    Flusberg, Deborah A.; Sorger, Peter K.

    2015-01-01

    Tissue development and homeostasis are regulated by opposing pro-survival and pro-death signals. An interesting feature of the Tumor Necrosis Factor (TNF) family of ligands is that they simultaneously activate opposing signals within a single cell via the same ligand-receptor complex. The magnitude of pro-death events such as caspase activation and pro-survival events such as NF-κB activation vary not only from one cell type to the next but also among individual cells of the same type due to intrinsic and extrinsic noise. The molecules involved in these pro-survival/pro-death pathways, and the different phenotypes that result from their activities, have been recently reviewed. Here we focus on the impact of cell-to-cell variability in the strength of these opposing signals on shaping cell fate decisions. PMID:25920803

  14. Hydrodynamic stretching of single cells for large population mechanical phenotyping

    PubMed Central

    Gossett, Daniel R.; Tse, Henry T. K.; Lee, Serena A.; Ying, Yong; Lindgren, Anne G.; Yang, Otto O.; Rao, Jianyu; Clark, Amander T.; Di Carlo, Dino

    2012-01-01

    Cell state is often assayed through measurement of biochemical and biophysical markers. Although biochemical markers have been widely used, intrinsic biophysical markers, such as the ability to mechanically deform under a load, are advantageous in that they do not require costly labeling or sample preparation. However, current techniques that assay cell mechanical properties have had limited adoption in clinical and cell biology research applications. Here, we demonstrate an automated microfluidic technology capable of probing single-cell deformability at approximately 2,000 cells/s. The method uses inertial focusing to uniformly deliver cells to a stretching extensional flow where cells are deformed at high strain rates, imaged with a high-speed camera, and computationally analyzed to extract quantitative parameters. This approach allows us to analyze cells at throughputs orders of magnitude faster than previously reported biophysical flow cytometers and single-cell mechanics tools, while creating easily observable larger strains and limiting user time commitment and bias through automation. Using this approach we rapidly assay the deformability of native populations of leukocytes and malignant cells in pleural effusions and accurately predict disease state in patients with cancer and immune activation with a sensitivity of 91% and a specificity of 86%. As a tool for biological research, we show the deformability we measure is an early biomarker for pluripotent stem cell differentiation and is likely linked to nuclear structural changes. Microfluidic deformability cytometry brings the statistical accuracy of traditional flow cytometric techniques to label-free biophysical biomarkers, enabling applications in clinical diagnostics, stem cell characterization, and single-cell biophysics. PMID:22547795

  15. Single-cell protein from waste cellulose

    NASA Technical Reports Server (NTRS)

    Dunlap, C. E.; Callihan, C. D.

    1973-01-01

    The recycle, reuse, or reclamation of single cell protein from liquid and solid agricultural waste fibers by a fermentation process is reported. It is shown that cellulose comprises the bulk of the fibers at 50% to 55% of the dry weight of the refuse and that its biodegradability is of prime importance in the choice of a substrate. The application of sodium hydroxide followed by heat and pressure serves to de-polymerize and disrupt lignin structure while swelling the cellulose to increase water uptake and pore volume. Some of the lignin, hemi-celluloses, ash, and cellulose of the material is hydrolized and solubilized. Introduction of microorganisms to the substrate fibers mixed with nutrients produces continuous fermentation of cellulose for further protein extraction and purification.

  16. Nanosensing at the single cell level

    NASA Astrophysics Data System (ADS)

    Vo-Dinh, Tuan

    2008-02-01

    This article presents an overview of the development, operation, and applications of optical nanobiosensors for use in in vivo detection of biotargets in individual living cells. The nanobiosensors are equipped with immobilized bioreceptor probes (e.g., antibodies, enzyme substrate) selective to specific molecular targets. Laser excitation is transmitted into the fiber producing an evanescent field at the tip of the fiber in order to excite target molecules bound to the bioreceptors immobilized at the fiber tips. A photometric system detects the optical signal (e.g., fluorescence) originated from the analyte molecules or from the analyte-bioreceptor reaction. Examples of detection of biospecies and molecular signaling pathways of apoptosis in a living cell are discussed to illustrate the potential of the nanobiosensor technology for single cell analysis.

  17. From single cells to social perception

    PubMed Central

    Barraclough, Nick E.; Perrett, David I.

    2011-01-01

    Research describing the cellular coding of faces in non-human primates often provides the underlying physiological framework for our understanding of face processing in humans. Models of face perception, explanations of perceptual after-effects from viewing particular types of faces, and interpretation of human neuroimaging data rely on monkey neurophysiological data and the assumption that neurophysiological responses of humans are comparable to those recorded in the non-human primate. Here, we review studies that describe cells that preferentially respond to faces, and assess the link between the physiological characteristics of single cells and social perception. Principally, we describe cells recorded from the non-human primate, although a limited number of cells have been recorded in humans, and are included in order to appraise the validity of non-human physiological data for our understanding of human face and social perception. PMID:21536557

  18. Single-Cell Transcriptomics of the Human Endocrine Pancreas.

    PubMed

    Wang, Yue J; Schug, Jonathan; Won, Kyoung-Jae; Liu, Chengyang; Naji, Ali; Avrahami, Dana; Golson, Maria L; Kaestner, Klaus H

    2016-10-01

    Human pancreatic islets consist of multiple endocrine cell types. To facilitate the detection of rare cellular states and uncover population heterogeneity, we performed single-cell RNA sequencing (RNA-seq) on islets from multiple deceased organ donors, including children, healthy adults, and individuals with type 1 or type 2 diabetes. We developed a robust computational biology framework for cell type annotation. Using this framework, we show that α- and β-cells from children exhibit less well-defined gene signatures than those in adults. Remarkably, α- and β-cells from donors with type 2 diabetes have expression profiles with features seen in children, indicating a partial dedifferentiation process. We also examined a naturally proliferating α-cell from a healthy adult, for which pathway analysis indicated activation of the cell cycle and repression of checkpoint control pathways. Importantly, this replicating α-cell exhibited activated Sonic hedgehog signaling, a pathway not previously known to contribute to human α-cell proliferation. Our study highlights the power of single-cell RNA-seq and provides a stepping stone for future explorations of cellular heterogeneity in pancreatic endocrine cells. © 2016 by the American Diabetes Association.

  19. Hand-Held and Integrated Single-Cell Pipettes

    PubMed Central

    2015-01-01

    Successful single-cell isolation is a primary step for subsequent chemical and biological analyses of single cells. Conventional single-cell isolation methods often encounter operational complexity, limited efficiency, deterioration of cell viability, incompetence in the isolation of a single-cell into nanoliter liquid, and/or inability to select single adherent cells with specific phenotypes. Here, we develop a hand-held single-cell pipet (hSCP) that is rapid, operationally simple, highly efficient, and inexpensive for unbiased isolation of single viable suspended cells directly from submicroliter cell suspensions into nanoliter droplets without the assistance of any additional equipment. An integrated SCP (iSCP) has also been developed for selective isolation of single suspended and adherent cells according to the fluorescence imaging and morphological features. The isolated single cells can be conveniently transferred into standard 96-/384-well plates, Petri dishes, or vials for cloning, PCR, and other single-cell biochemical assays. PMID:25036187

  20. Digital Microfluidics for Manipulation and Analysis of a Single Cell

    PubMed Central

    He, Jie-Long; Chen, An-Te; Lee, Jyong-Huei; Fan, Shih-Kang

    2015-01-01

    The basic structural and functional unit of a living organism is a single cell. To understand the variability and to improve the biomedical requirement of a single cell, its analysis has become a key technique in biological and biomedical research. With a physical boundary of microchannels and microstructures, single cells are efficiently captured and analyzed, whereas electric forces sort and position single cells. Various microfluidic techniques have been exploited to manipulate single cells through hydrodynamic and electric forces. Digital microfluidics (DMF), the manipulation of individual droplets holding minute reagents and cells of interest by electric forces, has received more attention recently. Because of ease of fabrication, compactness and prospective automation, DMF has become a powerful approach for biological application. We review recent developments of various microfluidic chips for analysis of a single cell and for efficient genetic screening. In addition, perspectives to develop analysis of single cells based on DMF and emerging functionality with high throughput are discussed. PMID:26389890

  1. Single-cell transcriptome sequencing: recent advances and remaining challenges

    PubMed Central

    Liu, Serena; Trapnell, Cole

    2016-01-01

    Single-cell RNA-sequencing methods are now robust and economically practical and are becoming a powerful tool for high-throughput, high-resolution transcriptomic analysis of cell states and dynamics. Single-cell approaches circumvent the averaging artifacts associated with traditional bulk population data, yielding new insights into the cellular diversity underlying superficially homogeneous populations. Thus far, single-cell RNA-sequencing has already shown great effectiveness in unraveling complex cell populations, reconstructing developmental trajectories, and modeling transcriptional dynamics. Ongoing technical improvements to single-cell RNA-sequencing throughput and sensitivity, the development of more sophisticated analytical frameworks for single-cell data, and an increasing array of complementary single-cell assays all promise to expand the usefulness and potential applications of single-cell transcriptomic profiling. PMID:26949524

  2. Digital Microfluidics for Manipulation and Analysis of a Single Cell.

    PubMed

    He, Jie-Long; Chen, An-Te; Lee, Jyong-Huei; Fan, Shih-Kang

    2015-09-15

    The basic structural and functional unit of a living organism is a single cell. To understand the variability and to improve the biomedical requirement of a single cell, its analysis has become a key technique in biological and biomedical research. With a physical boundary of microchannels and microstructures, single cells are efficiently captured and analyzed, whereas electric forces sort and position single cells. Various microfluidic techniques have been exploited to manipulate single cells through hydrodynamic and electric forces. Digital microfluidics (DMF), the manipulation of individual droplets holding minute reagents and cells of interest by electric forces, has received more attention recently. Because of ease of fabrication, compactness and prospective automation, DMF has become a powerful approach for biological application. We review recent developments of various microfluidic chips for analysis of a single cell and for efficient genetic screening. In addition, perspectives to develop analysis of single cells based on DMF and emerging functionality with high throughput are discussed.

  3. Single cell analysis and selection of living retrovirus vector-corrected mucopolysaccharidosis VII cells using a fluorescence-activated cell sorting-based assay for mammalian beta-glucuronidase enzymatic activity.

    PubMed

    Lorincz, M C; Parente, M K; Roederer, M; Nolan, G P; Diwu, Z; Martin, D I; Herzenberg, L A; Wolfe, J H

    1999-01-08

    Mutations in the acid beta-glucuronidase gene lead to systemic accumulation of undegraded glycosaminoglycans in lysosomes and ultimately to clinical manifestations of mucopolysaccharidosis VII (Sly disease). Gene transfer by retrovirus vectors into murine mucopolysaccharidosis VII hematopoietic stem cells or fibroblasts ameliorates glycosaminoglycan accumulation in some affected tissues. The efficacy of gene therapy for mucopolysaccharidosis VII depends on the levels of beta-glucuronidase secreted by gene-corrected cells; therefore, enrichment of transduced cells expressing high levels of enzyme prior to transplantation is desirable. We describe the development of a fluorescence-activated cell sorter-based assay for the quantitative analysis of beta-glucuronidase activity in viable cells. Murine mucopolysaccharidosis VII cells transduced with a beta-glucuronidase retroviral vector can be isolated by cell sorting on the basis of beta-glucuronidase activity and cultured for further use. In vitro analysis revealed that sorted cells have elevated levels of beta-glucuronidase activity and secrete higher levels of cross-correcting enzyme than the population from which they were sorted. Transduced fibroblasts stably expressing beta-glucuronidase after subcutaneous passage in the mucopolysaccharidosis VII mouse can be isolated by cell sorting and expanded ex vivo. A relatively high percentage of these cells maintain stable expression after secondary transplantation, yielding significantly higher levels of enzymatic activity than that generated in the primary transplant.

  4. Promise of Wearable Physical Activity Monitors in Oncology Practice.

    PubMed

    Beg, Muhammad S; Gupta, Arjun; Stewart, Tyler; Rethorst, Chad D

    2017-02-01

    Commercially available physical activity monitors provide clinicians an opportunity to obtain oncology patient health measures to an unprecedented degree. These devices can provide objective and quantifiable measures of physical activity, which are not subject to errors or bias of self-reporting or shorter duration of formal testing. Prior work on so-called quantified-self data was based on older-generation, research-grade accelerometers, which laid the foundation for consumer-based physical activity monitoring devices to be validated as a feasible and reliable tool in patients with cancer. Physical activity monitors are being used in chronic conditions including chronic obstructive pulmonary disease, congestive heart failure, diabetes mellitus, and obesity. Differing demographics, compounded with higher symptom and treatment burdens in patients with cancer, imply that additional work is needed to understand the unique strengths and weaknesses of physical activity monitors in this population. Oncology programs can systematically implement these tools into their workflows in an adaptable and iterative manner. Translating large amounts of data collected from an individual physical activity monitoring device into clinically relevant information requires sophisticated data compilation and reduction. In this article, we summarize the characteristics of older- and newer-generation physical activity monitors, review the validation of physical activity monitors with respect to health-related quality-of-life assessments, and describe the current role of these devices for the practicing oncologist. We also highlight the challenges and next steps needed for physical activity monitors to provide relevant information that can change the current state of oncology practice.

  5. Comparative studies on performance of single cell and PEMFC stack

    NASA Astrophysics Data System (ADS)

    Onggo, Holia; Irmawati, Yuyun; Yudianti, Rike

    2016-02-01

    Single-cell, 2-cell and 3-cell of polymer electrolyte membrane fuel cells (PEMFCs) with an active area of 9 cm2 per cell have been fabricated and characterized its performance and electrochemical properties. The membrane electrode assembly (MEA) was prepared by hot pressing commercial gas diffusion electrodes (Pt loading 0.5 mg/cm2) on pre-treated Nafion 117 membrane. The experimental results are presented as polarization and power output curves which show the effects of varying H2/O2 back pressures on the PEMFC performance. Three varying H2/O2 volumetric flow-rates for three different cell stacks were employed based on the optimal condition to produce good performance of stack. Cell performances for single cell, 2-cell, or 3-cell PEMFCs are slightly comparable with the power output 1.2 - 1.3 W in average. Enhancing back pressure induces increasing performance PEMFCs as indicated by changing the power from 1.19 W (open end) to 1.33W (15 psi).

  6. Single-cell sequencing for drug discovery and drug development.

    PubMed

    Wu, Hongjin; Wang, Charles; Wu, Shixiu

    2016-11-16

    Next-generation sequencing (NGS), particularly single-cell sequencing, has revolutionized the scale and scope of genomic and biomedical research. Recent technological advances in NGS and single-cell studies have made the deep whole-genome (DNA-seq), whole epigenome and whole-transcriptome sequencing (RNA-seq) at single-cell level feasible. NGS at the single-cell level expands our view of genome, epigenome and transcriptome and allows the genome, epigenome and transcriptome of any organism to be explored without a priori assumptions and with unprecedented throughput. And it does so with single-nucleotide resolution. NGS is also a very powerful tool for drug discovery and drug development. In this review, we describe the current state of single-cell sequencing techniques, which can provide a new, more powerful and precise approach for analyzing effects of drugs on treated cells and tissues. Our review discusses single-cell whole genome/exome sequencing (scWGS/scWES), single-cell transcriptome sequencing (scRNA-seq), single-cell bisulfite sequencing (scBS), and multiple omics of single-cell sequencing. We also highlight the advantages and challenges of each of these approaches. Finally, we describe, elaborate and speculate the potential applications of single-cell sequencing for drug discovery and drug development.

  7. Active Low Intrusion Hybrid Monitor for Wireless Sensor Networks.

    PubMed

    Navia, Marlon; Campelo, Jose C; Bonastre, Alberto; Ors, Rafael; Capella, Juan V; Serrano, Juan J

    2015-09-18

    Several systems have been proposed to monitor wireless sensor networks (WSN). These systems may be active (causing a high degree of intrusion) or passive (low observability inside the nodes). This paper presents the implementation of an active hybrid (hardware and software) monitor with low intrusion. It is based on the addition to the sensor node of a monitor node (hardware part) which, through a standard interface, is able to receive the monitoring information sent by a piece of software executed in the sensor node. The intrusion on time, code, and energy caused in the sensor nodes by the monitor is evaluated as a function of data size and the interface used. Then different interfaces, commonly available in sensor nodes, are evaluated: serial transmission (USART), serial peripheral interface (SPI), and parallel. The proposed hybrid monitor provides highly detailed information, barely disturbed by the measurement tool (interference), about the behavior of the WSN that may be used to evaluate many properties such as performance, dependability, security, etc. Monitor nodes are self-powered and may be removed after the monitoring campaign to be reused in other campaigns and/or WSNs. No other hardware-independent monitoring platforms with such low interference have been found in the literature.

  8. Active Low Intrusion Hybrid Monitor for Wireless Sensor Networks

    PubMed Central

    Navia, Marlon; Campelo, Jose C.; Bonastre, Alberto; Ors, Rafael; Capella, Juan V.; Serrano, Juan J.

    2015-01-01

    Several systems have been proposed to monitor wireless sensor networks (WSN). These systems may be active (causing a high degree of intrusion) or passive (low observability inside the nodes). This paper presents the implementation of an active hybrid (hardware and software) monitor with low intrusion. It is based on the addition to the sensor node of a monitor node (hardware part) which, through a standard interface, is able to receive the monitoring information sent by a piece of software executed in the sensor node. The intrusion on time, code, and energy caused in the sensor nodes by the monitor is evaluated as a function of data size and the interface used. Then different interfaces, commonly available in sensor nodes, are evaluated: serial transmission (USART), serial peripheral interface (SPI), and parallel. The proposed hybrid monitor provides highly detailed information, barely disturbed by the measurement tool (interference), about the behavior of the WSN that may be used to evaluate many properties such as performance, dependability, security, etc. Monitor nodes are self-powered and may be removed after the monitoring campaign to be reused in other campaigns and/or WSNs. No other hardware-independent monitoring platforms with such low interference have been found in the literature. PMID:26393604

  9. Passive and active structural monitoring experience: Civil engineering applications

    NASA Astrophysics Data System (ADS)

    Thompson, L. D.; Westermo, B. D.; Crum, D. B.; Law, W. R.; Trombi, R. G.

    2000-05-01

    State Departments of Transportation and regional city government officials are beginning to view the long-term monitoring of infrastructure as being beneficial for structural damage accumulation assessment, condition based maintenance, life extension, and post-earthquake or -hurricane (-tornado, -typhoon, etc.) damage assessment. Active and passive structural monitoring systems were installed over the last few years to monitor concerns in a wide range of civil infrastructure applications. This paper describes the monitoring technologies and systems employed for such applications. Bridge system applications were directed at monitoring corrosion damage accumulation, composite reinforcements for life extension, general service cracking damage related to fatigue and overloads, and post-earthquake damage. Residential system applications were directed primarily at identifying damage accumulation and post-earthquake damage assessment. A professional sports stadium was monitored for isolated ground instability problems and for post-earthquake damage assessment. Internet-based, remote, data acquisition system experience is discussed with examples of long-term passive and active system data collected from many of the individual sites to illustrate the potential for both passive and active structural health monitoring. A summary of system-based operating characteristics and key engineering recommendations are provided to achieve specific structural monitoring objectives for a wide range of civil infrastructure applications.

  10. Live Single-Cell Plant Hormone Analysis by Video-Mass Spectrometry.

    PubMed

    Shimizu, Takafumi; Miyakawa, Shinya; Esaki, Tsuyoshi; Mizuno, Hajime; Masujima, Tsutomu; Koshiba, Tomokazu; Seo, Mitsunori

    2015-07-01

    Studies have indicated that endogenous concentrations of plant hormones are regulated very locally within plants. To understand the mechanisms underlying hormone-mediated physiological processes, it is indispensable to know the exact hormone concentrations at cellular levels. In the present study, we established a system to determine levels of ABA and jasmonoyl-isoleucine (JA-Ile) from single cells. Samples taken from a cell of Vicia faba leaves using nano-electrospray ionization (ESI) tips under a microscope were directly introduced into mass spectrometers by infusion and subjected to tandem mass spectrometry (MS/MS) analysis. Stable isotope-labeled [D(6)]ABA or [(13)C(6)]JA-Ile was used as an internal standard to compensate ionization efficiencies, which determine the amount of ions introduced into mass spectrometers. We detected ABA and JA-Ile from single cells of water- and wound-stressed leaves, whereas they were almost undetectable in non-stressed single cells. The levels of ABA and JA-Ile found in the single-cell analysis were compared with levels found by analysis of purified extracts with liquid chromatography-tandem mass spectrometry (LC-MS/MS). These results demonstrated that stress-induced accumulation of ABA and JA-Ile could be monitored from living single cells.

  11. Photocardiography: a novel method for monitoring cardiac activity in fish.

    PubMed

    Yoshida, Masayuki; Hirano, Ruriko; Shima, Takao

    2009-05-01

    A non-invasive technique to monitor cardiac activity in small fish, such as goldfish, zebrafish, and medaka, is needed. In the present study, we developed photocardiography (PCG), a non-invasive optical method, to record cardiac activity in small fish. The method monitors changes in near-infrared light transmission through the heart using a phototransistor located outside the body. With this technique, heartbeats in fish of various sizes (14-218 mm) were stably recorded. PCG was applied to monitor the heartbeat during fear-related classical heart rate conditioning in goldfish wherein an electrical shock was used as an unconditioned stimulus. The heartbeats were continuously monitored, even when the beat coincided with the electrical shock, showing that PCG is robust even in an electrically noisy environment. This technique is particularly useful when monitoring the heartbeats of fish of small size or in the presence of ambient electrical noise, conditions in which the use of conventional electrocardiography (ECG) is difficult.

  12. Gravisensing in single-celled systems

    NASA Astrophysics Data System (ADS)

    Braun, M.; Limbach, C.

    Single-celled systems are favourable cell types for studying several aspects of gravisensing and gravitropic responses. Whether and how actin is involved in both processes in higher plant statocytes is still a matter of intensive debate. In single-celled and tip-growing characean rhizoids and protonemata, however, there is clear evidence that actin is a central keyplayer controlling polarized growth and the mechanisms of gravity sensing and growth reorientation. Both cell types exhibit a unique actin polymerization in the extending tip, strictly colocalized with the prominent ER-aggregate in the center of the Spitzenkoerper. The local accumulation of ADF and profilin in this central array suggest that actin polymerization is controlled by these actin-binding proteins, which can be regulated by calcium, pH and a variety of other parameters. Distinct actin filaments extend even into the outermost tip and form a dense meshwork in the apical and subapical region, before they become bundled by villin to form two populations of thick actin cables that generate rotational cytoplasmic streaming in the basal region. Actomyosin not only mediates the delivery of secretory vesicles to the growing tip and controls the incorporation pattern of cell wall material, but also coordinates the tip-focused distribution pattern of calcium channels in the apical membrane. They establish the tip-high calcium gradient, a prerequisite for exocytosis. Microgravity experiments have added much to our understanding that both cell types use an efficient actomyosin-based system to control and correct the position of their statoliths and to direct sedimenting statoliths to confined graviperception sites at the plasma membrane. Actin's involvement in the graviresponses is more indirect. The upward growth of negatively gravitropic protonemata was shown to be preceded by a statolith-induced relocalization the Ca2+-calcium gradient to the upper flank that does not occur in positively gravitropic

  13. Construction monitoring activities in the ESF starter tunnel

    SciTech Connect

    Pott, J.; Carlisle, S.

    1994-05-01

    In situ design verification activities am being conducted in the North Ramp Starter Tunnel of the Yucca Mountain Project Exploratory Studies Facility. These activities include: monitoring the peak particle velocities and evaluating the damage to the rock mass associated with construction blasting, assessing the rock mass quality surrounding the tunnel, monitoring the performance of the installed ground support, and monitoring the stability of the tunnel. In this paper, examples of the data that have been collected and preliminary conclusions from the data are presented.

  14. A transgenic zebrafish model for monitoring glucocorticoid receptor activity

    PubMed Central

    Krug, Randall G.; Poshusta, Tanya L.; Skuster, Kimberly J.; Berg, MaKayla R.; Gardner, Samantha L.; Clark, Karl J.

    2014-01-01

    Gene regulation resulting from glucocorticoid receptor and glucocorticoid response element interactions is a hallmark feature of stress response signaling. Imbalanced glucocorticoid production and glucocorticoid receptor activity have been linked to socio-economically crippling neuropsychiatric disorders, and accordingly there is a need to develop in vivo models to help understand disease progression and management. Therefore, we developed the transgenic SR4G zebrafish reporter line with six glucocorticoid response elements used to promote expression of a short half-life green fluorescent protein following glucocorticoid receptor activation. Herein, we document the ability of this reporter line to respond to both chronic and acute exogenous glucocorticoid treatment. The green fluorescent protein expression in response to transgene activation was high in a variety of tissues including the brain, and provided single cell resolution in the effected regions. The specificity of these responses is demonstrated using the partial agonist mifepristone and mutation of the glucocorticoid receptor. Importantly, the reporter line also modeled the temporal dynamics of endogenous stress response signaling, including the increased production of the glucocorticoid cortisol following hyperosmotic stress and the fluctuations of basal cortisol concentrations with the circadian rhythm. Taken together, these results characterize our newly developed reporter line for elucidating environmental or genetic modifiers of stress response signaling, which may provide insights to the neuronal mechanisms underlying neuropsychiatric disorders such as major depressive disorder. PMID:24679220

  15. Bigelow Expandable Activity Module (BEAM) Monitoring System

    NASA Technical Reports Server (NTRS)

    Wells, Nathan

    2017-01-01

    What is Bigelow Expandable Activity Module (BEAM)? The Bigelow Expandable Activity Module (BEAM) is an expandable habitat technology demonstration on ISS; increase human-rated inflatable structure Technology Readiness Level (TRL) to level 9. NASA managed ISS payload project in partnership with Bigelow Aerospace. Launched to ISS on Space X 8 (April 8th, 2016). Fully expanded on May 28th, 2016. Jeff Williams/Exp. 48 Commander first entered BEAM on June 5th, 2016.

  16. The potential of single-cell profiling in plants.

    PubMed

    Efroni, Idan; Birnbaum, Kenneth D

    2016-04-05

    Single-cell transcriptomics has been employed in a growing number of animal studies, but the technique has yet to be widely used in plants. Nonetheless, early studies indicate that single-cell RNA-seq protocols developed for animal cells produce informative datasets in plants. We argue that single-cell transcriptomics has the potential to provide a new perspective on plant problems, such as the nature of the stem cells or initials, the plasticity of plant cells, and the extent of localized cellular responses to environmental inputs. Single-cell experimental outputs require different analytical approaches compared with pooled cell profiles and new tools tailored to single-cell assays are being developed. Here, we highlight promising new single-cell profiling approaches, their limitations as applied to plants, and their potential to address fundamental questions in plant biology.

  17. The added value of single-cell gene expression profiling.

    PubMed

    Ståhlberg, Anders; Rusnakova, Vendula; Kubista, Mikael

    2013-03-01

    Cells are the basic unit of life and they have remarkable abilities to respond individually as well as in concert to internal and external stimuli in a specific manner. Studying complex tissues and whole organs requires understanding of cell heterogeneity and responses to stimuli at the single-cell level. In this review, we discuss the potential of single-cell gene expression profiling, focusing on data analysis and biological interpretation. We exemplify several aspects of the added value of single-cell analysis by comparing the same experimental data at both single-cell and cell population level. Data normalization and handling of missing data are two important steps in data analysis that are performed differently at single-cell level compared with cell population level. Furthermore, we discuss how single-cell gene expression data can be viewed and how subpopulations of cells can be identified and characterized.

  18. Single-cell sequencing technologies: current and future.

    PubMed

    Liang, Jialong; Cai, Wanshi; Sun, Zhongsheng

    2014-10-20

    Intensively developed in the last few years, single-cell sequencing technologies now present numerous advantages over traditional sequencing methods for solving the problems of biological heterogeneity and low quantities of available biological materials. The application of single-cell sequencing technologies has profoundly changed our understanding of a series of biological phenomena, including gene transcription, embryo development, and carcinogenesis. However, before single-cell sequencing technologies can be used extensively, researchers face the serious challenge of overcoming inherent issues of high amplification bias, low accuracy and reproducibility. Here, we simply summarize the techniques used for single-cell isolation, and review the current technologies used in single-cell genomic, transcriptomic, and epigenomic sequencing. We discuss the merits, defects, and scope of application of single-cell sequencing technologies and then speculate on the direction of future developments.

  19. Monitoring volcano activity through Hidden Markov Model

    NASA Astrophysics Data System (ADS)

    Cassisi, C.; Montalto, P.; Prestifilippo, M.; Aliotta, M.; Cannata, A.; Patanè, D.

    2013-12-01

    During 2011-2013, Mt. Etna was mainly characterized by cyclic occurrences of lava fountains, totaling to 38 episodes. During this time interval Etna volcano's states (QUIET, PRE-FOUNTAIN, FOUNTAIN, POST-FOUNTAIN), whose automatic recognition is very useful for monitoring purposes, turned out to be strongly related to the trend of RMS (Root Mean Square) of the seismic signal recorded by stations close to the summit area. Since RMS time series behavior is considered to be stochastic, we can try to model the system generating its values, assuming to be a Markov process, by using Hidden Markov models (HMMs). HMMs are a powerful tool in modeling any time-varying series. HMMs analysis seeks to recover the sequence of hidden states from the observed emissions. In our framework, observed emissions are characters generated by the SAX (Symbolic Aggregate approXimation) technique, which maps RMS time series values with discrete literal emissions. The experiments show how it is possible to guess volcano states by means of HMMs and SAX.

  20. Silicon dioxide thin film mediated single cell nucleic acid isolation.

    PubMed

    Bogdanov, Evgeny; Dominova, Irina; Shusharina, Natalia; Botman, Stepan; Kasymov, Vitaliy; Patrushev, Maksim

    2013-01-01

    A limited amount of DNA extracted from single cells, and the development of single cell diagnostics make it necessary to create a new highly effective method for the single cells nucleic acids isolation. In this paper, we propose the DNA isolation method from biomaterials with limited DNA quantity in sample, and from samples with degradable DNA based on the use of solid-phase adsorbent silicon dioxide nanofilm deposited on the inner surface of PCR tube.

  1. Silicon Dioxide Thin Film Mediated Single Cell Nucleic Acid Isolation

    PubMed Central

    Bogdanov, Evgeny; Dominova, Irina; Shusharina, Natalia; Botman, Stepan; Kasymov, Vitaliy; Patrushev, Maksim

    2013-01-01

    A limited amount of DNA extracted from single cells, and the development of single cell diagnostics make it necessary to create a new highly effective method for the single cells nucleic acids isolation. In this paper, we propose the DNA isolation method from biomaterials with limited DNA quantity in sample, and from samples with degradable DNA based on the use of solid-phase adsorbent silicon dioxide nanofilm deposited on the inner surface of PCR tube. PMID:23874571

  2. Monitoring dressing activity failures through RFID and video.

    PubMed

    Matic, A; Mehta, P; Rehg, J M; Osmani, V; Mayora, O

    2012-01-01

    Monitoring and evaluation of Activities of Daily Living in general, and dressing activity in particular, is an important indicator in the evaluation of the overall cognitive state of patients. In addition, the effectiveness of therapy in patients with motor impairments caused by a stroke, for example, can be measured through long-term monitoring of dressing activity. However, automatic monitoring of dressing activity has not received significant attention in the current literature. Considering the importance of monitoring dressing activity, the main goal of this work was to investigate the possibility of recognizing dressing activities and automatically identifying common failures exhibited by patients suffering from motor or cognitive impairments. The system developed for this purpose comprised analysis of RFID (radio frequency identification) tracking and computer vision processing. Eleven test subjects, not connected to the research, were recruited and asked to perform the dressing task by choosing any combination of clothes without further assistance. Initially the test subjects performed correct dressing and then they were free to choose from a set of dressing failures identified from the current research literature. The developed system was capable of automatically recognizing common dressing failures. In total, there were four dressing failures observed for upper garments and three failures for lower garments, in addition to recognizing successful dressing. The recognition rate for identified dressing failures was between 80% and 100%. We developed a robust system to monitor the dressing activity. Given the importance of monitoring the dressing activity as an indicator of both cognitive and motor skills the system allows for the possibility of long term tracking and continuous evaluation of the dressing task. Long term monitoring can be used in rehabilitation and cognitive skills evaluation.

  3. Management plan for Facility Effluent Monitoring Plan activities

    SciTech Connect

    Nickels, J.M.; Pratt, D.R.

    1991-08-01

    The DOE/RL 89-19, United States Department of Energy-Richland Operations Office Environmental Protection Implementation Plan (1989), requires the Hanford Site to prepare an Environmental Monitoring Plan (EMP) by November 9, 1991. The DOE/EH-0173T, Environmental Regulatory Guide for Radiological Effluent Monitoring and Environmental Surveillance (1991), provides additional guidance and requires implementation of the EMP within 36 months of the effective data of the rule. DOE Order 5400.1, General Environmental Protection Program, requires each US Department of Energy (DOE) site, facility, or activity that uses, generates, releases, or manages significant quantities of hazardous materials to prepare an EMP. This EMP is to identify and discuss two major activities: (1) effluent monitoring and (2) environmental surveillance. At the Hanford Site, the site-wide EMP will consist of the following elements: (1) A conceptual plan addressing effluent monitoring and environmental surveillance; (2) Pacific Northwest Laboratory (PNL) site-wide environmental surveillance program; (3) Westinghouse Hanford Company (Westinghouse Hanford) effluent monitoring program consisting of the near-field operations environmental monitoring activities and abstracts of each Facility Effluent Monitoring Plan (FEMP). This management plan addresses the third of these three elements of the EMP, the FEMPs.

  4. Single cell analysis: the new frontier in 'Omics'

    SciTech Connect

    Wang, Daojing; Bodovitz, Steven

    2010-01-14

    Cellular heterogeneity arising from stochastic expression of genes, proteins, and metabolites is a fundamental principle of cell biology, but single cell analysis has been beyond the capabilities of 'Omics' technologies. This is rapidly changing with the recent examples of single cell genomics, transcriptomics, proteomics, and metabolomics. The rate of change is expected to accelerate owing to emerging technologies that range from micro/nanofluidics to microfabricated interfaces for mass spectrometry to third- and fourth-generation automated DNA sequencers. As described in this review, single cell analysis is the new frontier in Omics, and single cell Omics has the potential to transform systems biology through new discoveries derived from cellular heterogeneity.

  5. Single-cell analysis of radiotracers' uptake by fluorescence microscopy: direct and droplet approach

    NASA Astrophysics Data System (ADS)

    Gallina, M. E.; Kim, T. J.; Vasquez, J.; Tuerkcan, S.; Abbyad, P.; Pratx, G.

    2017-02-01

    Radionuclides are used for sensitive and specific detection of small molecules in vivo and in vitro. Recently, radioluminescence microscopy extended their use to single-cell studies. Here we propose a new single-cell radioisotopic assay that improves throughput while adding sorting capabilities. The new method uses fluorescence-based sensor for revealing single-cell interactions with radioactive molecular markers. This study focuses on comparing two different experimental approaches. Several probes were tested and Dihydrorhodamine 123 was selected as the best compromise between sensitivity, brightness and stability. The sensor was incorporated either directly within the cell cytoplasm (direct approach), or it was coencapsulated with radiolabeled single-cells in oil-dispersed water droplets (droplet approach). Both approaches successfully activated the fluorescence signal following cellular uptake of 18F-fluorodeoxyglucose (FDG) and external Xrays exposure. The direct approach offered single-cell resolution and longtime stability ( > 20 hours), moreover it could discriminate FDG uptake at labelling concentration as low as 300 μCi/ml. In cells incubated with Dihydrorhodamine 123 after exposure to high radiation doses (8-16 Gy), the fluorescence signal was found to increase with the depletion of ROS quenchers. On the other side, the droplet approach required higher labelling concentrations (1.00 mCi/ml), and, at the current state of art, three cells per droplet are necessary to produce a fluorescent signal. This approach, however, is independent on cellular oxidative stress and, with further improvements, will be more suitable for studying heterogeneous populations. We anticipate this technology to pave the way for the analysis of single-cell interactions with radiomarkers by radiofluorogenic-activated single-cell sorting.

  6. Preparation of single cells from aggregated Taxus suspension cultures for population analysis.

    PubMed

    Naill, Michael C; Roberts, Susan C

    2004-06-30

    A method for the isolation of single plant cells from Taxus suspension cultures has been developed for the analysis of single cells via rapid throughput techniques such as flow cytometry. Several cell wall specific enzymes, such as pectinase, pectolyase Y-23, macerozyme, Driselase(R), and cellulase were tested for efficacy in producing single cell suspensions. The method was optimized for single cell yield, viability, time, and representivity of aggregated cell cultures. The best combination for single cell isolation was found to be 0.5% (w/v) pectolyase Y-23 and 0.04% (w/v) cellulase. High viability (>95%) and high yields of single cell aggregates (>90%) were obtained following 4 hours of digestion for four separate Taxus cell lines. In addition, methyl jasmonate elicitation (200 microM) was found to have no effect on three of the four tested Taxus lines. Isolated single cells were statistically similar to untreated cell cultures for peroxidase activity (model cell wall protein) and paclitaxel content (secondary metabolite produced in Taxus cell cultures). In comparison, protoplasts showed marked changes in both peroxidase activity and paclitaxel content as compared to untreated cultures. The use of flow cytometry was demonstrated with isolated cells that were found to have > 99% viability upon staining with fluorescein diacetate. The development of a method for the isolation of single plant cells will allow the study of population dynamics and culture variability on a single cell level for the development of population models of plant cell cultures and secondary metabolism. Copyright 2004 Wiley Periodicals, Inc.

  7. Single-cell Raman spectroscopy of irradiated tumour cells

    NASA Astrophysics Data System (ADS)

    Matthews, Quinn

    This work describes the development and application of a novel combination of single-cell Raman spectroscopy (RS), automated data processing, and principal component analysis (PCA) for investigating radiation induced biochemical responses in human tumour cells. The developed techniques are first validated for the analysis of large data sets (˜200 spectra) obtained from single cells. The effectiveness and robustness of the automated data processing methods is demonstrated, and potential pitfalls that may arise during the implementation of such methods are identified. The techniques are first applied to investigate the inherent sources of spectral variability between single cells of a human prostate tumour cell line (DU145) cultured in vitro. PCA is used to identify spectral differences that correlate with cell cycle progression and the changing confluency of a cell culture during the first 3-4 days after sub-culturing. Spectral variability arising from cell cycle progression is (i) expressed as varying intensities of protein and nucleic acid features relative to lipid features, (ii) well correlated with known biochemical changes in cells as they progress through the cell cycle, and (iii) shown to be the most significant source of inherent spectral variability between cells. This characterization provides a foundation for interpreting spectral variability in subsequent studies. The techniques are then applied to study the effects of ionizing radiation on human tumour cells. DU145 cells are cultured in vitro and irradiated to doses between 15 and 50 Gy with single fractions of 6 MV photons from a medical linear accelerator. Raman spectra are acquired from irradiated and unirradiated cells, up to 5 days post-irradiation. PCA is used to distinguish radiation induced spectral changes from inherent sources of spectral variability, such as those arising from cell cycle. Radiation induced spectral changes are found to correlate with both the irradiated dose and the

  8. Single cell genomics of subsurface microorganisms

    NASA Astrophysics Data System (ADS)

    Stepanauskas, R.; Onstott, T. C.; Lau, C.; Kieft, T. L.; Woyke, T.; Rinke, C.; Sczyrba, A.; van Heerden, E.

    2012-12-01

    Recent studies have revealed unexpected abundance and diversity of microorganisms in terrestrial and marine subsurface, providing new perspectives over their biogeochemical significance, evolution, and the limits of life. The now commonly used research tools, such as metagenomics and PCR-based gene surveys enabled cultivation-unbiased analysis of genes encoded by natural microbial communities. However, these methods seldom provide direct evidence for how the discovered genes are organized inside genomes and from which organisms do they come from. Here we evaluated the feasibility of an alternative, single cell genomics approach, in the analysis of subsurface microbial community composition, metabolic potential and microevolution at the Sanford Underground Research Facility (SURF), South Dakota, and the Witwaterstrand Basin, South Africa. We successfully recovered genomic DNA from individual microbial cells from multiple locations, including ultra-deep (down to 3,500 m) and low-biomass (down to 10^3 cells mL^-1) fracture water. The obtained single amplified genomes (SAGs) from SURF contained multiple representatives of the candidate divisions OP3, OP11, OD1 and uncharacterized archaea. By sequencing eight of these SAGs, we obtained the first genome content information for these phylum-level lineages that do not contain a single cultured representative. The Witwaterstrand samples were collected from deep fractures, biogeochemical dating of which suggests isolation from tens of thousands to tens of millions of years. Thus, these fractures may be viewed as "underground Galapagos", a natural, long-term experiment of microbial evolution within well-defined temporal and spatial boundaries. We are analyzing multiple SAGs from these environments, which will provide detailed information about adaptations to life in deep subsurface, mutation rates, selective pressures and gene flux within and across microbial populations.

  9. Single cell mechanics of keratinocyte cells.

    PubMed

    Lulevich, Valentin; Yang, Hsin-ya; Isseroff, R Rivkah; Liu, Gang-yu

    2010-11-01

    Keratinocytes represent the major cell type of the uppermost layer of human skin, the epidermis. Using AFM-based single cell compression, the ability of individual keratinocytes to resist external pressure and global rupturing forces is investigated and compared with various cell types. Keratinocytes are found to be 6-70 times stiffer than other cell types, such as white blood, breast epithelial, fibroblast, or neuronal cells, and in contrast to other cell types they retain high mechanic strength even after the cell's death. The absence of membrane rupturing peaks in the force-deformation profiles of keratinocytes and their high stiffness during a second load cycle suggests that their unique mechanical resistance is dictated by the cytoskeleton. A simple analytical model enables the quantification of Young's modulus of keratinocyte cytoskeleton, as high as 120-340 Pa. Selective disruption of the two major cytoskeletal networks, actin filaments and microtubules, does not significantly affect keratinocyte mechanics. F-actin is found to impact cell deformation under pressure. During keratinocyte compression, the plasma membrane stretches to form peripheral blebs. Instead of blebbing, cells with depolymerized F-actin respond to pressure by detaching the plasma membrane from the cytoskeleton underneath. On the other hand, the compression force of keratinocytes expressing a mutated keratin (cell line, KEB-7) is 1.6-2.2 times less than that for the control cell line that has normal keratin networks. Therefore, we infer that the keratin intermediate filament network is responsible for the extremely high keratinocyte stiffness and resilience. This could manifest into the rugged protective nature of the human epidermis.

  10. Active Sites Environmental Monitoring Program FY 1996 annual report

    SciTech Connect

    Morrissey, C.M.; Marshall, D.S.; Cunningham, G.R.

    1997-11-01

    This report summarizes the activities of the Active Sites Environmental Monitoring Program (ASEMP) from October 1995 through September 1996. The Radioactive Solid Waste Operations Group (RSWOG) of the Waste Management and Remedial Action Division (WMRAD) and the Environmental Sciences Division (ESD) at Oak Ridge National Laboratory (ORNL) established ASEMP in 1989. The purpose of the program is to provide early detection and performance monitoring at active low-level waste (LLW) disposal sites in Solid Waste Storage Area (SWSA) 6 and transuranic (TRU) waste storage sites in SWSA 5 North as required by Chapters 2 and 3 of US Department of Energy Order 5820.2A.

  11. Predicting Activity Energy Expenditure Using the Actical[R] Activity Monitor

    ERIC Educational Resources Information Center

    Heil, Daniel P.

    2006-01-01

    This study developed algorithms for predicting activity energy expenditure (AEE) in children (n = 24) and adults (n = 24) from the Actical[R] activity monitor. Each participant performed 10 activities (supine resting, three sitting, three house cleaning, and three locomotion) while wearing monitors on the ankle, hip, and wrist; AEE was computed…

  12. Predicting Activity Energy Expenditure Using the Actical[R] Activity Monitor

    ERIC Educational Resources Information Center

    Heil, Daniel P.

    2006-01-01

    This study developed algorithms for predicting activity energy expenditure (AEE) in children (n = 24) and adults (n = 24) from the Actical[R] activity monitor. Each participant performed 10 activities (supine resting, three sitting, three house cleaning, and three locomotion) while wearing monitors on the ankle, hip, and wrist; AEE was computed…

  13. Intra-Species Bacterial Quorum Sensing Studied at Single Cell Level in a Double Droplet Trapping System

    PubMed Central

    Bai, Yunpeng; Patil, Santoshkumar N.; Bowden, Steven D.; Poulter, Simon; Pan, Jie; Salmond, George P. C.; Welch, Martin; Huck, Wilhelm T. S.; Abell, Chris

    2013-01-01

    In this paper, we investigated the intra-species bacterial quorum sensing at the single cell level using a double droplet trapping system. Escherichia coli transformed to express the quorum sensing receptor protein, LasR, were encapsulated in microdroplets that were positioned adjacent to microdroplets containing the autoinducer, N-(3-oxododecanoyl)- l-homoserine lactone (OdDHL). Functional activation of the LasR protein by diffusion of the OdDHL across the droplet interface was measured by monitoring the expression of green fluorescent protein (GFP) from a LasR-dependent promoter. A threshold concentration of OdDHL was found to induce production of quorum-sensing associated GFP by E. coli. Additionally, we demonstrated that LasR-dependent activation of GFP expression was also initiated when the adjacent droplets contained single E. coli transformed with the OdDHL synthase gene, LasI, representing a simple quorum sensing circuit between two droplets. PMID:23698779

  14. Dashboard applications to monitor experiment activities at sites

    NASA Astrophysics Data System (ADS)

    Andreeva, Julia; Belforte, Stefano; Boehm, Max; Casajus, Adrian; Flix, Josep; Gaidioz, Benjamin; Grigoras, Costin; Kokoszkiewicz, Lukasz; Lanciotti, Elisa; Rocha, Ricardo; Saiz, Pablo; Santinelli, Roberto; Sidorova, Irina; Sciabà, Andrea; Tsaregorodtsev, Andrei

    2010-04-01

    In the framework of a distributed computing environment, such as WLCG, monitoring has a key role in order to keep under control activities going on in sites located in different countries and involving people based in many different sites. To be able to cope with such a large scale heterogeneous infrastructure, it is necessary to have monitoring tools providing a complete and reliable view of the overall performance of the sites. Moreover, the structure of a monitoring system critically depends on the object to monitor and on the users it is addressed to. In this article we will describe two different monitoring systems both aimed to monitor activities and services provided in the WLCG framework, but designed in order to meet the requirements of different users: Site Status Board has an overall view of the services available in all the sites supporting an experiment, whereas Siteview provides a complete view of all the activities going on at a site, for all the experiments supported by the site.

  15. Preparation of Single Cell Suspensions from Mouse Aorta

    PubMed Central

    Hu, Desheng; Yin, Changjun; Mohanta, Sarajo K.; Weber, Christian; Habenicht, Andreas J. R.

    2016-01-01

    fluorescence activated cell sorter (FACS) analyses from single cell suspensions to quantify the cells of interest. This protocol describes isolation of single cells from mouse aorta for FACS and other analysis. PMID:27335895

  16. Short Peptides Enhance Single Cell Adhesion and Viability on Microarrays

    PubMed Central

    Veiseh, Mandana; Veiseh, Omid; Martin, Michael C.; Asphahani, Fareid; Zhang, Miqin

    2011-01-01

    Single cell patterning holds important implications for biology, biochemistry, biotechnology, medicine, and bioinformatics. The challenge for single cell patterning is to produce small islands hosting only single cells and retaining their viability for a prolonged period of time. This study demonstrated a surface engineering approach that uses a covalently-bound short peptide as a mediator to pattern cells with improved single cell adhesion and prolonged cellular viability on gold patterned SiO2 substrates. The underlying hypothesis is that cell adhesion is regulated by the type, availability and stability of effective cell adhesion peptides, and thus covalently bound short peptides would promote cell spreading and thus, single cell adhesion and viability. The effectiveness of this approach and the underlying mechanism for the increased probability of single cell adhesion and prolonged cell viability by short peptides were studied by comparing cellular behavior of human umbilical cord vein endothelial cells on three model surfaces whose gold electrodes were immobilized with fibronectin, physically adsorbed Arg-Glu-Asp-Val-Tyr, and covalently-bound Lys-Arg-Glu-Asp-Val-Tyr, respectively. The surface chemistry and binding properties were characterized by reflectance Fourier transform infrared spectroscopy. Both short peptides were superior to fibronectin in producing adhesion of only single cells, while the covalently bound peptide also reduced apoptosis and necrosis of adhered cells. Controlling cell spreading by peptide binding domains to regulate apoptosis and viability represents a fundamental mechanism in cell-materials interaction and provides an effective strategy in engineering arrays of single cells. PMID:17371055

  17. Single Cell Characterization of Prostate Cancer Circulating Tumor Cells

    DTIC Science & Technology

    2011-08-01

    single cell sequencing protocol for CTCs (Figure 3). So far, using their protocol we have done whole transcriptome amplification and mRNA seq on 6 single...perform additional single cell sequencing profiles. In our application we also hypothesized that there would be heterogeneity in gene expression

  18. Activity monitoring in sleep research, medicine and psychopharmacology.

    PubMed

    Klösch, G; Gruber, G; Anderer, P; Saletu, B

    2001-04-17

    Motor activity as a diagnostic parameter has become an important feature in many fields of medicine and psychology. The concept of mobility and immobility implies the assumption that mental and behaviour disorders involve abnormal activity that can be measured to characterise the disorder itself, to diagnose its presence and to document the impact of treatment. In sleep research, activity monitoring by wrist actigraphs has proven its usefulness as an efficient method to assess the rest-activity cycle over long time periods and to estimate sleep-related features such as sleep efficiency and total sleep time. But like many other techniques and devices, activity monitoring has some limitations and drawbacks. This paper describes the basic features of wrist actigraphy in measuring nocturnal and daytime motor activity.

  19. Monitoring Neural Activity with Bioluminescence during Natural Behavior

    PubMed Central

    Naumann, Eva A.; Kampff, Adam R.; Prober, David A.; Schier, Alexander F.; Engert, Florian

    2010-01-01

    Existing techniques for monitoring neural activity in awake, freely behaving vertebrates are invasive and difficult to target to genetically identified neurons. Here we describe the use of bioluminescence to non-invasively monitor the activity of genetically specified neurons in freely behaving zebrafish. Transgenic fish expressing the Ca2+-sensitive photoprotein GFP-apoAequorin (GA) in most neurons generated large and fast bioluminescent signals related to neural activity, neuroluminescence, that could be recorded continuously for many days. To test the limits of this technique, GA was specifically targeted to the hypocretin-positive neurons of the hypothalamus. We found that neuroluminescence generated by this group of ~20 neurons was associated with periods of increased locomotor activity and identified two classes of neural activity corresponding to distinct swim latencies. Thus, our neuroluminescence assay can report, with high temporal resolution and sensitivity, the activity of small subsets of neurons during unrestrained behavior. PMID:20305645

  20. Active Sites Environmental Monitoring Program: Mid-FY 1991 report

    SciTech Connect

    Ashwood, T.L.; Wickliff, D.S.; Morrissey, C.M.

    1991-10-01

    This report summarizes the activities of the Active Sites Environmental Monitoring Program (ASEMP) from October 1990 through March 1991. The ASEMP was established in 1989 by Solid Waste Operations and the Environmental Sciences Division to provide early detection and performance monitoring at active low-level radioactive waste (LLW) disposal sites in Solid Waste Storage Area (SWSA) 6 and transuranic (TRU) waste storage sites in SWSA 5 as required by chapters II and III of US Department of Energy Order 5820.2A. Monitoring results continue to demonstrate the no LLW is being leached from the storage vaults on the tumulus pads. Loading of vaults on Tumulus II began during this reporting period and 115 vaults had been loaded by the end of March 1991.

  1. Single cell sequencing approaches for complex biological systems.

    PubMed

    Baslan, Timour; Hicks, James

    2014-06-01

    Biological phenotype is the output of complex interactions between heterogeneous cells within a specified niche. These interactions are tightly governed and regulated by the genetic, epigenetic, and transcriptional states of single cells, with deregulation of these states resulting in disease. As such, genome wide single cell investigations are bound to enhance our knowledge of the underlying principles that govern biological systems. Recent technological advances have enabled such investigations in the form of single-cell sequencing. Here, we review the most recent developments in genome wide profiling of single cells, discuss some of the novel biological observations gleaned by such investigations, and touch upon the promise of single cell sequencing in unraveling biological systems.

  2. Detection of Copy Number Alterations Using Single Cell Sequencing.

    PubMed

    Knouse, Kristin A; Wu, Jie; Hendricks, Austin

    2017-02-17

    Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and microbial populations. Traditional methods for assessing genetic heterogeneity have been limited by low resolution, low sensitivity, and/or low specificity. Single cell sequencing has emerged as a powerful tool for detecting genetic heterogeneity with high resolution, high sensitivity and, when appropriately analyzed, high specificity. Here we provide a protocol for the isolation, whole genome amplification, sequencing, and analysis of single cells. Our approach allows for the reliable identification of megabase-scale copy number variants in single cells. However, aspects of this protocol can also be applied to investigate other types of genetic alterations in single cells.

  3. Single-cell mechanogenetics using monovalent magnetoplasmonic nanoparticles.

    PubMed

    Kim, Ji-Wook; Seo, Daeha; Lee, Jung-Uk; Southard, Kaden M; Lim, Yongjun; Kim, Daehyun; Gartner, Zev J; Jun, Young-Wook; Cheon, Jinwoo

    2017-09-01

    Spatiotemporal interrogation of signal transduction at the single-cell level is necessary to answer a host of important biological questions. This protocol describes a nanotechnology-based single-cell and single-molecule perturbation tool, termed mechanogenetics, that enables precise spatial and mechanical control over genetically encoded cell-surface receptors in live cells. The key components of this tool are a magnetoplasmonic nanoparticle (MPN) actuator that delivers defined spatial and mechanical cues to receptors through target-specific one-to-one engagement and a micromagnetic tweezers (μMT) that remotely controls the magnitude of force exerted on a single MPN. In our approach, a SNAP-tagged cell-surface receptor of interest is conjugated with a single-stranded DNA oligonucleotide, which hybridizes to its complementary oligonucleotide on the MPN. This protocol consists of four major stages: (i) chemical synthesis of MPNs, (ii) conjugation with DNA and purification of monovalent MPNs, (iii) modular targeting of MPNs to cell-surface receptors, and (iv) control of spatial and mechanical properties of targeted mechanosensitive receptors in live cells by adjusting the μMT-to-MPN distance. Using benzylguanine (BG)-functionalized MPNs and model cell lines expressing either SNAP-tagged Notch or vascular endothelial cadherin (VE-cadherin), we provide stepwise instructions for mechanogenetic control of receptor clustering and for mechanical receptor activation. The ability of this method to differentially control spatial and mechanical inputs to targeted receptors makes it particularly useful for interrogating the differential contributions of each individual cue to cell signaling. The entire procedure takes up to 1 week.

  4. Relating Single Cell Heterogeneity To Genotype During Cancer Progression

    NASA Astrophysics Data System (ADS)

    Rajaram, Satwik

    2013-03-01

    Progression of normal cells towards cancer is driven by a series of genetic changes. Traditional population-averaged measurements have found that cell signalling activities are increasingly altered during this progression. Despite the fact that cancer cells are known to be highly heterogeneous, the response of individual pathways to specific genetic changes remains poorly characterized at a single cell level. Do signalling alterations in a pathway reflect a shift of the whole population, or changes to specific subpopulations? Are alterations to pathways independent, or are cells with alterations in one pathway more likely to be abnormal in another due to crosstalk? We are building a computational framework that analyzes immunofluorescence microscopy images of cells to identify alterations in individual pathways at a single-cell level. A primary novelty of our approach is a ``change of basis'' that allows us to understand signalling in cancer cells in terms of the much better understood patterns of signalling in normal cells. This allows us to model heterogeneous populations of cancer cells as a mixture of distinct subpopulations, each with a specific combination of signalling pathways altered beyond the normal baseline. We used this framework to analyze human bronchial epithelial cell lines containing a series of genetic modifications commonly seen in lung cancer. We confirmed expected trends (such as a population-wide epithelial mesenchymal transition following the last of our series of modifications) and are presently studying the relation between the mutational profiles of cancer cells and pathway crosstalk. Our framework will help establish a more natural basis for future investigations into the phenotype-genotype relationship in heterogeneous populations.

  5. Lab-on-chip device for single cell trapping and analysis.

    PubMed

    Shah, Pratikkumar; Zhu, Xuena; Chen, Chunying; Hu, Ye; Li, Chen-Zhong

    2014-02-01

    Traditional cell assay gives us an average result of multiple cells and it is assumed that the resultant is the outcome of all cells in population. However, single cell studies have revealed that individual cells of same type may differ dramatically and these differences may have important role to play in cells functionality. Such information can be obscured in only studying cell population experimental approach. To uncover biological principles and ultimately to improve the detection and treatment of disease, new approaches are highly required to single cell analysis. We propose to fabricate a lab on chip device to study high throughput single cell nanotoxicity analysis. The chip incorporates independently addressable active microwell electrodes for cell manipulation and analysis. We employed positive-dielectrophoresis approach to quickly and efficiently capture single cells in each wells with having control over individual microwells. We examined change in impedance properties to verify cell capture in microwell and its health and present a novel model of single cell assay for nanotoxicity, and drug testing.

  6. Single cell measurement of telomerase expression and splicing using microfluidic emulsion cultures

    PubMed Central

    Novak, Richard; Hart, Kristina; Mathies, Richard A.

    2015-01-01

    Telomerase is a reverse transcriptase that maintains telomeres on the ends of chromosomes, allowing rapidly dividing cells to proliferate while avoiding senescence and apoptosis. Understanding telomerase gene expression and splicing at the single cell level could yield insights into the roles of telomerase during normal cell growth as well as cancer development. Here we use droplet-based single cell culture followed by single cell or colony transcript abundance analysis to investigate the relationship between cell growth and transcript abundance of the telomerase genes encoding the RNA component (hTR) and protein component (hTERT) as well as hTERT splicing. Jurkat and K562 cells were examined under normal cell culture conditions and during exposure to curcumin, a natural compound with anti-carcinogenic and telomerase activity-reducing properties. Individual cells predominantly express single hTERT splice variants, with the α+/β− variant exhibiting significant transcript abundance bimodality that is sustained through cell division. Sub-lethal curcumin exposure results in reduced bimodality of all hTERT splice variants and significant upregulation of alpha splicing, suggesting a possible role in cellular stress response. The single cell culture and transcript abundance analysis method presented here provides the tools necessary for multiparameter single cell analysis which will be critical for understanding phenotypes of heterogeneous cell populations, disease cell populations and their drug response. PMID:26202962

  7. Solid oxide fuel cell anode degradation by the effect of hydrogen chloride in stack and single cell environments

    NASA Astrophysics Data System (ADS)

    Madi, Hossein; Lanzini, Andrea; Papurello, Davide; Diethelm, Stefan; Ludwig, Christian; Santarelli, Massimo; Van herle, Jan

    2016-09-01

    The poisoning effect by hydrogen chloride (HCl) on state-of-the-art Ni anode-supported solid oxide fuel cells (SOFCs) at 750 °C is evaluated in either hydrogen or syngas fuel. Experiments are performed on single cells and short stacks and HCl concentration in the fuel gas is increased from 1 ppm(v) up to 1000 ppm(v) at different current densities. Characterization methods such as cell voltage monitoring vs. time and electrochemical impedance response analysis (distribution of relaxation times (DRT), equivalent electrical circuit) are used to identify the prevailing degradation mechanism. Single cell experiments revealed that the poisoning is more severe when feeding with hydrogen than with syngas. Performance loss is attributed to the effects of HCl adsorption onto nickel surfaces, which lowered the catalyst activity. Interestingly, in syngas HCl does not affect stack performance even at concentrations up to 500 ppm(v), even when causing severe corrosion of the anode exhaust pipe. Furthermore, post-test analysis suggests that chlorine is present on the nickel particles in the form of adsorbed chlorine, rather than forming a secondary phase of nickel chlorine.

  8. EarthScope Content Module for IRIS Active Earth Monitor

    NASA Astrophysics Data System (ADS)

    McQuillan, P. J.; Welti, R.; Johnson, J. A.; Shiffman, C. R.; Olds, S. E.

    2012-12-01

    The Active Earth Monitor (AEM) is an interactive computer-based display for university lobbies, museums, visitor centers, schools and libraries. AEM runs in a standard Internet web browser in full screen mode. The display consists of a customizable set of content pages about plate tectonics, earthquakes, volcanoes and tsunamis. Low-cost and simple-to-implement, the Active Earth Monitor provides a way to engage audiences with earth science information without spending resources on a large exhibit. The EarthScope Active Earth Monitor content set highlights the connections between the landscape and the research and monitoring being conducted by EarthScope in partnership with regional monitoring networks. Modules consist of chapters that focus on What is EarthScope?, EarthScope Observatories, and EarthScope Research Results. Content topics are easily explored using a web page button type navigation interface via a touch screen or mouse. A formative evaluation of general public users informed the interface design. Chapters in the modules start with a general overview and proceed to detailed specifics. Each chapter utilizes at least one set of live or near real-time research data (often more than one). This exposes the general public to active ongoing research that is engaging, relevant to the individual user, and explained in easy to understand terms. All live content is updated each time a user accesses the individual page displaying the live data. Leading questions are presented allowing the user to examine the content before accessing the answer via pop-up box. Diagrams and charts of research data have explanatory keys that allow users to self explore all content. Content pages can be created and inserted in the Active Earth Monitor by utilizing the simple HTML/CSS coding.;

  9. Oligotrophic Bacterioplankton with a Novel Single-Cell Life Strategy

    PubMed Central

    Simu, Karin; Hagström, Åke

    2004-01-01

    A large fraction of the marine bacterioplankton community is unable to form colonies on agar surfaces, which so far no experimental evidence can explain. Here we describe a previously undescribed growth behavior of three non-colony-forming oligotrophic bacterioplankton, including a SAR11 cluster representative, the world's most abundant organism. We found that these bacteria exhibit a behavior that promotes growth and dispersal instead of colony formation. Although these bacteria do not form colonies on agar, it was possible to monitor growth on the surface of seawater agar slides containing a fluorescent stain, 4′,6′-diamidino-2-phenylindole (DAPI). Agar slides were prepared by pouring a solution containing 0.7% agar and 0.5 μg of DAPI per ml in seawater onto glass slides. Prompt dispersal of newly divided cells explained the inability to form colonies since immobilized cells (cells immersed in agar) formed microcolonies. The behavior observed suggests a life strategy intended to optimize access of individual cells to substrates. Thus, the inability to form colonies or biofilms appears to be part of a K-selected population strategy in which oligotrophic bacteria explore dissolved organic matter in seawater as single cells. PMID:15066843

  10. Chemical functionalization of polysilicon microparticles for single-cell studies.

    PubMed

    Fernández-Rosas, E; Baldi, A; Ibañez, E; Barrios, L; Novo, S; Esteve, J; Plaza, J A; Duch, M; Gómez, R; Castell, O; Nogués, C; Fernández-Sánchez, C

    2011-07-05

    In this work, two types of polycrystalline silicon (polysilicon) microparticles were modified with specific ligands in order to be selectively attached to chemical residues located at the plasma membrane and thus to be applied to study individual cells in culture. Two different functionalization approaches based on adsorption and covalent attachment were assayed. A comparative study of the efficiency of the ligand immobilization and stability of the modified particle in the culture medium was carried out using the selected ligands labeled with a fluorophore. Cylindrical microparticles (nonencoded microparticles) and shape-encoded microparticles (bar codes) were used with the aim of demonstrating the nondependence of the particle size and shape on the efficiency of the immobilization protocol. Fluorescence imaging and statistical analysis of the recorded fluorescence intensity showed that the covalent attachment of the ligand to the surface of the microparticle, previously modified with an aldehyde-terminated silane, gave the best results. As a proof of concept, Vero cells in culture were labeled with the covalently modified bar codes and successfully tracked for up to 1 week without observing any alteration in the viability of the cells. Bar code numbers could be easily read by eye using a bright-field optical microscope. It is anticipated that such modified microparticles could be feasible platforms for the introduction of other analytical functions of interest in single-cell monitoring and cell sorting in automatic analysis systems. © 2011 American Chemical Society

  11. Postictal single-cell firing patterns in the hippocampus.

    PubMed

    Zhou, Jun-Li; Lenck-Santini, Pierre-Pascal; Holmes, Gregory L

    2007-04-01

    Patients with epilepsy have varying degrees of postictal impairment including confusion and amnesia. This impairment adds substantially to the disease burden of epilepsy. However, the mechanism responsible for postictal cognitive impairment is unclear. The purpose of this study was to study single-cell firing patterns in hippocampal cells after spontaneous seizures in rats previously subjected to status epilepticus. In this study, we monitored place cells and interneurons in the CA1 region of the hippocampus before and after spontaneous seizures in six epileptic rats with a history of status epilepticus. Place cells fire action potentials when the animal is in a specific location in space, the so-called place field. Place cell function correlates well with performance in tasks of visual-spatial memory and appears to be an excellent surrogate measure of spatial memory. Twelve spontaneous seizures were recorded. After the seizures, a marked decrease in firing rate of action potentials from place cells was noted, whereas interneuron firing was unchanged. In addition, when place cell firing fields persisted or returned, they had aberrant firing fields with reduced coherence and information content. In addition to postictal suppression of firing patterns, seizures led to the emergence of firing fields in previously silent cells, demonstrating a postictal remapping of the hippocampus. These findings demonstrate that postictal alterations in behavior are not due solely to reduced neuronal firing. Rather, the postictal period is characterized by robust and dynamic changes in cell-firing patterns resulting in remapping of the hippocampal map.

  12. Oscope identifies oscillatory genes in unsynchronized single-cell RNA-seq experiments.

    PubMed

    Leng, Ning; Chu, Li-Fang; Barry, Chris; Li, Yuan; Choi, Jeea; Li, Xiaomao; Jiang, Peng; Stewart, Ron M; Thomson, James A; Kendziorski, Christina

    2015-10-01

    Oscillatory gene expression is fundamental to development, but technologies for monitoring expression oscillations are limited. We have developed a statistical approach called Oscope to identify and characterize the transcriptional dynamics of oscillating genes in single-cell RNA-seq data from an unsynchronized cell population. Applying Oscope to a number of data sets, we demonstrated its utility and also identified a potential artifact in the Fluidigm C1 platform.

  13. Probing single cells of purple sulfur bacteria with Raman spectroscopy: carotenoids and elemental sulfur.

    PubMed

    Oren, Aharon; Mana, Lily; Jehlička, Jan

    2015-03-01

    We explored the use of Raman spectroscopy to simultaneously monitor the presence of different biomarkers (carotenoids, elemental sulfur) within single cells of the purple sulfur photosynthetic bacteria Allochromatium vinosum and A. warmingii. Raman microspectrometry using excitation at 532 nm allowed the detection of different carotenoids. Raman signals of elemental sulfur appeared soon after feeding starved cells with sulfide. Raman spectroscopy is thus a convenient and sensitive technique to qualitatively and semiquantitatively assess the presence of different compounds of interest within single bacterial cells.

  14. Influence of Activity Monitor Location and Bout Duration on Free-Living Physical Activity

    ERIC Educational Resources Information Center

    Heil, Daniel P.; Bennett, Gary G.; Bond, Kathleen S.; Webster, Michael D.; Wolin, Kathleen Y.

    2009-01-01

    The purpose of this study was to evaluate the influence of the location (ankle, hip, wrist) where an activity monitor (AM) is worn and of the minimum bout duration (BD) on physical activity (PA) variables during free-living monitoring. Study 1 participants wore AMs at three locations for 1 day while wearing the Intelligent Device for Energy…

  15. Influence of Activity Monitor Location and Bout Duration on Free-Living Physical Activity

    ERIC Educational Resources Information Center

    Heil, Daniel P.; Bennett, Gary G.; Bond, Kathleen S.; Webster, Michael D.; Wolin, Kathleen Y.

    2009-01-01

    The purpose of this study was to evaluate the influence of the location (ankle, hip, wrist) where an activity monitor (AM) is worn and of the minimum bout duration (BD) on physical activity (PA) variables during free-living monitoring. Study 1 participants wore AMs at three locations for 1 day while wearing the Intelligent Device for Energy…

  16. Mapping cellular hierarchy by single cell analysis of the cell surface repertoire

    PubMed Central

    Guo, Guoji; Luc, Sidinh; Marco, Eugenio; Lin, Ta-Wei; Peng, Cong; Kerenyi, Marc A.; Beyaz, Semir; Kim, Woojin; Xu, Jian; Das, Partha Pratim; Neff, Tobias; Zou, Keyong; Yuan, Guo-Cheng; Orkin, Stuart H.

    2013-01-01

    SUMMARY Stem cell differentiation pathways are most often studied at the population level, whereas critical decisions are executed at the level of single cells. We have established a highly multiplexed, quantitative PCR assay to profile in an unbiased manner a panel of all commonly used cell surface markers (280 genes) from individual cells. With this method we analyzed over 1500 single cells throughout the mouse hematopoietic system, and illustrate its utility for revealing important biological insights. The comprehensive single cell dataset permits mapping of the mouse hematopoietic stem cell (HSC) differentiation hierarchy by computational lineage progression analysis. Further profiling of 180 intracellular regulators enabled construction of a genetic network to assign the earliest differentiation event during hematopoietic lineage specification. Analysis of acute myeloid leukemia elicited by MLL-AF9 uncovered a distinct cellular hierarchy containing two independent self-renewing lineages with different clonal activities. The strategy has broad applicability in other cellular systems. PMID:24035353

  17. What single-cell stimulation has told us about neural coding.

    PubMed

    Doron, Guy; Brecht, Michael

    2015-09-19

    In recent years, single-cell stimulation experiments have resulted in substantial progress towards directly linking single-cell activity to movement and sensation. Recent advances in electrical recording and stimulation techniques have enabled control of single neuron spiking in vivo and have contributed to our understanding of neuronal coding schemes in the brain. Here, we review single neuron stimulation effects in different brain structures and how they vary with artificially inserted spike patterns. We briefly compare single neuron stimulation with other brain stimulation techniques. A key advantage of single neuron stimulation is the precise control of the evoked spiking patterns. Systematically varying spike patterns and measuring evoked movements and sensations enables 'decoding' of the single-cell spike patterns and provides insights into the readout mechanisms of sensory and motor cortical spikes.

  18. What single-cell stimulation has told us about neural coding

    PubMed Central

    Doron, Guy; Brecht, Michael

    2015-01-01

    In recent years, single-cell stimulation experiments have resulted in substantial progress towards directly linking single-cell activity to movement and sensation. Recent advances in electrical recording and stimulation techniques have enabled control of single neuron spiking in vivo and have contributed to our understanding of neuronal coding schemes in the brain. Here, we review single neuron stimulation effects in different brain structures and how they vary with artificially inserted spike patterns. We briefly compare single neuron stimulation with other brain stimulation techniques. A key advantage of single neuron stimulation is the precise control of the evoked spiking patterns. Systematically varying spike patterns and measuring evoked movements and sensations enables ‘decoding’ of the single-cell spike patterns and provides insights into the readout mechanisms of sensory and motor cortical spikes. PMID:26240419

  19. In vivo single cell detection of metabolic oscillations in stem cells

    PubMed Central

    Stringari, Chiara; Wang, Hong; Geyfman, Mikhail; Crosignani, Viera; Kumar, Vivek; Takahashi, Joseph S.; Andersen, Bogi; Gratton, Enrico

    2015-01-01

    Summary Using bulk measurements in metabolic organs, the circadian clock was shown to play roles in organismal energy homeostasis. However, the relationship between metabolic and circadian oscillations has not been studied in vivo at a single cell level. Also, it is unknown whether the circadian clock controls metabolism in stem cells. We used a sensitive, non-invasive method to detect metabolic oscillations and circadian phase within epidermal stem cells in live mice at the single cell level. We observe a higher NADH/NAD+ ratio, reflecting an increased glycolysis/oxidative phosphorylation ratio, during the night compared to the day. Furthermore, we demonstrate that single cell metabolic heterogeneity within the basal cell layer correlates with the circadian clock and that diurnal fluctuations in NADH/NAD+ ratio are Bmal1 dependent. Our data show that in proliferating stem cells the circadian clock coordinates activities of oxidative phosphorylation and glycolysis with DNA synthesis, perhaps as a protective mechanism against genotoxicity. PMID:25543138

  20. Fast and high resolution single-cell BRET imaging

    PubMed Central

    Goyet, Elise; Bouquier, Nathalie; Ollendorff, Vincent; Perroy, Julie

    2016-01-01

    Resonance Energy Transfer (RET)-based technologies are used to report protein-protein interactions in living cells. Among them, Bioluminescence-initiated RET (BRET) provides excellent sensitivity but the low light intensity intrinsic to the bioluminescent process hampers its use for the localization of protein complexes at the sub-cellular level. Herein we have characterized the methodological conditions required to reliably perform single-cell BRET imaging using an extremely bright luciferase, Nanoluciferase (Nluc). With this, we achieved an unprecedented performance in the field of protein-protein interaction imaging in terms of temporal and spatial resolution, duration of signal stability, signal sensitivity and dynamic range. As proof-of-principle, an Nluc-containing BRET-based sensor of ERK activity enabled the detection of subtle, transient and localized variations in ERK activity in neuronal dendritic spines, induced by the activation of endogenous synaptic NMDA receptors. This development will improve our comprehension of both the spatio-temporal dynamics of protein-protein interactions and the activation patterns of specific signaling pathways. PMID:27302735

  1. Active air vs. passive air (settle plate) monitoring in routine environmental monitoring programs.

    PubMed

    Andon, Barbara M

    2006-01-01

    This article discusses the utility of active air versus passive air settle plate monitoring in a routine environmental monitoring program with an emphasis on the monitoring of the critical Grade A environments. It is recognized that there has been a long-standing historical use of settle plates in the pharmaceutical industry, and that European regulatory agencies have supported their use. However, current active air sampling technology can be more advantageous and effective in assessing airborne viable contamination in cleanrooms than settle plate monitoring. Given that both methods are designed to assess viable airborne contamination in cleanrooms, there may be no advantage in performing these two parallel methods for the detection of airborne contamination, especially if doing so increases the number of interventions into critical areas, which may in turn increase the risk of contamination without providing any added benefit in terms of data collection and/or process control. Therefore, the best use of settle plate monitoring may be as an optional test method for those applications where other, more efficient sampling methods may not be possible or may have limited applicability.

  2. Poly(3-hydroxybutyrate) anabolism in Cupriavidus necator cultivated at various carbon-to-nitrogen ratios: insights from single-cell Raman spectroscopy

    NASA Astrophysics Data System (ADS)

    Tao, Zhanhua; Zhang, Pengfei; Qin, Zhaojun; Li, Yong-Qing; Wang, Guiwen

    2016-09-01

    Cupriavidus necator accumulates large amounts of poly(3-hydroxybutyrate) (PHB), a biodegradable substitute for petroleum-based plastics, under certain nutrient conditions. Conventional solvent-extraction-based methods for PHB quantification only obtain average information from cell populations and, thus, mask the heterogeneity among individual cells. Laser tweezers Raman spectroscopy (LTRS) was used to monitor dynamic changes in the contents of PHB, nucleic acids, and proteins in C. necator at the population and single-cell levels when the microorganism cells were cultivated at various carbon-to-nitrogen ratios. The biosynthetic activities of nucleic acids and proteins were maintained at high levels, and only a small amount of PHB was produced when the bacterial cells were cultured under balanced growth conditions. By contrast, the syntheses of nucleic acids and proteins were blocked, and PHB was accumulated in massive amount inside the microbial cells under nitrogen-limiting growth circumstances. Single-cell analysis revealed a relatively high heterogeneity in PHB level at the early stage of the bacterial growth. Additionally, bacterial cells in populations at certain cultivation stages were composed of two or three subpopulations on the basis of their PHB abundance. Overall, LTRS is a reliable single-cell analysis tool that can provide insights into PHB fermentation.

  3. A Single-Cell Resolution Imaging Protocol of Mitochondrial DNA Dynamics in Physiopathology, mTRIP, Which Also Evaluates Sublethal Cytotoxicity.

    PubMed

    Chatre, Laurent; Montagne, Benjamin; Ricchetti, Miria

    2016-01-01

    Mitochondria autonomously replicate and transcribe their own genome, which is present in multiple copies in the organelle. Transcription and replication of the mitochondrial DNA (mtDNA), which are defined here as mtDNA processing, are essential for mitochondrial function. The extent, efficiency, and coordination of mtDNA processing are key parameters of the mitochondrial state in living cells. Recently, single-cell analysis of mtDNA processing revealed a large and dynamic heterogeneity of mitochondrial populations in single cells, which is linked to mitochondrial function and is altered during disease. This was achieved using mitochondrial Transcription and Replication Imaging Protocol (mTRIP), a modified fluorescence in situ hybridization (FISH) approach that simultaneously reveals the mitochondrial RNA content and mtDNA engaged in initiation of replication at the single-cell level. mTRIP can also be coupled to immunofluorescence or MitoTracker, resulting in the additional labeling of proteins or active mitochondria, respectively. Therefore, mTRIP detects quantitative and qualitative alterations of the dynamics of mtDNA processing in human cells that respond to physiological changes or result from diseases. In addition, we show here that mTRIP is a rather sensitive tool for detecting mitochondrial alterations that may lead to loss of cell viability, and is thereby a useful tool for monitoring sublethal cytotoxicity for instance during chronic drug treatment.

  4. CellStress - open source image analysis program for single-cell analysis

    NASA Astrophysics Data System (ADS)

    Smedh, Maria; Beck, Caroline; Sott, Kristin; Goksör, Mattias

    2010-08-01

    This work describes our image-analysis software, CellStress, which has been developed in Matlab and is issued under a GPL license. CellStress was developed in order to analyze migration of fluorescent proteins inside single cells during changing environmental conditions. CellStress can also be used to score information regarding protein aggregation in single cells over time, which is especially useful when monitoring cell signaling pathways involved in e.g. Alzheimer's or Huntington's disease. Parallel single-cell analysis of large numbers of cells is an important part of the research conducted in systems biology and quantitative biology in order to mathematically describe cellular processes. To quantify properties for single cells, large amounts of data acquired during extended time periods are needed. Manual analyses of such data involve huge efforts and could also include a bias, which complicates the use and comparison of data for further simulations or modeling. Therefore, it is necessary to have an automated and unbiased image analysis procedure, which is the aim of CellStress. CellStress utilizes cell contours detected by CellStat (developed at Fraunhofer-Chalmers Centre), which identifies cell boundaries using bright field images, and thus reduces the fluorescent labeling needed.

  5. Massively parallel single-cell RNA-seq for marker-free decomposition of tissues into cell types.

    PubMed

    Jaitin, Diego Adhemar; Kenigsberg, Ephraim; Keren-Shaul, Hadas; Elefant, Naama; Paul, Franziska; Zaretsky, Irina; Mildner, Alexander; Cohen, Nadav; Jung, Steffen; Tanay, Amos; Amit, Ido

    2014-02-14

    In multicellular organisms, biological function emerges when heterogeneous cell types form complex organs. Nevertheless, dissection of tissues into mixtures of cellular subpopulations is currently challenging. We introduce an automated massively parallel single-cell RNA sequencing (RNA-seq) approach for analyzing in vivo transcriptional states in thousands of single cells. Combined with unsupervised classification algorithms, this facilitates ab initio cell-type characterization of splenic tissues. Modeling single-cell transcriptional states in dendritic cells and additional hematopoietic cell types uncovers rich cell-type heterogeneity and gene-modules activity in steady state and after pathogen activation. Cellular diversity is thereby approached through inference of variable and dynamic pathway activity rather than a fixed preprogrammed cell-type hierarchy. These data demonstrate single-cell RNA-seq as an effective tool for comprehensive cellular decomposition of complex tissues.

  6. 21 CFR 884.2730 - Home uterine activity monitor.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Home uterine activity monitor. 884.2730 Section 884.2730 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES... previous preterm delivery to aid in the detection of preterm labor. (b) Classification. Class II (special...

  7. Embedded Triboelectric Active Sensors for Real-Time Pneumatic Monitoring.

    PubMed

    Fu, Xian Peng; Bu, Tian Zhao; Xi, Feng Ben; Cheng, Ting Hai; Zhang, Chi; Wang, Zhong Lin

    2017-09-20

    Pneumatic monitoring sensors have great demands for power supply in cylinder systems. Here, we present an embedded sliding triboelectric nanogenerator (TENG) in air cylinder as active sensors for position and velocity monitoring. The embedded TENG is composed of a circular poly(tetrafluoroethylene) polymer and a triangular copper electrode. The working mechanism as triboelectric active sensors and electric output performance are systematically investigated. By integrating into the pneumatic system, the embedded triboelectric active sensors have been used for real-time air pressure/flow monitoring and energy storage. Air pressures are measured from 0.04 to 0.12 MPa at a step of 0.02 MPa with a sensitivity of 49.235 V/MPa, as well as airflow from 50 to 250 L/min at a step of 50 L/min with a sensitivity of 0.002 μA·min/L. This work has first demonstrated triboelectric active sensors for pneumatic monitoring and may promote the development of TENG in intelligent pneumatic system.

  8. 21 CFR 884.2730 - Home uterine activity monitor.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Home uterine activity monitor. 884.2730 Section 884.2730 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES... the clinic. The HUAM system comprises a tocotransducer, an at-home recorder, a modem, and a...

  9. 21 CFR 884.2730 - Home uterine activity monitor.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Home uterine activity monitor. 884.2730 Section 884.2730 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES... the clinic. The HUAM system comprises a tocotransducer, an at-home recorder, a modem, and a...

  10. Permanent Infrasound Monitoring of Active Volcanoes in Ecuador

    NASA Astrophysics Data System (ADS)

    Ruiz, M. C.; Yepes, H. A.; Steele, A.; Segovia, M.; Vaca, S.; Cordova, A.; Enriquez, W.; Vaca, M.; Ramos, C.; Arrais, S.; Tapa, I.; Mejia, F.; Macias, C.

    2013-12-01

    Since 2006, infrasound monitoring has become a permanent tool for observing, analyzing and understanding volcanic activity in Ecuador. Within the framework of a cooperative project between the Japanese International Cooperation Agency (JICA) and the Instituto Geofísico to enhance volcano monitoring capabilities within the country, 10 infrasound sensors were deployed in conjunction with broadband seismic stations at Cotopaxi and Tungurahua volcanoes. Each station comprises 1 ACO microphone (model 7144) and an amplifier with a flat response down to 0.1 Hz. At Tungurahua, between July 2006 and July 2013, the network recorded more than 5,500 explosion events with peak-to-peak pressure amplitudes larger than 45 Pa at station Mason (BMAS) which is located ~ 5.5 km from the active crater. This includes 3 explosions with pressure amplitudes larger than 1,000 Pa and which all have exhibited clear shock wave components. Two seismic and infrasound arrays were also installed in 2006 under the Acoustic Surveillance for Hazardous Eruptions (ASHE) project, used in volcano monitoring at Tungurahua, Sangay, and Reventador. This venture was led by the Geological Survey of Canada and the University of Hawaii. Through the SENESCYT-IGEPN project, the Instituto Geofísico is currently installing a regional network of MB2005 microbarometers with the aim to enhance monitoring of active and potentially active volcanoes that include Reventador, Guagua Pichincha, Chimborazo, Antisana, Sangay, and Volcán Chico in the Galapagos Islands. Through the infrasound monitoring station at Volcán Chico it is also possible to extend observations to any activity initiated from Sierra Negra, Fernandina, Cerro Azul, and Alcedo volcanoes. During the past decade, a series of temporary acoustic arrays have also been deployed around Ecuador's most active volcanoes, helping to aid in short term volcanic monitoring and/or used in a series of research projects aimed at better understanding volcanic systems

  11. Fabric-based integrated energy devices for wearable activity monitors.

    PubMed

    Jung, Sungmook; Lee, Jongsu; Hyeon, Taeghwan; Lee, Minbaek; Kim, Dae-Hyeong

    2014-09-01

    A wearable fabric-based integrated power-supply system that generates energy triboelectrically using human activity and stores the generated energy in an integrated supercapacitor is developed. This system can be utilized as either a self-powered activity monitor or as a power supply for external wearable sensors. These demonstrations give new insights for the research of wearable electronics. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Massively multiplex single-cell Hi-C.

    PubMed

    Ramani, Vijay; Deng, Xinxian; Qiu, Ruolan; Gunderson, Kevin L; Steemers, Frank J; Disteche, Christine M; Noble, William S; Duan, Zhijun; Shendure, Jay

    2017-03-01

    We present single-cell combinatorial indexed Hi-C (sciHi-C), a method that applies combinatorial cellular indexing to chromosome conformation capture. In this proof of concept, we generate and sequence six sciHi-C libraries comprising a total of 10,696 single cells. We use sciHi-C data to separate cells by karyotypic and cell-cycle state differences and identify cell-to-cell heterogeneity in mammalian chromosomal conformation. Our results demonstrate that combinatorial indexing is a generalizable strategy for single-cell genomics.

  13. Delineating biological and technical variance in single cell expression data.

    PubMed

    Arzalluz-Luque, Ángeles; Devailly, Guillaume; Mantsoki, Anna; Joshi, Anagha

    2017-09-01

    Single cell transcriptomics is becoming a common technique to unravel new biological phenomena whose functional significance can only be understood in the light of differences in gene expression between single cells. The technology is still in its early days and therefore suffers from many technical challenges. This review discusses the continuous effort to identify and systematically characterise various sources of technical variability in single cell expression data and the need to further develop experimental and computational tools and resources to help deal with it. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

  14. Single-cell Transcriptome Study as Big Data

    PubMed Central

    Yu, Pingjian; Lin, Wei

    2016-01-01

    The rapid growth of single-cell RNA-seq studies (scRNA-seq) demands efficient data storage, processing, and analysis. Big-data technology provides a framework that facilitates the comprehensive discovery of biological signals from inter-institutional scRNA-seq datasets. The strategies to solve the stochastic and heterogeneous single-cell transcriptome signal are discussed in this article. After extensively reviewing the available big-data applications of next-generation sequencing (NGS)-based studies, we propose a workflow that accounts for the unique characteristics of scRNA-seq data and primary objectives of single-cell studies. PMID:26876720

  15. Single cell studies of the cell cycle and some models

    PubMed Central

    Mitchison, JM

    2005-01-01

    Analysis of growth and division often involves measurements made on cell populations, which tend to average data. The value of single cell analysis needs to be appreciated, and models based on findings from single cells should be taken into greater consideration in our understanding of the way in which cell size and division are co-ordinated. Examples are given of some single cell analyses in mammalian cells, yeast and other microorganisms. There is also a short discussion on how far the results are in accord with simple models. PMID:15703075

  16. Normalizing single-cell RNA sequencing data: Challenges and opportunities

    PubMed Central

    Dudoit, Sandrine; Marioni, John C.

    2017-01-01

    Single-cell transcriptomics is becoming an important component of the molecular biologist’s toolkit. A critical step when analyzing this type of data is normalization. However, normalization is typically performed using methods developed for bulk RNA sequencing or even microarray data, whose suitability for single-cell transcriptomics has not been assessed. In this perspective, we discuss commonly used normalization approaches and illustrate how these can lead to misleading results. Finally, we present alternative approaches and provide recommendations for single-cell RNA sequencing users. PMID:28504683

  17. Single Nanowire Probe for Single Cell Endoscopy and Sensing

    NASA Astrophysics Data System (ADS)

    Yan, Ruoxue

    The ability to manipulate light in subwavelength photonic and plasmonic structures has shown great potentials in revolutionizing how information is generated, transformed and processed. Chemically synthesized nanowires, in particular, offers a unique toolbox not only for highly compact and integrated photonic modules and devices, including coherent and incoherent light sources, waveguides, photodetectors and photovoltaics, but also for new types of nanoscopic bio-probes for spot cargo delivery and in-situ single cell endoscopy and sensing. Such nanowire probes would enable us to carry out intracellular imaging and probing with high spatial resolution, monitor in-vivo biological processes within single living cells and greatly improve our fundamental understanding of cell functions, intracellular physiological processes, and cellular signal pathways. My work is aimed at developing a material and instrumental platform for such single nanowire probe. Successful optical integration of Ag nanowire plasmonic waveguides, which offers deep subwavelength mode confinement, and conventional photonic waveguides was demonstrated on a single nanowire level. The highest plasmonic-photonic coupling efficiency coupling was found at small coupling angles and low input frequencies. The frequency dependent propagation loss was observed in Ag nanowire and was confirmed by quantitative measurement and in agreement with theoretical expectations. Rational integration of dielectric and Ag nanowire waveguide components into hybrid optical-plasmonic routing devices has been demonstrated. This capability is essential for incorporating sub-100nm Ag nanowire waveguides into optical fiber based nanoprobes for single cell endoscopy. The nanoprobe system based on single nanowire waveguides was demonstrated by optically coupling semiconductor or metal nanowire with an optical fiber with tapered tip. This nanoprobe design requires minimal instrumentation which makes it cost efficient and readily

  18. Optical control and study of biological processes at the single-cell level in a live organism

    NASA Astrophysics Data System (ADS)

    Feng, Zhiping; Zhang, Weiting; Xu, Jianmin; Gauron, Carole; Ducos, Bertrand; Vriz, Sophie; Volovitch, Michel; Jullien, Ludovic; Weiss, Shimon; Bensimon, David

    2013-07-01

    Living organisms are made of cells that are capable of responding to external signals by modifying their internal state and subsequently their external environment. Revealing and understanding the spatio-temporal dynamics of these complex interaction networks is the subject of a field known as systems biology. To investigate these interactions (a necessary step before understanding or modelling them) one needs to develop means to control or interfere spatially and temporally with these processes and to monitor their response on a fast timescale (< minute) and with single-cell resolution. In 2012, an EMBO workshop on ‘single-cell physiology’ (organized by some of us) was held in Paris to discuss those issues in the light of recent developments that allow for precise spatio-temporal perturbations and observations. This review will be largely based on the investigations reported there. We will first present a non-exhaustive list of examples of cellular interactions and developmental pathways that could benefit from these new approaches. We will review some of the novel tools that have been developed for the observation of cellular activity and then discuss the recent breakthroughs in optical super-resolution microscopy that allow for optical observations beyond the diffraction limit. We will review the various means to photo-control the activity of biomolecules, which allow for local perturbations of physiological processes. We will end up this review with a report on the current status of optogenetics: the use of photo-sensitive DNA-encoded proteins as sensitive reporters and efficient actuators to perturb and monitor physiological processes.

  19. Monitoring cell concentration and activity by multiple excitation fluorometry.

    PubMed

    Li, J K; Asali, E C; Humphrey, A E; Horvath, J J

    1991-01-01

    Four key cellular metabolic fluorophores--tryptophan, pyridoxine, NAD(P)H, and riboflavin--were monitored on-line by a multiple excitation fluorometric system (MEFS) and a modified SLM 8000C scanning spectrofluorometer in three model yeast fermentation systems--bakers' yeast growing on glucose, Candida utilis growing on ethanol, and Saccharomyces cerevisiae RTY110/pRB58 growing on glucose. The measured fluorescence signals were compared with cell concentration, protein concentration, and cellular activity. The results indicate that the behavior and fluorescence intensity of various fluorophores differ in the various fermentation systems. Tryptophan fluorescence is the best signal for the monitoring of cell concentration in bakers' yeast and C. utilis fermentations. Pyridoxine fluoresce is the best signal for the monitoring of cell concentration in the S. cerevisiae RTY110/pRB58 fermentation. In bakers' yeast fermentations the pyridoxine fluorescence signal can be used to monitor cellular activity. The NAD(P)H fluorescence signal is a good indicator of cellular activity in the C. utilis fermentation. For this fermentation NAD(P)H fluorescence can be used to control ethanol feeding in a fed-batch process.

  20. Parallel measurement of dynamic changes in translation rates in single cells

    PubMed Central

    Han, Kyuho; Jaimovich, Ariel; Dey, Gautam; Ruggero, Davide; Meyuhas, Oded; Sonenberg, Nahum; Meyer, Tobias

    2014-01-01

    Protein concentrations are often regulated by dynamic changes in translation rates. Nevertheless, it has been challenging to directly monitor changes in translation in living cells. We have developed a reporter system to measure real-time changes of translation rates in human or mouse individual cells by conjugating translation regulatory motifs to sequences encoding a nuclear targeted fluorescent protein and a controllable destabilization domain. Application of the method showed that individual cells undergo marked fluctuations in the translation rate of mRNAs whose 5′ terminal oligopyrimidine (5′ TOP) motif regulates the synthesis of ribosomal proteins. Furthermore, we show that small reductions in amino acid levels signal through different mTOR-dependent pathways to control TOP mRNA translation, whereas larger reductions in amino acid levels control translation through eIF2A. Our study demonstrates that dynamic measurements of single-cell activities of translation regulatory motifs can be used to identify and investigate fundamental principles of translation. PMID:24213167

  1. Bet-hedging in bacteriocin producing Escherichia coli populations: the single cell perspective

    PubMed Central

    Bayramoglu, Bihter; Toubiana, David; van Vliet, Simon; Inglis, R. Fredrik; Shnerb, Nadav; Gillor, Osnat

    2017-01-01

    Production of public goods in biological systems is often a collaborative effort that may be detrimental to the producers. It is therefore sustainable only if a small fraction of the population shoulders the cost while the majority reap the benefits. We modelled this scenario using Escherichia coli populations producing colicins, an antibiotic that kills producer cells’ close relatives. Colicin expression is a costly trait, and it has been proposed that only a small fraction of the population actively expresses the antibiotic. Colicinogenic populations were followed at the single-cell level using time-lapse microscopy, and showed two distinct, albeit dynamic, subpopulations: the majority silenced colicin expression, while a small fraction of elongated, slow-growing cells formed colicin-expressing hotspots, placing a significant burden on expressers. Moreover, monitoring lineages of individual colicinogenic cells showed stochastic switching between expressers and non-expressers. Hence, colicin expressers may be engaged in risk-reducing strategies—or bet-hedging—as they balance the cost of colicin production with the need to repel competitors. To test the bet-hedging strategy in colicin-mediated interactions, competitions between colicin-sensitive and producer cells were simulated using a numerical model, demonstrating a finely balanced expression range that is essential to sustaining the colicinogenic population. PMID:28165017

  2. Bet-hedging in bacteriocin producing Escherichia coli populations: the single cell perspective

    NASA Astrophysics Data System (ADS)

    Bayramoglu, Bihter; Toubiana, David; van Vliet, Simon; Inglis, R. Fredrik; Shnerb, Nadav; Gillor, Osnat

    2017-02-01

    Production of public goods in biological systems is often a collaborative effort that may be detrimental to the producers. It is therefore sustainable only if a small fraction of the population shoulders the cost while the majority reap the benefits. We modelled this scenario using Escherichia coli populations producing colicins, an antibiotic that kills producer cells’ close relatives. Colicin expression is a costly trait, and it has been proposed that only a small fraction of the population actively expresses the antibiotic. Colicinogenic populations were followed at the single-cell level using time-lapse microscopy, and showed two distinct, albeit dynamic, subpopulations: the majority silenced colicin expression, while a small fraction of elongated, slow-growing cells formed colicin-expressing hotspots, placing a significant burden on expressers. Moreover, monitoring lineages of individual colicinogenic cells showed stochastic switching between expressers and non-expressers. Hence, colicin expressers may be engaged in risk-reducing strategies—or bet-hedging—as they balance the cost of colicin production with the need to repel competitors. To test the bet-hedging strategy in colicin-mediated interactions, competitions between colicin-sensitive and producer cells were simulated using a numerical model, demonstrating a finely balanced expression range that is essential to sustaining the colicinogenic population.

  3. Revealing Assembly of a Pore-Forming Complex Using Single-Cell Kinetic Analysis and Modeling.

    PubMed

    Bischofberger, Mirko; Iacovache, Ioan; Boss, Daniel; Naef, Felix; van der Goot, F Gisou; Molina, Nacho

    2016-04-12

    Many biological processes depend on the sequential assembly of protein complexes. However, studying the kinetics of such processes by direct methods is often not feasible. As an important class of such protein complexes, pore-forming toxins start their journey as soluble monomeric proteins, and oligomerize into transmembrane complexes to eventually form pores in the target cell membrane. Here, we monitored pore formation kinetics for the well-characterized bacterial pore-forming toxin aerolysin in single cells in real time to determine the lag times leading to the formation of the first functional pores per cell. Probabilistic modeling of these lag times revealed that one slow and seven equally fast rate-limiting reactions best explain the overall pore formation kinetics. The model predicted that monomer activation is the rate-limiting step for the entire pore formation process. We hypothesized that this could be through release of a propeptide and indeed found that peptide removal abolished these steps. This study illustrates how stochasticity in the kinetics of a complex process can be exploited to identify rate-limiting mechanisms underlying multistep biomolecular assembly pathways. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  4. Limited activity monitoring in toddlers with autism spectrum disorder.

    PubMed

    Shic, Frederick; Bradshaw, Jessica; Klin, Ami; Scassellati, Brian; Chawarska, Katarzyna

    2011-03-22

    This study used eye-tracking to examine how 20-month-old toddlers with autism spectrum disorder (ASD) (n=28), typical development (TD) (n=34), and non-autistic developmental delays (DD) (n=16) monitored the activities occurring in a context of an adult-child play interaction. Toddlers with ASD, in comparison to control groups, showed less attention to the activities of others and focused more on background objects (e.g., toys). In addition, while all groups spent the same time overall looking at people, toddlers with ASD looked less at people's heads and more at their bodies. In ASD, these patterns were associated with cognitive deficits and greater autism severity. These results suggest that the monitoring of the social activities of others is disrupted early in the developmental progression of autism, limiting future avenues for observational learning.

  5. Monitoring and evaluating school nutrition and physical activity policies.

    PubMed

    Taylor, Jennifer P; McKenna, Mary L; Butler, Gregory P

    2010-01-01

    Given the increase in the number of Canadian jurisdictions with school nutrition and/or physical activity policies, there is a need to assess the effectiveness of such policies. The objectives of this paper are to 1) provide an overview of key issues in monitoring and evaluating school nutrition and physical activity policies in Canada and 2) identify areas for further research needed to strengthen the evidence base and inform the development of effective approaches to monitoring and evaluation. Evaluation indicators, data sources and existing tools for evaluating nutrition and physical activity are reviewed. This paper has underscored the importance of identifying common indicators and approaches, using a comprehensive approach based on the WHO framework and ensuring that research capacity and funding is in place to facilitate high-quality evaluation efforts in the future.

  6. Atomic force microscopy for the examination of single cell rheology.

    PubMed

    Okajima, Takaharu

    2012-11-01

    Rheological properties of living cells play important roles in regulating their various biological functions. Therefore, measuring cell rheology is crucial for not only elucidating the relationship between the cell mechanics and functions, but also mechanical diagnosis of single cells. Atomic force microscopy (AFM) is becoming a useful technique for single cell diagnosis because it allows us to measure the rheological properties of adherent cells at any region on the surface without any modifications. In this review, we summarize AFM techniques for examining single cell rheology in frequency and time domains. Recent applications of AFM for investigating the statistical analysis of single cell rheology in comparison to other micro-rheological techniques are reviewed, and we discuss what specificity and universality of cell rheology are extracted using AFM.

  7. Virtual Microfluidics for digital quantification and single-cell sequencing

    PubMed Central

    Xu, Liyi; Brito, Ilana L.; Alm, Eric J.; Blainey, Paul C.

    2016-01-01

    Interest in highly parallelized analysis of single molecules and single cells is growing rapidly. Here we develop hydrogel-based virtual microfluidics as a simple alternative to complex engineered microfluidic systems for the compartmentalization of nucleic acid amplification reactions. We applied digital multiple displacement amplification (dMDA) to purified DNA templates, cultured bacterial cells, and human microbiome samples in the virtual microfluidics system and demonstrated recovery and whole-genome sequencing of single-cell MDA products. Our results from control samples showed excellent coverage uniformity and markedly reduced chimerism compared with single-cell data obtained from conventional liquid MDA reactions. We also demonstrate the applicability of the hydrogel method for genomic studies of naturally occurring microbes in human microbiome samples. The virtual microfluidics approach is a simple and robust method that will enable many laboratories to perform single-cell genomic analyses. PMID:27479330

  8. Droplet microfluidics--a tool for single-cell analysis.

    PubMed

    Joensson, Haakan N; Andersson Svahn, Helene

    2012-12-03

    Droplet microfluidics allows the isolation of single cells and reagents in monodisperse picoliter liquid capsules and manipulations at a throughput of thousands of droplets per second. These qualities allow many of the challenges in single-cell analysis to be overcome. Monodispersity enables quantitative control of solute concentrations, while encapsulation in droplets provides an isolated compartment for the single cell and its immediate environment. The high throughput allows the processing and analysis of the tens of thousands to millions of cells that must be analyzed to accurately describe a heterogeneous cell population so as to find rare cell types or access sufficient biological space to find hits in a directed evolution experiment. The low volumes of the droplets make very large screens economically viable. This Review gives an overview of the current state of single-cell analysis involving droplet microfluidics and offers examples where droplet microfluidics can further biological understanding.

  9. The value to the anaesthetist of monitoring cerebral activity.

    PubMed

    Langford, R M; Thomsen, C E

    1994-03-01

    The administration rate of general anaesthetic drugs is at present guided by clinical experience, and indirect indicators such as haemodynamic parameters. In the presence of muscle relaxants most of the clinical signs of inadequate anaesthesia are lost and accidental awareness may occur. A number of monitoring modalities, primarily based on analysis of the electroencephalogram (EEG), have been proposed for measurement of the anaesthetic depth. Moreover intraoperative cerebral monitoring may also provide the anaesthetist with early warning of cerebral ischaemia, or information on specific neurological pathways. To facilitate this, it is essential to combine analysis of the spontaneous EEG with recording of evoked potentials, to assess both cortical and subcortical activity/events. None of the reviewed methods, however promising, can alone meet all of the requirements for intraoperative monitoring of cerebral function. We suggest that the future direction should be to integrate several modalities in a single device, to provide valuable new information, upon which to base clinical management decisions.

  10. An overview of existing raptor contaminant monitoring activities in Europe.

    PubMed

    Gómez-Ramírez, P; Shore, R F; van den Brink, N W; van Hattum, B; Bustnes, J O; Duke, G; Fritsch, C; García-Fernández, A J; Helander, B O; Jaspers, V; Krone, O; Martínez-López, E; Mateo, R; Movalli, P; Sonne, C

    2014-06-01

    Biomonitoring using raptors as sentinels can provide early warning of the potential impacts of contaminants on humans and the environment and also a means of tracking the success of associated mitigation measures. Examples include detection of heavy metal-induced immune system impairment, PCB-induced altered reproductive impacts, and toxicity associated with lead in shot game. Authorisation of such releases and implementation of mitigation is now increasingly delivered through EU-wide directives but there is little established pan-European monitoring to quantify outcomes. We investigated the potential for EU-wide coordinated contaminant monitoring using raptors as sentinels. We did this using a questionnaire to ascertain the current scale of national activity across 44 European countries. According to this survey, there have been 52 different contaminant monitoring schemes with raptors over the last 50years. There were active schemes in 15 (predominantly western European) countries and 23 schemes have been running for >20years; most monitoring was conducted for >5years. Legacy persistent organic compounds (specifically organochlorine insecticides and PCBs), and metals/metalloids were monitored in most of the 15 countries. Fungicides, flame retardants and anticoagulant rodenticides were also relatively frequently monitored (each in at least 6 countries). Common buzzard (Buteo buteo), common kestrel (Falco tinnunculus), golden eagle (Aquila chrysaetos), white-tailed sea eagle (Haliaeetus albicilla), peregrine falcon (Falco peregrinus), tawny owl (Strix aluco) and barn owl (Tyto alba) were most commonly monitored (each in 6-10 countries). Feathers and eggs were most widely analysed although many schemes also analysed body tissues. Our study reveals an existing capability across multiple European countries for contaminant monitoring using raptors. However, coordination between existing schemes and expansion of monitoring into Eastern Europe is needed. This would enable

  11. On-line Monitoring and Active Control for Transformer Noise

    NASA Astrophysics Data System (ADS)

    Liang, Jiabi; Zhao, Tong; Tian, Chun; Wang, Xia; He, Zhenhua; Duan, Lunfeng

    This paper introduces the system for on-line monitoring and active noise control towards the transformer noise based on LabVIEW and the hardware equipment including the hardware and software. For the hardware part, it is mainly focused on the composition and the role of hardware devices, as well as the mounting location in the active noise control experiment. And the software part introduces the software flow chats, the measurement and analysis module for the sound pressure level including A, B, C weighting methods, the 1/n octave spectrum and the power spectrum, active noise control module and noise data access module.

  12. New Array Approaches to Explore Single Cells Genomes

    PubMed Central

    Vanneste, Evelyne; Bittman, Lilach; Van der Aa, Niels; Voet, Thierry; Vermeesch, Joris Robert

    2011-01-01

    Microarray analysis enables the genome-wide detection of copy number variations and the investigation of chromosomal instability. Whereas array techniques have been well established for the analysis of unamplified DNA derived from many cells, it has been more challenging to enable the accurate analysis of single cell genomes. In this review, we provide an overview of single cell DNA amplification techniques, the different array approaches, and discuss their potential applications to study human embryos. PMID:22509179

  13. Remote monitoring of biodynamic activity using electric potential sensors

    NASA Astrophysics Data System (ADS)

    Harl, C. J.; Prance, R. J.; Prance, H.

    2008-12-01

    Previous work in applying the electric potential sensor to the monitoring of body electrophysiological signals has shown that it is now possible to monitor these signals without needing to make any electrical contact with the body. Conventional electrophysiology makes use of electrodes which are placed in direct electrical contact with the skin. The electric potential sensor requires no cutaneous electrical contact, it operates by sensing the displacement current using a capacitive coupling. When high resolution body electrophysiology is required a strong (capacitive) coupling is used to maximise the collected signal. However, in remote applications where there is typically an air-gap between the body and the sensor only a weak coupling can be achieved. In this paper we demonstrate that the electric potential sensor can be successfully used for the remote sensing and monitoring of bioelectric activity. We show examples of heart-rate measurements taken from a seated subject using sensors mounted in the chair. We also show that it is possible to monitor body movements on the opposite side of a wall to the sensor. These sensing techniques have biomedical applications for non-contact monitoring of electrophysiological conditions and can be applied to passive through-the-wall surveillance systems for security applications.

  14. Single Cell Analysis: From Technology to Biology and Medicine.

    PubMed

    Pan, Xinghua

    2014-01-01

    Single-cell analysis heralds a new era that allows "omics" analysis, notably genomics, transcriptomics, epigenomics and proteomics at the single-cell level. It enables the identification of the minor subpopulations that may play a critical role in a biological process of a population of cells, which conventionally are regarded as homogeneous. It provides an ultra-sensitive tool to clarify specific molecular mechanisms and pathways and reveal the nature of cell heterogeneity. It also facilitates the clinical investigation of patients when a very low quantity or a single cell is available for analysis, such as noninvasive prenatal diagnosis and cancer screening, and genetic evaluation for in vitro fertilization. Within a few short years, single-cell analysis, especially whole genomic sequencing and transcriptomic sequencing, is becoming robust and broadly accessible, although not yet a routine practice. Here, with single cell RNA-seq emphasized, an overview of the discipline, progresses, and prospects of single-cell analysis and its applications in biology and medicine are given with a series of logic and theoretical considerations.

  15. Single cell analysis of normal and leukemic hematopoiesis.

    PubMed

    Povinelli, Benjamin J; Rodriguez-Meira, Alba; Mead, Adam J

    2017-09-07

    The hematopoietic system is well established as a paradigm for the study of cellular hierarchies, their disruption in disease and therapeutic use in regenerative medicine. Traditional approaches to study hematopoiesis involve purification of cell populations based on a small number of surface markers. However, such population-based analysis obscures underlying heterogeneity contained within any phenotypically defined cell population. This heterogeneity can only be resolved through single cell analysis. Recent advances in single cell techniques allow analysis of the genome, transcriptome, epigenome and proteome in single cells at an unprecedented scale. The application of these new single cell methods to investigate the hematopoietic system has led to paradigm shifts in our understanding of cellular heterogeneity in hematopoiesis and how this is disrupted in disease. In this review, we summarize how single cell techniques have been applied to the analysis of hematopoietic stem/progenitor cells in normal and malignant hematopoiesis, with a particular focus on recent advances in single-cell genomics, including how these might be utilized for clinical application. Copyright © 2017. Published by Elsevier Ltd.

  16. Active Sites Environmental Monitoring Program: Program plan. Revision 1

    SciTech Connect

    Ashwood, T.L.; Wickliff, D.S.; Morrissey, C.M.

    1992-02-01

    The Active Sites Environmental Monitoring Program (ASEMP), initiated in 1989, provides early detection and performance monitoring of transuranic (TRU) waste and active low-level waste (LLW) facilities at Oak Ridge National Laboratory (ORNL) in accordance with US Department of Energy (DOE) Order 5820.2A. Active LLW facilities in Solid Waste Storage Area (SWSA) 6 include Tumulus I and Tumulus II, the Interim Waste Management Facility (IWMF), LLW silos, high-range wells, asbestos silos, and fissile wells. The tumulus pads and IWMF are aboveground, high-strength concrete pads on which concrete vaults containing metal boxes of LLW are placed; the void space between the boxes and vaults is filled with grout. Eventually, these pads and vaults will be covered by an engineered multilayered cap. All other LLW facilities in SWSA 6 are below ground. In addition, this plan includes monitoring of the Hillcut Disposal Test Facility (HDTF) in SWSA 6, even though this facility was completed prior to the data of the DOE order. In SWSA 5 North, the TRU facilities include below-grade engineered caves, high-range wells, and unlined trenches. All samples from SWSA 6 are screened for alpha and beta activity, counted for gamma-emitting isotopes, and analyzed for tritium. In addition to these analytes, samples from SWSA 5 North are analyzed for specific transuranic elements.

  17. Sentinel-1 Contribution to Monitoring Maritime Activity in the Arctic

    NASA Astrophysics Data System (ADS)

    Santamaria, Carlos; Greidanus, Harm; Fournier, Melanie; Eriksen, Torkild; Vespe, Michele; Alvarez, Marlene; Arguedas, Virginia Fernandez; Delaney, Conor; Argentieri, Pietro

    2016-08-01

    This paper presents results on the use of Sentinel-1 combined with satellite AIS to monitor maritime activity in the Arctic. Such activities are expected to increase, even if not uniformly across the Arctic, as the ice cover in the region retreats due to changes in climate. The objectives of monitoring efforts in the region can vary from country to country, but are generally related to increasing awareness on non- cooperative, small and cruise ships, fisheries, safety at sea, and Search and Rescue. A ship monitoring study has been conducted, involving more than 2,000 Sentinel-1 images acquired during one year in the central Arctic, where the ship densities are high. The main challenges to SAR-based monitoring in this area are described, solutions for some of them are proposed, and analyses of the results are shown. With the high detection thresholds needed to prevent false alarms from sea ice, 16% of the ships detected overall in the Sentinel-1 images have not been correlated to AIS- transmitting ships, and 48% of the AIS-transmitting ships are not correlated to ships detected in the images.

  18. Efficient propagation of single cells Accutase-dissociated human embryonic stem cells.

    PubMed

    Bajpai, Ruchi; Lesperance, Jacqueline; Kim, Min; Terskikh, Alexey V

    2008-05-01

    Human embryonic stem cells (hESCs) hold great promise for cell-based therapies and drug screening applications. However, growing and processing large quantities of undifferentiated hESCs is a challenging task. Conventionally, hESCs are passaged as clusters, which can limit their growth efficiency and use in downstream applications. This study demonstrates that hESCs can be passaged as single cells using Accutase, a formulated mixture of digestive enzymes. In contrast to trypsin treatment, Accutase treatment does not significantly affect the viability and proliferation rate of hESC dissociation into single cells. Accutase-dissociated single cells can be separated by FACS and proliferate as fully pluripotent hESCs. An Oct4-eGFP reporter construct engineered into hESCs was used to monitor the pluripotency of hESCs passaged with Accutase. Compared to collagenase-passaged hESCs, Accutase-treated cultures contained a larger proportion of undifferentiated (Oct4-positive) cells. Additionally, Accutase-passaged undifferentiated hESCs could be grown as monolayers without the need for monitoring and/or selection for quality hESC colonies. (c) 2007 Wiley-Liss, Inc.

  19. Active sites environmental monitoring program. Annual report FY 1992

    SciTech Connect

    Morrissey, C.M.; Ashwood, T.L.; Hicks, D.S.

    1994-04-01

    This report summarizes the activities of the Active Sites Environmental Monitoring Program (ASEMP) at ORNL from October 1991 through September 1992. Solid Waste Operations and the Environmental Sciences Division established ASEMP in 1989 to provide early detection and performance monitoring at active low-level waste (LLW) disposal sites in Solid Waste Storage Area (SWSA) 6 and transuranic (TRU) waste storage sites in SWSA 5 as required by Chapter 2 and 3 of US Department of Energy Order 5820.2A. The Interim Waste Management Facility (IWMF) began operation in December 1991. Monitoring results from the tumulus and IWMF disposal pads continue to indicate that no LLW is leaching from the storage vaults. Storm water falling on the IWMF active pad was collected and transported to the Process Waste Treatment Plant while operators awaited approval of the National Pollutant Discharge Elimination System (NPDES) permit. Several of the recent samples collected from the active IWMF pad had pH levels above the NPDES limit of 9.0 because of alkali leached from the concrete. The increase in gross beta activity has been slight; only 1 of the 21 samples collected contained activity above the 5.0 Bq/L action level. Automated sample-collection and flow-measurement equipment has been installed at IWMF and is being tested. The flume designed to electronically measure flow from the IWMF pads and underpads is too large to be of practical value for measuring most flows at this site. Modification of this system will be necessary. A CO{sub 2} bubbler system designed to reduce the pH of water from the pads is being tested at IWMF.

  20. Energy monitoring system based on human activity in the workplace

    NASA Astrophysics Data System (ADS)

    Mustafa, Nur Hanim; Husain, Mohd Nor; Aziz, Mohamad Zoinol Abidin Abdul; Othman, Mohd Azlishah; Malek, Fareq

    2015-05-01

    Human behaviors always related to day routine activities in a smart house directly give the significant factor to manage energy usage in human life. An Addition that, the factor will contribute to the best efficiency of the system. This paper will focus on the monitoring efficiency based on duration time in office hours around 8am until 5pm which depend on human behavior at working place. Besides that, the correlation coefficient method is used to show the relation between energy consumption and energy saving based on the total hours of time energy spent. In future, the percentages of energy monitoring system usage will be increase to manage energy saving based on human behaviors. This scenario will help to see the human activity in the workplace in order to get the energy saving and support world green environment.

  1. Active Cavity Irradiance Monitor Satellite ACRIMSAT Artist Concept

    NASA Image and Video Library

    1999-12-21

    The Active Cavity Irradiance Monitor Satellite, or ACRIMSAT, mission is a climate change investigation that measures changes in how much of the sun's energy reaches Earth's atmosphere. This energy, called solar irradience, creates winds, heats the land and drives ocean currents, and therefore contains significant data that climatologists can use to improve predictions of climate change and global warming. The satellite's Active Cavity Irradiance Monitor III instrument, now in its third generation, has been used since the 1980s to study solar irradiance and its impacts on global warming. Scientists, using data from the instrument, now theorize that there is a significant correlation between solar radiation and global warming. ACRIMSAT completed its five-year primary mission in 2005 when it began operating under its extended mission. http://photojournal.jpl.nasa.gov/catalog/PIA18157

  2. Method for monitoring stack gases for uranium activity

    DOEpatents

    Beverly, C.R.; Ernstberger, E.G.

    1985-07-03

    A method for monitoring the stack gases of a purge cascade of gaseous diffusion plant for uranium activity. A sample stream is taken from the stack gases and contacted with a volume of moisture-laden air for converting trace levels of uranium hexafluoride, if any, in the stack gases into particulate uranyl fluoride. A continuous strip of filter paper from a supply roll is passed through this sampling stream to intercept and gather any uranyl fluoride in the sampling stream. This filter paper is then passed by an alpha scintillation counting device where any radioactivity on the filter paper is sensed so as to provide a continuous monitoring of the gas stream for activity indicative of the uranium content in the stack gases. 1 fig.

  3. Method for monitoring stack gases for uranium activity

    DOEpatents

    Beverly, Claude R.; Ernstberger, Harold G.

    1988-01-01

    A method for monitoring the stack gases of a purge cascade of a gaseous diffusion plant for uranium activity. A sample stream is taken from the stack gases and contacted with a volume of moisture-laden air for converting trace levels of uranium hexafluoride, if any, in the stack gases into particulate uranyl fluoride. A continuous strip of filter paper from a supply roll is passed through this sampling stream to intercept and gather any uranyl fluoride in the sampling stream. This filter paper is then passed by an alpha scintillation counting device where any radioactivity on the filter paper is sensed so as to provide a continuous monitoring of the gas stream for activity indicative of the uranium content in the stack gases.

  4. Toward Active Monitoring of Piping Using Ultrasonic Guided Waves

    SciTech Connect

    Park, Joon-Soo; Kim, Young H.; Song, Sung-Jin; Kim, Jae-Hee; Eom, Heung-Seop; Im, Kwang-Hee

    2004-02-26

    Piping in nuclear power plants is exposed to severe environmental conditions so that it is very susceptible to failure caused by the growth of defects. Thus, it is necessary to have thorough inspection of piping in order to detect defects before failure. Unfortunately, however, inspection of piping in nuclear power plants is not easy in practice because of its long length as well as the radioactive environment. To take care of this difficulty, a research endeavor to develop techniques to monitor piping in nuclear power plants continuously and actively using ultrasonic guided wave is currently undertaken. This paper reports initial results of our endeavor including design of an ultrasonic array system for active monitoring of piping.

  5. Single-cell level methods for studying the effect of antibiotics on bacteria during infection.

    PubMed

    Kogermann, Karin; Putrinš, Marta; Tenson, Tanel

    2016-12-01

    Considerable evidence about phenotypic heterogeneity among bacteria during infection has accumulated during recent years. This heterogeneity has to be considered if the mechanisms of infection and antibiotic action are to be understood, so we need to implement existing and find novel methods to monitor the effects of antibiotics on bacteria at the single-cell level. This review provides an overview of methods by which this aim can be achieved. Fluorescence label-based methods and Raman scattering as a label-free approach are discussed in particular detail. Other label-free methods that can provide single-cell level information, such as impedance spectroscopy and surface plasmon resonance, are briefly summarized. The advantages and disadvantages of these different methods are discussed in light of a challenging in vivo environment.

  6. Active Geophysical Monitoring in Oil and Gas Industry

    NASA Astrophysics Data System (ADS)

    Bakulin, A.; Calvert, R.

    2005-12-01

    Effective reservoir management is a Holy Grail of the oil and gas industry. Quest for new technologies is never ending but most often they increase effectiveness and decrease the costs. None of the newcomers proved to be a silver bullet in such a key metric of the industry as average oil recovery factor. This factor is still around 30 %, meaning that 70 % of hydrocarbon reserves are left in the ground in places where we already have expensive infrastructure (platforms, wells) to extract them. Main reason for this inefficiency is our inability to address realistic reservoir complexity. Most of the time we fail to properly characterize our reservoirs before production. As a matter of fact, one of the most important parameters -- permeability -- can not be mapped from remote geophysical methods. Therefore we always start production blind even though reservoir state before production is the simplest one. Once first oil is produced, we greatly complicate the things and quickly become unable to estimate the state and condition of the reservoir (fluid, pressures, faults etc) or oilfield hardware (wells, platforms, pumps) to make a sound next decision in the chain of reservoir management. Our modeling capabilities are such that if we know true state of the things - we can make incredibly accurate predictions and make extremely efficient decisions. Thus the bottleneck is our inability to properly describe the state of the reservoirs in real time. Industry is starting to recognize active monitoring as an answer to this critical issue. We will highlight industry strides in active geophysical monitoring from well to reservoir scale. It is worth noting that when one says ``monitoring" production technologists think of measuring pressures at the wellhead or at the pump, reservoir engineers think of measuring extracted volumes and pressures, while geophysicist may think of change in elastic properties. We prefer to think of monitoring as to measuring those parameters of the

  7. Single-cell analysis of dihydroartemisinin-induced apoptosis through reactive oxygen species-mediated caspase-8 activation and mitochondrial pathway in ASTC-a-1 cells using fluorescence imaging techniques

    NASA Astrophysics Data System (ADS)

    Lu, Ying-Ying; Chen, Tong-Sheng; Wang, Xiao-Ping; Li, Li

    2010-07-01

    Dihydroartemisinin (DHA), a front-line antimalarial herbal compound, has been shown to possess promising anticancer activity with low toxicity. We have previously reported that DHA induced caspase-3-dependent apoptosis in human lung adenocarcinoma cells. However, the cellular target and molecular mechanism of DHA-induced apoptosis is still poorly defined. We use confocal fluorescence microscopy imaging, fluorescence resonance energy transfer, and fluorescence recovery after photobleaching techniques to explore the roles of DHA-elicited reactive oxygen species (ROS) in the DHA-induced Bcl-2 family proteins activation, mitochondrial dysfunction, caspase cascade, and cell death. Cell Counting Kit-8 assay and flow cytometry analysis showed that DHA induced ROS-mediated apoptosis. Confocal imaging analysis in a single living cell and Western blot assay showed that DHA triggered ROS-dependent Bax translocation, mitochondrial membrane depolarization, alteration of mitochondrial morphology, cytochrome c release, caspase-9, caspase-8, and caspase-3 activation, indicating the coexistence of ROS-mediated mitochondrial and death receptor pathway. Collectively, our findings demonstrate for the first time that DHA induces cell apoptosis by triggering ROS-mediated caspase-8/Bid activation and the mitochondrial pathway, which provides some novel insights into the application of DHA as a potential anticancer drug and a new therapeutic strategy by targeting ROS signaling in lung adenocarcinoma therapy in the future.

  8. Feasibility of Automating FIWC Website Noncompliance Monitoring and Enforcement Activities

    DTIC Science & Technology

    2003-06-01

    At the outset, we had only a general idea of the problem we were attacking , and little notion of the form our final implementation would take. But...access, and to verify security procedures, survivability, and operational security. Monitoring includes active attacks by authorized DoD entities to test...Original Message ----- From: "Vickie" < Seahorse @Redshift.com> To: <clarion@attryde.com> Sent: Saturday, May 31, 2003 20:14 Subject: Clarion

  9. A new approach to monitoring seismic activity: California case study

    NASA Astrophysics Data System (ADS)

    Dzeboev, B. A.

    2017-03-01

    Preliminary work on the creation of a method for monitoring of seismic activity, on the basis of discrete mathematical analysis for further seismic hazard assessment of a territory, is presented. The method developed is tested for the territory of California and adjacent areas of the United States. The results obtained are analyzed for the period of 1980-2015. The nonrandomness of the results is shown by means of an error chart.

  10. Integrated active sensor system for real time vibration monitoring

    NASA Astrophysics Data System (ADS)

    Liang, Qijie; Yan, Xiaoqin; Liao, Xinqin; Cao, Shiyao; Lu, Shengnan; Zheng, Xin; Zhang, Yue

    2015-11-01

    We report a self-powered, lightweight and cost-effective active sensor system for vibration monitoring with multiplexed operation based on contact electrification between sensor and detected objects. The as-fabricated sensor matrix is capable of monitoring and mapping the vibration state of large amounts of units. The monitoring contents include: on-off state, vibration frequency and vibration amplitude of each unit. The active sensor system delivers a detection range of 0-60 Hz, high accuracy (relative error below 0.42%), long-term stability (10000 cycles). On the time dimension, the sensor can provide the vibration process memory by recording the outputs of the sensor system in an extend period of time. Besides, the developed sensor system can realize detection under contact mode and non-contact mode. Its high performance is not sensitive to the shape or the conductivity of the detected object. With these features, the active sensor system has great potential in automatic control, remote operation, surveillance and security systems.

  11. Integrated active sensor system for real time vibration monitoring.

    PubMed

    Liang, Qijie; Yan, Xiaoqin; Liao, Xinqin; Cao, Shiyao; Lu, Shengnan; Zheng, Xin; Zhang, Yue

    2015-11-05

    We report a self-powered, lightweight and cost-effective active sensor system for vibration monitoring with multiplexed operation based on contact electrification between sensor and detected objects. The as-fabricated sensor matrix is capable of monitoring and mapping the vibration state of large amounts of units. The monitoring contents include: on-off state, vibration frequency and vibration amplitude of each unit. The active sensor system delivers a detection range of 0-60 Hz, high accuracy (relative error below 0.42%), long-term stability (10000 cycles). On the time dimension, the sensor can provide the vibration process memory by recording the outputs of the sensor system in an extend period of time. Besides, the developed sensor system can realize detection under contact mode and non-contact mode. Its high performance is not sensitive to the shape or the conductivity of the detected object. With these features, the active sensor system has great potential in automatic control, remote operation, surveillance and security systems.

  12. Integrated active sensor system for real time vibration monitoring

    PubMed Central

    Liang, Qijie; Yan, Xiaoqin; Liao, Xinqin; Cao, Shiyao; Lu, Shengnan; Zheng, Xin; Zhang, Yue

    2015-01-01

    We report a self-powered, lightweight and cost-effective active sensor system for vibration monitoring with multiplexed operation based on contact electrification between sensor and detected objects. The as-fabricated sensor matrix is capable of monitoring and mapping the vibration state of large amounts of units. The monitoring contents include: on-off state, vibration frequency and vibration amplitude of each unit. The active sensor system delivers a detection range of 0–60 Hz, high accuracy (relative error below 0.42%), long-term stability (10000 cycles). On the time dimension, the sensor can provide the vibration process memory by recording the outputs of the sensor system in an extend period of time. Besides, the developed sensor system can realize detection under contact mode and non-contact mode. Its high performance is not sensitive to the shape or the conductivity of the detected object. With these features, the active sensor system has great potential in automatic control, remote operation, surveillance and security systems. PMID:26538293

  13. Single-cell PCR of genomic DNA enabled by automated single-cell printing for cell isolation.

    PubMed

    Stumpf, F; Schoendube, J; Gross, A; Rath, C; Niekrawietz, S; Koltay, P; Roth, G

    2015-07-15

    Single-cell analysis has developed into a key topic in cell biology with future applications in personalized medicine, tumor identification as well as tumor discovery (Editorial, 2013). Here we employ inkjet-like printing to isolate individual living single human B cells (Raji cell line) and load them directly into standard PCR tubes. Single cells are optically detected in the nozzle of the microfluidic piezoelectric dispenser chip to ensure printing of droplets with single cells only. The printing process has been characterized by using microbeads (10µm diameter) resulting in a single bead delivery in 27 out of 28 cases and relative positional precision of ±350µm at a printing distance of 6mm between nozzle and tube lid. Process-integrated optical imaging enabled to identify the printing failure as void droplet and to exclude it from downstream processing. PCR of truly single-cell DNA was performed without pre-amplification directly from single Raji cells with 33% success rate (N=197) and Cq values of 36.3±2.5. Additionally single cell whole genome amplification (WGA) was employed to pre-amplify the single-cell DNA by a factor of >1000. This facilitated subsequent PCR for the same gene yielding a success rate of 64% (N=33) which will allow more sophisticated downstream analysis like sequencing, electrophoresis or multiplexing.

  14. Single cell detection using a magnetic zigzag nanowire biosensor.

    PubMed

    Huang, Hao-Ting; Ger, Tzong-Rong; Lin, Ya-Hui; Wei, Zung-Hang

    2013-08-07

    A magnetic zigzag nanowire device was designed for single cell biosensing. Nanowires with widths of 150, 300, 500, and 800 nm were fabricated on silicon trenches by electron beam lithography, electron beam evaporation, and lift-off processes. Magnetoresistance measurements were performed before and after the attachment of a single magnetic cell to the nanowires to characterize the magnetic signal change due to the influence of the magnetic cell. Magnetoresistance responses were measured in different magnetic field directions, and the results showed that this nanowire device can be used for multi-directional detection. It was observed that the highest switching field variation occurred in a 150 nm wide nanowire when the field was perpendicular to the substrate plane. On the other hand, the highest magnetoresistance ratio variation occurred in a 800 nm wide nanowire also when the field was perpendicular to the substrate plane. Besides, the trench-structured substrate proposed in this study can fix the magnetic cell to the sensor in a fluid environment, and the stray field generated by the corners of the magnetic zigzag nanowires has the function of actively attracting the magnetic cells for detection.

  15. Gravity perception and signal transduction in single cells

    NASA Astrophysics Data System (ADS)

    Block, I.; Wolke, A.; Briegleb, W.; Ivanova, K.

    Cellular signal processing in multi-, as well as in unicellular organisms, has to rely on fundamentally similar mechanisms. Free-living single cells often use the gravity vector for their spatial orientation (gravitaxis) and show distinct gravisensitivities. In this investigation the gravisensitive giant ameboid cell Physarum polycephalum (Myxomycetes, acellular slime molds) is used. Its gravitaxis and the modulation of its intrinsic rhythmic contraction activity by gravity was demonstrated in 180 °turn experiments and in simulated, as well as in actual, near-weightlessness studies (fast-rotating clinostat; Spacelab D1, IML-1). The stimulus perception was addressed in an IML-2 experiment, which provided information on the gravireceptor itself by the determination of the cell's acceleration-sensitivity threshold. Ground-based experiments designed to elucidate the subsequent steps in signal transduction leading to a motor response, suggest that an acceleration stimulus induces changes in the level of second messenger, adenosine 3',5'-cyclic monophosphate (cAMP), indicating also that the acceleration-stimulus signal transduction chain of Physarum uses an ubiquitous second messenger pathway.

  16. T cell fate and clonality inference from single cell transcriptomes

    PubMed Central

    Proserpio, Valentina; Clare, Simon; Speak, Anneliese O.; Dougan, Gordon; Teichmann, Sarah A.

    2016-01-01

    The enormous sequence diversity within T cell receptor (TCR) repertoires allows specific TCR sequences to be used as lineage markers for T cells that derive from a common progenitor. We have developed a computational method, called TraCeR, to reconstruct full-length, paired TCR sequences from T lymphocyte single-cell RNA-seq by combining existing assembly and alignment programs with “combinatorial recombinome” sequences comprising all possible TCR combinations. We validate this method to quantify its accuracy and sensitivity. Inferred TCR sequences reveal clonal relationships between T cells whilst the cells’ complete transcriptional landscapes can be quantified from the remaining RNA-seq data. This provides a powerful tool to link T cell specificity with functional response and we demonstrate this by determining the distribution of members of expanded T cell clonotypes in a mouse Salmonella infection model. Members of the same clonotype span early activated CD4+ T cells, as well as mature effector and memory cells. PMID:26950746

  17. Microfluidic single-cell whole-transcriptome sequencing

    PubMed Central

    Streets, Aaron M.; Zhang, Xiannian; Cao, Chen; Pang, Yuhong; Wu, Xinglong; Xiong, Liang; Yang, Lu; Fu, Yusi; Zhao, Liang; Tang, Fuchou; Huang, Yanyi

    2014-01-01

    Single-cell whole-transcriptome analysis is a powerful tool for quantifying gene expression heterogeneity in populations of cells. Many techniques have, thus, been recently developed to perform transcriptome sequencing (RNA-Seq) on individual cells. To probe subtle biological variation between samples with limiting amounts of RNA, more precise and sensitive methods are still required. We adapted a previously developed strategy for single-cell RNA-Seq that has shown promise for superior sensitivity and implemented the chemistry in a microfluidic platform for single-cell whole-transcriptome analysis. In this approach, single cells are captured and lysed in a microfluidic device, where mRNAs with poly(A) tails are reverse-transcribed into cDNA. Double-stranded cDNA is then collected and sequenced using a next generation sequencing platform. We prepared 94 libraries consisting of single mouse embryonic cells and technical replicates of extracted RNA and thoroughly characterized the performance of this technology. Microfluidic implementation increased mRNA detection sensitivity as well as improved measurement precision compared with tube-based protocols. With 0.2 M reads per cell, we were able to reconstruct a majority of the bulk transcriptome with 10 single cells. We also quantified variation between and within different types of mouse embryonic cells and found that enhanced measurement precision, detection sensitivity, and experimental throughput aided the distinction between biological variability and technical noise. With this work, we validated the advantages of an early approach to single-cell RNA-Seq and showed that the benefits of combining microfluidic technology with high-throughput sequencing will be valuable for large-scale efforts in single-cell transcriptome analysis. PMID:24782542

  18. Microfluidic single-cell whole-transcriptome sequencing.

    PubMed

    Streets, Aaron M; Zhang, Xiannian; Cao, Chen; Pang, Yuhong; Wu, Xinglong; Xiong, Liang; Yang, Lu; Fu, Yusi; Zhao, Liang; Tang, Fuchou; Huang, Yanyi

    2014-05-13

    Single-cell whole-transcriptome analysis is a powerful tool for quantifying gene expression heterogeneity in populations of cells. Many techniques have, thus, been recently developed to perform transcriptome sequencing (RNA-Seq) on individual cells. To probe subtle biological variation between samples with limiting amounts of RNA, more precise and sensitive methods are still required. We adapted a previously developed strategy for single-cell RNA-Seq that has shown promise for superior sensitivity and implemented the chemistry in a microfluidic platform for single-cell whole-transcriptome analysis. In this approach, single cells are captured and lysed in a microfluidic device, where mRNAs with poly(A) tails are reverse-transcribed into cDNA. Double-stranded cDNA is then collected and sequenced using a next generation sequencing platform. We prepared 94 libraries consisting of single mouse embryonic cells and technical replicates of extracted RNA and thoroughly characterized the performance of this technology. Microfluidic implementation increased mRNA detection sensitivity as well as improved measurement precision compared with tube-based protocols. With 0.2 M reads per cell, we were able to reconstruct a majority of the bulk transcriptome with 10 single cells. We also quantified variation between and within different types of mouse embryonic cells and found that enhanced measurement precision, detection sensitivity, and experimental throughput aided the distinction between biological variability and technical noise. With this work, we validated the advantages of an early approach to single-cell RNA-Seq and showed that the benefits of combining microfluidic technology with high-throughput sequencing will be valuable for large-scale efforts in single-cell transcriptome analysis.

  19. Bioinformatics approaches to single-cell analysis in developmental biology.

    PubMed

    Yalcin, Dicle; Hakguder, Zeynep M; Otu, Hasan H

    2016-03-01

    Individual cells within the same population show various degrees of heterogeneity, which may be better handled with single-cell analysis to address biological and clinical questions. Single-cell analysis is especially important in developmental biology as subtle spatial and temporal differences in cells have significant associations with cell fate decisions during differentiation and with the description of a particular state of a cell exhibiting an aberrant phenotype. Biotechnological advances, especially in the area of microfluidics, have led to a robust, massively parallel and multi-dimensional capturing, sorting, and lysis of single-cells and amplification of related macromolecules, which have enabled the use of imaging and omics techniques on single cells. There have been improvements in computational single-cell image analysis in developmental biology regarding feature extraction, segmentation, image enhancement and machine learning, handling limitations of optical resolution to gain new perspectives from the raw microscopy images. Omics approaches, such as transcriptomics, genomics and epigenomics, targeting gene and small RNA expression, single nucleotide and structural variations and methylation and histone modifications, rely heavily on high-throughput sequencing technologies. Although there are well-established bioinformatics methods for analysis of sequence data, there are limited bioinformatics approaches which address experimental design, sample size considerations, amplification bias, normalization, differential expression, coverage, clustering and classification issues, specifically applied at the single-cell level. In this review, we summarize biological and technological advancements, discuss challenges faced in the aforementioned data acquisition and analysis issues and present future prospects for application of single-cell analyses to developmental biology. © The Author 2015. Published by Oxford University Press on behalf of the European

  20. Image analysis driven single-cell analytics for systems microbiology.

    PubMed

    Balomenos, Athanasios D; Tsakanikas, Panagiotis; Aspridou, Zafiro; Tampakaki, Anastasia P; Koutsoumanis, Konstantinos P; Manolakos, Elias S

    2017-04-04

    Time-lapse microscopy is an essential tool for capturing and correlating bacterial morphology and gene expression dynamics at single-cell resolution. However state-of-the-art computational methods are limited in terms of the complexity of cell movies that they can analyze and lack of automation. The proposed Bacterial image analysis driven Single Cell Analytics (BaSCA) computational pipeline addresses these limitations thus enabling high throughput systems microbiology. BaSCA can segment and track multiple bacterial colonies and single-cells, as they grow and divide over time (cell segmentation and lineage tree construction) to give rise to dense communities with thousands of interacting cells in the field of view. It combines advanced image processing and machine learning methods to deliver very accurate bacterial cell segmentation and tracking (F-measure over 95%) even when processing images of imperfect quality with several overcrowded colonies in the field of view. In addition, BaSCA extracts on the fly a plethora of single-cell properties, which get organized into a database summarizing the analysis of the cell movie. We present alternative ways to analyze and visually explore the spatiotemporal evolution of single-cell properties in order to understand trends and epigenetic effects across cell generations. The robustness of BaSCA is demonstrated across different imaging modalities and microscopy types. BaSCA can be used to analyze accurately and efficiently cell movies both at a high resolution (single-cell level) and at a large scale (communities with many dense colonies) as needed to shed light on e.g. how bacterial community effects and epigenetic information transfer play a role on important phenomena for human health, such as biofilm formation, persisters' emergence etc. Moreover, it enables studying the role of single-cell stochasticity without losing sight of community effects that may drive it.

  1. Human psychophysiological activity monitoring methods using fiber optic sensors

    NASA Astrophysics Data System (ADS)

    Zyczkowski, M.; Uzieblo-Zyczkowska, B.

    2010-10-01

    The paper presents the concept of fiber optic sensor system for human psycho-physical activity detection. A fiber optic sensor that utilizes optical phase interferometry or intensity in modalmetric to monitor a patient's vital signs such as respiration cardiac activity, blood pressure and body's physical movements. The sensor, which is non-invasive, comprises an optical fiber interferometer that includes an optical fiber proximately situated to the patient so that time varying acusto-mechanical signals from the patient are coupled into the optical fiber. The system can be implemented in embodiments ranging form a low cost in-home to a high end product for in hospital use.

  2. In-vessel activation monitors in JET: Progress in modeling

    SciTech Connect

    Bonheure, Georges; Lengar, I.; Syme, B.; Popovichev, S.; Arnold, Dirk; Laubenstein, Matthias

    2008-10-15

    Activation studies were performed in JET with new in-vessel activation monitors. Though primarily dedicated to R and D in the challenging issue of lost {alpha} diagnostics for ITER, which is being addressed at JET with several techniques, these monitors provide for both neutron and charged particle fluences. A set of samples with different orientation with respect to the magnetic field is transported inside the torus by means of a manipulator arm (in contrast with the conventional JET activation system with pneumatic transport system). In this case, radionuclides with longer half-life were selected and ultralow background gamma-ray measurements were needed. The irradiation was closer to the plasma and this potentially reduces the neutron scattering problem. This approach could also be of interest for ITER, where the calibration methods have yet to be developed. The MCNP neutron transport model for JET was modified to include the activation probe and so provide calculations to help assess the new data. The neutron induced activity on the samples are well reproduced by the calculations.

  3. Characterizing heterogeneity in leukemic cells using single-cell gene expression analysis.

    PubMed

    Saadatpour, Assieh; Guo, Guoji; Orkin, Stuart H; Yuan, Guo-Cheng

    2014-12-03

    A fundamental challenge for cancer therapy is that each tumor contains a highly heterogeneous cell population whose structure and mechanistic underpinnings remain incompletely understood. Recent advances in single-cell gene expression profiling have created new possibilities to characterize this heterogeneity and to dissect the potential intra-cancer cellular hierarchy. Here, we apply single-cell analysis to systematically characterize the heterogeneity within leukemic cells using the MLL-AF9 driven mouse model of acute myeloid leukemia. We start with fluorescence-activated cell sorting analysis with seven surface markers, and extend by using a multiplexing quantitative polymerase chain reaction approach to assay the transcriptional profile of a panel of 175 carefully selected genes in leukemic cells at the single-cell level. By employing a set of computational tools we find striking heterogeneity within leukemic cells. Mapping to the normal hematopoietic cellular hierarchy identifies two distinct subtypes of leukemic cells; one similar to granulocyte/monocyte progenitors and the other to macrophage and dendritic cells. Further functional experiments suggest that these subtypes differ in proliferation rates and clonal phenotypes. Finally, co-expression network analysis reveals similarities as well as organizational differences between leukemia and normal granulocyte/monocyte progenitor networks. Overall, our single-cell analysis pinpoints previously uncharacterized heterogeneity within leukemic cells and provides new insights into the molecular signatures of acute myeloid leukemia.

  4. Advanced Performance Modeling with Combined Passive and Active Monitoring

    SciTech Connect

    Dovrolis, Constantine; Sim, Alex

    2015-04-15

    To improve the efficiency of resource utilization and scheduling of scientific data transfers on high-speed networks, the "Advanced Performance Modeling with combined passive and active monitoring" (APM) project investigates and models a general-purpose, reusable and expandable network performance estimation framework. The predictive estimation model and the framework will be helpful in optimizing the performance and utilization of networks as well as sharing resources with predictable performance for scientific collaborations, especially in data intensive applications. Our prediction model utilizes historical network performance information from various network activity logs as well as live streaming measurements from network peering devices. Historical network performance information is used without putting extra load on the resources by active measurement collection. Performance measurements collected by active probing is used judiciously for improving the accuracy of predictions.

  5. Optical sensor based system to monitor caries activity

    NASA Astrophysics Data System (ADS)

    Shrestha, A.; Tahir, R.; Kishen, A.

    2007-07-01

    The aim of the study is to evaluate the ability of a visible light based spectroscopic sensor system to monitor caries activity in saliva. In this study an optical sensor is utilized to monitor the bacterial-mediated acidogenic profile of stimulated saliva using a photosensitive pH indicator. Microbiological assessment of the saliva samples were carried out using the conventional culture methods. In addition, the shifts in the pH of saliva-sucrose samples were recorded using a pH meter. The absorption spectra obtained from the optical sensor showed peak maxima at 595nm, which decreased as a function of time. The microbiological assessment showed increase in the bacterial count as a function of time. A strong positive correlation was also observed between the rates of decrease in the absorption intensity measured using the optical sensor and the decrease in pH measured using the pH meter. This study highlights the potential advantages of using the optical sensor as a sensitive and rapid chairside system for monitoring caries activity by quantification of the acidogenic profile of saliva.

  6. A novel permalloy based magnetic single cell micro array.

    PubMed

    Liu, William; Dechev, Nikolai; Foulds, Ian G; Burke, Robert; Parameswaran, Ash; Park, Edward J

    2009-08-21

    Devices capable of automatically aligning cells onto geometrical arrays are of great interest to biomedical researchers. Such devices can facilitate the study of numerous cells while the cells remain physically separated from one another. In this way, cell arrays reduce cell-to-cell interactions while the cells are all subjected to common stimuli, which allows individual cell behaviour to be revealed. The use of arrays allows for the parallel analysis of single cells, facilitates data logging, and opens the door to the use of automated machine-based single cell analysis techniques. A novel permalloy based magnetic single cell micro array (MSCMA) is presented in this paper. The MSCMA creates an array of magnetic traps by generating magnetic flux density peaks at predefined locations. When using cells labelled with immunomagnetic labels, the cells will interact with the magnetic fields, and can be captured at the magnetic trap sites. Prototypes of the MSCMA have been successfully fabricated and tested using both fixed and live Jurkat cells (10 microm average diameter) that were labelled. The prototypes performed as predicted during experimental trials. The experimental results show that the MSCMA can randomly array up to 136 single cells per square mm. The results also show that the number of single cells captured is a function of the trap site density of the MSCMA design and the cell density in the fluid sample.

  7. Isolation and Characterization of Single Cells from Zebrafish Embryos

    PubMed Central

    Samsa, Leigh Ann; Fleming, Nicole; Magness, Scott; Qian, Li; Liu, Jiandong

    2017-01-01

    The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been described at the morphological level, little is known about the molecular basis of cellular changes that occur as the organism develops. With recent advancements in microfluidics and multiplexing technologies, it is now possible to characterize gene expression in single cells. This allows for investigation of heterogeneity between individual cells of specific cell populations to identify and classify cell subtypes, characterize intermediate states that occur during cell differentiation, and explore differential cellular responses to stimuli. This study describes a protocol to isolate viable, single cells from zebrafish embryos for high throughput multiplexing assays. This method may be rapidly applied to any zebrafish embryonic cell type with fluorescent markers. An extension of this method may also be used in combination with high throughput sequencing technologies to fully characterize the transcriptome of single cells. As proof of principle, the relative abundance of cardiac differentiation markers was assessed in isolated, single cells derived from nkx2.5 positive cardiac progenitors. By evaluation of gene expression at the single cell level and at a single time point, the data support a model in which cardiac progenitors coexist with differentiating progeny. The method and work flow described here is broadly applicable to the zebrafish research community, requiring only a labeled transgenic fish line and access to microfluidics technologies. PMID:27022828

  8. [Recent progress in single-cell RNA-Seq analysis].

    PubMed

    Lu, Wen; Fuchou, Tang

    2014-11-01

    Cell heterogeneity is a general feature of biological tissues. Standard transcriptome analysis approaches require tens of thousands of cells to provide an average view of gene expression and ignore the information of gene expression heterogeneity. The single-cell RNA-Seq technologies profile gene expression at the single-cell level and serve as powerful tools to identify distinct phenotypic cell types within a heterogeneous population. The single-cell RNA-Seq technologies have been developed rapidly in recent years. The methodological progress includes a variety of cDNA amplification methods, the quantitative analysis of the sensitivity and noise of the technologies, and the development of the low-coverage high-throughput single-cell RNA-Seq and the in situ RNA-Seq technologies. Furthermore, the scope of application is extended from early embryonic development to tissue and organ development, immunology and oncology. In this review, we discuss recent progress in methodology and applications of the single-cell RNA-Seq technologies.

  9. Detection of aneuploidy in single cells using comparative genomic hybridization.

    PubMed

    Voullaire, L; Wilton, L; Slater, H; Williamson, R

    1999-09-01

    The ability of comparative genomic hybridization (CGH) to detect aneuploidy following universal amplification of DNA from a single cell, or a small number of cells, was investigated with a view to preimplantation diagnosis following in vitro fertilization, and prenatal diagnosis using fetal erythroblasts obtained from maternal blood. The DNA obtained from lysed single cells was amplified using degenerate oligonucleotide-primed PCR (DOP-PCR). This product was labelled using nick translation and hybridized together with normal reference genomic DNA. The CGH fluorescent ratio profiles obtained could be used to determine aneuploidy with cut-off thresholds of 0.75 and 1.25. Deviation in the profiles in the heterochromatic regions was reduced by using, as a reference sample, normal genomic DNA that had also undergone DOP-PCR. Single cells known to be trisomic for chromosomes 13, 18 or 21 were analysed using this technique. The resolution of CGH with amplified DNA from a single cell is of the order of 40 Mb, sufficient for the diagnosis of trisomy 21, and possibly segmental aneuploidy of equivalent size. These results, and those of others, demonstrate that diagnosis of chromosomal aneuploidy in single cells is possible using CGH with DOP-PCR amplified DNA.

  10. Landslide Activity Monitoring with the Help of Unmanned Aerial Vehicle

    NASA Astrophysics Data System (ADS)

    Peterman, V.

    2015-08-01

    This paper presents a practical example of a landslide monitoring through the use of a UAV - tracking and monitoring the movements of the Potoska Planina landslide located above the village of Koroska Bela in the western Karavanke Mountains in north-western Slovenia. Past geological research in this area indicated slope landmass movement of more than 10 cm per year. However, much larger movements have been detected since - significant enough to be observed photogrammetrically with the help of a UAV. With the intention to assess the dynamics of the landslide we have established a system of periodic observations carried out twice per year - in mid-spring and mid-autumn. This paper offers an activity summary along with the presentation of data acquisition, data processing and results.

  11. Oogenesis: single cell development and differentiation.

    PubMed

    Song, Jia L; Wong, Julian L; Wessel, Gary M

    2006-12-01

    Oocytes express a unique set of genes that are essential for their growth, for meiotic recombination and division, for storage of nutrients, and for fertilization. We have utilized the newly sequenced genome of Strongylocentrotus purpuratus to identify genes that help the oocyte accomplish each of these tasks. This study emphasizes four classes of genes that are specialized for oocyte function: (1) Transcription factors: many of these factors are not significantly expressed in embryos, but are shared by other adult tissues, namely the ovary, testis, and gut. (2) Meiosis: A full set of meiotic genes is present in the sea urchin, including those involved in cohesion, in synaptonemal complex formation, and in meiotic recombination. (3) Yolk uptake and storage: Nutrient storage for use during early embryogenesis is essential to oocyte function in most animals; the sea urchin accomplishes this task by using the major yolk protein and a family of accessory proteins called YP30. Comparison of the YP30 family members across their conserved, tandem fasciclin domains with their intervening introns reveals an incongruence in the evolution of its major clades. (4) Fertilization: This set of genes includes many of the cell surface proteins involved in sperm interaction and in the physical block to polyspermy. The majority of these genes are active only in oocytes, and in many cases, their anatomy reflects the tandem repeating interaction domains essential for the function of these proteins. Together, the expression profile of these four gene classes highlights the transitions of the oocyte from a stem cell precursor, through stages of development, to the clearing and re-programming of gene expression necessary to transition from oocyte, to egg, to embryo.

  12. Live single cell functional phenotyping in droplet nano-liter reactors

    NASA Astrophysics Data System (ADS)

    Konry, Tania; Golberg, Alexander; Yarmush, Martin

    2013-11-01

    While single cell heterogeneity is present in all biological systems, most studies cannot address it due to technical limitations. Here we describe a nano-liter droplet microfluidic-based approach for stimulation and monitoring of surfaceand secreted markers of live single immune dendritic cells (DCs) as well as monitoring the live T cell/DC interaction. This nano-liter in vivo simulating microenvironment allows delivering various stimuli reagents to each cell and appropriate gas exchanges which are necessary to ensure functionality and viability of encapsulated cells. Labeling bioassay and microsphere sensors were integrated into nano-liter reaction volume of the droplet to monitor live single cell surface markers and secretion analysis in the time-dependent fashion. Thus live cell stimulation, secretion and surface monitoring can be obtained simultaneously in distinct microenvironment, which previously was possible using complicated and multi-step in vitro and in vivo live-cell microscopy, together with immunological studies of the outcome secretion of cellular function.

  13. Live single cell functional phenotyping in droplet nano-liter reactors.

    PubMed

    Konry, Tania; Golberg, Alexander; Yarmush, Martin

    2013-11-11

    While single cell heterogeneity is present in all biological systems, most studies cannot address it due to technical limitations. Here we describe a nano-liter droplet microfluidic-based approach for stimulation and monitoring of surface and secreted markers of live single immune dendritic cells (DCs) as well as monitoring the live T cell/DC interaction. This nano-liter in vivo simulating microenvironment allows delivering various stimuli reagents to each cell and appropriate gas exchanges which are necessary to ensure functionality and viability of encapsulated cells. Labeling bioassay and microsphere sensors were integrated into nano-liter reaction volume of the droplet to monitor live single cell surface markers and secretion analysis in the time-dependent fashion. Thus live cell stimulation, secretion and surface monitoring can be obtained simultaneously in distinct microenvironment, which previously was possible using complicated and multi-step in vitro and in vivo live-cell microscopy, together with immunological studies of the outcome secretion of cellular function.

  14. Live single cell functional phenotyping in droplet nano-liter reactors

    PubMed Central

    Konry, Tania; Golberg, Alexander; Yarmush, Martin

    2013-01-01

    While single cell heterogeneity is present in all biological systems, most studies cannot address it due to technical limitations. Here we describe a nano-liter droplet microfluidic-based approach for stimulation and monitoring of surfaceand secreted markers of live single immune dendritic cells (DCs) as well as monitoring the live T cell/DC interaction. This nano-liter in vivo simulating microenvironment allows delivering various stimuli reagents to each cell and appropriate gas exchanges which are necessary to ensure functionality and viability of encapsulated cells. Labeling bioassay and microsphere sensors were integrated into nano-liter reaction volume of the droplet to monitor live single cell surface markers and secretion analysis in the time-dependent fashion. Thus live cell stimulation, secretion and surface monitoring can be obtained simultaneously in distinct microenvironment, which previously was possible using complicated and multi-step in vitro and in vivo live-cell microscopy, together with immunological studies of the outcome secretion of cellular function. PMID:24212247

  15. Ambulation monitoring of transtibial amputation subjects with patient activity monitor versus pedometer.

    PubMed

    Dudek, Nancy L; Khan, Omar D; Lemaire, Edward D; Marks, Meridith B; Saville, Leyana

    2008-01-01

    Our study aimed to compare the accuracy of step count and ambulation distance determined with the Yamax Digi-Walker SW-700 pedometer (DW) and the Ossur patient activity monitor (PAM) in 20 transtibial amputation subjects who were functioning at the K3 Medicare Functional Classification Level. Subjects completed four simulated household tasks in an apartment setup and a gymnasium walking course designed to simulate outdoor walking without the presence of environmental barriers or varied terrain. The mean step count accuracy of the DW and the PAM was equivalent for both the household activity (75.3% vs 70.6%) and the walking course (93.8% vs 94.0%). The mean distance measurement accuracy was better with the DW than with the PAM (household activity: 72.8% vs 0%, walking course: 92.5% vs 86.3%; p < 0.05). With acceptable step count accuracy, both devices are appropriate for assessing relatively continuous ambulation. The DW may be preferred for its more accurate distance measurements. Neither device is ideal for monitoring in-home ambulation.

  16. Design considerations in an active medical product safety monitoring system.

    PubMed

    Gagne, Joshua J; Fireman, Bruce; Ryan, Patrick B; Maclure, Malcolm; Gerhard, Tobias; Toh, Sengwee; Rassen, Jeremy A; Nelson, Jennifer C; Schneeweiss, Sebastian

    2012-01-01

    Active medical product monitoring systems, such as the Sentinel System, will utilize electronic healthcare data captured during routine health care. Safety signals that arise from these data may be spurious because of chance or bias, particularly confounding bias, given the observational nature of the data. Applying appropriate monitoring designs can filter out many false-positive and false-negative associations from the outset. Designs can be classified by whether they produce estimates based on between-person or within-person comparisons. In deciding which approach is more suitable for a given monitoring scenario, stakeholders must consider the characteristics of the monitored product, characteristics of the health outcome of interest (HOI), and characteristics of the potential link between these. Specifically, three factors drive design decisions: (i) strength of within-person and between-person confounding; (ii) whether circumstances exist that may predispose to misclassification of exposure or misclassification of the timing of the HOI; and (iii) whether the exposure of interest is predominantly transient or sustained. Additional design considerations include whether to focus on new users, the availability of appropriate active comparators, the presence of an exposure time trend, and the measure of association of interest. When the key assumptions of self-controlled designs are fulfilled (i.e., lack of within-person, time-varying confounding; abrupt HOI onset; and transient exposure), within-person comparisons are preferred because they inherently avoid confounding by fixed factors. The cohort approach generally is preferred in other situations and particularly when timing of exposure or outcome is uncertain because cohort approaches are less vulnerable to biases resulting from misclassification.

  17. Genetic programs constructed from layered logic gates in single cells.

    PubMed

    Moon, Tae Seok; Lou, Chunbo; Tamsir, Alvin; Stanton, Brynne C; Voigt, Christopher A

    2012-11-08

    Genetic programs function to integrate environmental sensors, implement signal processing algorithms and control expression dynamics. These programs consist of integrated genetic circuits that individually implement operations ranging from digital logic to dynamic circuits, and they have been used in various cellular engineering applications, including the implementation of process control in metabolic networks and the coordination of spatial differentiation in artificial tissues. A key limitation is that the circuits are based on biochemical interactions occurring in the confined volume of the cell, so the size of programs has been limited to a few circuits. Here we apply part mining and directed evolution to build a set of transcriptional AND gates in Escherichia coli. Each AND gate integrates two promoter inputs and controls one promoter output. This allows the gates to be layered by having the output promoter of an upstream circuit serve as the input promoter for a downstream circuit. Each gate consists of a transcription factor that requires a second chaperone protein to activate the output promoter. Multiple activator-chaperone pairs are identified from type III secretion pathways in different strains of bacteria. Directed evolution is applied to increase the dynamic range and orthogonality of the circuits. These gates are connected in different permutations to form programs, the largest of which is a 4-input AND gate that consists of 3 circuits that integrate 4 inducible systems, thus requiring 11 regulatory proteins. Measuring the performance of individual gates is sufficient to capture the behaviour of the complete program. Errors in the output due to delays (faults), a common problem for layered circuits, are not observed. This work demonstrates the successful layering of orthogonal logic gates, a design strategy that could enable the construction of large, integrated circuits in single cells.

  18. Single-cell technologies to study the immune system.

    PubMed

    Proserpio, Valentina; Mahata, Bidesh

    2016-02-01

    The immune system is composed of a variety of cells that act in a coordinated fashion to protect the organism against a multitude of different pathogens. The great variability of existing pathogens corresponds to a similar high heterogeneity of the immune cells. The study of individual immune cells, the fundamental unit of immunity, has recently transformed from a qualitative microscopic imaging to a nearly complete quantitative transcriptomic analysis. This shift has been driven by the rapid development of multiple single-cell technologies. These new advances are expected to boost the detection of less frequent cell types and transient or intermediate cell states. They will highlight the individuality of each single cell and greatly expand the resolution of current available classifications and differentiation trajectories. In this review we discuss the recent advancement and application of single-cell technologies, their limitations and future applications to study the immune system. © 2015 The Authors. Immunology Published by John Wiley & Sons Ltd.

  19. Review of methods to probe single cell metabolism and bioenergetics

    PubMed Central

    Vasdekis, Andreas E.; Stephanopoulos, Gregory

    2015-01-01

    Single cell investigations have enabled unexpected discoveries, such as the existence of biological noise and phenotypic switching in infection, metabolism and treatment. Herein, we review methods that enable such single cell investigations specific to metabolism and bioenergetics. Firstly, we discuss how to isolate and immobilize individuals from a cell suspension, including both permanent and reversible approaches. We also highlight specific advances in microbiology for its implications in metabolic engineering. Methods for probing single cell physiology and metabolism are subsequently reviewed. The primary focus therein is on dynamic and high-content profiling strategies based on label-free and fluorescence microspectroscopy and microscopy. Non-dynamic approaches, such as mass spectrometry and nuclear magnetic resonance, are also briefly discussed. PMID:25448400

  20. Single-cell RNA-seq: advances and future challenges

    PubMed Central

    Saliba, Antoine-Emmanuel; Westermann, Alexander J.; Gorski, Stanislaw A.; Vogel, Jörg

    2014-01-01

    Phenotypically identical cells can dramatically vary with respect to behavior during their lifespan and this variation is reflected in their molecular composition such as the transcriptomic landscape. Single-cell transcriptomics using next-generation transcript sequencing (RNA-seq) is now emerging as a powerful tool to profile cell-to-cell variability on a genomic scale. Its application has already greatly impacted our conceptual understanding of diverse biological processes with broad implications for both basic and clinical research. Different single-cell RNA-seq protocols have been introduced and are reviewed here—each one with its own strengths and current limitations. We further provide an overview of the biological questions single-cell RNA-seq has been used to address, the major findings obtained from such studies, and current challenges and expected future developments in this booming field. PMID:25053837

  1. Spatial reconstruction of single-cell gene expression

    PubMed Central

    Satija, Rahul; Farrell, Jeffrey A.; Gennert, David; Schier, Alexander F.; Regev, Aviv

    2015-01-01

    Spatial localization is a key determinant of cellular fate and behavior, but spatial RNA assays traditionally rely on staining for a limited number of RNA species. In contrast, single-cell RNA-seq allows for deep profiling of cellular gene expression, but established methods separate cells from their native spatial context. Here we present Seurat, a computational strategy to infer cellular localization by integrating single-cell RNA-seq data with in situ RNA patterns. We applied Seurat to spatially map 851 single cells from dissociated zebrafish (Danio rerio) embryos, inferring a transcriptome-wide map of spatial patterning. We confirmed Seurat’s accuracy using several experimental approaches, and used it to identify a set of archetypal expression patterns and spatial markers. Additionally, Seurat correctly localizes rare subpopulations, accurately mapping both spatially restricted and scattered groups. Seurat will be applicable to mapping cellular localization within complex patterned tissues in diverse systems. PMID:25867923

  2. Dense transcript profiling in single cells by image correlation decoding

    PubMed Central

    Coskun, Ahmet F.; Cai, Long

    2016-01-01

    Recent work in sequential fluorescent in-situ hybridization (FISH) has demonstrated the ability to uniquely encode a large number of molecular species in single cells. However, the multiplexing capacity is practically limited by the density of the barcoded objects in the cell. Here, we present a general method using image correlation to resolve the temporal barcodes in sequential hybridization experiments, allowing high density objects to be decoded. Using this correlation FISH (corrFISH) approach, we profiled the gene expression of ribosomal proteins in single cells in cell cultures and in mouse thymus tissue sections. In tissues, corrFISH revealed cell type specific gene expression of ribosomal proteins. The combination of sequential barcoding FISH and correlation analyses provides a general strategy for multiplexing a large number of RNA molecules and potentially other high copy number molecules in single cells. PMID:27271198

  3. Wishbone identifies bifurcating developmental trajectories from single-cell data.

    PubMed

    Setty, Manu; Tadmor, Michelle D; Reich-Zeliger, Shlomit; Angel, Omer; Salame, Tomer Meir; Kathail, Pooja; Choi, Kristy; Bendall, Sean; Friedman, Nir; Pe'er, Dana

    2016-06-01

    Recent single-cell analysis technologies offer an unprecedented opportunity to elucidate developmental pathways. Here we present Wishbone, an algorithm for positioning single cells along bifurcating developmental trajectories with high resolution. Wishbone uses multi-dimensional single-cell data, such as mass cytometry or RNA-Seq data, as input and orders cells according to their developmental progression, and it pinpoints bifurcation points by labeling each cell as pre-bifurcation or as one of two post-bifurcation cell fates. Using 30-channel mass cytometry data, we show that Wishbone accurately recovers the known stages of T-cell development in the mouse thymus, including the bifurcation point. We also apply the algorithm to mouse myeloid differentiation and demonstrate its generalization to additional lineages. A comparison of Wishbone to diffusion maps, SCUBA and Monocle shows that it outperforms these methods both in the accuracy of ordering cells and in the correct identification of branch points.

  4. Single cell electroporation using proton beam fabricated biochips

    NASA Astrophysics Data System (ADS)

    Homhuan, S.; Zhang, B.; Sheu, F.-S.; Bettiol, A. A.; Watt, F.

    2010-05-01

    We report the design and fabrication of a novel single cell electroporation biochip fabricated by the Proton Beam Writing technique (PBW), a new technique capable of direct-writing high-aspect-ratio nano and microstructures. The biochip features nickel micro-electrodes with straight-side walls between which individual cells are positioned. By applying electrical impulses across the electrodes, SYTOX® Green nucleic acid stain is incorporated into mouse neuroblastoma (N2a) cells. When the stain binds with DNA inside the cell nucleus, green fluorescence is observed upon excitation from a halogen lamp. Three parameters; electric field strength, pulse duration, and the number of pulses have been considered and optimized for the single cell electroporation. The results show that our biochip gives successfully electroporated cells . This single cell electroporation system represents a promising method for investigating the introduction of a wide variety of fluorophores, nanoparticles, quantum dots, DNAs and proteins into cells.

  5. Single-cell RNA-seq: advances and future challenges.

    PubMed

    Saliba, Antoine-Emmanuel; Westermann, Alexander J; Gorski, Stanislaw A; Vogel, Jörg

    2014-08-01

    Phenotypically identical cells can dramatically vary with respect to behavior during their lifespan and this variation is reflected in their molecular composition such as the transcriptomic landscape. Single-cell transcriptomics using next-generation transcript sequencing (RNA-seq) is now emerging as a powerful tool to profile cell-to-cell variability on a genomic scale. Its application has already greatly impacted our conceptual understanding of diverse biological processes with broad implications for both basic and clinical research. Different single-cell RNA-seq protocols have been introduced and are reviewed here-each one with its own strengths and current limitations. We further provide an overview of the biological questions single-cell RNA-seq has been used to address, the major findings obtained from such studies, and current challenges and expected future developments in this booming field.

  6. Massively parallel digital transcriptional profiling of single cells.

    PubMed

    Zheng, Grace X Y; Terry, Jessica M; Belgrader, Phillip; Ryvkin, Paul; Bent, Zachary W; Wilson, Ryan; Ziraldo, Solongo B; Wheeler, Tobias D; McDermott, Geoff P; Zhu, Junjie; Gregory, Mark T; Shuga, Joe; Montesclaros, Luz; Underwood, Jason G; Masquelier, Donald A; Nishimura, Stefanie Y; Schnall-Levin, Michael; Wyatt, Paul W; Hindson, Christopher M; Bharadwaj, Rajiv; Wong, Alexander; Ness, Kevin D; Beppu, Lan W; Deeg, H Joachim; McFarland, Christopher; Loeb, Keith R; Valente, William J; Ericson, Nolan G; Stevens, Emily A; Radich, Jerald P; Mikkelsen, Tarjei S; Hindson, Benjamin J; Bielas, Jason H

    2017-01-16

    Characterizing the transcriptome of individual cells is fundamental to understanding complex biological systems. We describe a droplet-based system that enables 3' mRNA counting of tens of thousands of single cells per sample. Cell encapsulation, of up to 8 samples at a time, takes place in ∼6 min, with ∼50% cell capture efficiency. To demonstrate the system's technical performance, we collected transcriptome data from ∼250k single cells across 29 samples. We validated the sensitivity of the system and its ability to detect rare populations using cell lines and synthetic RNAs. We profiled 68k peripheral blood mononuclear cells to demonstrate the system's ability to characterize large immune populations. Finally, we used sequence variation in the transcriptome data to determine host and donor chimerism at single-cell resolution from bone marrow mononuclear cells isolated from transplant patients.

  7. Exploring viral infection using single-cell sequencing.

    PubMed

    Rato, Sylvie; Golumbeanu, Monica; Telenti, Amalio; Ciuffi, Angela

    2016-11-02

    Single-cell sequencing (SCS) has emerged as a valuable tool to study cellular heterogeneity in diverse fields, including virology. By studying the viral and cellular genome and/or transcriptome, the dynamics of viral infection can be investigated at single cell level. Most studies have explored the impact of cell-to-cell variation on the viral life cycle from the point of view of the virus, by analyzing viral sequences, and from the point of view of the cell, mainly by analyzing the cellular host transcriptome. In this review, we will focus on recent studies that use single-cell sequencing to explore viral diversity and cell variability in response to viral replication.

  8. Wishbone identifies bifurcating developmental trajectories from single-cell data

    PubMed Central

    Setty, Manu; Tadmor, Michelle D; Reich-Zeliger, Shlomit; Angel, Omer; Salame, Tomer Meir; Kathail, Pooja; Choi, Kristy; Bendall, Sean; Friedman, Nir; Pe’er, Dana

    2016-01-01

    Recent single-cell analysis technologies offer an unprecedented opportunity to elucidate developmental pathways. Here we present Wishbone, an algorithm for positioning single cells along bifurcating developmental trajectories with high resolution. Wishbone uses multi-dimensional single-cell data, such as mass cytometry or RNA-seq data, as input and orders cells according to their developmental progression by pinpointing bifurcation points and labeling each cell as pre-bifurcation or as one of two post-bifurcation cell fates. Using 30-channel mass cytometry data, we show that Wishbone accurately recovers the known stages of T cell development in the mouse thymus, including the bifurcation point. We also apply the algorithm to mouse myeloid differentiation and demonstrate its generalization to additional lineages. A comparison of Wishbone to diffusion maps, SCUBA and Monocle shows that it outperforms these methods both in the accuracy of ordering cells and in the correct identification of branch points. PMID:27136076

  9. Massively parallel digital transcriptional profiling of single cells

    PubMed Central

    Zheng, Grace X. Y.; Terry, Jessica M.; Belgrader, Phillip; Ryvkin, Paul; Bent, Zachary W.; Wilson, Ryan; Ziraldo, Solongo B.; Wheeler, Tobias D.; McDermott, Geoff P.; Zhu, Junjie; Gregory, Mark T.; Shuga, Joe; Montesclaros, Luz; Underwood, Jason G.; Masquelier, Donald A.; Nishimura, Stefanie Y.; Schnall-Levin, Michael; Wyatt, Paul W.; Hindson, Christopher M.; Bharadwaj, Rajiv; Wong, Alexander; Ness, Kevin D.; Beppu, Lan W.; Deeg, H. Joachim; McFarland, Christopher; Loeb, Keith R.; Valente, William J.; Ericson, Nolan G.; Stevens, Emily A.; Radich, Jerald P.; Mikkelsen, Tarjei S.; Hindson, Benjamin J.; Bielas, Jason H.

    2017-01-01

    Characterizing the transcriptome of individual cells is fundamental to understanding complex biological systems. We describe a droplet-based system that enables 3′ mRNA counting of tens of thousands of single cells per sample. Cell encapsulation, of up to 8 samples at a time, takes place in ∼6 min, with ∼50% cell capture efficiency. To demonstrate the system's technical performance, we collected transcriptome data from ∼250k single cells across 29 samples. We validated the sensitivity of the system and its ability to detect rare populations using cell lines and synthetic RNAs. We profiled 68k peripheral blood mononuclear cells to demonstrate the system's ability to characterize large immune populations. Finally, we used sequence variation in the transcriptome data to determine host and donor chimerism at single-cell resolution from bone marrow mononuclear cells isolated from transplant patients. PMID:28091601

  10. Single-cell sequencing in stem cell biology.

    PubMed

    Wen, Lu; Tang, Fuchou

    2016-04-15

    Cell-to-cell variation and heterogeneity are fundamental and intrinsic characteristics of stem cell populations, but these differences are masked when bulk cells are used for omic analysis. Single-cell sequencing technologies serve as powerful tools to dissect cellular heterogeneity comprehensively and to identify distinct phenotypic cell types, even within a 'homogeneous' stem cell population. These technologies, including single-cell genome, epigenome, and transcriptome sequencing technologies, have been developing rapidly in recent years. The application of these methods to different types of stem cells, including pluripotent stem cells and tissue-specific stem cells, has led to exciting new findings in the stem cell field. In this review, we discuss the recent progress as well as future perspectives in the methodologies and applications of single-cell omic sequencing technologies.

  11. Single-cell resolution of intracellular T cell Ca(2+) dynamics in response to frequency-based H2O2 stimulation.

    PubMed

    Kniss-James, Ariel S; Rivet, Catherine A; Chingozha, Loice; Lu, Hang; Kemp, Melissa L

    2017-03-01

    Adaptive immune cells, such as T cells, integrate information from their extracellular environment through complex signaling networks with exquisite sensitivity in order to direct decisions on proliferation, apoptosis, and cytokine production. These signaling networks are reliant on the interplay between finely tuned secondary messengers, such as Ca(2+) and H2O2. Frequency response analysis, originally developed in control engineering, is a tool used for discerning complex networks. This analytical technique has been shown to be useful for understanding biological systems and facilitates identification of the dominant behaviour of the system. We probed intracellular Ca(2+) dynamics in the frequency domain to investigate the complex relationship between two second messenger signaling molecules, H2O2 and Ca(2+), during T cell activation with single cell resolution. Single-cell analysis provides a unique platform for interrogating and monitoring cellular processes of interest. We utilized a previously developed microfluidic device to monitor individual T cells through time while applying a dynamic input to reveal a natural frequency of the system at approximately 2.78 mHz stimulation. Although our network was much larger with more unknown connections than previous applications, we are able to derive features from our data, observe forced oscillations associated with specific amplitudes and frequencies of stimuli, and arrive at conclusions about potential transfer function fits as well as the underlying population dynamics.

  12. A single-cell resolution map of mouse hematopoietic stem and progenitor cell differentiation.

    PubMed

    Nestorowa, Sonia; Hamey, Fiona K; Pijuan Sala, Blanca; Diamanti, Evangelia; Shepherd, Mairi; Laurenti, Elisa; Wilson, Nicola K; Kent, David G; Göttgens, Berthold

    2016-08-25

    Maintenance of the blood system requires balanced cell fate decisions by hematopoietic stem and progenitor cells (HSPCs). Because cell fate choices are executed at the individual cell level, new single-cell profiling technologies offer exciting possibilities for mapping the dynamic molecular changes underlying HSPC differentiation. Here, we have used single-cell RNA sequencing to profile more than 1600 single HSPCs, and deep sequencing has enabled detection of an average of 6558 protein-coding genes per cell. Index sorting, in combination with broad sorting gates, allowed us to retrospectively assign cells to 12 commonly sorted HSPC phenotypes while also capturing intermediate cells typically excluded by conventional gating. We further show that independently generated single-cell data sets can be projected onto the single-cell resolution expression map to directly compare data from multiple groups and to build and refine new hypotheses. Reconstruction of differentiation trajectories reveals dynamic expression changes associated with early lymphoid, erythroid, and granulocyte-macrophage differentiation. The latter two trajectories were characterized by common upregulation of cell cycle and oxidative phosphorylation transcriptional programs. By using external spike-in controls, we estimate absolute messenger RNA (mRNA) levels per cell, showing for the first time that despite a general reduction in total mRNA, a subset of genes shows higher expression levels in immature stem cells consistent with active maintenance of the stem-cell state. Finally, we report the development of an intuitive Web interface as a new community resource to permit visualization of gene expression in HSPCs at single-cell resolution for any gene of choice.

  13. A single-cell resolution map of mouse hematopoietic stem and progenitor cell differentiation

    PubMed Central

    Nestorowa, Sonia; Hamey, Fiona K.; Pijuan Sala, Blanca; Diamanti, Evangelia; Shepherd, Mairi; Laurenti, Elisa; Wilson, Nicola K.; Kent, David G.

    2016-01-01

    Maintenance of the blood system requires balanced cell fate decisions by hematopoietic stem and progenitor cells (HSPCs). Because cell fate choices are executed at the individual cell level, new single-cell profiling technologies offer exciting possibilities for mapping the dynamic molecular changes underlying HSPC differentiation. Here, we have used single-cell RNA sequencing to profile more than 1600 single HSPCs, and deep sequencing has enabled detection of an average of 6558 protein-coding genes per cell. Index sorting, in combination with broad sorting gates, allowed us to retrospectively assign cells to 12 commonly sorted HSPC phenotypes while also capturing intermediate cells typically excluded by conventional gating. We further show that independently generated single-cell data sets can be projected onto the single-cell resolution expression map to directly compare data from multiple groups and to build and refine new hypotheses. Reconstruction of differentiation trajectories reveals dynamic expression changes associated with early lymphoid, erythroid, and granulocyte-macrophage differentiation. The latter two trajectories were characterized by common upregulation of cell cycle and oxidative phosphorylation transcriptional programs. By using external spike-in controls, we estimate absolute messenger RNA (mRNA) levels per cell, showing for the first time that despite a general reduction in total mRNA, a subset of genes shows higher expression levels in immature stem cells consistent with active maintenance of the stem-cell state. Finally, we report the development of an intuitive Web interface as a new community resource to permit visualization of gene expression in HSPCs at single-cell resolution for any gene of choice. PMID:27365425

  14. Genome-wide Detection of DNase I Hypersensitive Sites in Single Cells and FFPE Samples

    PubMed Central

    Jin, Wenfei; Tang, Qingsong; Wan, Mimi; Cui, Kairong; Zhang, Yi; Ren, Gang; Ni, Bing; Sklar, Jeffrey; Przytycka, Teresa M.; Childs, Richard; Levens, David; Zhao, Keji

    2015-01-01

    DNase I hypersensitive sites (DHSs) provide important information on the presence of transcriptional regulatory elements and the state of chromatin in mammalian cells1–3. Conventional DNase-Seq for genome-wide DHSs profiling is limited by the requirement of millions of cells4,5. Here we report an ultrasensitive strategy, called Pico-Seq, for detection of genome-wide DHSs in single cells. We show that DHS patterns at the single cell level are highly reproducible among individual cells. Among different single cells, highly expressed gene promoters and the enhancers associated with multiple active histone modifications display constitutive DHS while chromatin regions with fewer histone modifications exhibit high variation of DHS. Furthermore, the single-cell DHSs predict enhancers that regulate cell-specific gene expression programs and the cell-to-cell variations of DHS are predictive of gene expression. Finally, we apply Pico-Seq to pools of tumor cells and pools of normal cells, dissected from formalin-fixed paraffin-embedded (FFPE) tissue slides from thyroid cancer patients, and detect thousands of tumor-specific DHSs. Many of these DHSs are associated with promoters and enhancers critically involved in cancer development. Analysis of the DHS sequences uncovers one single-nucleotide variant (chr18:52417839 G>C) in the tumor cells of a follicular thyroid carcinoma patient, which affects the binding of the tumor suppressor protein p53 and correlates with decreased expression of its target gene TXNL1. In conclusion, Pico-Seq can reliably detect DHSs in single cells, greatly extending the range of applications of DHS analysis for both basic and translational research and may provide critical information for personalized medicine. PMID:26605532

  15. Automated single cell sorting and deposition in submicroliter drops

    NASA Astrophysics Data System (ADS)

    Salánki, Rita; Gerecsei, Tamás; Orgovan, Norbert; Sándor, Noémi; Péter, Beatrix; Bajtay, Zsuzsa; Erdei, Anna; Horvath, Robert; Szabó, Bálint

    2014-08-01

    Automated manipulation and sorting of single cells are challenging, when intact cells are needed for further investigations, e.g., RNA or DNA sequencing. We applied a computer controlled micropipette on a microscope admitting 80 PCR (Polymerase Chain Reaction) tubes to be filled with single cells in a cycle. Due to the Laplace pressure, fluid starts to flow out from the micropipette only above a critical pressure preventing the precise control of drop volume in the submicroliter range. We found an anomalous pressure additive to the Laplace pressure that we attribute to the evaporation of the drop. We have overcome the problem of the critical dropping pressure with sequentially operated fast fluidic valves timed with a millisecond precision. Minimum drop volume was 0.4-0.7 μl with a sorting speed of 15-20 s per cell. After picking NE-4C neuroectodermal mouse stem cells and human primary monocytes from a standard plastic Petri dish we could gently deposit single cells inside tiny drops. 94 ± 3% and 54 ± 7% of the deposited drops contained single cells for NE-4C and monocytes, respectively. 7.5 ± 4% of the drops contained multiple cells in case of monocytes. Remaining drops were empty. Number of cells deposited in a drop could be documented by imaging the Petri dish before and after sorting. We tuned the adhesion force of cells to make the manipulation successful without the application of microstructures for trapping cells on the surface. We propose that our straightforward and flexible setup opens an avenue for single cell isolation, critically needed for the rapidly growing field of single cell biology.

  16. Impedimetric and optical interrogation of single cells in a microfluidic device for real-time viability and chemical response assessment.

    PubMed

    James, Conrad D; Reuel, Nigel; Lee, Eunice S; Davalos, Rafael V; Mani, Seethambal S; Carroll-Portillo, Amanda; Rebeil, Roberto; Martino, Anthony; Apblett, Christopher A

    2008-01-18

    We report here a non-invasive, reversible method for interrogating single cells in a microfluidic flow-through system. Impedance spectroscopy of cells held at a micron-sized pore under negative pressure is demonstrated and used to determine the presence and viability of the captured cell. The cell capture pore is optimized for electrical response and mechanical interfacing to a cell using a deposited layer of parylene. Changes in the mechanical interface between the cell and the chip due to chemical exposure or environmental changes can also be assessed. Here, we monitored the change in adhesion/spreading of RAW264.7 macrophages in response to the immune stimulant lipopolysaccharide (LPS). This method enables selective, reversible, and quantitative long-term impedance measurements on single cells. The fully sealed electrofluidic assembly is compatible with long-term cell culturing, and could be modified to incorporate single cell lysis and subsequent intracellular separation and analysis.

  17. Ferroelectric thin-film active sensors for structural health monitoring

    NASA Astrophysics Data System (ADS)

    Lin, Bin; Giurgiutiu, Victor; Yuan, Zheng; Liu, Jian; Chen, Chonglin; Jiang, Jiechao; Bhalla, Amar S.; Guo, Ruyan

    2007-04-01

    Piezoelectric wafer active sensors (PWAS) have been proven a valuable tool in structural health monitoring. Piezoelectric wafer active sensors are able to send and receive guided Lamb/Rayleigh waves that scan the structure and detect the presence of incipient cracks and structural damage. In-situ thin-film active sensor deposition can eliminate the bonding layer to improve the durability issue and reduce the acoustic impedance mismatch. Ferroelectric thin films have been shown to have piezoelectric properties that are close to those of single-crystal ferroelectrics but the fabrication of ferroelectric thin films on structural materials (steel, aluminum, titanium, etc.) has not been yet attempted. In this work, in-situ fabrication method of piezoelectric thin-film active sensors arrays was developed using the nano technology approach. Specification for the piezoelectric thin-film active sensors arrays was based on electro-mechanical-acoustical model. Ferroelectric BaTiO3 (BTO) thin films were successfully deposited on Ni tapes by pulsed laser deposition under the optimal synthesis conditions. Microstructural studies by X-ray diffractometer and transmission electron microscopy reveal that the as-grown BTO thin films have the nanopillar structures with an average size of approximately 80 nm in diameter and the good interface structures with no inter-diffusion or reaction. The dielectric and ferroelectric property measurements exhibit that the BTO films have a relatively large dielectric constant, a small dielectric loss, and an extremely large piezoelectric response with a symmetric hysteresis loop. The research objective is to develop the fabrication and optimum design of thin-film active sensor arrays for structural health monitoring applications. The short wavelengths of the micro phased arrays will permit the phased-array imaging of smaller parts and smaller damage than is currently not possible with existing technology.

  18. Photoacoustic bio-quantification of graphene based nanomaterials at a single cell level (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Nedosekin, Dmitry A.; Nolan, Jacqueline; Biris, Alexandru S.; Zharov, Vladimir P.

    2017-03-01

    Arkansas Nanomedicine Center at the University of Arkansas for Medical Sciences in collaboration with other Arkansas Universities and the FDA-based National Center of Toxicological Research in Jefferson, AR is developing novel techniques for rapid quantification of graphene-based nanomaterials (GBNs) in various biological samples. All-carbon GBNs have wide range of potential applications in industry, agriculture, food processing and medicine; however, quantification of GBNs is difficult in carbon reach biological tissues. The accurate quantification of GBNs is essential for research on material toxicity and the development of GBNs-based drug delivery platforms. We have developed microscopy and cytometry platforms for detection and quantification of GBNs in single cells, tissue and blood samples using photoacoustic contrast of GBNs. We demonstrated PA quantification of individual graphene uptake by single cells. High-resolution PA microscopy provided mapping of GBN distribution within live cells to establish correlation with intracellular toxic phenomena using apoptotic and necrotic assays. This new methodology and corresponding technical platform provide the insight on possible toxicological risks of GBNs at singe cells levels. In addition, in vivo PA image flow cytometry demonstrated the capability to monitor of GBNs pharmacokinetics in mouse model and to map the resulting biodistribution of GBNs in mouse tissues. The integrated PA platform provided an unprecedented sensitivity toward GBNs and allowed to enhance conventional toxicology research by providing a direct correlation between uptake of GBNs at a single cell level and cell viability status.

  19. Single-Cell Sequencing for Precise Cancer Research: Progress and Prospects.

    PubMed

    Zhang, Xiaoyan; Marjani, Sadie L; Hu, Zhaoyang; Weissman, Sherman M; Pan, Xinghua; Wu, Shixiu

    2016-03-15

    Advances in genomic technology have enabled the faithful detection and measurement of mutations and the gene expression profile of cancer cells at the single-cell level. Recently, several single-cell sequencing methods have been developed that permit the comprehensive and precise analysis of the cancer-cell genome, transcriptome, and epigenome. The use of these methods to analyze cancer cells has led to a series of unanticipated discoveries, such as the high heterogeneity and stochastic changes in cancer-cell populations, the new driver mutations and the complicated clonal evolution mechanisms, and the novel identification of biomarkers of variant tumors. These methods and the knowledge gained from their utilization could potentially improve the early detection and monitoring of rare cancer cells, such as circulating tumor cells and disseminated tumor cells, and promote the development of personalized and highly precise cancer therapy. Here, we discuss the current methods for single cancer-cell sequencing, with a strong focus on those practically used or potentially valuable in cancer research, including single-cell isolation, whole genome and transcriptome amplification, epigenome profiling, multi-dimensional sequencing, and next-generation sequencing and analysis. We also examine the current applications, challenges, and prospects of single cancer-cell sequencing.

  20. Single cell transcriptomic analysis of prostate cancer cells.

    PubMed

    Welty, Christopher J; Coleman, Ilsa; Coleman, Roger; Lakely, Bryce; Xia, Jing; Chen, Shu; Gulati, Roman; Larson, Sandy R; Lange, Paul H; Montgomery, Bruce; Nelson, Peter S; Vessella, Robert L; Morrissey, Colm

    2013-02-16

    The ability to interrogate circulating tumor cells (CTC) and disseminated tumor cells (DTC) is restricted by the small number detected and isolated (typically <10). To determine if a commercially available technology could provide a transcriptomic profile of a single prostate cancer (PCa) cell, we clonally selected and cultured a single passage of cell cycle synchronized C4-2B PCa cells. Ten sets of single, 5-, or 10-cells were isolated using a micromanipulator under direct visualization with an inverted microscope. Additionally, two groups of 10 individual DTC, each isolated from bone marrow of 2 patients with metastatic PCa were obtained. RNA was amplified using the WT-Ovation™ One-Direct Amplification System. The amplified material was hybridized on a 44K Whole Human Gene Expression Microarray. A high stringency threshold, a mean Alexa Fluor® 3 signal intensity above 300, was used for gene detection. Relative expression levels were validated for select genes using real-time PCR (RT-qPCR). Using this approach, 22,410, 20,423, and 17,009 probes were positive on the arrays from 10-cell pools, 5-cell pools, and single-cells, respectively. The sensitivity and specificity of gene detection on the single-cell analyses were 0.739 and 0.972 respectively when compared to 10-cell pools, and 0.814 and 0.979 respectively when compared to 5-cell pools, demonstrating a low false positive rate. Among 10,000 randomly selected pairs of genes, the Pearson correlation coefficient was 0.875 between the single-cell and 5-cell pools and 0.783 between the single-cell and 10-cell pools. As expected, abundant transcripts in the 5- and 10-cell samples were detected by RT-qPCR in the single-cell isolates, while lower abundance messages were not. Using the same stringency, 16,039 probes were positive on the patient single-cell arrays. Cluster analysis showed that all 10 DTC grouped together within each patient. A transcriptomic profile can be reliably obtained from a single cell using

  1. A single cell penetration system by ultrasonic driving

    NASA Astrophysics Data System (ADS)

    Zhou, Zhaoying; Xiao, Mingfei; Yang, Xing; Wu, Ting

    2008-12-01

    The researches of single cell's control and operation are the hotspots in whole world. Among the various technologies, the transmission of ectogenic genetic materials between cell membrane is very significant. Imitating the Chinese traditional acupuncture therapy, a new ultrasonic resonance driving method, is imported to drive a cell's penetration probe. A set of the single cell penetration system was established to perform this function. This system includes four subsystems: driving part, micromanipulation part, observation and measurement part, and actuation part. Some fish egg experiments indicate that this system is workable and effective.

  2. Pooled CRISPR screening with single-cell transcriptome readout.

    PubMed

    Datlinger, Paul; Rendeiro, André F; Schmidl, Christian; Krausgruber, Thomas; Traxler, Peter; Klughammer, Johanna; Schuster, Linda C; Kuchler, Amelie; Alpar, Donat; Bock, Christoph

    2017-03-01

    CRISPR-based genetic screens are accelerating biological discovery, but current methods have inherent limitations. Widely used pooled screens are restricted to simple readouts including cell proliferation and sortable marker proteins. Arrayed screens allow for comprehensive molecular readouts such as transcriptome profiling, but at much lower throughput. Here we combine pooled CRISPR screening with single-cell RNA sequencing into a broadly applicable workflow, directly linking guide RNA expression to transcriptome responses in thousands of individual cells. Our method for CRISPR droplet sequencing (CROP-seq) enables pooled CRISPR screens with single-cell transcriptome resolution, which will facilitate high-throughput functional dissection of complex regulatory mechanisms and heterogeneous cell populations.

  3. Molecular circuits for associative learning in single-celled organisms

    PubMed Central

    Fernando, Chrisantha T.; Liekens, Anthony M.L.; Bingle, Lewis E.H.; Beck, Christian; Lenser, Thorsten; Stekel, Dov J.; Rowe, Jonathan E.

    2008-01-01

    We demonstrate how a single-celled organism could undertake associative learning. Although to date only one previous study has found experimental evidence for such learning, there is no reason in principle why it should not occur. We propose a gene regulatory network that is capable of associative learning between any pre-specified set of chemical signals, in a Hebbian manner, within a single cell. A mathematical model is developed, and simulations show a clear learned response. A preliminary design for implementing this model using plasmids within Escherichia coli is presented, along with an alternative approach, based on double-phosphorylated protein kinases. PMID:18835803

  4. Single-cell epigenomics: techniques and emerging applications.

    PubMed

    Schwartzman, Omer; Tanay, Amos

    2015-12-01

    Epigenomics is the study of the physical modifications, associations and conformations of genomic DNA sequences, with the aim of linking these with epigenetic memory, cellular identity and tissue-specific functions. While current techniques in the field are characterizing the average epigenomic features across large cell ensembles, the increasing interest in the epigenetics within complex and heterogeneous tissues is driving the development of single-cell epigenomics. We review emerging single-cell methods for capturing DNA methylation, chromatin accessibility, histone modifications, chromosome conformation and replication dynamics. Together, these techniques are rapidly becoming a powerful tool in studies of cellular plasticity and diversity, as seen in stem cells and cancer.

  5. Single-cell growth analysis in a mixed cell culture

    NASA Astrophysics Data System (ADS)

    Ando, Jun; Bato, Mary Grace P.; Daria, Vincent Ricardo

    2008-06-01

    We perform single cell analysis of cell growth in a mixed cell culture. Two species of yeast cells: Saccharomyces cerevisiae and Candida albicans, are optically trapped using focused continuous-wave near infrared laser. Cell growth for both cells is inhibited only when the two species of cells are in contact with each other. This indicates cell-cell interaction mediated cell growth inhibition mechanism. Single cell level analysis of cell growth studied here contributes to the further understanding of yeast growth arrest in a mixed yeast culture.

  6. A microfluidic device enabling high-efficiency single cell trapping

    PubMed Central

    Jin, D.; Deng, B.; Cai, W.; Tu, L.; Chen, J.; Wu, Q.; Wang, W. H.

    2015-01-01

    Single cell trapping increasingly serves as a key manipulation technique in single cell analysis for many cutting-edge cell studies. Due to their inherent advantages, microfluidic devices have been widely used to enable single cell immobilization. To further improve the single cell trapping efficiency, this paper reports on a passive hydrodynamic microfluidic device based on the “least flow resistance path” principle with geometry optimized in line with corresponding cell types. Different from serpentine structure, the core trapping structure of the micro-device consists of a series of concatenated T and inverse T junction pairs which function as bypassing channels and trapping constrictions. This new device enhances the single cell trapping efficiency from three aspects: (1) there is no need to deploy very long or complicated channels to adjust flow resistance, thus saving space for each trapping unit; (2) the trapping works in a “deterministic” manner, thus saving a great deal of cell samples; and (3) the compact configuration allows shorter flowing path of cells in multiple channels, thus increasing the speed and throughput of cell trapping. The mathematical model of the design was proposed and optimization of associated key geometric parameters was conducted based on computational fluid dynamics (CFD) simulation. As a proof demonstration, two types of PDMS microfluidic devices were fabricated to trap HeLa and HEK-293T cells with relatively significant differences in cell sizes. Experimental results showed 100% cell trapping and 90% single cell trapping over 4 × 100 trap sites for these two cell types, respectively. The space saving is estimated to be 2-fold and the cell trapping speed enhancement to be 3-fold compared to previously reported devices. This device can be used for trapping various types of cells and expanded to trap cells in the order of tens of thousands on 1-cm2 scale area, as a promising tool to pattern large-scale single cells on

  7. Imaging of anticancer drug action in single cells.

    PubMed

    Miller, Miles A; Weissleder, Ralph

    2017-06-23

    Imaging is widely used in anticancer drug development, typically for whole-body tracking of labelled drugs to different organs or to assess drug efficacy through volumetric measurements. However, increasing attention has been drawn to pharmacology at the single-cell level. Diverse cell types, including cancer-associated immune cells, physicochemical features of the tumour microenvironment and heterogeneous cell behaviour all affect drug delivery, response and resistance. This Review summarizes developments in the imaging of in vivo anticancer drug action, with a focus on microscopy approaches at the single-cell level and translational lessons for the clinic.

  8. Single-Cell Isolation and Gene Analysis: Pitfalls and Possibilities

    PubMed Central

    Hodne, Kjetil; Weltzien, Finn-Arne

    2015-01-01

    During the last two decades single-cell analysis (SCA) has revealed extensive phenotypic differences within homogenous cell populations. These phenotypic differences are reflected in the stochastic nature of gene regulation, which is often masked by qualitatively and quantitatively averaging in whole tissue analyses. The ability to isolate transcripts and investigate how genes are regulated at the single cell level requires highly sensitive and refined methods. This paper reviews different strategies currently used for SCA, including harvesting, reverse transcription, and amplification of the RNA, followed by methods for transcript quantification. The review provides the historical background to SCA, discusses limitations, and current and future possibilities in this exciting field of research. PMID:26569222

  9. Single-cell analysis tools for drug discovery and development.

    PubMed

    Heath, James R; Ribas, Antoni; Mischel, Paul S

    2016-03-01

    The genetic, functional or compositional heterogeneity of healthy and diseased tissues presents major challenges in drug discovery and development. Such heterogeneity hinders the design of accurate disease models and can confound the interpretation of biomarker levels and of patient responses to specific therapies. The complex nature of virtually all tissues has motivated the development of tools for single-cell genomic, transcriptomic and multiplex proteomic analyses. Here, we review these tools and assess their advantages and limitations. Emerging applications of single cell analysis tools in drug discovery and development, particularly in the field of oncology, are discussed.

  10. FRET-based optical assay for monitoring riboswitch activation.

    PubMed

    Harbaugh, Svetlana; Kelley-Loughnane, Nancy; Davidson, Molly; Narayanan, Latha; Trott, Sandra; Chushak, Yaroslav G; Stone, Morley O

    2009-05-11

    Riboswitches are regulatory RNAs located in the 5'-untranslated region of mRNA sequences that recognize and bind to small molecules and regulate the expression of downstream genes. Creation of synthetic riboswitches to novel ligands depends on the ability to monitor riboswitch activation in the presence of analyte. In our work, we have coupled a synthetic riboswitch to an optical reporter assay based on fluorescence resonance energy transfer (FRET) between two genetically encoded fluorescent proteins. The theophylline-sensitive riboswitch was placed upstream of the Tobacco Etch Virus (TEV) protease coding sequence. Our FRET construct was composed of eGFP and a nonfluorescent yellow fluorescent protein mutant called REACh (for resonance energy-accepting chromoprotein) connected with a peptide linker containing a TEV protease cleavage site. Addition of theophylline to the E. coli cells activates the riboswitch and initiates the translation of mRNA. Synthesized protease cleaves the linker in the FRET-based fusion protein causing a change in the fluorescence signal. By this method, we observed an 11-fold increase in cellular extract fluorescence in the presence of theophylline. The advantage of using an eGFP-REACh pair is the elimination of acceptor fluorescence. This leads to an improved detection of FRET via better signal-to-noise ratio, allowing us to monitor riboswitch activation in a wide range of analyte concentrations from 0.01 to 2.5 mM.

  11. Passive and Active Sensing Technologies for Structural Health Monitoring

    NASA Astrophysics Data System (ADS)

    Do, Richard

    A combination of passive and active sensing technologies is proposed as a structural health monitoring solution for several applications. Passive sensing is differentiated from active sensing in that with the former, no energy is intentionally imparted into the structure under test; sensors are deployed in a pure detection mode for collecting data mined for structural health monitoring purposes. In this thesis, passive sensing using embedded fiber Bragg grating optical strain gages was used to detect varying degrees of impact damage using two different classes of features drawn from traditional spectral analysis and auto-regressive time series modeling. The two feature classes were compared in detail through receiver operating curve performance analysis. The passive detection problem was then augmented with an active sensing system using ultrasonic guided waves (UGWs). This thesis considered two main challenges associated with UGW SHM including in-situ wave propagation property determination and thermal corruption of data. Regarding determination of wave propagation properties, of which dispersion characteristics are the most important, a new dispersion curve extraction method called sparse wavenumber analysis (SWA) was experimentally validated. Also, because UGWs are extremely sensitive to ambient temperature changes on the structure, it significantly affects the wave propagation properties by causing large errors in the residual error in the processing of the UGWs from an array. This thesis presented a novel method that compensates for uniform temperature change by considering the magnitude and phase of the signal separately and applying a scalable transformation.

  12. Monitoring Heparin Therapy with the Activated Partial Thromboplastin Time

    PubMed Central

    Stuart, R. K.; Michel, A.

    1971-01-01

    Difficulties associated with the whole blood clotting time (W.B.C.T.) as a method of monitoring heparin therapy have led to the investigation of the activated partial thromboplastin time (A.P.T.T.) as an alternative. The conclusion is reached that the latter procedure possesses several advantages. Using the method described and a citrate-preserved blood sample collected just prior to the administration of the next serial dose of heparin, the suggested therapeutic duration of the A.P.T.T. is 70 seconds or twice the mean control value. A practical range for this method is 60 to 70 seconds. PMID:5557913

  13. Monitoring rice farming activities in the Mekong Delta region

    NASA Astrophysics Data System (ADS)

    Nguyen, S. T.; Chen, C. F.; Chen, C. R.; Chiang, S. H.; Chang, L. Y.; Khin, L. V.

    2015-12-01

    Half of the world's population depends on rice for survival. Rice agriculture thus plays an important role in the developing world's economy. Vietnam is one of the largest rice producers and suppliers on earth and more than 80% of the exported rice was produced from the Mekong Delta region, which is situated in the southwestern Vietnam and encompasses approximately 40,000 km2. Changes in climate conditions could likely trigger the increase of insect populations and rice diseases, causing the potential loss of rice yields. Monitoring rice-farming activities through crop phenology detection can provide policymakers with timely strategies to mitigate possible impacts on the potential yield as well as rice grain exports to ensure food security for the region. The main objective of this study is to develop a logistic-based algorithm to investigate rice sowing and harvesting activities from the multi-temporal Moderate Resolution Imaging Spectroradiometer (MODIS)-Landsat fusion data. We processed the data for two main cropping seasons (i.e., winter-spring and summer-autumn seasons) through a three-step procedure: (1) MODIS-Landsat data fusion, (2) construction of the time-series enhanced vegetation index 2 (EVI2) data, (3) rice crop phenology detection. The EVI2 data derived from the fusion results between MODIS and Landsat data were compared with that of Landsat data indicated close correlation between the two datasets (R2 = 0.93). The time-series EVI2 data were processed using the double logistic method to detect the progress of sowing and harvesting activities in the region. The comparisons between the estimated sowing and harvesting dates and the field survey data revealed the root mean squared error (RMSE) values of 8.4 and 5.5 days for the winter-spring crop and 9.4 and 12.8 days for the summer-autumn crop, respectively. This study demonstrates the effectiveness of the double logistic-based algorithm for rice crop monitoring from temporal MODIS-Landsat fusion data

  14. Environmental Monitoring Networks Optimization Using Advanced Active Learning Algorithms

    NASA Astrophysics Data System (ADS)

    Kanevski, Mikhail; Volpi, Michele; Copa, Loris

    2010-05-01

    The problem of environmental monitoring networks optimization (MNO) belongs to one of the basic and fundamental tasks in spatio-temporal data collection, analysis, and modeling. There are several approaches to this problem, which can be considered as a design or redesign of monitoring network by applying some optimization criteria. The most developed and widespread methods are based on geostatistics (family of kriging models, conditional stochastic simulations). In geostatistics the variance is mainly used as an optimization criterion which has some advantages and drawbacks. In the present research we study an application of advanced techniques following from the statistical learning theory (SLT) - support vector machines (SVM) and the optimization of monitoring networks when dealing with a classification problem (data are discrete values/classes: hydrogeological units, soil types, pollution decision levels, etc.) is considered. SVM is a universal nonlinear modeling tool for classification problems in high dimensional spaces. The SVM solution is maximizing the decision boundary between classes and has a good generalization property for noisy data. The sparse solution of SVM is based on support vectors - data which contribute to the solution with nonzero weights. Fundamentally the MNO for classification problems can be considered as a task of selecting new measurement points which increase the quality of spatial classification and reduce the testing error (error on new independent measurements). In SLT this is a typical problem of active learning - a selection of the new unlabelled points which efficiently reduce the testing error. A classical approach (margin sampling) to active learning is to sample the points closest to the classification boundary. This solution is suboptimal when points (or generally the dataset) are redundant for the same class. In the present research we propose and study two new advanced methods of active learning adapted to the solution of

  15. C3N4 Nanosheet Modified Microwell Array with Enhanced Electrochemiluminescence for Total Analysis of Cholesterol at Single Cells.

    PubMed

    Xu, Jingjing; Jiang, Depeng; Qin, Yanling; Xia, Juan; Jiang, Dechen; Chen, Hong-Yuan

    2017-02-21

    Here, a g-C3N4 nanosheet modified microwell array providing enhanced electrochemiluminescence (ECL) and better visible sensitivity was prepared to simultaneously analyze total (membrane and intracellular) cholesterol at single cells. The detection limit for ECL visualization of hydrogen peroxide at microwell array was improved to be 500 nM that guaranteed the detection of low concentration cholesterol at single cells in parallel. To achieve single cell cholesterol analysis, the individual cells cultured at the microwell array were exposed to cholesterol oxidase generating hydrogen peroxide for luminescence analysis of membrane cholesterol, and then treated with triton X-100, cholesterol esterase, and cholesterol oxidase to produce hydrogen peroxide from intracellular cholesterol for luminescence determination. The observation of the luminescence spots at microwells in these two steps confirmed the codetection of membrane and intracellular cholesterol at single cells. The inhibition of intracellular acyl-coA/cholesterol acyltransferase (ACAT) resulted in less intracellular cholesterol storage (less luminescence) and more membrane cholesterol (more luminescence). The correlation of the luminescence intensity with the amount of cholesterol confirmed that our assay could simultaneously monitor membrane and intracellular cholesterol pools at different cellular states, which should offer more information for the study of cholesterol-related pathways at single cells.

  16. Stress monitoring versus microseismic ruptures in an active deep mine

    NASA Astrophysics Data System (ADS)

    Tonnellier, Alice; Bouffier, Christian; Bigarré, Pascal; Nyström, Anders; Österberg, Anders; Fjellström, Peter

    2015-04-01

    monitoring data coming from the mine in quasi-real time and facilitates information exchanges and decision making for experts and stakeholders. On the basis of these data acquisition and sharing, preliminary analysis has been started to highlight whether stress variations and seismic sources behaviour might be directly bound with mine working evolution and could improve the knowledge on the equilibrium states inside the mine. Knowing such parameters indeed will be a potential solution to understand better the response of deep mining activities to the exploitation solicitations and to develop, if possible, methods to prevent from major hazards such as rock bursts and other ground failure phenomena.

  17. Single cell Hi-C reveals cell-to-cell variability in chromosome structure

    PubMed Central

    Schoenfelder, Stefan; Yaffe, Eitan; Dean, Wendy; Laue, Ernest D.; Tanay, Amos; Fraser, Peter

    2013-01-01

    Large-scale chromosome structure and spatial nuclear arrangement have been linked to control of gene expression and DNA replication and repair. Genomic techniques based on chromosome conformation capture assess contacts for millions of loci simultaneously, but do so by averaging chromosome conformations from millions of nuclei. Here we introduce single cell Hi-C, combined with genome-wide statistical analysis and structural modeling of single copy X chromosomes, to show that individual chromosomes maintain domain organisation at the megabase scale, but show variable cell-to-cell chromosome territory structures at larger scales. Despite this structural stochasticity, localisation of active gene domains to boundaries of territories is a hallmark of chromosomal conformation. Single cell Hi-C data bridge current gaps between genomics and microscopy studies of chromosomes, demonstrating how modular organisation underlies dynamic chromosome structure, and how this structure is probabilistically linked with genome activity patterns. PMID:24067610

  18. Single-cell analysis challenges the connection between autophagy and senescence induced by DNA damage.

    PubMed

    Filippi-Chiela, Eduardo Cremonese; Bueno e Silva, Mardja Manssur; Thomé, Marcos Paulo; Lenz, Guido

    2015-01-01

    Autophagy and senescence have been described as central features of cell biology, but the interplay between these mechanisms remains obscure. Using a therapeutically relevant model of DNA damage-induced senescence in human glioma cells, we demonstrated that acute treatment with temozolomide induces DNA damage, a transitory activation of PRKAA/AMPK-ULK1 and MAPK14/p38 and the sustained inhibition of AKT-MTOR. This produced a transient induction of autophagy, which was followed by senescence. However, at the single cell level, this coordinated transition was not observed, and autophagy and senescence were triggered in a very heterogeneous manner. Indeed, at a population level, autophagy was highly negatively correlated with senescence markers, while in single cells this correlation did not exist. The inhibition of autophagy triggered apoptosis and decreased senescence, while its activation increased temozolomide-induced senescence, showing that DNA damage-induced autophagy acts by suppressing apoptosis.

  19. Single-cell Hi-C reveals cell-to-cell variability in chromosome structure.

    PubMed

    Nagano, Takashi; Lubling, Yaniv; Stevens, Tim J; Schoenfelder, Stefan; Yaffe, Eitan; Dean, Wendy; Laue, Ernest D; Tanay, Amos; Fraser, Peter

    2013-10-03

    Large-scale chromosome structure and spatial nuclear arrangement have been linked to control of gene expression and DNA replication and repair. Genomic techniques based on chromosome conformation capture (3C) assess contacts for millions of loci simultaneously, but do so by averaging chromosome conformations from millions of nuclei. Here we introduce single-cell Hi-C, combined with genome-wide statistical analysis and structural modelling of single-copy X chromosomes, to show that individual chromosomes maintain domain organization at the megabase scale, but show variable cell-to-cell chromosome structures at larger scales. Despite this structural stochasticity, localization of active gene domains to boundaries of chromosome territories is a hallmark of chromosomal conformation. Single-cell Hi-C data bridge current gaps between genomics and microscopy studies of chromosomes, demonstrating how modular organization underlies dynamic chromosome structure, and how this structure is probabilistically linked with genome activity patterns.

  20. Single-Cell RNA-Seq with Waterfall Reveals Molecular Cascades underlying Adult Neurogenesis.

    PubMed

    Shin, Jaehoon; Berg, Daniel A; Zhu, Yunhua; Shin, Joseph Y; Song, Juan; Bonaguidi, Michael A; Enikolopov, Grigori; Nauen, David W; Christian, Kimberly M; Ming, Guo-li; Song, Hongjun

    2015-09-03

    Somatic stem cells contribute to tissue ontogenesis, homeostasis, and regeneration through sequential processes. Systematic molecular analysis of stem cell behavior is challenging because classic approaches cannot resolve cellular heterogeneity or capture developmental dynamics. Here we provide a comprehensive resource of single-cell transcriptomes of adult hippocampal quiescent neural stem cells (qNSCs) and their immediate progeny. We further developed Waterfall, a bioinformatic pipeline, to statistically quantify singe-cell gene expression along a de novo reconstructed continuous developmental trajectory. Our study reveals molecular signatures of adult qNSCs, characterized by active niche signaling integration and low protein translation capacity. Our analyses further delineate molecular cascades underlying qNSC activation and neurogenesis initiation, exemplified by decreased extrinsic signaling capacity, primed translational machinery, and regulatory switches in transcription factors, metabolism, and energy sources. Our study reveals the molecular continuum underlying adult neurogenesis and illustrates how Waterfall can be used for single-cell omics analyses of various continuous biological processes.

  1. [TCD monitoring during intravenous administration of recombinant tissue plasminogen activator].

    PubMed

    Aoki, Junya; Iguchi, Yasuyuki; Kobayashi, Kazuto; Sakai, Kenichiro; Shibazaki, Kensaku; Sakamoto, Yuki; Kimura, Kazumi

    2010-08-01

    Our aim is to investigate the utility of transcranial Doppler (TCD) monitoring during intravenous administration of 0.6 mg/kg recombinant tissue plasminogen activator (IV rt-PA) which is governmental approved in Japan. Acute ischemic stroke patients with M1 portion of the middle cerebral artery (M1) occlusion treated with IV rt-PA were prospectively enrolled. M1 occlusion was diagnosed before IV rt-PA using magnetic resonance angiography (MRA). Patients without sufficient temporal window of TCD were excluded. TCD monitoring was conducted for 1 hour (h) during IV rt-PA. Recanalization on TCD was defined using thrombolysis in brain ischemia (TIBI) flow grades. After all patients were classified into two groups according to the presence of TCD recanalization (TCD recanalization and TCD non-recanalization group), three-month patients outcome, recanalization rate on MRA 1 h of IV rt-PA, and symptomatic cerebral hemorrhage within 24 h were compared between two groups. We enrolled 16 patients. Eight patients (50%, 7 men [88%]; age, 70 years [interquartile range. 55-81]; NIHSS score, 18 [12-22]) were in the TCD recanalization group and 8 (50%, 6 men [75%]; age, 72 years [62-79]; NIHSS score 19 [15-23] were in the TCD non-recanalization group. Symptomatic cerebral hemorrhage was not seen in both groups at all. MRA 1 h of IV rt-PA revealed recanalization in all 8 (100%) patients with TCD recanalization group and 2 (25%) with TCD non-recanalization group (agreement, 88%; and kappa value, 0.75, P = 0.002). At three months, 5 (63%) of 8 patients in the TCD recanalization group had favorable outcome, and 0 (0%) of 8 in the TCD non-recanalization group (P = 0.026). TCD monitoring for 1 h during IV rt-PA can diagnose the recanalization based on MRA. TCD monitoring should predict good clinical outcome at three months.

  2. A microchip integrating cell array positioning with in situ single-cell impedance measurement.

    PubMed

    Guo, Xiaoliang; Zhu, Rong; Zong, Xianli

    2015-10-07

    This paper presents a novel microarray chip integrating cell positioning with in situ, real-time and long-time impedance measurement on a single cell. The microchip integrates a plurality of quadrupole-electrode units (termed positioning electrodes) patterned into an array with pairs of planar electrodes (termed measuring electrodes) located at the centers of each quadrupole-electrode unit. The positioning electrodes are utilized to trap and position living cells onto the measuring electrodes based on negative dielectrophoresis (nDEP), while the measuring electrodes are used to measure impedances of the trapped single cells. Each measuring electrode has a small footprint area of 7 × 7 μm(2) to ensure inhabiting only one single cell on it. However, the electrode with a small surface area has a low double-layer capacitance when it is immersed in a liquid solution, thus generating a large double-layer impedance, which reduces the sensitivity for impedance measurement on the single cell. To enlarge the effective surface areas of the measuring electrodes, a novel surface-modification process is proposed to controllably construct gold nanostructures on the surfaces of the measuring electrodes while the positioning electrodes are unstained. The double layer capacitances of the modified electrodes are increased by about one order after surface-modification. The developed microchip is used to monitor the adhering behavior of a single HeLa cell by measuring its impedance spectra in real time. The measured impedance is analyzed and used to extract cellular electrical parameters, which demonstrated that the cell compresses the electrical double layer in the process of adherence and adheres onto the measuring electrodes after 4-5 hours.

  3. Comparability of measured acceleration from accelerometry-based activity monitors.

    PubMed

    Rowlands, Alex V; Fraysse, FranÇois; Catt, Mike; Stiles, Victoria H; Stanley, Rebecca M; Eston, Roger G; Olds, Tim S

    2015-01-01

    Accelerometers that provide triaxial measured acceleration data are now available. However, equivalence of output between brands cannot be assumed and testing is necessary to determine whether features of the acceleration signal are interchangeable. This study aimed to establish the equivalence of output between two brands of monitor in a laboratory and in a free-living environment. For part 1, 38 adults performed nine laboratory-based activities while wearing an ActiGraph GT3X+ and GENEActiv (Gravity Estimator of Normal Everyday Activity) at the hip. For part 2, 58 children age 10-12 yr wore a GT3X+ and GENEActiv at the hip for 7 d in a free-living setting. For part 1, the magnitude of time domain features from the GENEActiv was greater than that from the GT3X+. However, frequency domain features compared well, with perfect agreement of the dominant frequency for 97%-100% of participants for most activities. For part 2, mean daily acceleration measured by the two brands was correlated (r = 0.93, P < 0.001, respectively) but the magnitude was approximately 15% lower for the GT3X+ than that for the GENEActiv at the hip. Frequency domain-based classification algorithms should be transferable between monitors, and it should be possible to apply time domain-based classification algorithms developed for one device to the other by applying an affine conversion on the measured acceleration values. The strong relation between accelerations measured by the two brands suggests that habitual activity level and activity patterns assessed by the GENE and GT3X+ may compare well if analyzed appropriately.

  4. Single Cell Measurements of Vacuolar Rupture Caused by Intracellular Pathogens

    PubMed Central

    Danckaert, Anne; Simeone, Roxane; Brosch, Roland; Enninga, Jost; Bobard, Alexandre

    2013-01-01

    Shigella flexneri are pathogenic bacteria that invade host cells entering into an endocytic vacuole. Subsequently, the rupture of this membrane-enclosed compartment allows bacteria to move within the cytosol, proliferate and further invade neighboring cells. Mycobacterium tuberculosis is phagocytosed by immune cells, and has recently been shown to rupture phagosomal membrane in macrophages. We developed a robust assay for tracking phagosomal membrane disruption after host cell entry of Shigella flexneri or Mycobacterium tuberculosis. The approach makes use of CCF4, a FRET reporter sensitive to β-lactamase that equilibrates in the cytosol of host cells. Upon invasion of host cells by bacterial pathogens, the probe remains intact as long as the bacteria reside in membrane-enclosed compartments. After disruption of the vacuole, β-lactamase activity on the surface of the intracellular pathogen cleaves CCF4 instantly leading to a loss of FRET signal and switching its emission spectrum. This robust ratiometric assay yields accurate information about the timing of vacuolar rupture induced by the invading bacteria, and it can be coupled to automated microscopy and image processing by specialized algorithms for the detection of the emission signals of the FRET donor and acceptor. Further, it allows investigating the dynamics of vacuolar disruption elicited by intracellular bacteria in real time in single cells. Finally, it is perfectly suited for high-throughput analysis with a spatio-temporal resolution exceeding previous methods. Here, we provide the experimental details of exemplary protocols for the CCF4 vacuolar rupture assay on HeLa cells and THP-1 macrophages for time-lapse experiments or end points experiments using Shigella flexneri as well as multiple mycobacterial strains such as Mycobacterium marinum, Mycobacterium bovis, and Mycobacterium tuberculosis. PMID:23792688

  5. Laser capture microdissection of single cells from complex tissues.

    PubMed

    Suarez-Quian, C A; Goldstein, S R; Pohida, T; Smith, P D; Peterson, J I; Wellner, E; Ghany, M; Bonner, R F

    1999-02-01

    Laser capture microdissection (LCM) is a new method used to select and procure cell clusters from tissue sections. Once captured, the DNA, RNA or protein can be easily extracted from the isolated cells and analyzed by conventional PCR, reverse transcription (RT)-PCR or polyacrylamide gel electrophoresis, including protein zymography for specific macromolecular changes. In LCM, a thermoplastic polymer coating [ethylene vinyl acetate (EVA)] attached to a rigid support is placed in contact with a tissue section. The EVA polymer over microscopically selected cell clusters is precisely activated by a near-infrared laser pulse and then bonds to the targeted area. Removal of the EVA and its support from the tissue section procures the selected cell aggregates for molecular analysis. This initial NIH LCM approach using a flat transfer EVA film has been recently commercialized and has proven to be an effective routine microdissection technique for subsequent macromolecular analysis in many laboratories around the world. However, reliable and precise capture of individual cells from tissue sections has been difficult to perform with the current LCM instruments. In this report, we describe the capture of individual cells with a new NIH LCM microscope, which epi-irradiates the EVA polymer overlying individual cells with 1-ms laser pulses focused to 6 microns. A computer-controlled arm precisely positions a 40-micron-wide strip of a cylindrical EVA surface onto a sample with a light contact force (ca. 0.1 g). The small contact force and contact area on the film on the sample diminishes nonspecific transfer to negligible levels. By slightly rotating the cylinder to provide a renewable transfer surface, concentration of a distinct cell type on a single cylinder is possible. Using this novel adaptation, we demonstrate the rapid and practical capture of single cells from different types of tissue sections, including immunostained cells.

  6. Actively Heated Fiber Optic Method for Distributed Soil Moisture Monitoring

    NASA Astrophysics Data System (ADS)

    Sayde, C.; Selker, J. S.; Rodriguez-Sinobas, L.; Gil-Rodriguez, M.; Cuenca, R. H.; Tyler, S. W.; English, M.

    2010-12-01

    The temporal and spatial distribution of soil water at scales from 1 to 10,000m is both poorly understood and critical to terrestrial processes. Areas of great uncertainty include the spatio-temporal patterns of: soil water; evapo-transpiration; recharge during and following rainfalls. Observation of dynamics at these scales requires an innovative measurement approach. A novel in-situ distributed measurement of soil water content using temperature measured with Raman scattering in fiber optic cables is presented. This technology, called “Actively Heated Fiber Optic Method,” demonstrated in the lab setting by Sayde et al. 2010 in Water Resources Research involves use of a heat pulse method with fiber optic temperature sensing to obtain precise, distributed measurements of soil water content, with high temporal resolution and sub-meter scale spatial resolution, along a fiber optic cable that can exceed several km in length. The method is based on the influence of water content on soil thermal properties as observed with a buried fiber optical cable monitored by a laser Raman backscatter DTS system. The buried fiber optic is actively heated via electrical resistance, using the steel elements that surround the fiber, and the optical fiber is used as a sub-meter scale thermal sensor to monitor the changes in soil thermal responses every meter along the fiber optic cable. A response metric that has not been previously employed “the time integral of temperature deviation” is used as a simple interpretation of heat data that takes advantage of the characteristics of fiber optic measurements. Validation of the method based on large-column laboratory tests, and field testing results using and 750 m of fiber optic cable buried at 30, 60, and 90 cm depth in the field are presented. The results indicate the feasibility of using the actively heated fiber optic method to monitor soil water content at temporal resolution well under one hour and spatial resolution of 1 m

  7. A process activity monitor for AOS/VS

    NASA Technical Reports Server (NTRS)

    Mckosky, R. A.; Lindley, S. W.; Chapman, J. S.

    1986-01-01

    With the ever increasing concern for computer security, users of computer systems are becoming more sensitive to unauthorized access. One of the initial security concerns for the Shuttle Management Information System was the problem of users leaving their workstations unattended while still connected to the system. This common habit was a concern for two reasons: it ties up resources unnecessarily and it opens the way for unauthorized access to the system. The Data General MV/10000 does not come equipped with an automatic time-out option on interactive peripherals. The purpose of this memorandum is to describe a system which monitors process activity on the system and disconnects those users who show no activity for some time quantum.

  8. Single Cell Characterization of Prostate Cancer-Circulating Tumor Cells

    DTIC Science & Technology

    2013-09-01

    CONTRACTING ORGANIZATION: Stanford University Stanford, CA , 94305 REPORT DATE: September 2013 TYPE OF REPORT: Final PREPARED FOR: U.S...ORGANIZATION NAME(S) AND ADDRESS(ES) Stanford University Stanford, CA , 94305 8. PERFORMING ORGANIZATION REPORT NUMBER...of technology and cost. We have begun using NexGen single cell sequencing methodology developed at Illumina Inc. (Hayward, CA ) to interrogate gene

  9. Cloning of Plasmodium falciparum by single-cell sorting

    PubMed Central

    Miao, Jun; Li, Xiaolian; Cui, Liwang

    2010-01-01

    Malaria parasite cloning is traditionally carried out mainly by using the limiting dilution method, which is laborious, imprecise, and unable to distinguish multiply-infected RBCs. In this study, we used a parasite engineered to express green fluorescent protein (GFP) to evaluate a single-cell sorting method for rapidly cloning Plasmodium falciparum. By dividing a two dimensional scattergram from a cell sorter into 17 gates, we determined the parameters for isolating singly-infected erythrocytes and sorted them into individual cultures. Pre-gating of the engineered parasites for GFP allowed the isolation of almost 100% GFP-positive clones. Compared with the limiting dilution method, the number of parasite clones obtained by single-cell sorting was much higher. Molecular analyses showed that parasite isolates obtained by single-cell sorting were highly homogenous. This highly efficient single-cell sorting method should prove very useful for cloning both P. falciparum laboratory populations from genetic manipulation experiments and clinical samples. PMID:20435038

  10. Single-cell chromatin accessibility reveals principles of regulatory variation.

    PubMed

    Buenrostro, Jason D; Wu, Beijing; Litzenburger, Ulrike M; Ruff, Dave; Gonzales, Michael L; Snyder, Michael P; Chang, Howard Y; Greenleaf, William J

    2015-07-23

    Cell-to-cell variation is a universal feature of life that affects a wide range of biological phenomena, from developmental plasticity to tumour heterogeneity. Although recent advances have improved our ability to document cellular phenotypic variation, the fundamental mechanisms that generate variability from identical DNA sequences remain elusive. Here we reveal the landscape and principles of mammalian DNA regulatory variation by developing a robust method for mapping the accessible genome of individual cells by assay for transposase-accessible chromatin using sequencing (ATAC-seq) integrated into a programmable microfluidics platform. Single-cell ATAC-seq (scATAC-seq) maps from hundreds of single cells in aggregate closely resemble accessibility profiles from tens of millions of cells and provide insights into cell-to-cell variation. Accessibility variance is systematically associated with specific trans-factors and cis-elements, and we discover combinations of trans-factors associated with either induction or suppression of cell-to-cell variability. We further identify sets of trans-factors associated with cell-type-specific accessibility variance across eight cell types. Targeted perturbations of cell cycle or transcription factor signalling evoke stimulus-specific changes in this observed variability. The pattern of accessibility variation in cis across the genome recapitulates chromosome compartments de novo, linking single-cell accessibility variation to three-dimensional genome organization. Single-cell analysis of DNA accessibility provides new insight into cellular variation of the 'regulome'.

  11. Cloning of Plasmodium falciparum by single-cell sorting.

    PubMed

    Miao, Jun; Li, Xiaolian; Cui, Liwang

    2010-10-01

    Malaria parasite cloning is traditionally carried out mainly by using the limiting dilution method, which is laborious, imprecise, and unable to distinguish multiply-infected RBCs. In this study, we used a parasite engineered to express green fluorescent protein (GFP) to evaluate a single-cell sorting method for rapidly cloning Plasmodium falciparum. By dividing a two-dimensional scattergram from a cell sorter into 17 gates, we determined the parameters for isolating singly-infected erythrocytes and sorted them into individual cultures. Pre-gating of the engineered parasites for GFP allowed the isolation of almost 100% GFP-positive clones. Compared with the limiting dilution method, the number of parasite clones obtained by single-cell sorting was much higher. Molecular analyses showed that parasite isolates obtained by single-cell sorting were highly homogenous. This highly efficient single-cell sorting method should prove very useful for cloning both P. falciparum laboratory populations from genetic manipulation experiments and clinical samples. Copyright 2010 Elsevier Inc. All rights reserved.

  12. Selective single cell isolation for genomics using microraft arrays

    PubMed Central

    Welch, Joshua D.; Williams, Lindsay A.; DiSalvo, Matthew; Brandt, Alicia T.; Marayati, Raoud; Sims, Christopher E.; Allbritton, Nancy L.; Prins, Jan F.; Yeh, Jen Jen; Jones, Corbin D.

    2016-01-01

    Genomic methods are used increasingly to interrogate the individual cells that compose specific tissues. However, current methods for single cell isolation struggle to phenotypically differentiate specific cells in a heterogeneous population and rely primarily on the use of fluorescent markers. Many cellular phenotypes of interest are too complex to be measured by this approach, making it difficult to connect genotype and phenotype at the level of individual cells. Here we demonstrate that microraft arrays, which are arrays containing thousands of individual cell culture sites, can be used to select single cells based on a variety of phenotypes, such as cell surface markers, cell proliferation and drug response. We then show that a common genomic procedure, RNA-seq, can be readily adapted to the single cells isolated from these rafts. We show that data generated using microrafts and our modified RNA-seq protocol compared favorably with the Fluidigm C1. We then used microraft arrays to select pancreatic cancer cells that proliferate in spite of cytotoxic drug treatment. Our single cell RNA-seq data identified several expected and novel gene expression changes associated with early drug resistance. PMID:27530426

  13. Enhanced single-cell printing by acoustophoretic cell focusing

    PubMed Central

    Leibacher, I.; Schoendube, J.; Dual, J.; Zengerle, R.; Koltay, P.

    2015-01-01

    Recent years have witnessed a strong trend towards analysis of single-cells. To access and handle single-cells, many new tools are needed and have partly been developed. Here, we present an improved version of a single-cell printer which is able to deliver individual single cells and beads encapsulated in free-flying picoliter droplets at a single-bead efficiency of 96% and with a throughput of more than 10 beads per minute. By integration of acoustophoretic focusing, the cells could be focused in x and y direction. This way, the cells were lined-up in front of a 40 μm nozzle, where they were analyzed individually by an optical system prior to printing. In agreement with acoustic simulations, the focusing of 10 μm beads and Raji cells has been achieved with an efficiency of 99% (beads) and 86% (Raji cells) to a 40 μm wide center region in the 1 mm wide microfluidic channel. This enabled improved optical analysis and reduced bead losses. The loss of beads that ended up in the waste (because printing them as single beads arrangements could not be ensured) was reduced from 52% ± 6% to 28% ± 1%. The piezoelectric transducer employed for cell focusing could be positioned on an outer part of the device, which proves the acoustophoretic focusing to be versatile and adaptable. PMID:25945135

  14. Integral Equation Solution for Biopotentials of Single Cells

    PubMed Central

    Klee, Maurice; Plonsey, Robert

    1972-01-01

    A Fredholm integral equation of the second type is developed for the biopotentials of single cells. Two singularities arise in the numerical solution of this integral equation and methods for handling them are presented. The problem of a spherical cell in an applied uniform field is used to illustrate the technique. PMID:4655666

  15. Inkjet-like printing of single-cells.

    PubMed

    Yusof, Azmi; Keegan, Helen; Spillane, Cathy D; Sheils, Orla M; Martin, Cara M; O'Leary, John J; Zengerle, Roland; Koltay, Peter

    2011-07-21

    Cell sorting and separation techniques are essential tools for cell biology research and for many diagnostic and therapeutic applications. For many of these applications, it is imperative that heterogeneous populations of cells are segregated according to their cell type and that individual cells can be isolated and analysed. We present a novel technique to isolate single cells encapsulated in a picolitre sized droplet that are then deposited by inkjet-like printing at defined locations for downstream genomic analysis. The single-cell-manipulator (SCM) developed for this purpose consists of a dispenser chip to print cells contained in a free flying droplet, a computer vision system to detect single-cells inside the dispenser chip prior to printing, and appropriate automation equipment to print single-cells onto defined locations on a substrate. This technique is spatially dynamic, enabling cell printing on a wide range of commonly used substrates such as microscope slides, membranes and microtiter plates. Demonstration experiments performed using the SCM resulted in a printing efficiency of 87% for polystyrene microbeads of 10 μm size. When the SCM was applied to a cervical cancer cell line (HeLa), a printing efficiency of 87% was observed and a post-SCM cell viability rate of 75% was achieved.

  16. Single-cell genomics for dissection of complex malaria infections

    PubMed Central

    Nair, Shalini; Nkhoma, Standwell C.; Serre, David; Zimmerman, Peter A.; Gorena, Karla; Daniel, Benjamin J.; Nosten, François; Anderson, Timothy J.C.; Cheeseman, Ian H.

    2014-01-01

    Most malaria infections contain complex mixtures of distinct parasite lineages. These multiple-genotype infections (MGIs) impact virulence evolution, drug resistance, intra-host dynamics, and recombination, but are poorly understood. To address this we have developed a single-cell genomics approach to dissect MGIs. By combining cell sorting and whole-genome amplification (WGA), we are able to generate high-quality material from parasite-infected red blood cells (RBCs) for genotyping and next-generation sequencing. We optimized our approach through analysis of >260 single-cell assays. To quantify accuracy, we decomposed mixtures of known parasite genotypes and obtained highly accurate (>99%) single-cell genotypes. We applied this validated approach directly to infections of two major malaria species, Plasmodium falciparum, for which long term culture is possible, and Plasmodium vivax, for which no long-term culture is feasible. We demonstrate that our single-cell genomics approach can be used to generate parasite genome sequences directly from patient blood in order to unravel the complexity of P. vivax and P. falciparum infections. These methods open the door for large-scale analysis of within-host variation of malaria infections, and reveal information on relatedness and drug resistance haplotypes that is inaccessible through conventional sequencing of infections. PMID:24812326

  17. Single cell metastatic phenotyping using pulsed nanomechanical indentations

    NASA Astrophysics Data System (ADS)

    Babahosseini, Hesam; Strobl, Jeannine S.; Agah, Masoud

    2015-09-01

    The existing approach to characterize cell biomechanical properties typically utilizes switch-like models of mechanotransduction in which cell responses are analyzed in response to a single nanomechanical indentation or a transient pulsed stress. Although this approach provides effective descriptors at population-level, at a single-cell-level, there are significant overlaps in the biomechanical descriptors of non-metastatic and metastatic cells which precludes the use of biomechanical markers for single cell metastatic phenotyping. This study presents a new promising marker for biosensing metastatic and non-metastatic cells at a single-cell-level using the effects of a dynamic microenvironment on the biomechanical properties of cells. Two non-metastatic and two metastatic epithelial breast cell lines are subjected to a pulsed stresses regimen exerted by atomic force microscopy. The force-time data obtained for the cells revealed that the non-metastatic cells increase their resistance against deformation and become more stiffened when subjected to a series of nanomechanical indentations. On the other hand, metastatic cells become slightly softened when their mechanical microenvironment is subjected to a similar dynamical changes. This distinct behavior of the non-metastatic and metastatic cells to the pulsed stresses paradigm provided a signature for single-cell-level metastatic phenotyping with a high confidence level of ∼95%.

  18. Modeling genome coverage in single-cell sequencing

    PubMed Central

    Daley, Timothy; Smith, Andrew D.

    2014-01-01

    Motivation: Single-cell DNA sequencing is necessary for examining genetic variation at the cellular level, which remains hidden in bulk sequencing experiments. But because they begin with such small amounts of starting material, the amount of information that is obtained from single-cell sequencing experiment is highly sensitive to the choice of protocol employed and variability in library preparation. In particular, the fraction of the genome represented in single-cell sequencing libraries exhibits extreme variability due to quantitative biases in amplification and loss of genetic material. Results: We propose a method to predict the genome coverage of a deep sequencing experiment using information from an initial shallow sequencing experiment mapped to a reference genome. The observed coverage statistics are used in a non-parametric empirical Bayes Poisson model to estimate the gain in coverage from deeper sequencing. This approach allows researchers to know statistical features of deep sequencing experiments without actually sequencing deeply, providing a basis for optimizing and comparing single-cell sequencing protocols or screening libraries. Availability and implementation: The method is available as part of the preseq software package. Source code is available at http://smithlabresearch.org/preseq. Contact: andrewds@usc.edu Supplementary information: Supplementary material is available at Bioinformatics online. PMID:25107873

  19. Active sensors for health monitoring of aging aerospace structures

    SciTech Connect

    GIURGIUTIU,VICTOR; REDMOND,JAMES M.; ROACH,DENNIS P.; RACKOW,KIRK A.

    2000-02-29

    A project to develop non-intrusive active sensors that can be applied on existing aging aerospace structures for monitoring the onset and progress of structural damage (fatigue cracks and corrosion) is presented. The state of the art in active sensors structural health monitoring and damage detection is reviewed. Methods based on (a) elastic wave propagation and (b) electro-mechanical (E/M) impedance technique are cited and briefly discussed. The instrumentation of these specimens with piezoelectric active sensors is illustrated. The main detection strategies (E/M impedance for local area detection and wave propagation for wide area interrogation) are discussed. The signal processing and damage interpretation algorithms are tuned to the specific structural interrogation method used. In the high-frequency E/M impedance approach, pattern recognition methods are used to compare impedance signatures taken at various time intervals and to identify damage presence and progression from the change in these signatures. In the wave propagation approach, the acousto-ultrasonic methods identifying additional reflection generated from the damage site and changes in transmission velocity and phase are used. Both approaches benefit from the use of artificial intelligence neural networks algorithms that can extract damage features based on a learning process. Design and fabrication of a set of structural specimens representative of aging aerospace structures is presented. Three built-up specimens (pristine, with cracks, and with corrosion damage) are used. The specimen instrumentation with active sensors fabricated at the University of South Carolina is illustrated. Preliminary results obtained with the E/M impedance method on pristine and cracked specimens are presented.

  20. Differential actigraphy for monitoring asymmetry in upper limb motor activities.

    PubMed

    Rabuffetti, M; Meriggi, P; Pagliari, C; Bartolomeo, P; Ferrarin, M

    2016-09-21

    Most applications of accelerometry-based actigraphy require a single sensor, properly located onto the body, to estimate, for example, the level of activity or the energy expenditure. Some approaches adopt a multi-sensor setup to improve those analyses or to classify different types of activity. The specific case of two symmetrically placed actigraphs allowing, by some kind of differential analysis, for the assessment of asymmetric motor behaviors, has been considered in relatively few studies. This article presents a novel method for differential actigraphy, which requires the synchronized measurements of two triaxial accelerometers (programmable eZ430-Chronos, Texas Instruments, USA) placed symmetrically on both wrists. The method involved the definition of a robust epoch-related activity index and its implementation on-board the adopted programmable platform. Finally, the activity recordings from both sensors allowed us to define a novel asymmetry index AR24 h ranging from  -100% (only the left arm moves) to  +100% (only the right arm moves) with null value marking a perfect symmetrical behavior. The accuracy of the AR24 h index was 1.3%. Round-the-clock monitoring on 31 healthy participants (20-79 years old, 10 left handed) provided for the AR24 h reference data (range  -5% to 21%) and a fairly good correlation to the clinical handedness index (r  =  0.66, p  <  0.001). A subset of 20 participants repeated the monitoring one week apart evidencing an excellent test-retest reliability (r  =  0.70, p  <  0.001). Such figures support future applications of the methodology for the study of pathologies involving motor asymmetries, such as in patients with motor hemisyndromes and, in general, for those subjects for whom a quantification of the asymmetry in daily motor performances is required to complement laboratory tests.

  1. Active sensors for health monitoring of aging aerospace structures

    SciTech Connect

    GIURGIUTIU,VICTOR; REDMOND,JAMES M.; ROACH,DENNIS P.; RACKOW,KIRK A.

    2000-03-08

    A project to develop non-intrusive active sensors that can be applied on existing aging aerospace structures for monitoring the onset and progress of structural damage (fatigue cracks and corrosion) is presented. The state of the art in active sensors structural health monitoring and damage detection is reviewed. Methods based on (a) elastic wave propagation and (b) electro-mechanical (NM) impedance technique are sighted and briefly discussed. The instrumentation of these specimens with piezoelectric active sensors is illustrated. The main detection strategies (E/M impedance for local area detection and wave propagation for wide area interrogation) are discussed. The signal processing and damage interpretation algorithms are tuned to the specific structural interrogation method used. In the high-frequency EIM impedance approach, pattern recognition methods are used to compare impedance signatures taken at various time intervals and to identify damage presence and progression from the change in these signatures. In the wave propagation approach, the acoustic-ultrasonic methods identifying additional reflection generated from the damage site and changes in transmission velocity and phase are used. Both approaches benefit from the use of artificial intelligence neural networks algorithms that can extract damage features based on a learning process. Design and fabrication of a set of structural specimens representative of aging aerospace structures is presented. Three built-up specimens, (pristine, with cracks, and with corrosion damage) are used. The specimen instrumentation with active sensors fabricated at the University of South Carolina is illustrated. Preliminary results obtained with the E/M impedance method on pristine and cracked specimens are presented.

  2. Active Volcano Monitoring using a Space-based Hyperspectral Imager

    NASA Astrophysics Data System (ADS)

    Cipar, J. J.; Dunn, R.; Cooley, T.

    2010-12-01

    Active volcanoes occur on every continent, often in close proximity to heavily populated areas. While ground-based studies are essential for scientific research and disaster mitigation, remote sensing from space can provide rapid and continuous monitoring of active and potentially active volcanoes [Ramsey and Flynn, 2004]. In this paper, we report on hyperspectral measurements of Kilauea volcano, Hawaii. Hyperspectral images obtained by the US Air Force TacSat-3/ARTEMIS sensor [Lockwood et al, 2006] are used to obtain estimates of the surface temperatures for the volcano. ARTEMIS measures surface-reflected light in the visible, near-infrared, and short-wave infrared bands (VNIR-SWIR). The SWIR bands are known to be sensitive to thermal radiation [Green, 1996]. For example, images from the NASA Hyperion hyperspectral sensor have shown the extent of wildfires and active volcanoes [Young, 2009]. We employ the methodology described by Dennison et al, (2006) to obtain an estimate of the temperature of the active region of Kilauea. Both day and night-time images were used in the analysis. To improve the estimate, we aggregated neighboring pixels. The active rim of the lava lake is clearly discernable in the temperature image, with a measured temperature exceeding 1100o C. The temperature decreases markedly on the exterior of the summit crater. While a long-wave infrared (LWIR) sensor would be ideal for volcano monitoring, we have shown that the thermal state of an active volcano can be monitored using the SWIR channels of a reflective hyperspectral imager. References: Dennison, Philip E., Kraivut Charoensiri, Dar A. Roberts, Seth H. Peterson, and Robert O. Green (2006). Wildfire temperature and land cover modeling using hyperspectral data, Remote Sens. Environ., vol. 100, pp. 212-222. Green, R. O. (1996). Estimation of biomass fire temperature and areal extent from calibrated AVIRIS spectra, in Summaries of the 6th Annual JPL Airborne Earth Science Workshop, Pasadena, CA

  3. Accelerometer's position independent physical activity recognition system for long-term activity monitoring in the elderly.

    PubMed

    Khan, Adil Mehmood; Lee, Young-Koo; Lee, Sungyoung; Kim, Tae-Seong

    2010-12-01

    Mobility is a good indicator of health status and thus objective mobility data could be used to assess the health status of elderly patients. Accelerometry has emerged as an effective means for long-term physical activity monitoring in the elderly. However, the output of an accelerometer varies at different positions on a subject's body, even for the same activity, resulting in high within-class variance. Existing accelerometer-based activity recognition systems thus require firm attachment of the sensor to a subject's body. This requirement makes them impractical for long-term activity monitoring during unsupervised free-living as it forces subjects into a fixed life pattern and impede their daily activities. Therefore, we introduce a novel single-triaxial-accelerometer-based activity recognition system that reduces the high within-class variance significantly and allows subjects to carry the sensor freely in any pocket without its firm attachment. We validated our system using seven activities: resting (lying/sitting/standing), walking, walking-upstairs, walking-downstairs, running, cycling, and vacuuming, recorded from five positions: chest pocket, front left trousers pocket, front right trousers pocket, rear trousers pocket, and inner jacket pocket. Its simplicity, ability to perform activities unimpeded, and an average recognition accuracy of 94% make our system a practical solution for continuous long-term activity monitoring in the elderly.

  4. Single-cell intracellular nano-pH probes.

    PubMed

    Özel, Rıfat Emrah; Lohith, Akshar; Mak, Wai Han; Pourmand, Nader

    2015-01-01

    Within a large clonal population, such as cancerous tumor entities, cells are not identical, and the differences between intracellular pH levels of individual cells may be important indicators of heterogeneity that could be relevant in clinical practice, especially in personalized medicine. Therefore, the detection of the intracellular pH at the single-cell level is of great importance to identify and study outlier cells. However, quantitative and real-time measurements of the intracellular pH of individual cells within a cell population is challenging with existing technologies, and there is a need to engineer new methodologies. In this paper, we discuss the use of nanopipette technology to overcome the limitations of intracellular pH measurements at the single-cell level. We have developed a nano-pH probe through physisorption of chitosan onto hydroxylated quartz nanopipettes with extremely small pore sizes (~100 nm). The dynamic pH range of the nano-pH probe was from 2.6 to 10.7 with a sensitivity of 0.09 units. We have performed single-cell intracellular pH measurements using non-cancerous and cancerous cell lines, including human fibroblasts, HeLa, MDA-MB-231 and MCF-7, with the pH nanoprobe. We have further demonstrated the real-time continuous single-cell pH measurement capability of the sensor, showing the cellular pH response to pharmaceutical manipulations. These findings suggest that the chitosan-functionalized nanopore is a powerful nano-tool for pH sensing at the single-cell level with high temporal and spatial resolution.

  5. Single cell sequencing reveals heterogeneity within ovarian cancer epithelium and cancer associated stromal cells.

    PubMed

    Winterhoff, Boris J; Maile, Makayla; Mitra, Amit Kumar; Sebe, Attila; Bazzaro, Martina; Geller, Melissa A; Abrahante, Juan E; Klein, Molly; Hellweg, Raffaele; Mullany, Sally A; Beckman, Kenneth; Daniel, Jerry; Starr, Timothy K

    2017-03-01

    The purpose of this study was to determine the level of heterogeneity in high grade serous ovarian cancer (HGSOC) by analyzing RNA expression in single epithelial and cancer associated stromal cells. In addition, we explored the possibility of identifying subgroups based on pathway activation and pre-defined signatures from cancer stem cells and chemo-resistant cells. A fresh, HGSOC tumor specimen derived from ovary was enzymatically digested and depleted of immune infiltrating cells. RNA sequencing was performed on 92 single cells and 66 of these single cell datasets passed quality control checks. Sequences were analyzed using multiple bioinformatics tools, including clustering, principle components analysis, and geneset enrichment analysis to identify subgroups and activated pathways. Immunohistochemistry for ovarian cancer, stem cell and stromal markers was performed on adjacent tumor sections. Analysis of the gene expression patterns identified two major subsets of cells characterized by epithelial and stromal gene expression patterns. The epithelial group was characterized by proliferative genes including genes associated with oxidative phosphorylation and MYC activity, while the stromal group was characterized by increased expression of extracellular matrix (ECM) genes and genes associated with epithelial-to-mesenchymal transition (EMT). Neither group expressed a signature correlating with published chemo-resistant gene signatures, but many cells, predominantly in the stromal subgroup, expressed markers associated with cancer stem cells. Single cell sequencing provides a means of identifying subpopulations of cancer cells within a single patient. Single cell sequence analysis may prove to be critical for understanding the etiology, progression and drug resistance in ovarian cancer. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. Evaluation of activity monitors in manual wheelchair users with paraplegia

    PubMed Central

    Hiremath, Shivayogi V.; Ding, Dan

    2011-01-01

    Objective The aim of this study was to evaluate the performance of SenseWear® (SW) and RT3 activity monitors (AMs) in estimating energy expenditure (EE) in manual wheelchair users (MWUs) with paraplegia for a variety of physical activities. Methods Twenty-four subjects completed four activities including resting, wheelchair propulsion, arm-ergometry exercise, and deskwork. The criterion EE was measured by a K4b2 portable metabolic cart. The EE estimated by the SW and RT3 were compared with the criterion EE by the absolute differences and absolute percentage errors. Intraclass correlations and the Bland and Altman plots were also used to assess the agreements between the two AMs and the metabolic cart. Correlations between the criterion EE and the estimated EE and sensors data from the AMs were evaluated. Results The EE estimation errors for the AMs varied from 24.4 to 125.8% for the SW and from 22.0 to 52.8% for the RT3. The intraclass correlation coefficients (ICCs) between the criterion EE and the EE estimated by the two AMs for each activity and all activities as a whole were considered poor with all the ICCs smaller than 0.75. Except for deskwork, the EE from the SW was more correlated to the criterion EE than the EE from the RT3. Conclusion The results indicate that neither of the AMs is an appropriate tool for quantifying physical activity in MWUs with paraplegia. However, the accuracy of EE estimation could be potentially improved by building new regression models based on wheelchair-related activities. PMID:21528634

  7. Passive and Active Monitoring on a High Performance Research Network.

    SciTech Connect

    Matthews, Warren

    2001-05-0