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Sample records for measuring single-cell oxygen

  1. Single-cell measurement of red blood cell oxygen affinity

    PubMed Central

    Di Caprio, Giuseppe; Stokes, Chris; Higgins, John M.; Schonbrun, Ethan

    2015-01-01

    Oxygen is transported throughout the body by hemoglobin (Hb) in red blood cells (RBCs). Although the oxygen affinity of blood is well-understood and routinely assessed in patients by pulse oximetry, variability at the single-cell level has not been previously measured. In contrast, single-cell measurements of RBC volume and Hb concentration are taken millions of times per day by clinical hematology analyzers, and they are important factors in determining the health of the hematologic system. To better understand the variability and determinants of oxygen affinity on a cellular level, we have developed a system that quantifies the oxygen saturation, cell volume, and Hb concentration for individual RBCs in high throughput. We find that the variability in single-cell saturation peaks at an oxygen partial pressure of 2.9%, which corresponds to the maximum slope of the oxygen–Hb dissociation curve. In addition, single-cell oxygen affinity is positively correlated with Hb concentration but independent of osmolarity, which suggests variation in the Hb to 2,3-diphosphoglycerate (2–3 DPG) ratio on a cellular level. By quantifying the functional behavior of a cellular population, our system adds a dimension to blood cell analysis and other measurements of single-cell variability. PMID:26216973

  2. Metabolic oxygen consumption measurement with a single-cell biosensor after particle microbeam irradiation.

    PubMed

    Xu, Yanping; Zhang, Bo; Messerli, Mark; Randers-Pehrson, Gerhard; Hei, Tom K; Brenner, David J

    2015-03-01

    A noninvasive, self-referencing biosensor/probe system has been integrated into the Columbia University Radiological Research Accelerator Facility Microbeam II end station. A single-cell oxygen consumption measurement has been conducted with this type of oxygen probe in 37° C Krebs-Ringer Bicarbonate buffer immediately before and after a single-cell microbeam irradiation. It is the first such measurement made for a microbeam irradiation, and a six fold increment of oxygen flux induced during a 15-s period of time has been observed following radiation exposure. The experimental procedure and the results are discussed.

  3. A microwell array device capable of measuring single-cell oxygen consumption rates

    PubMed Central

    Molter, Timothy W.; McQuaide, Sarah C.; Suchorolski, Martin T.; Strovas, Tim J.; Burgess, Lloyd W.; Meldrum, Deirdre R.; Lidstrom, Mary E.

    2009-01-01

    Due to interest in cell population heterogeneity, the development of new technology and methodologies for studying single cells has dramatically increased in recent years. The ideal single cell measurement system would be high throughput for statistical relevance, would measure the most important cellular parameters, and minimize disruption of normal cell function. We have developed a microwell array device capable of measuring single cell oxygen consumption rates (OCR). This OCR device is able to diffusionally isolate single cells and enables the quantitative measurement of oxygen consumed by a single cell with fmol/min resolution in a non-invasive and relatively high throughput manner. A glass microwell array format containing fixed luminescent sensors allows for future incorporation of additional cellular parameter sensing capabilities. To demonstrate the utility of the OCR device, we determined the oxygen consumption rates of a small group of single cells (12 to 18) for three different cells lines: murine macrophage cell line RAW264.7, human epithelial lung cancer cell line A549, and human Barrett’s esophagus cell line CP-D. PMID:20084089

  4. A New Approach for Measuring Single-Cell Oxygen Consumption Rates

    PubMed Central

    Molter, Timothy W.; McQuaide, Sarah C.; Holl, Mark R.; Meldrum, Deirdre R.; Dragavon, Joseph M.; Anderson, Judith B.; Young, A. Cody; Burgess, Lloyd W.; Lidstrom, Mary E.

    2010-01-01

    A novel system that has enabled the measurement of single-cell oxygen consumption rates is presented. The experimental apparatus includes a temperature controlled environmental chamber, an array of microwells etched in glass, and a lid actuator used to seal cells in the microwells. Each microwell contains an oxygen sensitive platinum phosphor sensor used to monitor the cellular metabolic rates. Custom automation software controls the digital image data collection for oxygen sensor measurements, which are analyzed using an image-processing program to yield the oxygen concentration within each microwell versus time. Two proof-of-concept experiments produced oxygen consumption rate measurements for A549 human epithelial lung cancer cells of 5.39 and 5.27 fmol/min/cell, closely matching published oxygen consumption rates for bulk A549 populations. PMID:21057593

  5. Advances in measuring single-cell pharmacology in vivo.

    PubMed

    Vinegoni, Claudio; Dubach, J Matthew; Thurber, Greg M; Miller, Miles A; Mazitschek, Ralph; Weissleder, Ralph

    2015-09-01

    Measuring key pharmacokinetic and pharmacodynamic parameters in vivo at the single cell level is likely to enhance drug discovery and development. In this review, we summarize recent advances in this field and highlight current and future capabilities. PMID:26024776

  6. Propellant production on Mars - Single cell oxygen production test bed

    NASA Technical Reports Server (NTRS)

    Colvin, James; Schallhorn, Paul; Ramohalli, Kumar

    1991-01-01

    A study focusing on oxygen production using resources indigenous to Mars is presented. A bank of solid zirconia electrolytic cells that will electrochemically separate oxygen from a high temperature stream of carbon dioxide is at the center of the oxygen production system. The experimental data are discussed with attention given to the cell operating temperature, the carbon dioxide flow rate, and the voltage applied across the cell.

  7. A cellular isolation system for real-time single-cell oxygen consumption monitoring

    PubMed Central

    Dragavon, Joe; Molter, Tim; Young, Cody; Strovas, Tim; McQuaide, Sarah; Holl, Mark; Zhang, Meng; Cookson, Brad; Jen, Alex; Lidstrom, Mary; Meldrum, Deirdre; Burgess, Lloyd

    2008-01-01

    The development of a cellular isolation system (CIS) that enables the monitoring of single-cell oxygen consumption rates in real time is presented. The CIS was developed through a multidisciplinary effort within the Microscale Life Sciences Center (MLSC) at the University of Washington. The system comprises arrays of microwells containing Pt-porphyrin-embedded polystyrene microspheres as the reporter chemistry, a lid actuator system and a gated intensified imaging camera, all mounted on a temperature-stabilized confocal microscope platform. Oxygen consumption determination experiments were performed on RAW264.7 mouse macrophage cells as proof of principle. Repeatable and consistent measurements indicate that the oxygen measurements did not adversely affect the physiological state of the cells measured. The observation of physiological rates in real time allows studies of cell-to-cell heterogeneity in oxygen consumption rate to be performed. Such studies have implications in understanding the role of mitochondrial function in the progression of inflammatory-based diseases, and in diagnosing and treating such diseases. PMID:18522927

  8. Development of Nano/Micro Probes for Femtoliter Volume and Single Cell Measurements

    NASA Astrophysics Data System (ADS)

    Gao, Yang

    Single cell analysis has recently emerged as an important field of biomedical re- search. It is now clear that heterogeneity of cell metabolism functions in complex biological systems is correlated to changes in biological function and disease processes. A variety of nano/micro probes were developed to enable investigation of cells properties such as membrane stiffness, pH value. However, very few designs were focused on single cell metabolic function studies. There is a critical need for technologies that provide analysis of heterogeneity of cell metabolic functions, especially on metabolism. Nevertheless, the few existing approaches suffer from fundamental defects and need to be improved. This work focused on developing nano/micro probes that are suitable for single cell functionality investigation. Both types of probes are designed to measure cell-to-cell/time-to-time heterogeneity in metabolic functions over a long period of time. Lab-made carbon nanoprobes were developed especially for electro-physiological measurement. The unique structure of the carbon nanoprobes makes them suitable for important intracellular applications like trans-membrane potential measurements and various electrochemical measurement for cell function studies. While it is important of have ability to carry out intracellular measure, there are also occasions where the information of a cell as a whole is collected. One of the most important indicator of a cells metabolic functions is cell respiration rate/oxygen consumption rate. A micro-perfusion based multi-functional single cell sensing probe was the developed to carry out measurements on cell as a whole. Formed by a double-barrel theta pipette, the perfusion flow enables the direct measurement of the metabolic flux for example oxygen consumption rate. In conclusion, this work developed nano/micro-probes as novel single cell investigation tools. The data acquired from these tools could provide valuable assistance on applications

  9. Microtrap electrode devices for single cell trapping and impedance measurement.

    PubMed

    Mondal, D; Roychaudhuri, C; Das, L; Chatterjee, J

    2012-10-01

    This paper reports the design and fabrication of electrode microtraps for single cell trapping and impedance measurement. In this work, the microtrap electrodes of parallel and elliptical geometry have been fabricated by electroplating of gold electrodes of optimum thickness. This has enabled the formation of electrode traps without requiring any precision alignment between separate insulating traps like PDMS and the bottom gold electrodes. Further the improved uniformity of the electric field between the trapping electrodes as observed from COVENTORWARE simulation significantly reduces the effect of cell position inside the microwell on the electrical measurement unlike previous reports. This makes it possible to directly extract the equivalent cell parameters from the electrical measurement without introducing any correction factor corresponding to cell position. We have performed impedance spectroscopy with both the microwell electrode structures with single HeLa cell at two different positions of trapping. It has been observed that there is almost no change in the extracted values of cell resistance and capacitance for different positions within parallel electrodes and there is only 0.7 % and 0.85 % change in cell resistance and capacitance for the two positions within elliptical electrodes. Thus these microwell electrode structures can be used as an improved and a more convenient platform for single cell electrical characterization. PMID:22767244

  10. Reactive oxygen species production in single cells following laser irradiation (Presentation Recording)

    NASA Astrophysics Data System (ADS)

    Duquette, Michelle L.; Kim, Justine; Shi, Linda Z.; Berns, Michael W.

    2015-08-01

    Region specific DNA breaks can be created in single cells using laser light that damages DNA but does not directly generate reactive oxygen species (ROS). We have examined the cellular response to directly generated DNA breaks in single cells. Using a combination of ROS specific dyes and oxidase inhibitors we have found that the oxidase and chromatin remodeling protein Lysine demethylase I (LSD1) generates detectable ROS as a byproduct of its chromatin remodeling activity during the initial DNA damage response. ROS is produced at detectable amounts primarily within the first 3 minutes post irradiation. LSD1 activity has been previously associated with transcriptional regulation therefore these findings have implications for regulation of gene expression following DNA damage particularly in cells with altered redox states.

  11. A combined electrochemical and optical trapping platform for measuring single cell respiration rates at electrode interfaces

    NASA Astrophysics Data System (ADS)

    Gross, Benjamin J.; El-Naggar, Mohamed Y.

    2015-06-01

    Metal-reducing bacteria gain energy by extracellular electron transfer to external solids, such as naturally abundant minerals, which substitute for oxygen or the other common soluble electron acceptors of respiration. This process is one of the earliest forms of respiration on earth and has significant environmental and technological implications. By performing electron transfer to electrodes instead of minerals, these microbes can be used as biocatalysts for conversion of diverse chemical fuels to electricity. Understanding such a complex biotic-abiotic interaction necessitates the development of tools capable of probing extracellular electron transfer down to the level of single cells. Here, we describe an experimental platform for single cell respiration measurements. The design integrates an infrared optical trap, perfusion chamber, and lithographically fabricated electrochemical chips containing potentiostatically controlled transparent indium tin oxide microelectrodes. Individual bacteria are manipulated using the optical trap and placed on the microelectrodes, which are biased at a suitable oxidizing potential in the absence of any chemical electron acceptor. The potentiostat is used to detect the respiration current correlated with cell-electrode contact. We demonstrate the system with single cell measurements of the dissimilatory-metal reducing bacterium Shewanella oneidensis MR-1, which resulted in respiration currents ranging from 15 fA to 100 fA per cell under our measurement conditions. Mutants lacking the outer-membrane cytochromes necessary for extracellular respiration did not result in any measurable current output upon contact. In addition to the application for extracellular electron transfer studies, the ability to electronically measure cell-specific respiration rates may provide answers for a variety of fundamental microbial physiology questions.

  12. A combined electrochemical and optical trapping platform for measuring single cell respiration rates at electrode interfaces

    SciTech Connect

    Gross, Benjamin J.; El-Naggar, Mohamed Y.

    2015-06-15

    Metal-reducing bacteria gain energy by extracellular electron transfer to external solids, such as naturally abundant minerals, which substitute for oxygen or the other common soluble electron acceptors of respiration. This process is one of the earliest forms of respiration on earth and has significant environmental and technological implications. By performing electron transfer to electrodes instead of minerals, these microbes can be used as biocatalysts for conversion of diverse chemical fuels to electricity. Understanding such a complex biotic-abiotic interaction necessitates the development of tools capable of probing extracellular electron transfer down to the level of single cells. Here, we describe an experimental platform for single cell respiration measurements. The design integrates an infrared optical trap, perfusion chamber, and lithographically fabricated electrochemical chips containing potentiostatically controlled transparent indium tin oxide microelectrodes. Individual bacteria are manipulated using the optical trap and placed on the microelectrodes, which are biased at a suitable oxidizing potential in the absence of any chemical electron acceptor. The potentiostat is used to detect the respiration current correlated with cell-electrode contact. We demonstrate the system with single cell measurements of the dissimilatory-metal reducing bacterium Shewanella oneidensis MR-1, which resulted in respiration currents ranging from 15 fA to 100 fA per cell under our measurement conditions. Mutants lacking the outer-membrane cytochromes necessary for extracellular respiration did not result in any measurable current output upon contact. In addition to the application for extracellular electron transfer studies, the ability to electronically measure cell-specific respiration rates may provide answers for a variety of fundamental microbial physiology questions.

  13. A combined electrochemical and optical trapping platform for measuring single cell respiration rates at electrode interfaces.

    PubMed

    Gross, Benjamin J; El-Naggar, Mohamed Y

    2015-06-01

    Metal-reducing bacteria gain energy by extracellular electron transfer to external solids, such as naturally abundant minerals, which substitute for oxygen or the other common soluble electron acceptors of respiration. This process is one of the earliest forms of respiration on earth and has significant environmental and technological implications. By performing electron transfer to electrodes instead of minerals, these microbes can be used as biocatalysts for conversion of diverse chemical fuels to electricity. Understanding such a complex biotic-abiotic interaction necessitates the development of tools capable of probing extracellular electron transfer down to the level of single cells. Here, we describe an experimental platform for single cell respiration measurements. The design integrates an infrared optical trap, perfusion chamber, and lithographically fabricated electrochemical chips containing potentiostatically controlled transparent indium tin oxide microelectrodes. Individual bacteria are manipulated using the optical trap and placed on the microelectrodes, which are biased at a suitable oxidizing potential in the absence of any chemical electron acceptor. The potentiostat is used to detect the respiration current correlated with cell-electrode contact. We demonstrate the system with single cell measurements of the dissimilatory-metal reducing bacterium Shewanella oneidensis MR-1, which resulted in respiration currents ranging from 15 fA to 100 fA per cell under our measurement conditions. Mutants lacking the outer-membrane cytochromes necessary for extracellular respiration did not result in any measurable current output upon contact. In addition to the application for extracellular electron transfer studies, the ability to electronically measure cell-specific respiration rates may provide answers for a variety of fundamental microbial physiology questions.

  14. Single-cell-based computer simulation of the oxygen-dependent tumour response to irradiation

    NASA Astrophysics Data System (ADS)

    Harting, Christine; Peschke, Peter; Borkenstein, Klaus; Karger, Christian P.

    2007-08-01

    Optimization of treatment plans in radiotherapy requires the knowledge of tumour control probability (TCP) and normal tissue complication probability (NTCP). Mathematical models may help to obtain quantitative estimates of TCP and NTCP. A single-cell-based computer simulation model is presented, which simulates tumour growth and radiation response on the basis of the response of the constituting cells. The model contains oxic, hypoxic and necrotic tumour cells as well as capillary cells which are considered as sources of a radial oxygen profile. Survival of tumour cells is calculated by the linear quadratic model including the modified response due to the local oxygen concentration. The model additionally includes cell proliferation, hypoxia-induced angiogenesis, apoptosis and resorption of inactivated tumour cells. By selecting different degrees of angiogenesis, the model allows the simulation of oxic as well as hypoxic tumours having distinctly different oxygen distributions. The simulation model showed that poorly oxygenated tumours exhibit an increased radiation tolerance. Inter-tumoural variation of radiosensitivity flattens the dose response curve. This effect is enhanced by proliferation between fractions. Intra-tumoural radiosensitivity variation does not play a significant role. The model may contribute to the mechanistic understanding of the influence of biological tumour parameters on TCP. It can in principle be validated in radiation experiments with experimental tumours.

  15. Microfluidic devices for measuring gene network dynamics in single cells

    PubMed Central

    Bennett, Matthew R.; Hasty, Jeff

    2010-01-01

    The dynamics governing gene regulation have an important role in determining the phenotype of a cell or organism. From processing extracellular signals to generating internal rhythms, gene networks are central to many time-dependent cellular processes. Recent technological advances now make it possible to track the dynamics of gene networks in single cells under various environmental conditions using microfluidic ‘lab-on-a-chip’ devices, and researchers are using these new techniques to analyse cellular dynamics and discover regulatory mechanisms. These technologies are expected to yield novel insights and allow the construction of mathematical models that more accurately describe the complex dynamics of gene regulation. PMID:19668248

  16. Low-pressure airlift fermenter for single cell protein production. I. Design and oxygen transfer studies

    SciTech Connect

    Chen, N.Y.; Kondis, E.F.; Srinivasan, S.

    1987-03-01

    The energy consumption of a fermenter constitutes a major part of the operating expense of a single cell protein process. A low-pressure airlift fermenter was designed to reduce this cost. In this new design, the fermenter broth is kept below 120 cm in depth, and air alone is employed to fulfil the need of supplying oxygen, and cooling and agitating the broth. The use of low-pressure air from air blowers instead of air compressors lowers the capital cost of air delivery and reduces the energy consumption in the fermenter section to below 1 kWh/kg protein, a saving of over 70% as compared to a conventional stirred tank fermenter. It also eliminates the investment of mechanical agitators, heat exchangers, and air compressors. Sulfite oxidation studies confirmed the design concepts. 30 references.

  17. Single Cell Magnetic Measurements with a Superconducting Quantum Interference Device

    NASA Astrophysics Data System (ADS)

    Palmstrom, Johanna C.; Arps, Jennifer; Dwyer, Bo; Kalisky, Beena; Kirtley, John R.; Moler, Kathryn A.; Qian, Lisa C.; Rosenberg, Aaron J.; Rutt, Brian; Tee, Sui Seng; Theis, Eric; Urbach, Elana; Wang, Yihua

    2014-03-01

    Magnetic nanoparticles play an important role in numerous biomedical applications such as magnetic resonance imaging and targeted drug delivery. There is a need for tools to characterize individual magnetic nanoparticles and the magnetic properties of individual cells. We use a scanning superconducting quantum interference device (SQUID) to observe the magnetic fields from single mammalian cells loaded with superparamagnetic iron oxide nanoparticles. We show that the SQUID is a useful tool for imaging biological magnetism and is capable of resolving cell to cell variations in magnetic dipole moments. We hope to correlate these magnetic images with real space imaging techniques such as optical and scanning electron microscopy. The visualization of single cell magnetism can be used to optimize biological magnetic imaging techniques, such as MRI, by quantifying the strength of magnetic dipole moments of in vitro magnetic labeling. This work is supported by a National Science Foundation Graduate Research Fellowship and a Gabilan Stanford Graduate Fellowship.

  18. Single cell measurements of vacuolar rupture caused by intracellular pathogens.

    PubMed

    Keller, Charlotte; Mellouk, Nora; Danckaert, Anne; Simeone, Roxane; Brosch, Roland; Enninga, Jost; Bobard, Alexandre

    2013-06-12

    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.

  19. 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

  20. DESIGN, PROTOTYPE AND MEASUREMENT OF A SINGLE-CELL DEFLECTING CAVITY FOR THE ADVANCED PHOTON SOURCE

    SciTech Connect

    Haipeng Wang, Guangfeng Cheng, Gianluigi Ciovati, Peter Kneisel, Robert Rimmer, Kai Tian, Larry Turlington, Alireza Nassiri, Geoff Waldschmidt

    2009-05-01

    After the design optimization of a squashed elliptical shape, single-cell, superconducting (SC) deflecting cavity at 2.815 GHz, a copper prototype has been bench measured to determine its rf properties and the effectiveness of waveguide damping of parasitic modes [1]. RF cold tests were also performed at 2K on niobium single-cell and two-cell prototype cavities. Details of impedance calculation using wakefiled analysis of the single-cell cavity are shown to meet the strict 200 mA beam stability requirement of the Advanced Photon Source (APS) at Argonne National Lab where a total of 16 single-cell cavities will be divided into two cryomodule. The design of higher-order mode (HOM) waveguide damping, the simulations of the Lorenz force detuning, and the prototype of on-cell damping are presented.

  1. Single Cell Mass Measurement Using Drag Force Inside Lab-on-Chip Microfluidics System.

    PubMed

    Rahman, Md Habibur; Ahmad, Mohd Ridzuan; Takeuchi, Masaru; Nakajima, Masahiro; Hasegawa, Yasuhisa; Fukuda, Toshio

    2015-12-01

    Single cell mass (SCM) is an intrinsic property of single cell, it arouses a great interest among scientists as cell mass depends on the synthesis of proteins, DNA replication, cell wall stiffness, cell cytoplasm density, cell growth, ribosome, and other analogous of organisms. To date, several great strides have been taken to the advancements of SCM measurement techniques. Nevertheless, more works are required to enable the technology to push frontier in deep analysis of SCM measurement, hence to elucidate intracellular properties. In this paper, we present a lab-on-chip microfluidics system for SCM measurement, related with the force required to drag a single cell and Newton's law of motion inside microfluidics channel. Drag force on the cell was generated by a pressure driven syringe micropump and the motion of the cell was measured using optical observation under an inverted microscope. This approach of measuring SCM was calibrated using known mass (77.3 pg) of a polystyrene particle of 5.2 μm diameter. Furthermore, we used Saccharomyces cerevisiae baker's yeast cells of different sizes ([Formula: see text] diameter) for SCM measurement. Mass of 4.4 μm diameter of single yeast cell was measured as 2.12 pg which is in the range of previously reported single yeast cell mass (2-3 pg). In addition, we also studied the relation between SCM and single cell size. Results showed that single yeast cell mass increases exponentially with the increasing of single cell size.

  2. Single Cell Mass Measurement Using Drag Force Inside Lab-on-Chip Microfluidics System.

    PubMed

    Rahman, Md Habibur; Ahmad, Mohd Ridzuan; Takeuchi, Masaru; Nakajima, Masahiro; Hasegawa, Yasuhisa; Fukuda, Toshio

    2015-12-01

    Single cell mass (SCM) is an intrinsic property of single cell, it arouses a great interest among scientists as cell mass depends on the synthesis of proteins, DNA replication, cell wall stiffness, cell cytoplasm density, cell growth, ribosome, and other analogous of organisms. To date, several great strides have been taken to the advancements of SCM measurement techniques. Nevertheless, more works are required to enable the technology to push frontier in deep analysis of SCM measurement, hence to elucidate intracellular properties. In this paper, we present a lab-on-chip microfluidics system for SCM measurement, related with the force required to drag a single cell and Newton's law of motion inside microfluidics channel. Drag force on the cell was generated by a pressure driven syringe micropump and the motion of the cell was measured using optical observation under an inverted microscope. This approach of measuring SCM was calibrated using known mass (77.3 pg) of a polystyrene particle of 5.2 μm diameter. Furthermore, we used Saccharomyces cerevisiae baker's yeast cells of different sizes ([Formula: see text] diameter) for SCM measurement. Mass of 4.4 μm diameter of single yeast cell was measured as 2.12 pg which is in the range of previously reported single yeast cell mass (2-3 pg). In addition, we also studied the relation between SCM and single cell size. Results showed that single yeast cell mass increases exponentially with the increasing of single cell size. PMID:26761952

  3. Heat conduction nanocalorimeter for pl-scale single cell measurements

    NASA Astrophysics Data System (ADS)

    Johannessen, E. A.; Weaver, J. M. R.; Cobbold, P. H.; Cooper, J. M.

    2002-03-01

    An ultrasensitive nanocalorimeter for use with pl-scale biological samples using silicon microfabrication technology has been developed in which a 720 pl reaction vessel, a calibration heater, and a thermoelectric transducer of 125 μK sensitivity were integrated into a single multilayer thin-film configuration. The resolution of the system ranged from 10 to 25 nW depending on the heat capacity, conductance and power density of the samples studied. The device has been used in heat conduction measurements of the energy released from the enzyme catalyzed hydrolysis of hydrogen peroxide using purified catalase, and for the determination of the catalase activity within a single mouse hepatocyte. The nanocalorimeter has the potential for integration in a high-density array format, where the change in temperature from ultralow volume cellular assays could be used as a generic analytical tool for high throughput screening of bioactive compounds.

  4. Kinetics of small molecule interactions with membrane proteins in single cells measured with mechanical amplification

    PubMed Central

    Guan, Yan; Shan, Xiaonan; Zhang, Fenni; Wang, Shaopeng; Chen, Hong-Yuan; Tao, Nongjian

    2015-01-01

    Measuring small molecule interactions with membrane proteins in single cells is critical for understanding many cellular processes and for screening drugs. However, developing such a capability has been a difficult challenge. We show that molecular interactions with membrane proteins induce a mechanical deformation in the cellular membrane, and real-time monitoring of the deformation with subnanometer resolution allows quantitative analysis of small molecule–membrane protein interaction kinetics in single cells. This new strategy provides mechanical amplification of small binding signals, making it possible to detect small molecule interactions with membrane proteins. This capability, together with spatial resolution, also allows the study of the heterogeneous nature of cells by analyzing the interaction kinetics variability between different cells and between different regions of a single cell. PMID:26601298

  5. Noninvasive measurement of hydrogen and potassium ion flux from single cells and epithelial structures.

    PubMed

    Smith, P J; Trimarchi, J

    2001-01-01

    This review introduces new developments in a technique for measuring the movement of ions across the plasma membrane. With the use of a self-referencing ion-selective (Seris) probe, transport mechanisms can be studied on a variety of preparations ranging from tissues to single cells. In this paper we illustrate this versatility with examples from the vas deferens and inner ear epithelium to large and small single cells represented by mouse single-cell embryos and rat microglia. Potassium and hydrogen ion fluxes are studied and pharmacological manipulation of the signals are reported. The strengths of the self-referencing technique are reviewed with regard to biological applications, and the expansion of self-referencing probes to include electrochemical and enzyme-based sensors is discussed.

  6. Single Cell Spectroscopy: Noninvasive Measures of Small-Scale Structure and Function

    PubMed Central

    Mousoulis, Charilaos; Xu, Xin; Reiter, David A.; Neu, Corey P.

    2013-01-01

    The advancement of spectroscopy methods attained through increases in sensitivity, and often with the coupling of complementary techniques, has enabled real-time structure and function measurements of single cells. The purpose of this review is to illustrate, in light of advances, the strengths and the weaknesses of these methods. Included also is an assessment of the impact of the experimental setup and conditions of each method on cellular function and integrity. A particular emphasis is placed on noninvasive and nondestructive techniques for achieving single cell detection, including nuclear magnetic resonance, in addition to physical, optical, and vibrational methods. PMID:23886910

  7. DNA-Based Sensor Particles Enable Measuring Light Intensity in Single Cells.

    PubMed

    Mikutis, Gediminas; Mora, Carlos A; Puddu, Michela; Paunescu, Daniela; Grass, Robert N; Stark, Wendelin J

    2016-04-13

    "Lab on a particle" architecture is employed in designing a light nanosensor. Light-sensitive protecting groups are installed on DNA, which is encapsulated in silica particles, qualifying as a self-sufficient light sensor. The nanosensors allow measuring light intensity and duration in very small volumes, such as single cells, and store the irradiation information until readout.

  8. A microchip integrating cell array positioning with in situ single-cell impedance measurement.

    PubMed

    Guo, Xiaoliang; Zhu, Rong; Zong, Xianli

    2015-10-01

    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. PMID:26282920

  9. A microchip integrating cell array positioning with in situ single-cell impedance measurement.

    PubMed

    Guo, Xiaoliang; Zhu, Rong; Zong, Xianli

    2015-10-01

    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.

  10. Measuring tissue oxygenation

    NASA Technical Reports Server (NTRS)

    Soyemi, Olusola O. (Inventor); Soller, Babs R. (Inventor); Yang, Ye (Inventor)

    2009-01-01

    Methods and systems for calculating tissue oxygenation, e.g., oxygen saturation, in a target tissue are disclosed. In some embodiments, the methods include: (a) directing incident radiation to a target tissue and determining reflectance spectra of the target tissue by measuring intensities of reflected radiation from the target tissue at a plurality of radiation wavelengths; (b) correcting the measured intensities of the reflectance spectra to reduce contributions thereto from skin and fat layers through which the incident radiation propagates; (c) determining oxygen saturation in the target tissue based on the corrected reflectance spectra; and (d) outputting the determined value of oxygen saturation.

  11. Evaluating quantitative methods for measuring plasmid copy numbers in single cells

    PubMed Central

    Tal, Shay; Paulsson, Johan

    2013-01-01

    The life of plasmids is a constant battle against fluctuations: failing to correct copy number fluctuations can increase the plasmid loss rate by many orders of magnitude, as can a failure to more evenly divide the copies between daughters at cell division. Plasmids are therefore long-standing model systems for stochastic processes in cells, much thanks to the efforts of Kurt Nordström to whose memory this issue is dedicated. Here we analyze a range of experimental methods for measuring plasmid copy numbers in single cells, focusing on challenges, trade-offs and necessary experimental controls. In particular we analyze published and unpublished strategies to infer copy numbers from expression of plasmid-encoded reporters, direct labeling of plasmids with fluorescent probes or DNA binding proteins fused to fluorescent reporters, PCR based methods applied to single cell lysates, and plasmid-specific replication arrest. We conclude that no method currently exists to measure plasmid copy numbers in single cells, and that most methods instead inadvertently measure various types of experimental noise. We also discuss how accurate methods can be developed. PMID:22305922

  12. Sensitive thermal microsensor with pn junction for heat measurement of a single cell

    NASA Astrophysics Data System (ADS)

    Yamada, Taito; Inomata, Naoki; Ono, Takahito

    2016-02-01

    A sensitive thermal microsensor based on a pn junction diode for heat measurements of biological single cells is developed and evaluated. Using a fabricated device, we demonstrated the heat measurement of a single brown fat cell. The principle of the sensor relies on the temperature dependence of the pn junction diode resistance. This method has a capability of the highly thermal sensitivity by downsizing and the advantage of a simple experimental setup using electrical circuits without any special equipment. To achieve highly sensitive heat measurement of single cells, downsizing of the sensor is necessary to reduce the heat capacity of the sensor itself. The sensor with the pn junction diode can be downsized by microfabrication. A bridge beam structure with the pn junction diode as a thermal sensor is placed in vacuum using a microfludic chip to decrease the heat loss to the surroundings. A temperature coefficient of resistance of 1.4%/K was achieved. The temperature and thermal resolutions of the fabricated device are 1.1 mK and 73.6 nW, respectively. The heat measurements of norepinephrine stimulated and nonstimulated single brown fat cells were demonstrated, and different behaviors in heat generation were observed.

  13. Robust organelle size extractions from elastic scattering measurements of single cells (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Cannaday, Ashley E.; Draham, Robert; Berger, Andrew J.

    2016-04-01

    The goal of this project is to estimate non-nuclear organelle size distributions in single cells by measuring angular scattering patterns and fitting them with Mie theory. Simulations have indicated that the large relative size distribution of organelles (mean:width≈2) leads to unstable Mie fits unless scattering is collected at polar angles less than 20 degrees. Our optical system has therefore been modified to collect angles down to 10 degrees. Initial validations will be performed on polystyrene bead populations whose size distributions resemble those of cell organelles. Unlike with the narrow bead distributions that are often used for calibration, we expect to see an order-of-magnitude improvement in the stability of the size estimates as the minimum angle decreases from 20 to 10 degrees. Scattering patterns will then be acquired and analyzed from single cells (EMT6 mouse cancer cells), both fixed and live, at multiple time points. Fixed cells, with no changes in organelle sizes over time, will be measured to determine the fluctuation level in estimated size distribution due to measurement imperfections alone. Subsequent measurements on live cells will determine whether there is a higher level of fluctuation that could be attributed to dynamic changes in organelle size. Studies on unperturbed cells are precursors to ones in which the effects of exogenous agents are monitored over time.

  14. Apparatus and method for measuring single cell and sub-cellular photosynthetic efficiency

    DOEpatents

    Davis, Ryan Wesley; Singh, Seema; Wu, Huawen

    2013-07-09

    Devices for measuring single cell changes in photosynthetic efficiency in algal aquaculture are disclosed that include a combination of modulated LED trans-illumination of different intensities with synchronized through objective laser illumination and confocal detection. Synchronization and intensity modulation of a dual illumination scheme were provided using a custom microcontroller for a laser beam block and constant current LED driver. Therefore, single whole cell photosynthetic efficiency, and subcellular (diffraction limited) photosynthetic efficiency measurement modes are permitted. Wide field rapid light scanning actinic illumination is provided for both by an intensity modulated 470 nm LED. For the whole cell photosynthetic efficiency measurement, the same LED provides saturating pulses for generating photosynthetic induction curves. For the subcellular photosynthetic efficiency measurement, a switched through objective 488 nm laser provides saturating pulses for generating photosynthetic induction curves. A second near IR LED is employed to generate dark adapted states in the system under study.

  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. PMID:26682814

  16. 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.

  17. 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.

  18. Ultrasonic Scattering Measurements of a Live Single Cell at 86 MHz

    PubMed Central

    Lee, Changyang; Jung, Hayong; Lam, Kwok Ho; Yoon, Changhan; Shung, K. Kirk

    2016-01-01

    Cell separation and sorting techniques have been employed biomedical applications such as cancer diagnosis and cell gene expression analysis. The capability to accurately measure ultrasonic scattering properties from cells is crucial in making an ultrasonic cell sorter a reality if ultrasound scattering is to be used as the sensing mechanism as well. To assess the performance of sensing and identifying live single cells with high-frequency ultrasound, an 86-MHz lithium niobate press-focused single-element acoustic transducer was used in a high-frequency ultrasound scattering measurement system that was custom designed and developed for minimizing noise and allowing better mobility. Peak-to-peak echo amplitude, integrated backscatter (IB) coefficient, spectral parameters including spectral slope and intercept, and midband fit from spectral analysis of the backscattered echoes were measured and calculated from a live single cell of two different types on an agar surface: leukemia cells (K562 cells) and red blood cells (RBCs). The amplitudes of echo signals from K562 cells and RBCs were 48.25 ± 11.98 mVpp and 56.97 ± 7.53 mVpp, respectively. The IB coefficient was −89.39 ± 2.44 dB for K562 cells and −89.00 ± 1.19 dB for RBCs. The spectral slope and intercept were 0.30 ± 0.19 dB/MHz and −56.07 ± 17.17 dB, respectively, for K562 cells and 0.78 ± 0.092 dB/MHz and −98.18 ± 8.80 dB, respectively, for RBCs. Midband fits of K562 cells and RBCs were −31.02 ± 3.04 dB and −33.51 ± 1.55 dB, respectively. Acoustic cellular discrimination via these parameters was tested by Student’s t-test. Their values, except for the IB value, showed statistically significant difference (p < 0.001). This paper reports for the first time that ultrasonic scattering measurements can be made on a live single cell with a highly focused high-frequency ultrasound microbeam at 86 MHz. These results also suggest the feasibility of ultrasonic scattering as a sensing mechanism in

  19. Quantitative photoacoustics to measure single cell melanin production and nanoparticle attachment

    NASA Astrophysics Data System (ADS)

    Bhattacharyya, Kiran; Eshein, Adam; Chandrasekhar, Anand; Viator, John A.

    2015-04-01

    Photoacoustics can be used as a label-free spectroscopic method of identifying pigmented proteins and characterizing their intracellular concentration over time in a single living cell. The authors use a microscopic laser irradiation system with a 5 ns, Q-switched laser focused onto single cells in order to collect photoacoustic responses of melanoma cells from the HS936 cell line and gold nanoparticle labeled breast cancer cells from the T47D cell line. The volume averaged intracellular concentration of melanin is found to range from 29-270 mM for single melanoma cells and the number of gold nanoparticles (AuNP) is shown to range from 850-5900 AuNPs/cell. Additionally, the melanin production response to UV-A light stimulus is measured in four melanoma cells to find a mass production rate of 5.7 pg of melanin every 15 min.

  20. Quantitative photoacoustics to measure single cell melanin production and nanoparticle attachment

    PubMed Central

    Bhattacharyya, Kiran; Eshein, Adam; Chandrasekhar, Anand; Viator, John A.

    2015-01-01

    Photoacoustics can be used as a label-free spectroscopic method of identifying pigmented proteins and characterizing their intracellular concentration over time in a single living cell. The authors use a microscopic laser irradiation system with a 5 ns, Q-switched laser focused onto single cells in order to collect photoacoustic responses of melanoma cells from the HS936 cell line and gold nanoparticle labeled breast cancer cells from the T47D cell line. The volume averaged intracellular concentration of melanin is found to range from 29–270mM for single melanoma cells and the number of gold nanoparticles (AuNP) is shown to range from 850–5900 AuNPs/cell. Additionally, the melanin production response to UV-A light stimulus is measured in four melanoma cells to find a mass production rate of 5.7 pg of melanin every 15 minutes. PMID:25803095

  1. PolyMUMPs MEMS device to measure mechanical stiffness of single cells in aqueous media

    NASA Astrophysics Data System (ADS)

    Warnat, S.; King, H.; Forbrigger, C.; Hubbard, T.

    2015-02-01

    A method of experimentally determining the mechanical stiffness of single cells by using differential displacement measurements in a two stage spring system is presented. The spring system consists of a known MEMS reference spring and an unknown cellular stiffness: the ratio of displacements is related to the ratio of stiffness. A polyMUMPs implementation for aqueous media is presented and displacement measurements made from optical microphotographs using a FFT based displacement method with a repeatability of ~20 nm. The approach was first validated on a MEMS two stage spring system of known stiffness. The measured stiffness ratios of control structures (i) MEMS spring systems and (ii) polystyrene microspheres were found to agree with theoretical values. Mechanical tests were then performed on Saccharomyces cerevisiae (Baker’s yeast) in aqueous media. Cells were placed (using a micropipette) inside MEMS measuring structures and compressed between two jaws using an electrostatic actuator and displacements measured. Tested cells showed stiffness values between 5.4 and 8.4 N m-1 with an uncertainty of 11%. In addition, non-viable cells were tested by exposing viable cells to methanol. The resultant mean cell stiffness dropped by factor of 3 × and an explicit discrimination between viable and non-viable cells based on mechanical stiffness was seen.

  2. The physical origins of transit time measurements for rapid, single cell mechanotyping.

    PubMed

    Nyberg, Kendra D; Scott, Michael B; Bruce, Samuel L; Gopinath, Ajay B; Bikos, Dimitri; Mason, Thomas G; Kim, Jin Woong; Choi, Hong Sung; Rowat, Amy C

    2016-08-16

    The mechanical phenotype or 'mechanotype' of cells is emerging as a potential biomarker for cell types ranging from pluripotent stem cells to cancer cells. Using a microfluidic device, cell mechanotype can be rapidly analyzed by measuring the time required for cells to deform as they flow through constricted channels. While cells typically exhibit deformation timescales, or transit times, on the order of milliseconds to tens of seconds, transit times can span several orders of magnitude and vary from day to day within a population of single cells; this makes it challenging to characterize different cell samples based on transit time data. Here we investigate how variability in transit time measurements depends on both experimental factors and heterogeneity in physical properties across a population of single cells. We find that simultaneous transit events that occur across neighboring constrictions can alter transit time, but only significantly when more than 65% of channels in the parallel array are occluded. Variability in transit time measurements is also affected by the age of the device following plasma treatment, which could be attributed to changes in channel surface properties. We additionally investigate the role of variability in cell physical properties. Transit time depends on cell size; by binning transit time data for cells of similar diameters, we reduce measurement variability by 20%. To gain further insight into the effects of cell-to-cell differences in physical properties, we fabricate a panel of gel particles and oil droplets with tunable mechanical properties. We demonstrate that particles with homogeneous composition exhibit a marked reduction in transit time variability, suggesting that the width of transit time distributions reflects the degree of heterogeneity in subcellular structure and mechanical properties within a cell population. Our results also provide fundamental insight into the physical underpinnings of transit measurements

  3. Oxygen pressure measurement using singlet oxygen emission

    SciTech Connect

    Khalil, Gamal E.; Chang, Alvin; Gouterman, Martin; Callis, James B.; Dalton, Larry R.; Turro, Nicholas J.; Jockusch, Steffen

    2005-05-15

    Pressure sensitive paint (PSP) provides a visualization of two-dimensional pressure distributions on airfoil and model automobile surfaces. One type of PSP utilizes platinum tetra(pentafluorophenyl)porphine (PtTFPP) dissolved in a fluoro-polymer film. Since the intense 650 nm triplet emission of PtTFPP is quenched by ground state oxygen, it is possible to measure two-dimensional oxygen concentration from the 650 nm emission intensity using a Stern-Volmer-type relationship. This article reports an alternative luminescence method to measure oxygen concentration based on the porphyrin-sensitized 1270 nm singlet oxygen emission, which can be imaged with an InGaAs near infrared camera. This direct measurement of oxygen emission complements and further validates the oxygen measurement based on PtTFPP phosphorescence quenching. Initial success at obtaining a negative correlation between the 650 nm PtTFPP emission and the 1270 nm O{sub 2} emission in solution led us to additional two-dimensional film studies using surfaces coated with PtTFPP, MgTFPP, and H{sub 2}TFPP in polymers in a pressure and temperature controlled chamber.

  4. Oxygen pressure measurement using singlet oxygen emission

    NASA Astrophysics Data System (ADS)

    Khalil, Gamal E.; Chang, Alvin; Gouterman, Martin; Callis, James B.; Dalton, Larry R.; Turro, Nicholas J.; Jockusch, Steffen

    2005-05-01

    Pressure sensitive paint (PSP) provides a visualization of two-dimensional pressure distributions on airfoil and model automobile surfaces. One type of PSP utilizes platinum tetra(pentafluorophenyl)porphine (PtTFPP) dissolved in a fluoro-polymer film. Since the intense 650nm triplet emission of PtTFPP is quenched by ground state oxygen, it is possible to measure two-dimensional oxygen concentration from the 650nm emission intensity using a Stern-Volmer-type relationship. This article reports an alternative luminescence method to measure oxygen concentration based on the porphyrin-sensitized 1270nm singlet oxygen emission, which can be imaged with an InGaAs near infrared camera. This direct measurement of oxygen emission complements and further validates the oxygen measurement based on PtTFPP phosphorescence quenching. Initial success at obtaining a negative correlation between the 650nm PtTFPP emission and the 1270nm O2 emission in solution led us to additional two-dimensional film studies using surfaces coated with PtTFPP, MgTFPP, and H2TFPP in polymers in a pressure and temperature controlled chamber.

  5. Battery-powered portable instrument system for single-cell trapping, impedance measurements, and modeling analyses.

    PubMed

    Tsai, Sung-Lin; Chiang, Yang; Wang, Min-Haw; Chen, Ming-Kun; Jang, Ling-Sheng

    2014-08-01

    A battery-powered portable instrument system for the single-HeLa-cell trapping and analyses is developed. A method of alternating current electrothermal (ACET) and DEP are employed for the cell trapping and the method of impedance spectroscopy is employed for cell characterizations. The proposed instrument (160 mm × 170 mm × 110 mm, 1269 g) equips with a highly efficient energy-saving design that promises approximately 120 h of use. It includes an impedance analyzer performing an excitation voltage of 0.2-2 Vpp and a frequency sweep of 11-101 kHz, function generator with the sine wave output at an operating voltage of 1-50 Vpp with a frequency of 4-12 MHz, cell-trapping biochip, microscope, and input/output interface. The biochip for the single cell trapping is designed and simulated based on a combination of ACET and DEP forces. In order to improve measurement accuracy, the curve fitting method is adopted to calibrate the proposed impedance spectroscopy. Measurement results from the proposed system are compared with results from a precision impedance analyzer. The trapped cell can be modeled for numerical analyses. Many advantages are offered in the proposed instrument such as the small volume, real-time monitoring, rapid analysis, low cost, low-power consumption, and portable application.

  6. Measuring fast gene dynamics in single cells with time-lapse luminescence microscopy

    PubMed Central

    Mazo-Vargas, Anyimilehidi; Park, Heungwon; Aydin, Mert; Buchler, Nicolas E.

    2014-01-01

    Time-lapse fluorescence microscopy is an important tool for measuring in vivo gene dynamics in single cells. However, fluorescent proteins are limited by slow chromophore maturation times and the cellular autofluorescence or phototoxicity that arises from light excitation. An alternative is luciferase, an enzyme that emits photons and is active upon folding. The photon flux per luciferase is significantly lower than that for fluorescent proteins. Thus time-lapse luminescence microscopy has been successfully used to track gene dynamics only in larger organisms and for slower processes, for which more total photons can be collected in one exposure. Here we tested green, yellow, and red beetle luciferases and optimized substrate conditions for in vivo luminescence. By combining time-lapse luminescence microscopy with a microfluidic device, we tracked the dynamics of cell cycle genes in single yeast with subminute exposure times over many generations. Our method was faster and in cells with much smaller volumes than previous work. Fluorescence of an optimized reporter (Venus) lagged luminescence by 15–20 min, which is consistent with its known rate of chromophore maturation in yeast. Our work demonstrates that luciferases are better than fluorescent proteins at faithfully tracking the underlying gene expression. PMID:25232010

  7. 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

  8. Nanowell-based immunoassays for measuring single-cell secretion: characterization of transport and surface binding.

    PubMed

    Torres, Alexis J; Hill, Abby S; Love, J Christopher

    2014-12-01

    Arrays of subnanoliter wells (nanowells) provide a useful system to isolate single cells and analyze their secreted proteins. Two general approaches have emerged: one that uses open arrays and local capture of secreted proteins, and a second (called microengraving) that relies on closed arrays to capture secreted proteins on a solid substrate, which is subsequently removed from the array. However, the design and operating parameters for efficient capture from these two approaches to analyze single-cell secretion have not been extensively considered. Using numerical simulations, we analyzed the operational envelope for both open and closed formats, as a function of the spatial distribution of capture ligands, their affinities for the protein, and the rates of single-cell secretion. Based on these analyses, we present a modified approach to capture secreted proteins in-well for highly active secreting cells. This simple method for in-well detection should facilitate rapid identification of cell lines with high specific productivities.

  9. Time-resolved ICP-MS measurement: a new method for elemental and multiparametric analysis of single cells.

    PubMed

    Miyashita, Shin-ichi; Groombridge, Alexander S; Fujii, Shin-ichiro; Takatsu, Akiko; Chiba, Koichi; Inagaki, Kazumi

    2014-01-01

    Time-resolved inductively coupled plasma mass spectrometry (ICP-MS) has attracted much attention for elemental and multiparametric analysis of single cells, instead of a classical bulk analysis of large amount of cells after a dissolution. In the time-resolved measurement, cells are directly introduced into the plasma via nebulizing or micro drop dispensing, and then ion plumes corresponding to single cells are individually detected with a high time resolution. The sensitivity and cell throughput in the measurement strongly depend on the time resolution. A high cell introduction efficiency into the plasma supports for a reduction of cell consumption. Biomolecules can also be measured through the attachment of elemental tags, and then the amount distribution of elements and biomolecules in single cells can be evaluated, while providing information concerning cell-to-cell variations. By applying ICP time-of-flight mass spectrometry (ICP-TOFMS), multiparametric analysis of elements and biomolecules can be achieved similar to that by a flow cytometer. This article highlights the technical aspects of the time-resolved ICP-MS measurement technique for elemental and multiparametric analysis of single cells.

  10. Electroporation followed by electrochemical measurement of quantal transmitter release from single cells using a patterned microelectrode

    PubMed Central

    Ghosh, Jaya; Liu, Xin; Gillis, Kevin D.

    2013-01-01

    An electrochemical microelectrode located immediately adjacent to a single neuroendocrine cell can record spikes of amperometric current that result from exocytosis of oxidizable transmitter from individual vesicles, i.e., quantal exocytosis. Here, we report the development of an efficient method where the same electrochemical microelectrode is used to electropermeabilize an adjacent chromaffin cell and then measure the consequent quantal catecholamine release using amperometry. Trains of voltage pulses, 5–7 V in amplitude and 0.1–0.2 ms in duration, were used to reliably trigger release from cells using gold electrodes. Amperometric spikes induced by electropermeabilization had similar areas, peak heights and durations as amperometric spikes elicited by depolarizing high K+ solutions, therefore release occurs from individual secretory granules. Uptake of trypan blue stain into cells demonstrated that the plasma membrane is permeabilized by the voltage stimulus. Voltage pulses did not degrade the electrochemical sensitivity of the electrodes assayed using a test analyte. Surprisingly, robust quantal release was elicited upon electroporation in the absence of Ca2+ in the bath solution (0 Ca2+/5 mM EGTA). In contrast, electropermeabilization-induced transmitter release required Cl− in the bath solution in that bracketed experiments demonstrated a steep dependence of the rate of electropermeabilization-induced transmitter release on [Cl−] between 2 and 32 mM. Using the same electrochemical electrode to electroporate and record quantal release of catecholamines from an individual chromaffin cell allows precise timing of the stimulus, stimulation of a single cell at a time, and can be used to load membrane-impermeant substances into a cell. PMID:23598689

  11. An automatic measure for classifying clusters of suspected spikes into single cells versus multiunits

    PubMed Central

    Tankus, Ariel; Yeshurun, Yehezkel; Fried, Itzhak

    2010-01-01

    While automatic spike sorting has been investigated for decades, little attention has been allotted to consistent evaluation criteria that will automatically determine whether a cluster of spikes represents the activity of a single cell or a multiunit. Consequently, the main tool for evaluation has remained visual inspection by a human. This paper quantifies the visual inspection process. The results are well-defined criteria for evaluation, which are mainly based on visual features of the spike waveform, and an automatic adaptive algorithm that learns the classification by a given human and can apply similar visual characteristics for classification of new data. To evaluate the suggested criteria, we recorded the activity of 1652 units (single cells and multiunits) from the cerebrum of 12 human patients undergoing evaluation for epilepsy surgery requiring implantation of chronic intracranial depth electrodes. The proposed method performed similar to human classifiers and obtained significantly higher accuracy than two existing methods (three variants of each). Evaluation on two synthetic datasets is also provided. The criteria are suggested as a standard for evaluation of the quality of separation that will allow comparison between different studies. The proposed algorithm is suitable for real-time operation and as such may allow brain–computer interfaces to treat single cells differently than multiunits. PMID:19667458

  12. An automatic measure for classifying clusters of suspected spikes into single cells versus multiunits

    NASA Astrophysics Data System (ADS)

    Tankus, Ariel; Yeshurun, Yehezkel; Fried, Itzhak

    2009-10-01

    While automatic spike sorting has been investigated for decades, little attention has been allotted to consistent evaluation criteria that will automatically determine whether a cluster of spikes represents the activity of a single cell or a multiunit. Consequently, the main tool for evaluation has remained visual inspection by a human. This paper quantifies the visual inspection process. The results are well-defined criteria for evaluation, which are mainly based on visual features of the spike waveform, and an automatic adaptive algorithm that learns the classification by a given human and can apply similar visual characteristics for classification of new data. To evaluate the suggested criteria, we recorded the activity of 1652 units (single cells and multiunits) from the cerebrum of 12 human patients undergoing evaluation for epilepsy surgery requiring implantation of chronic intracranial depth electrodes. The proposed method performed similar to human classifiers and obtained significantly higher accuracy than two existing methods (three variants of each). Evaluation on two synthetic datasets is also provided. The criteria are suggested as a standard for evaluation of the quality of separation that will allow comparison between different studies. The proposed algorithm is suitable for real-time operation and as such may allow brain-computer interfaces to treat single cells differently than multiunits.

  13. Single cell studies of mouse embryonic stem cell (mESC) differentiation by electrical impedance measurements in a microfluidic device.

    PubMed

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

    2016-07-15

    Biological populations of cells show considerable cell-to-cell variability. Study of single cells and analysis of cell heterogeneity are considered to be critical in understanding biological processes such as stem cell differentiation and cancer development. Recent advances in lab-on-a-chip techniques have allowed single-cell capture in microfluidic channels with the possibility of precise environmental control and high throughput of experiments with minimal usage of samples and reagents. In recent years, label-free techniques such as electrical impedance spectroscopy have emerged as a non-invasive approach to studying cell properties. In this study, we have designed and fabricated a microfluidic device that combines hydrodynamic trapping of single cells in pre-defined locations with the capability of running electrical impedance measurements within the same device. We have measured mouse embryonic stem cells (mESCs) at different states during differentiation (t=0h, 24h and 48h) and quantitatively analysed the changes in electrical parameters of cells during differentiation. A marked increase in the magnitude of the cell impedance is found during cell differentiation, which can be attributed to an increase in cell size. The analysis of the measurements shows that the nucleus-to-cytoplasm ratio decreases during this process. The degree of cell heterogeneity is observed to be the highest when the cells are at the transition state (24h), compare with cells at undifferentiated (0h) and fully differentiated (48h) states. The device enables highly efficient single cell trapping and provides sensitive, label-free electrical impedance measurements of individual cells, enabling the possibility of quantitatively analysing their physical state as well as studying the associated heterogeneity of a cell population.

  14. Single cell studies of mouse embryonic stem cell (mESC) differentiation by electrical impedance measurements in a microfluidic device

    PubMed Central

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

    2016-01-01

    Biological populations of cells show considerable cell-to-cell variability. Study of single cells and analysis of cell heterogeneity are considered to be critical in understanding biological processes such as stem cell differentiation and cancer development. Recent advances in lab-on-a-chip techniques have allowed single-cell capture in microfluidic channels with the possibility of precise environmental control and high throughput of experiments with minimal usage of samples and reagents. In recent years, label-free techniques such as electrical impedance spectroscopy have emerged as a non-invasive approach to studying cell properties. In this study, we have designed and fabricated a microfluidic device that combines hydrodynamic trapping of single cells in pre-defined locations with the capability of running electrical impedance measurements within the same device. We have measured mouse embryonic stem cells (mESCs) at different states during differentiation (t=0 h, 24 h and 48 h) and quantitatively analysed the changes in electrical parameters of cells during differentiation. A marked increase in the magnitude of the cell impedance is found during cell differentiation, which can be attributed to an increase in cell size. The analysis of the measurements shows that the nucleus-to-cytoplasm ratio decreases during this process. The degree of cell heterogeneity is observed to be the highest when the cells are at the transition state (24 h), compare with cells at undifferentiated (0 h) and fully differentiated (48 h) states. The device enables highly efficient single cell trapping and provides sensitive, label-free electrical impedance measurements of individual cells, enabling the possibility of quantitatively analysing their physical state as well as studying the associated heterogeneity of a cell population. PMID:26963790

  15. Tissue oxygen measurement system

    NASA Technical Reports Server (NTRS)

    Soller, Babs R. (Inventor)

    2004-01-01

    A device and method in accordance with the invention for determining the oxygen partial pressure (PO.sub.2) of a tissue by irradiating the tissue with optical radiation such that the light is emitted from the tissue, and by collecting the reflected or transmitted light from the tissue to form an optical spectrum. A spectral processor determines the PO.sub.2 level in tissue by processing this spectrum with a previously-constructed spectral calibration model. The tissue may, for example, be disposed underneath a covering tissue, such as skin, of a patient, and the tissue illuminated and light collected through the skin. Alternatively, direct tissue illumination and collection may be effected with a hand-held or endoscopic probe. A preferred system also determines pH from the same spectrum, and the processor may determine critical conditions and issue warnings based on parameter values.

  16. Comparison between steady-state and dynamic I-V measurements from a single-cell thermionic fuel element

    SciTech Connect

    Wernsman, B.

    1997-01-01

    A comparison between steady-state and dynamic I-V measurements from a single-cell thermionic fuel element (TFE) is made. The single-cell TFE used in this study is the prototype for the 40kW{sub e} space nuclear power system that is similar to the 6kW{sub e} TOPAZ-II. The steady-state I-V measurements influence the emitter temperature due to electron cooling. Therefore, to eliminate the steady-state I-V measurement influence on the TFE and provide a better understanding of the behavior of the thermionic energy converter and TFE characteristics, dynamic I-V measurements are made. The dynamic I-V measurements are made at various input power levels, cesium pressures, collector temperatures, and steady-state current levels. From these measurements, it is shown that the dynamic I-V{close_quote}s do not change the TFE characteristics at a given operating point. Also, the evaluation of the collector work function from the dynamic I-V measurements shows that the collector optimization is not due to a minimum in the collector work function but due to an emission optimization. Since the dynamic I-V measurements do not influence the TFE characteristics, it is believed that these measurements can be done at a system level to understand the influence of TFE placement in the reactor as a function of the core thermal distribution. {copyright} {ital 1997 American Institute of Physics.}

  17. Comparison between steady-state and dynamic I-V measurements from a single-cell thermionic fuel element

    SciTech Connect

    Wernsman, Bernard

    1997-01-10

    A comparison between steady-state and dynamic I-V measurements from a single-cell thermionic fuel element (TFE) is made. The single-cell TFE used in this study is the prototype for the 40 kW{sub e} space nuclear power system that is similar to the 6 kW{sub e} TOPAZ-II. The steady-state I-V measurements influence the emitter temperature due to electron cooling. Therefore, to eliminate the steady-state I-V measurement influence on the TFE and provide a better understanding of the behavior of the thermionic energy converter and TFE characteristics, dynamic I-V measurements are made. The dynamic I-V measurements are made at various input power levels, cesium pressures, collector temperatures, and steady-state current levels. From these measurements, it is shown that the dynamic I-V's do not change the TFE characteristics at a given operating point. Also, the evaluation of the collector work function from the dynamic I-V measurements shows that the collector optimization is not due to a minimum in the collector work function but due to an emission optimization. Since the dynamic I-V measurements do not influence the TFE characteristics, it is believed that these measurements can be done at a system level to understand the influence of TFE placement in the reactor as a function of the core thermal distribution.

  18. Measuring single-cell gene expression dynamics in bacteria using fluorescence time-lapse microscopy

    PubMed Central

    Young, Jonathan W; Locke, James C W; Altinok, Alphan; Rosenfeld, Nitzan; Bacarian, Tigran; Swain, Peter S; Mjolsness, Eric; Elowitz, Michael B

    2014-01-01

    Quantitative single-cell time-lapse microscopy is a powerful method for analyzing gene circuit dynamics and heterogeneous cell behavior. We describe the application of this method to imaging bacteria by using an automated microscopy system. This protocol has been used to analyze sporulation and competence differentiation in Bacillus subtilis, and to quantify gene regulation and its fluctuations in individual Escherichia coli cells. The protocol involves seeding and growing bacteria on small agarose pads and imaging the resulting microcolonies. Images are then reviewed and analyzed using our laboratory's custom MATLAB analysis code, which segments and tracks cells in a frame-to-frame method. This process yields quantitative expression data on cell lineages, which can illustrate dynamic expression profiles and facilitate mathematical models of gene circuits. With fast-growing bacteria, such as E. coli or B. subtilis, image acquisition can be completed in 1 d, with an additional 1–2 d for progressing through the analysis procedure. PMID:22179594

  19. Single-Cell Measurements of IgE-Mediated FcεRI Signaling Using an Integrated Microfluidic Platform

    SciTech Connect

    Liu, Yanli; Barua, Dipak; Liu, Peng; Wilson, Bridget S.; Oliver, Janet M.; Hlavacek, William S.; Singh, Anup K.

    2013-03-27

    Heterogeneity in responses of cells to a stimulus, such as a pathogen or allergen, can potentially play an important role in deciding the fate of the responding cell population and the overall systemic response. Measuring heterogeneous responses requires tools capable of interrogating individual cells. Cell signaling studies commonly do not have single-cell resolution because of the limitations of techniques used such as Westerns, ELISAs, mass spectrometry, and DNA microarrays. Microfluidics devices are increasingly being used to overcome these limitations. In this paper, we report on a microfluidic platform for cell signaling analysis that combines two orthogonal single-cell measurement technologies: on-chip flow cytometry and optical imaging. The device seamlessly integrates cell culture, stimulation, and preparation with downstream measurements permitting hands-free, automated analysis to minimize experimental variability. The platform was used to interrogate IgE receptor (FcεRI) signaling, which is responsible for triggering allergic reactions, in RBL-2H3 cells. Following on-chip crosslinking of IgE-FcεRI complexes by multivalent antigen, we monitored signaling events including protein phosphorylation, calcium mobilization and the release of inflammatory mediators. The results demonstrate the ability of our platform to produce quantitative measurements on a cell-by-cell basis from just a few hundred cells. Finally, model-based analysis of the Syk phosphorylation data suggests that heterogeneity in Syk phosphorylation can be attributed to protein copy number variations, with the level of Syk phosphorylation being particularly sensitive to the copy number of Lyn.

  20. Single-Cell Measurements of IgE-Mediated FcεRI Signaling Using an Integrated Microfluidic Platform

    PubMed Central

    Liu, Yanli; Barua, Dipak; Liu, Peng; Wilson, Bridget S.; Oliver, Janet M.; Hlavacek, William S.; Singh, Anup K.

    2013-01-01

    Heterogeneity in responses of cells to a stimulus, such as a pathogen or allergen, can potentially play an important role in deciding the fate of the responding cell population and the overall systemic response. Measuring heterogeneous responses requires tools capable of interrogating individual cells. Cell signaling studies commonly do not have single-cell resolution because of the limitations of techniques used such as Westerns, ELISAs, mass spectrometry, and DNA microarrays. Microfluidics devices are increasingly being used to overcome these limitations. Here, we report on a microfluidic platform for cell signaling analysis that combines two orthogonal single-cell measurement technologies: on-chip flow cytometry and optical imaging. The device seamlessly integrates cell culture, stimulation, and preparation with downstream measurements permitting hands-free, automated analysis to minimize experimental variability. The platform was used to interrogate IgE receptor (FcεRI) signaling, which is responsible for triggering allergic reactions, in RBL-2H3 cells. Following on-chip crosslinking of IgE-FcεRI complexes by multivalent antigen, we monitored signaling events including protein phosphorylation, calcium mobilization and the release of inflammatory mediators. The results demonstrate the ability of our platform to produce quantitative measurements on a cell-by-cell basis from just a few hundred cells. Model-based analysis of the Syk phosphorylation data suggests that heterogeneity in Syk phosphorylation can be attributed to protein copy number variations, with the level of Syk phosphorylation being particularly sensitive to the copy number of Lyn. PMID:23544131

  1. A microfluidic platform for systems pathology: multiparameter single-cell signaling measurements of clinical brain tumor specimens.

    PubMed

    Sun, Jing; Masterman-Smith, Michael D; Graham, Nicholas A; Jiao, Jing; Mottahedeh, Jack; Laks, Dan R; Ohashi, Minori; DeJesus, Jason; Kamei, Ken-ichiro; Lee, Ki-Bum; Wang, Hao; Yu, Zeta T F; Lu, Yi-Tsung; Hou, Shuang; Li, Keyu; Liu, Max; Zhang, Nangang; Wang, Shutao; Angenieux, Brigitte; Panosyan, Eduard; Samuels, Eric R; Park, Jun; Williams, Dirk; Konkankit, Vera; Nathanson, David; van Dam, R Michael; Phelps, Michael E; Wu, Hong; Liau, Linda M; Mischel, Paul S; Lazareff, Jorge A; Kornblum, Harley I; Yong, William H; Graeber, Thomas G; Tseng, Hsian-Rong

    2010-08-01

    The clinical practice of oncology is being transformed by molecular diagnostics that will enable predictive and personalized medicine. Current technologies for quantitation of the cancer proteome are either qualitative (e.g., immunohistochemistry) or require large sample sizes (e.g., flow cytometry). Here, we report a microfluidic platform-microfluidic image cytometry (MIC)-capable of quantitative, single-cell proteomic analysis of multiple signaling molecules using only 1,000 to 2,800 cells. Using cultured cell lines, we show simultaneous measurement of four critical signaling proteins (EGFR, PTEN, phospho-Akt, and phospho-S6) within the oncogenic phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling pathway. To show the clinical application of the MIC platform to solid tumors, we analyzed a panel of 19 human brain tumor biopsies, including glioblastomas. Our MIC measurements were validated by clinical immunohistochemistry and confirmed the striking intertumoral and intratumoral heterogeneity characteristic of glioblastoma. To interpret the multiparameter, single-cell MIC measurements, we adapted bioinformatic methods including self-organizing maps that stratify patients into clusters that predict tumor progression and patient survival. Together with bioinformatic analysis, the MIC platform represents a robust, enabling in vitro molecular diagnostic technology for systems pathology analysis and personalized medicine. PMID:20631065

  2. A microfluidic platform for systems pathology: multiparameter single-cell signaling measurements of clinical brain tumor specimens

    PubMed Central

    Sun, Jing; Masterman-Smith, Michael; Graham, Nicholas A.; Jiao, Jing; Mottahedeh, Jack; Laks, Dan R.; Ohashi, Minori; DeJesus, Jason; Kamei, Ken-ichiro; Lee, Ki-Bum; Wang, Hao; Yu, Zeta T.F.; Lu, Yi-Tsung; Hou, Shuang; Li, Keyu; Liu, Max; Zhang, Nangang; Wang, Shutao; Angenieux, Brigitte; Panosyan, Eduard; Samuels, Eric R.; Park, Jun; Williams, Dirk; Konkankit, Vera; Nathanson, David; van Dam, R. Michael; Phelps, Michael E.; Wu, Hong; Liau, Linda M.; Mischel, Paul S.; Lazareff, Jorge A.; Kornblum, Harley I.; Yong, William H.; Graeber, Thomas G.; Tseng, Hsian-Rong

    2011-01-01

    The clinical practice of oncology is being transformed by molecular diagnostics that will enable predictive and personalized medicine. Current technologies for quantitation of the cancer proteome are either qualitative (e.g., immunohistochemistry) or require large sample sizes (e.g., flow cytometry). Here, we report a microfluidic platform, Microfluidic Image Cytometry (MIC), capable of quantitative, single-cell proteomic analysis of multiple signaling molecules using only 1,000-2,800 cells. Using cultured cell lines, we demonstrate simultaneous measurement of four critical signaling proteins (EGFR, PTEN, phospho-Akt and phospho-S6) within the oncogenic PI3K/Akt/mTOR signaling pathway. To demonstrate the clinical application of the MIC platform to solid tumors, we analyzed a panel of 19 human brain tumor biopsies, including glioblastomas. Our MIC measurements were validated by clinical immunohistochemistry and confirmed the striking inter- and intra-tumoral heterogeneity characteristic of glioblastoma. To interpret the multiparameter, single-cell MIC measurements, we adapted bioinformatic methods including self-organizing maps that stratify patients into clusters which predict tumor progression and patient survival. Together with bioinformatic analysis, the MIC platform represents a robust, enabling in vitro molecular diagnostic technology for systems pathology analysis and personalized medicine. PMID:20631065

  3. A microfluidic platform for systems pathology: multiparameter single-cell signaling measurements of clinical brain tumor specimens.

    PubMed

    Sun, Jing; Masterman-Smith, Michael D; Graham, Nicholas A; Jiao, Jing; Mottahedeh, Jack; Laks, Dan R; Ohashi, Minori; DeJesus, Jason; Kamei, Ken-ichiro; Lee, Ki-Bum; Wang, Hao; Yu, Zeta T F; Lu, Yi-Tsung; Hou, Shuang; Li, Keyu; Liu, Max; Zhang, Nangang; Wang, Shutao; Angenieux, Brigitte; Panosyan, Eduard; Samuels, Eric R; Park, Jun; Williams, Dirk; Konkankit, Vera; Nathanson, David; van Dam, R Michael; Phelps, Michael E; Wu, Hong; Liau, Linda M; Mischel, Paul S; Lazareff, Jorge A; Kornblum, Harley I; Yong, William H; Graeber, Thomas G; Tseng, Hsian-Rong

    2010-08-01

    The clinical practice of oncology is being transformed by molecular diagnostics that will enable predictive and personalized medicine. Current technologies for quantitation of the cancer proteome are either qualitative (e.g., immunohistochemistry) or require large sample sizes (e.g., flow cytometry). Here, we report a microfluidic platform-microfluidic image cytometry (MIC)-capable of quantitative, single-cell proteomic analysis of multiple signaling molecules using only 1,000 to 2,800 cells. Using cultured cell lines, we show simultaneous measurement of four critical signaling proteins (EGFR, PTEN, phospho-Akt, and phospho-S6) within the oncogenic phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling pathway. To show the clinical application of the MIC platform to solid tumors, we analyzed a panel of 19 human brain tumor biopsies, including glioblastomas. Our MIC measurements were validated by clinical immunohistochemistry and confirmed the striking intertumoral and intratumoral heterogeneity characteristic of glioblastoma. To interpret the multiparameter, single-cell MIC measurements, we adapted bioinformatic methods including self-organizing maps that stratify patients into clusters that predict tumor progression and patient survival. Together with bioinformatic analysis, the MIC platform represents a robust, enabling in vitro molecular diagnostic technology for systems pathology analysis and personalized medicine.

  4. Yeast Replicator: A High-Throughput Multiplexed Microfluidics Platform for Automated Measurements of Single-Cell Aging.

    PubMed

    Liu, Ping; Young, Thomas Z; Acar, Murat

    2015-10-20

    The yeast Saccharomyces cerevisiae is a model organism for replicative aging studies; however, conventional lifespan measurement platforms have several limitations. Here, we present a microfluidics platform that facilitates simultaneous lifespan and gene expression measurements of aging yeast cells. Our multiplexed high-throughput platform offers the capability to perform independent lifespan experiments using different yeast strains or growth media. Using this platform in minimal media environments containing glucose, we measured the full lifespan of individual yeast cells in wild-type and canonical gene deletion backgrounds. Compared to glucose, in galactose we observed a 16.8% decrease in replicative lifespan accompanied by an ∼2-fold increase in single-cell oxidative stress levels reported by PSOD1-mCherry. Using PGAL1-YFP to measure the activity of the bistable galactose network, we saw that OFF and ON cells are similar in their lifespan. Our work shows that aging cells are committed to a single phenotypic state throughout their lifespan.

  5. Simultaneous measurement of water volume and pH in single cells using BCECF and fluorescence imaging microscopy.

    PubMed

    Alvarez-Leefmans, Francisco J; Herrera-Pérez, José J; Márquez, Martín S; Blanco, Víctor M

    2006-01-15

    Regulation and maintenance of cell water volume and intracellular pH (pHi) are vital functions that are interdependent; cell volume regulation affects, and is in turn affected by, changes in pHi. Disruption of either function underlies various pathologies. To study the interaction and kinetics of these two mechanisms, we developed and validated a quantitative fluorescence imaging microscopy method to measure simultaneous changes in pHi and volume in single cells loaded with the fluorescent probe BCECF. CWV is measured at the excitation isosbestic wavelength, whereas pHi is determined ratiometrically. The method has a time resolution of <1 s and sensitivity to osmotic changes of approximately 1%. It can be applied in real time to virtually any cell type attached to a coverslip, independently of cellular shape and geometry. Calibration procedures and algorithms developed to transform fluorescence signals into changes in cell water volume (CWV) and examples of applications are presented. PMID:16258035

  6. Single-Cell Measurements of IgE-Mediated FcεRI Signaling Using an Integrated Microfluidic Platform

    DOE PAGESBeta

    Liu, Yanli; Barua, Dipak; Liu, Peng; Wilson, Bridget S.; Oliver, Janet M.; Hlavacek, William S.; Singh, Anup K.

    2013-03-27

    Heterogeneity in responses of cells to a stimulus, such as a pathogen or allergen, can potentially play an important role in deciding the fate of the responding cell population and the overall systemic response. Measuring heterogeneous responses requires tools capable of interrogating individual cells. Cell signaling studies commonly do not have single-cell resolution because of the limitations of techniques used such as Westerns, ELISAs, mass spectrometry, and DNA microarrays. Microfluidics devices are increasingly being used to overcome these limitations. In this paper, we report on a microfluidic platform for cell signaling analysis that combines two orthogonal single-cell measurement technologies: on-chipmore » flow cytometry and optical imaging. The device seamlessly integrates cell culture, stimulation, and preparation with downstream measurements permitting hands-free, automated analysis to minimize experimental variability. The platform was used to interrogate IgE receptor (FcεRI) signaling, which is responsible for triggering allergic reactions, in RBL-2H3 cells. Following on-chip crosslinking of IgE-FcεRI complexes by multivalent antigen, we monitored signaling events including protein phosphorylation, calcium mobilization and the release of inflammatory mediators. The results demonstrate the ability of our platform to produce quantitative measurements on a cell-by-cell basis from just a few hundred cells. Finally, model-based analysis of the Syk phosphorylation data suggests that heterogeneity in Syk phosphorylation can be attributed to protein copy number variations, with the level of Syk phosphorylation being particularly sensitive to the copy number of Lyn.« less

  7. Reversible coupling of individual phycobiliprotein isoforms during state transitions in the cyanobacterium Trichodesmium analysed by single-cell fluorescence kinetic measurements.

    PubMed

    Küpper, Hendrik; Andresen, Elisa; Wiegert, Susanna; Simek, Miloslav; Leitenmaier, Barbara; Setlík, Ivan

    2009-03-01

    In the non-heterocyst, marine cyanobacterium Trichodesmium nitrogen fixation is confined to the photoperiod and occurs coevally with oxygenic photosynthesis although nitrogenase is irreversibly inactivated by oxygen. In previous studies it was found that regulation of photosynthesis for nitrogen fixation involves Mehler reaction and various activity states with reversible coupling of photosynthetic components. We now investigated these activity states in more detail. Spectrally resolved fluorescence kinetic measurements of single cells revealed that they were related to alternate uncoupling and coupling of phycobilisomes from and to the photosystems, changing the effective cross-section of PSII. Therefore, we isolated and purified the phycobiliproteins of Trichodesmium via ion exchange chromatography and recorded their UV/VIS absorption, fluorescence excitation and fluorescence emission spectra. After describing these spectra by mathematical equations via the Gauss-Peak-Spectra method, we used them to deconvolute the in vivo fluorescence spectra of Trichodesmium cells. This revealed that the contribution of different parts of the phycobilisome antenna to fluorescence quenching changed during the daily activity cycle, and that individual phycobiliproteins can be reversibly coupled to the photosystems, while the expression levels of these proteins did not change much during the daily activity cycle. Thus we propose that variable phycobilisome coupling plays a key role in the regulation of photosynthesis for nitrogen fixation in Trichodesmium.

  8. Measurement of Single Cell Refractive Index, Dry Mass, Volume, and Density Using a Transillumination Microscope

    NASA Astrophysics Data System (ADS)

    Phillips, Kevin G.; Jacques, Steven L.; McCarty, Owen J. T.

    2012-09-01

    Phase contrast microscopy has become ubiquitous in the field of biology, particularly in qualitative investigations of cellular morphology. However, the use of quantitative phase retrieval methods and their connection to cellular refractive index and dry mass density remain under utilized. This is due in part to the restriction of phase and cellular mass determination to custom built instruments, involved mathematical analysis, and prohibitive sample perturbations. We introduce tomographic bright field imaging, an accessible optical imaging technique enabling the three dimensional measurement of cellular refractive index and dry mass density using a standard transillumination optical microscope. The validity of the technique is demonstrated on polystyrene spheres. The technique is then applied to the measurement of the refractive index, dry mass, volume, and density of red blood cells. This optical technique enables a simple and robust means to perform quantitative investigations of engineered and biological specimens in three dimensions using standard optical microscopes.

  9. Deformation measurement of individual cells in large populations using a single-cell microchamber array chip

    NASA Astrophysics Data System (ADS)

    Doh, I.; Lee, W. C.; Cho, Y.-H.; Pisano, A. P.; Kuypers, F. A.

    2012-04-01

    We analyze the deformability of individual red blood cells (RBCs) using SiCMA technology. Our approach is adequate to quickly measure large numbers of individual cells in heterogeneous populations. Individual cells are trapped in a large-scale array of micro-wells, and dielectrophoretic (DEP) force is applied to deform the cells. The simple structures of micro-wells and DEP electrodes facilitate the analysis of thousands of RBCs in parallel. This unique method allows the correlation of red cell deformation with cell surface and cytosolic characteristics to define the distribution of individual cellular characteristics in heterogeneous populations.

  10. Spatiotemporal activity patterns detected from single cell measurements from behaving animals

    NASA Astrophysics Data System (ADS)

    Villa, Alessandro E. P.; Tetko, Igor V.

    1999-03-01

    Precise temporal patterning of activity within and between neurons has been predicted on theoretical grounds, and found in the spike trains of neurons recorded from anesthetized and conscious animals, in association with sensor stimuli and particular phases of task performance. However, the functional significance of such patterning in the generation of behavior has not been confirmed. We recorded from multiple single neurons in regions of rat auditory cortex during the waiting period of a Go/NoGo task. During this time the animal waited for an auditory signal with high cognitive load. Of note is the fact that neural activity during the period analyzed was essentially stationary, with no event related variability in firing. Detected patterns therefore provide a measure of brain state that could not be addressed by standard methods relying on analysis of changes in mean discharge rate. The possibility is discussed that some patterns might reflect a preset bias to a particular response, formed in the waiting period. Others patterns might reflect a state of prior preparation of appropriate neural assemblies for analyzing a signal that is expected but of unknown behavioral valence.

  11. Direct Measurement of Intranuclear Strain Distributions and RNA Synthesis in Single Cells Embedded within Native Tissue

    PubMed Central

    Henderson, Jonathan T.; Shannon, Garrett; Veress, Alexander I.; Neu, Corey P.

    2013-01-01

    Nuclear structure and mechanics play a critical role in diverse cellular functions, such as organizing direct access of chromatin to transcriptional regulators. Here, we use a new, to our knowledge, hybrid method, based on microscopy and hyperelastic warping, to determine three-dimensional strain distributions inside the nuclei of single living cells embedded within their native extracellular matrix. During physiologically relevant mechanical loading to tissue samples, strain was transferred to individual nuclei, resulting in submicron distributions of displacements, with compressive and tensile strain patterns approaching a fivefold magnitude increase in some locations compared to tissue-scale stimuli. Moreover, nascent RNA synthesis was observed in the interchromatin regions of the cells studied and spatially corresponded to strain patterns. Our ability to measure large strains in the interchromatin space, which reveals that movement of chromatin in the nucleus may not be due to random or biochemical mechanisms alone, but may result from the transfer of mechanical force applied at a distant tissue surface. PMID:24268137

  12. Multiphoton photochemical crosslinking-based fabrication of protein micropatterns with controllable mechanical properties for single cell traction force measurements

    PubMed Central

    Tong, Ming Hui; Huang, Nan; Zhang, Wei; Zhou, Zhuo Long; Ngan, Alfonso Hing Wan; Du, Yanan; Chan, Barbara Pui

    2016-01-01

    Engineering 3D microstructures with predetermined properties is critical for stem cell niche studies. We have developed a multiphoton femtosecond laser-based 3D printing platform, which generates complex protein microstructures in minutes. Here, we used the platform to test a series of fabrication and reagent parameters in precisely controlling the mechanical properties of protein micropillars. Atomic force microscopy was utilized to measure the reduced elastic modulus of the micropillars, and transmission electron microscopy was used to visualize the porosity of the structures. The reduced elastic modulus of the micropillars associated positively and linearly with the scanning power. On the other hand, the porosity and pore size of the micropillars associated inversely and linearly with the scanning power and reagent concentrations. While keeping the elastic modulus constant, the stiffness of the micropillars was controlled by varying their height. Subsequently, the single cell traction forces of rabbit chondrocytes, human dermal fibroblasts, human mesenchymal stem cells, and bovine nucleus pulposus cells (bNPCs) were successfully measured by culturing the cells on micropillar arrays of different stiffness. Our results showed that the traction forces of all groups showed positive relationship with stiffness, and that the chondrocytes and bNPCs generated the highest and lowest traction forces, respectively. PMID:26817674

  13. Multiphoton photochemical crosslinking-based fabrication of protein micropatterns with controllable mechanical properties for single cell traction force measurements.

    PubMed

    Tong, Ming Hui; Huang, Nan; Zhang, Wei; Zhou, Zhuo Long; Ngan, Alfonso Hing Wan; Du, Yanan; Chan, Barbara Pui

    2016-01-01

    Engineering 3D microstructures with predetermined properties is critical for stem cell niche studies. We have developed a multiphoton femtosecond laser-based 3D printing platform, which generates complex protein microstructures in minutes. Here, we used the platform to test a series of fabrication and reagent parameters in precisely controlling the mechanical properties of protein micropillars. Atomic force microscopy was utilized to measure the reduced elastic modulus of the micropillars, and transmission electron microscopy was used to visualize the porosity of the structures. The reduced elastic modulus of the micropillars associated positively and linearly with the scanning power. On the other hand, the porosity and pore size of the micropillars associated inversely and linearly with the scanning power and reagent concentrations. While keeping the elastic modulus constant, the stiffness of the micropillars was controlled by varying their height. Subsequently, the single cell traction forces of rabbit chondrocytes, human dermal fibroblasts, human mesenchymal stem cells, and bovine nucleus pulposus cells (bNPCs) were successfully measured by culturing the cells on micropillar arrays of different stiffness. Our results showed that the traction forces of all groups showed positive relationship with stiffness, and that the chondrocytes and bNPCs generated the highest and lowest traction forces, respectively.

  14. Multiphoton photochemical crosslinking-based fabrication of protein micropatterns with controllable mechanical properties for single cell traction force measurements

    NASA Astrophysics Data System (ADS)

    Tong, Ming Hui; Huang, Nan; Zhang, Wei; Zhou, Zhuo Long; Ngan, Alfonso Hing Wan; Du, Yanan; Chan, Barbara Pui

    2016-01-01

    Engineering 3D microstructures with predetermined properties is critical for stem cell niche studies. We have developed a multiphoton femtosecond laser-based 3D printing platform, which generates complex protein microstructures in minutes. Here, we used the platform to test a series of fabrication and reagent parameters in precisely controlling the mechanical properties of protein micropillars. Atomic force microscopy was utilized to measure the reduced elastic modulus of the micropillars, and transmission electron microscopy was used to visualize the porosity of the structures. The reduced elastic modulus of the micropillars associated positively and linearly with the scanning power. On the other hand, the porosity and pore size of the micropillars associated inversely and linearly with the scanning power and reagent concentrations. While keeping the elastic modulus constant, the stiffness of the micropillars was controlled by varying their height. Subsequently, the single cell traction forces of rabbit chondrocytes, human dermal fibroblasts, human mesenchymal stem cells, and bovine nucleus pulposus cells (bNPCs) were successfully measured by culturing the cells on micropillar arrays of different stiffness. Our results showed that the traction forces of all groups showed positive relationship with stiffness, and that the chondrocytes and bNPCs generated the highest and lowest traction forces, respectively.

  15. Dielectrophoretic Microfluidic Chip Enables Single-Cell Measurements for Multidrug Resistance in Heterogeneous Acute Myeloid Leukemia Patient Samples.

    PubMed

    Khamenehfar, Avid; Gandhi, Maher K; Chen, Yuchun; Hogge, Donna E; Li, Paul C H

    2016-06-01

    The front-line treatment for adult acute myeloid leukemia (AML) is anthracycline-based combination chemotherapy. However, treatment outcomes remain suboptimal with relapses frequently observed. Among the mechanisms of treatment failure is multidrug resistance (MDR) mediated by the ABCB1, ABCC1, and ABCG2 drug-efflux transporters. Although genetic and phenotypic heterogeneity between leukemic blast cells is a well-recognized phenomenon, there remains minimal data on differences in MDR activity at the individual cell level. Specifically, functional assays that can distinguish the variability in MDR activity between individual leukemic blasts are lacking. Here, we outline a new dielectrophoretic (DEP) chip-based assay. This assay permits measurement of drug accumulation in single cells, termed same-single-cell analysis in the accumulation mode (SASCA-A). Initially, the assay was optimized in pretherapy samples from 20 adults with AML whose leukemic blasts had MDR activity against the anthracyline daunorubicin (DNR) tested using multiple MDR inhibitors. Parameters tested were initial drug accumulation, time to achieve signal saturation, fold-increase of DNR accumulation with MDR inhibition, ease of cell trapping, and ease of maintaining the trapped cells stationary. This enabled categorization into leukemic blast cells with MDR activity (MDR(+)) and leukemic blast cells without MDR activity (MDR(-ve)). Leukemic blasts could also be distinguished from benign white blood cells (notably these also lacked MDR activity). MDR(-ve) blasts were observed to be enriched in samples taken from patients who went on to enter complete remission (CR), whereas MDR(+) blasts were frequently observed in patients who failed to achieve CR following front-line chemotherapy. However, pronounced variability in functional MDR activity between leukemic blasts was observed, with MDR(+) cells not infrequently seen in some patients that went on to achieve CR. Next, we tested MDR activity in two

  16. Single cell heterogeneity

    PubMed Central

    Abdallah, Batoul Y; Horne, Steven D; Stevens, Joshua B; Liu, Guo; Ying, Andrew Y; Vanderhyden, Barbara; Krawetz, Stephen A; Gorelick, Root; Heng, Henry HQ

    2013-01-01

    Multi-level heterogeneity is a fundamental but underappreciated feature of cancer. Most technical and analytical methods either completely ignore heterogeneity or do not fully account for it, as heterogeneity has been considered noise that needs to be eliminated. We have used single-cell and population-based assays to describe an instability-mediated mechanism where genome heterogeneity drastically affects cell growth and cannot be accurately measured using conventional averages. First, we show that most unstable cancer cell populations exhibit high levels of karyotype heterogeneity, where it is difficult, if not impossible, to karyotypically clone cells. Second, by comparing stable and unstable cell populations, we show that instability-mediated karyotype heterogeneity leads to growth heterogeneity, where outliers dominantly contribute to population growth and exhibit shorter cell cycles. Predictability of population growth is more difficult for heterogeneous cell populations than for homogenous cell populations. Since “outliers” play an important role in cancer evolution, where genome instability is the key feature, averaging methods used to characterize cell populations are misleading. Variances quantify heterogeneity; means (averages) smooth heterogeneity, invariably hiding it. Cell populations of pathological conditions with high genome instability, like cancer, behave differently than karyotypically homogeneous cell populations. Single-cell analysis is thus needed when cells are not genomically identical. Despite increased attention given to single-cell variation mediated heterogeneity of cancer cells, continued use of average-based methods is not only inaccurate but deceptive, as the “average” cancer cell clearly does not exist. Genome-level heterogeneity also may explain population heterogeneity, drug resistance, and cancer evolution. PMID:24091732

  17. Cell-specific localization of alkaloids in Catharanthus roseus stem tissue measured with Imaging MS and Single-cell MS

    PubMed Central

    Yamamoto, Kotaro; Takahashi, Katsutoshi; Mizuno, Hajime; Anegawa, Aya; Ishizaki, Kimitsune; Fukaki, Hidehiro; Ohnishi, Miwa; Yamazaki, Mami; Masujima, Tsutomu; Mimura, Tetsuro

    2016-01-01

    Catharanthus roseus (L.) G. Don is a medicinal plant well known for producing antitumor drugs such as vinblastine and vincristine, which are classified as terpenoid indole alkaloids (TIAs). The TIA metabolic pathway in C. roseus has been extensively studied. However, the localization of TIA intermediates at the cellular level has not been demonstrated directly. In the present study, the metabolic pathway of TIA in C. roseus was studied with two forefront metabolomic techniques, that is, Imaging mass spectrometry (MS) and live Single-cell MS, to elucidate cell-specific TIA localization in the stem tissue. Imaging MS indicated that most TIAs localize in the idioblast and laticifer cells, which emit blue fluorescence under UV excitation. Single-cell MS was applied to four different kinds of cells [idioblast (specialized parenchyma cell), laticifer, parenchyma, and epidermal cells] in the stem longitudinal section. Principal component analysis of Imaging MS and Single-cell MS spectra of these cells showed that similar alkaloids accumulate in both idioblast cell and laticifer cell. From MS/MS analysis of Single-cell MS spectra, catharanthine, ajmalicine, and strictosidine were found in both cell types in C. roseus stem tissue, where serpentine was also accumulated. Based on these data, we discuss the significance of TIA synthesis and accumulation in the idioblast and laticifer cells of C. roseus stem tissue. PMID:27001858

  18. The Measurement of Dissolved Oxygen

    ERIC Educational Resources Information Center

    Thistlethwayte, D.; And Others

    1974-01-01

    Describes an experiment in environmental chemistry which serves to determine the dissolved oxygen concentration in both fresh and saline water. Applications of the method at the undergraduate and secondary school levels are recommended. (CC)

  19. 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

  20. Fiber-Optic-Based Micro-Probe Using Hexagonal 1-in-6 Fiber Configuration for Intracellular Single-Cell pH Measurement.

    PubMed

    Yang, Qingbo; Wang, Hanzheng; Chen, Sisi; Lan, Xinwei; Xiao, Hai; Shi, Honglan; Ma, Yinfa

    2015-07-21

    Single-cell research is essential for understanding cell heterogeneity, cell differentiation, and carcinogenesis, among other important cellular processes. New techniques for intracellular pH monitoring are urgently needed to gain new insights into single-cell responses to external stimuli. In this study, fiber-optic reflection-based pH micro (μ)-probes (tip diameter: 500-3000 nm) were designed and fabricated using a novel hexagonal 1-in-6 fiber configuration. An organic-modified silicate (OrMoSils) sol-gel doped with a pH-sensitive dye, 2',7'-bis(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF), were coated onto the probe sensing tip for pH detection. These probes enabled neutral pH monitoring and quasi-real-time data acquisition (response time: 20 ± 5 s). The fluorescence signals of the newly developed probes were found to correlate linearly with pH (R(2) = 0.9869 when coupling laser power was at 8.2 mW) within a biologically relevant pH range (6.18-7.80). The pH resolution was 0.038 pH unit. The miniaturized probes were validated in single human lung cancer A549 cells to demonstrate applicability in single-cell experiments. In summary, novel pH μ-probes with excellent resolution and response times within a biologically relevant pH range were developed, and they can be used for measuring pH changes in single cells.

  1. A New Toolbox for Assessing Single Cells

    PubMed Central

    Tsioris, Konstantinos; Torres, Alexis J.; Douce, Thomas B.; Love, J. Christopher

    2015-01-01

    Unprecedented access to the biology of single cells is now feasible, enabled by recent technological advancements that allow us to manipulate and measure sparse samples and achieve a new level of resolution in space and time. This review focuses on advances in tools to study single cells for specific areas of biology. We examine both mature and nascent techniques to study single cells at the genomics, transcriptomics, and proteomics level. In addition, we provide an overview of tools that are well suited for following biological responses to defined perturbations with single-cell resolution. Techniques to analyze and manipulate single cells through soluble and chemical ligands, the microenvironment, and cell-cell interactions are provided. For each of these topics, we highlight the biological motivation, applications, methods, recent advances, and opportunities for improvement. The toolbox presented in this review can function as a starting point for the design of single-cell experiments. PMID:24910919

  2. A new toolbox for assessing single cells.

    PubMed

    Tsioris, Konstantinos; Torres, Alexis J; Douce, Thomas B; Love, J Christopher

    2014-01-01

    Unprecedented access to the biology of single cells is now feasible, enabled by recent technological advancements that allow us to manipulate and measure sparse samples and achieve a new level of resolution in space and time. This review focuses on advances in tools to study single cells for specific areas of biology. We examine both mature and nascent techniques to study single cells at the genomics, transcriptomics, and proteomics level. In addition, we provide an overview of tools that are well suited for following biological responses to defined perturbations with single-cell resolution. Techniques to analyze and manipulate single cells through soluble and chemical ligands, the microenvironment, and cell-cell interactions are provided. For each of these topics, we highlight the biological motivation, applications, methods, recent advances, and opportunities for improvement. The toolbox presented in this review can function as a starting point for the design of single-cell experiments.

  3. A new toolbox for assessing single cells.

    PubMed

    Tsioris, Konstantinos; Torres, Alexis J; Douce, Thomas B; Love, J Christopher

    2014-01-01

    Unprecedented access to the biology of single cells is now feasible, enabled by recent technological advancements that allow us to manipulate and measure sparse samples and achieve a new level of resolution in space and time. This review focuses on advances in tools to study single cells for specific areas of biology. We examine both mature and nascent techniques to study single cells at the genomics, transcriptomics, and proteomics level. In addition, we provide an overview of tools that are well suited for following biological responses to defined perturbations with single-cell resolution. Techniques to analyze and manipulate single cells through soluble and chemical ligands, the microenvironment, and cell-cell interactions are provided. For each of these topics, we highlight the biological motivation, applications, methods, recent advances, and opportunities for improvement. The toolbox presented in this review can function as a starting point for the design of single-cell experiments. PMID:24910919

  4. A Modular Probe Strategy for Drug Localization, Target Identification and Target Occupancy Measurement on Single Cell Level.

    PubMed

    Rutkowska, Anna; Thomson, Douglas W; Vappiani, Johanna; Werner, Thilo; Mueller, Katrin M; Dittus, Lars; Krause, Jana; Muelbaier, Marcel; Bergamini, Giovanna; Bantscheff, Marcus

    2016-09-16

    Late stage failures of candidate drug molecules are frequently caused by off-target effects or inefficient target engagement in vivo. In order to address these fundamental challenges in drug discovery, we developed a modular probe strategy based on bioorthogonal chemistry that enables the attachment of multiple reporters to the same probe in cell extracts and live cells. In a systematic evaluation, we identified the inverse electron demand Diels-Alder reaction between trans-cyclooctene labeled probe molecules and tetrazine-tagged reporters to be the most efficient bioorthogonal reaction for this strategy. Bioorthogonal biotinylation of the probe allows the identification of drug targets in a chemoproteomics competition binding assay using quantitative mass spectrometry. Attachment of a fluorescent reporter enables monitoring of spatial localization of probes as well as drug-target colocalization studies. Finally, direct target occupancy of unlabeled drugs can be determined at single cell resolution by competitive binding with fluorescently labeled probe molecules. The feasibility of the modular probe strategy is demonstrated with noncovalent PARP inhibitors.

  5. A Modular Probe Strategy for Drug Localization, Target Identification and Target Occupancy Measurement on Single Cell Level.

    PubMed

    Rutkowska, Anna; Thomson, Douglas W; Vappiani, Johanna; Werner, Thilo; Mueller, Katrin M; Dittus, Lars; Krause, Jana; Muelbaier, Marcel; Bergamini, Giovanna; Bantscheff, Marcus

    2016-09-16

    Late stage failures of candidate drug molecules are frequently caused by off-target effects or inefficient target engagement in vivo. In order to address these fundamental challenges in drug discovery, we developed a modular probe strategy based on bioorthogonal chemistry that enables the attachment of multiple reporters to the same probe in cell extracts and live cells. In a systematic evaluation, we identified the inverse electron demand Diels-Alder reaction between trans-cyclooctene labeled probe molecules and tetrazine-tagged reporters to be the most efficient bioorthogonal reaction for this strategy. Bioorthogonal biotinylation of the probe allows the identification of drug targets in a chemoproteomics competition binding assay using quantitative mass spectrometry. Attachment of a fluorescent reporter enables monitoring of spatial localization of probes as well as drug-target colocalization studies. Finally, direct target occupancy of unlabeled drugs can be determined at single cell resolution by competitive binding with fluorescently labeled probe molecules. The feasibility of the modular probe strategy is demonstrated with noncovalent PARP inhibitors. PMID:27384741

  6. Single-cell irradiation from [211At] astatine-labeled C215 monoclonal antibody: improved estimates of radiosensitivity from measurements on cellular uptake and retention.

    PubMed

    Palm, Stig; Bäck, Tom; Claesson, Ingela; Delle, Ulla; Hultborn, Ragnar; Lindegren, Sture; Jacobsson, Lars

    2003-01-01

    New data on the biological effect of 211At-C215 monoclonal antibody in a slowly rotating, widely dispersed single-cell suspension of the human cancer cell line Colo-205 is presented. Cell growth curves of each experiment were used to calculate an apparent cell survival after irradiation. Uptake measurements provided the data needed to calculate the average number of 211At decays per cell in the cell suspension. The results from each experiment were then fit to a mono-exponential function. From the exponential fit, an average of 35 +/- 2 (SD) astatine-211 decays per cell are required for 37% apparent cell survival (D0). PMID:12820374

  7. Single-cell irradiation from [211At] astatine-labeled C215 monoclonal antibody: improved estimates of radiosensitivity from measurements on cellular uptake and retention.

    PubMed

    Palm, Stig; Bäck, Tom; Claesson, Ingela; Delle, Ulla; Hultborn, Ragnar; Lindegren, Sture; Jacobsson, Lars

    2003-01-01

    New data on the biological effect of 211At-C215 monoclonal antibody in a slowly rotating, widely dispersed single-cell suspension of the human cancer cell line Colo-205 is presented. Cell growth curves of each experiment were used to calculate an apparent cell survival after irradiation. Uptake measurements provided the data needed to calculate the average number of 211At decays per cell in the cell suspension. The results from each experiment were then fit to a mono-exponential function. From the exponential fit, an average of 35 +/- 2 (SD) astatine-211 decays per cell are required for 37% apparent cell survival (D0).

  8. Measurement of Atomic-Oxygen Flux Distribution

    NASA Astrophysics Data System (ADS)

    Hisashiba, Takuya; Kuroda, Kazutaka; Masui, Hirokazu; Iwata, Minoru; Toyoda, Kazuhiro; Cho, Mengu

    Since material surfaces on the outside of spacecraft are exposed directly to the space environment, material having high tolerance to atomic oxygen, ultraviolet rays and radiation are preferred for long time spacecraft operation. In order to examine the influence of atomic oxygen (AO) on space grade materials, an atomic oxygen simulation chamber was developed. A system was developed that generates AO using a laser detonation method. A CO2 gas laser (5 J) irradiates the oxygen gas in a vacuum chamber (2 MPa) to dissociate the molecular oxygen into atomic. The velocity of AO can be controlled based on the timing between the laser and the gas pulse valve that injects oxygen gas into the chamber. The AO velocity generated by this system is measured using a QMASS (Quadruple Mass Spectrometer) and found to be 8 km/s. It was necessary to measure the AO flux distribution in the chamber at the location where the material samples are exposed to AO. The AO flux distribution was evaluated by measuring the mass gain on a QCM (QuartzCrystal Microbalance) coated with a silver film upon exposure to the AO.

  9. Single-Cell Measurements of Enzyme Levels as a Predictive Tool for Cellular Fates during Organic Acid Production

    PubMed Central

    Zdraljevic, Stefan; Wagner, Drew; Cheng, Kevin; Ruohonen, Laura; Jäntti, Jussi; Penttilä, Merja; Resnekov, Orna

    2013-01-01

    Organic acids derived from engineered microbes can replace fossil-derived chemicals in many applications. Fungal hosts are preferred for organic acid production because they tolerate lignocellulosic hydrolysates and low pH, allowing economic production and recovery of the free acid. However, cell death caused by cytosolic acidification constrains productivity. Cytosolic acidification affects cells asynchronously, suggesting that there is an underlying cell-to-cell heterogeneity in acid productivity and/or in resistance to toxicity. We used fluorescence microscopy to investigate the relationship between enzyme concentration, cytosolic pH, and viability at the single-cell level in Saccharomyces cerevisiae engineered to synthesize xylonic acid. We found that cultures producing xylonic acid accumulate cells with cytosolic pH below 5 (referred to here as “acidified”). Using live-cell time courses, we found that the probability of acidification was related to the initial levels of xylose dehydrogenase and sharply increased from 0.2 to 0.8 with just a 60% increase in enzyme abundance (Hill coefficient, >6). This “switch-like” relationship likely results from an enzyme level threshold above which the produced acid overwhelms the cell's pH buffering capacity. Consistent with this hypothesis, we showed that expression of xylose dehydrogenase from a chromosomal locus yields ∼20 times fewer acidified cells and ∼2-fold more xylonic acid relative to expression of the enzyme from a plasmid with variable copy number. These results suggest that strategies that further reduce cell-to-cell heterogeneity in enzyme levels could result in additional gains in xylonic acid productivity. Our results demonstrate a generalizable approach that takes advantage of the cell-to-cell variation of a clonal population to uncover causal relationships in the toxicity of engineered pathways. PMID:24038690

  10. SINGLE CELL GENOME SEQUENCING

    PubMed Central

    Yilmaz, Suzan; Singh, Anup K.

    2011-01-01

    Whole genome amplification and next-generation sequencing of single cells has become a powerful approach for studying uncultivated microorganisms that represent 90–99 % of all environmental microbes. Single cell sequencing enables not only the identification of microbes but also linking of functions to species, a feat not achievable by metagenomic techniques. Moreover, it allows the analysis of low abundance species that may be missed in community-based analyses. It has also proved very useful in complementing metagenomics in the assembly and binning of single genomes. With the advent of drastically cheaper and higher throughput sequencing technologies, it is expected that single cell sequencing will become a standard tool in studying the genome and transcriptome of microbial communities. PMID:22154471

  11. 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.

  12. On-Chip Single-Cell-Shape Control Technology for Understanding Contractile Motion of Cardiomyocytes Measured Using Optical Image Analysis System

    NASA Astrophysics Data System (ADS)

    Kaneko, Tomoyuki; Takizawa, Eikei; Nomura, Fumimasa; Hamada, Tomoyo; Hattori, Akihiro; Yasuda, Kenji

    2013-06-01

    Quantitative evaluation of mechanophysiological responses of cardiomyocytes has become more important for more precise prediction of cardiotoxicity. For the accurate detection of cardiomyocyte contraction, we have developed an on-chip single-cell-shape control technology on the basis of an agarose microchamber system and an on-chip optical image analysis system that records the contractile motions of cardiomyocytes with noninvasive/nondestructive measurement for long-term experiments. Using this on-chip single-cell-shape control technology, the shape of single cardiomyocytes was controlled by seeding the cells in 21-µm-radius (circular) or 20×70 µm2 (rectangular) agarose microchambers. To detect the contractility of cardiomyocytes, the cells were labeled with microbeads attached onto the surface of target cells and the motion of beads was acquired and analyzed using a newly developed wider-depth-of-field optics equipped with a 1/100 s high-speed digital camera. Mechanophysiological properties such as displacement and direction of movement were obtained using a real-time processing system module at spatial and temporal resolutions of 0.15 µm and 10 ms, respectively. Comparisons of displacement and direction of contraction between circular and rectangular cardiomyocytes indicated that the rectangular cardiomyocytes tended to contract along the longitudinal direction as in a real heart. This result suggests that the shape of cells affected the function of cells. The on-chip single-cell-shape control technology and optical image analysis system enable the detection of the motion of contraction of single-shape-controlled cardiomyocytes, and are expected to be applicable to the more precise prediction of cardiotoxicity.

  13. 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

  14. A New Radio Frequency Plasma Oxygen Primary Ion Source on Nano Secondary Ion Mass Spectrometry for Improved Lateral Resolution and Detection of Electropositive Elements at Single Cell Level.

    PubMed

    Malherbe, Julien; Penen, Florent; Isaure, Marie-Pierre; Frank, Julia; Hause, Gerd; Dobritzsch, Dirk; Gontier, Etienne; Horréard, François; Hillion, François; Schaumlöffel, Dirk

    2016-07-19

    An important application field of secondary ion mass spectrometry at the nanometer scale (NanoSIMS) is the detection of chemical elements and, in particular, metals at the subcellular level in biological samples. The detection of many trace metals requires an oxygen primary ion source to allow the generation of positive secondary ions with high yield in the NanoSIMS. The duoplasmatron oxygen source is commonly used in this ion microprobe but cannot achieve the same quality of images as the cesium primary ion source used to produce negative secondary ions (C(-), CN(-), S(-), P(-)) due to a larger primary ion beam size. In this paper, a new type of an oxygen ion source using a rf plasma is fitted and characterized on a NanoSIMS50L. The performances of this primary ion source in terms of current density and achievable lateral resolution have been characterized and compared to the conventional duoplasmatron and cesium sources. The new rf plasma oxygen source offered a net improvement in terms of primary beam current density compared to the commonly used duoplasmatron source, which resulted in higher ultimate lateral resolutions down to 37 nm and which provided a 5-45 times higher apparent sensitivity for electropositive elements. Other advantages include a better long-term stability and reduced maintenance. This new rf plasma oxygen primary ion source has been applied to the localization of essential macroelements and trace metals at basal levels in two biological models, cells of Chlamydomonas reinhardtii and Arabidopsis thaliana. PMID:27291826

  15. A microfluidic systems biology approach for live single-cell mitochondrial ROS imaging.

    PubMed

    Kniss, Ariel; Lu, Hang; Jones, Dean P; Kemp, Melissa L

    2013-01-01

    Most current studies of reactive oxygen species (ROS) production report globally averaged measurements within the cell; however, ROS can be produced in distinct subcellular locations and have local effects in their immediate vicinity. A microfluidic platform for high-throughput single-cell imaging allows mitochondrial ROS production to be monitored as varying in both space and time. Using this systems biology approach, single-cell variability can be viewed within a population. We discuss single-cell monitoring of contributors to mitochondrial redox state-mitochondrial hydrogen peroxide or superoxide-through the use of a small molecule probe or targeted fluorescent reporter protein. Jurkat T lymphoma cells were stimulated with antimycin A and imaged in an arrayed microfluidic device over time. Differences in single-cell responses were observed as a function of both inhibitor concentration and type of ROS measurement used.

  16. Oxygen fugacities directly measured in magmatic gases

    USGS Publications Warehouse

    Sato, M.; Wright, T.L.

    1966-01-01

    An electrochemical device was used to measure the fugacity of oxygen (fO2) in holes drilled through the crust of Makaopuhi lava lake, Kilauea Volcano, Hawaii. Results obtained within 6 months of the lake formation show that log fO2 normally varies linearly with the reciprocal of the absolute temperature, and that chemical changes occurring in the cooling tholeiitic basalt are reflected in the fO2 values measured in the holes.

  17. Quantitative measurement of oxygen in microgravity combustion

    NASA Technical Reports Server (NTRS)

    Silver, Joel A.

    1995-01-01

    This research combines two innovations in an experimental system which should result in a new capability for quantitative, nonintrusive measurement of major combustion species. Using a newly available vertical cavity surface-emitting diode laser (VCSEL) and an improved spatial scanning method, we plan to measure the temporal and spatial profiles of the concentrations and temperatures of molecular oxygen in a candle flame and in a solid fuel (cellulose sheet) system. The required sensitivity for detecting oxygen is achieved by the use of high frequency wavelength modulation spectroscopy (WMS). Measurements will be performed in the NASA Lewis 2.2-second Drop Tower Facility. The objective of this research is twofold. First, we want to develop a better understanding of the relative roles of diffusion and reaction of oxygen in microgravity combustion. As the primary oxidizer species, oxygen plays a major role in controlling the observed properties of flames, including flame front speed (in solid or liquid flames), extinguishment characteristics, flame size, and flame temperature. The second objective is to develop better diagnostics based on diode laser absorption which can be of real value in microgravity combustion research. We will also demonstrate diode lasers' potential usefulness for compact, intrinsically-safe monitoring sensors aboard spacecraft. Such sensors could be used to monitor any of the major cabin gases as well as important pollutants.

  18. Tracer monitored titrations: measurement of dissolved oxygen.

    PubMed

    Martz, Todd; Takeshita, Yuichiro; Rolph, Rebecca; Bresnahan, Philip

    2012-01-01

    The tracer monitored titration (TMT) technique is evaluated for measurement of dissolved oxygen. The TMT developed in this work uses a simple apparatus consisting of a low-precision pump for titrant delivery and an optical detector based on a white LED and two photodiodes with interference filters. It is shown that the classic Winkler method can be made free of routine volumetric and gravimetric measurements by application of TMT theory, which allows tracking the amounts of titrant and sample using a chemical tracer. The measurement precision of the prototype setup was 0.3% RSD.

  19. 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.

  20. [New Method of Measuring Arterial Oxygen Saturation].

    PubMed

    Li, Gang; Bao, Lei; Zhou, Mei; Lin, Ling; Liu, Rui; Zhao, Chun-jie

    2016-01-01

    The traditional method of measuring arterial oxygen saturation is that R value, the ratio of alternating component of the logarithmic photoplethysmography, is firstly computed and then the linear regression model is established by experiment. The R value computation is a dimension reduction process based on Lambert-beer law, which aims at eliminating the influence of optical path and minimizing the impact of individual differences. When taking scattering into consideration, the dimension reduction process loses information, introduces the system error and limits the precision of measurement. In order to reduce the measurement error resulting from the scattering effects, this paper presents a new method that the peak and valley values of dual-wavelength logarithmic photoplethysmography waves are used as the independent variables to develop a linear regression model to predict the arterial oxygen saturation. During the experiment, the in-vivo measurements were carried out on 23 healthy volunteer and 133 samples of photoplethysmography waves and the reference value of oxygen saturation were recorded. To compare the predictive performance between the new method and the R value method, 90 samples were randomly selected as modeling sets and the remaining 43 samples were used as prediction sets. Random selection of modeling sets and prediction are executed 10 times. The average related coefficients of the prediction sets of the new method and the R value method are 0.890 6 and 0.846 8, and the average root mean square errors are 0.889 6% and 1.037 3% respectively. Results indicate that the performance of the new method is better than the one of the R value method, and the predictivemodel based on 4 parameters can improve the stability and accuracy of measurement. And the new method has guiding significance to the measurement of human body's blood physiological information based on limited wavelength spectrum data. PMID:27228767

  1. Temperature-Dependent Model of Multi-step Transcription Initiation in Escherichia coli Based on Live Single-Cell Measurements

    PubMed Central

    Lloyd-Price, Jason; Tran, Huy; Ribeiro, Andre S.

    2016-01-01

    Transcription kinetics is limited by its initiation steps, which differ between promoters and with intra- and extracellular conditions. Regulation of these steps allows tuning both the rate and stochasticity of RNA production. We used time-lapse, single-RNA microscopy measurements in live Escherichia coli to study how the rate-limiting steps in initiation of the Plac/ara-1 promoter change with temperature and induction scheme. For this, we compared detailed stochastic models fit to the empirical data in maximum likelihood sense using statistical methods. Using this analysis, we found that temperature affects the rate limiting steps unequally, as nonlinear changes in the closed complex formation suffice to explain the differences in transcription dynamics between conditions. Meanwhile, a similar analysis of the PtetA promoter revealed that it has a different rate limiting step configuration, with temperature regulating different steps. Finally, we used the derived models to explore a possible cause for why the identified steps are preferred as the main cause for behavior modifications with temperature: we find that transcription dynamics is either insensitive or responds reciprocally to changes in the other steps. Our results suggests that different promoters employ different rate limiting step patterns that control not only their rate and variability, but also their sensitivity to environmental changes. PMID:27792724

  2. Single-Cell Analysis in Cancer Genomics.

    PubMed

    Saadatpour, Assieh; Lai, Shujing; Guo, Guoji; Yuan, Guo-Cheng

    2015-10-01

    Genetic changes and environmental differences result in cellular heterogeneity among cancer cells within the same tumor, thereby complicating treatment outcomes. Recent advances in single-cell technologies have opened new avenues to characterize the intra-tumor cellular heterogeneity, identify rare cell types, measure mutation rates, and, ultimately, guide diagnosis and treatment. In this paper we review the recent single-cell technological and computational advances at the genomic, transcriptomic, and proteomic levels, and discuss their applications in cancer research. PMID:26450340

  3. Oxygen Measurements in Liposome Encapsulated Hemoglobin

    NASA Astrophysics Data System (ADS)

    Phiri, Joshua Benjamin

    Liposome encapsulated hemoglobins (LEH's) are of current interest as blood substitutes. An analytical methodology for rapid non-invasive measurements of oxygen in artificial oxygen carriers is examined. High resolution optical absorption spectra are calculated by means of a one dimensional diffusion approximation. The encapsulated hemoglobin is prepared from fresh defibrinated bovine blood. Liposomes are prepared from hydrogenated soy phosphatidylcholine (HSPC), cholesterol and dicetylphosphate using a bath sonication method. An integrating sphere spectrophotometer is employed for diffuse optics measurements. Data is collected using an automated data acquisition system employing lock-in -amplifiers. The concentrations of hemoglobin derivatives are evaluated from the corresponding extinction coefficients using a numerical technique of singular value decomposition, and verification of the results is done using Monte Carlo simulations. In situ measurements are required for the determination of hemoglobin derivatives because most encapsulation methods invariably lead to the formation of methemoglobin, a nonfunctional form of hemoglobin. The methods employed in this work lead to high resolution absorption spectra of oxyhemoglobin and other derivatives in red blood cells and liposome encapsulated hemoglobin (LEH). The analysis using singular value decomposition method offers a quantitative means of calculating the fractions of oxyhemoglobin and other hemoglobin derivatives in LEH samples. The analytical methods developed in this work will become even more useful when production of LEH as a blood substitute is scaled up to large volumes.

  4. 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.

  5. Single cell optical transfection.

    PubMed

    Stevenson, David J; Gunn-Moore, Frank J; Campbell, Paul; Dholakia, Kishan

    2010-06-01

    The plasma membrane of a eukaryotic cell is impermeable to most hydrophilic substances, yet the insertion of these materials into cells is an extremely important and universal requirement for the cell biologist. To address this need, many transfection techniques have been developed including viral, lipoplex, polyplex, capillary microinjection, gene gun and electroporation. The current discussion explores a procedure called optical injection, where a laser field transiently increases the membrane permeability to allow species to be internalized. If the internalized substance is a nucleic acid, such as DNA, RNA or small interfering RNA (siRNA), then the process is called optical transfection. This contactless, aseptic, single cell transfection method provides a key nanosurgical tool to the microscopist-the intracellular delivery of reagents and single nanoscopic objects. The experimental possibilities enabled by this technology are only beginning to be realized. A review of optical transfection is presented, along with a forecast of future applications of this rapidly developing and exciting technology. PMID:20064901

  6. EVALUATING AN INNOVATIVE OXYGEN SENSOR FOR REMOTE SUBSURFACE OXYGEN MEASUREMENTS

    SciTech Connect

    Millings, M; Brian Riha, B; Warren Hyde, W; Karen Vangelas, K; Brian02 Looney, B

    2006-10-12

    Oxygen is a primary indicator of whether anaerobic reductive dechlorination and similar redox based processes contribute to natural attenuation remedies at chlorinated solvent contaminated sites. Thus, oxygen is a viable indicator parameter for documenting that a system is being sustained in an anaerobic condition. A team of researchers investigated the adaptation of an optical sensor that was developed for oceanographic applications. The optical sensor, because of its design and operating principle, has potential for extended deployment and sensitivity at the low oxygen levels relevant to natural attenuation. The results of the research indicate this tool will be useful for in situ long-term monitoring applications, but that the traditional characterization tools continue to be appropriate for characterization activities.

  7. A single-cell technique for the measurement of membrane potential, membrane conductance, and the efflux of rapidly penetrating solutes in Amphiuma erythrocytes.

    PubMed

    Stoner, L C; Kregenow, F M

    1980-10-01

    We describe a single-cell technique for measuring membrane potential, membrane resistance, and the efflux of rapidly penetrating solutes such as Cl and H2O. Erythrocytes from Amphiuma means were aspirated into a Sylgard (Dow Corning Corp.)-coated capillary. The aspirated cell separated a solution within the capillary from a solution in the bath. Each of these two solutions was contiguous with approximately 5% of the total membrane surface. Microelectrodes placed concentrically within the capillary permit the measurement of intracellular voltage, specific membrane resistance, and the electrical seal between the two solutions. The intracellular voltage averaged -17.7 mV (pH 7.6) and changed as either intra- or extracellular chloride was varied. The average specific membrane resistance measured by passing current across the exposed membrane surface was 110 ohm-cm2. 36Cl and tritiated H2O fluxes (0.84 +/- 0.05 x 10(-6) M . cm-2 . min-1 and 6.4 +/- 1.5 x 10(-3) M . cm-2 . min-1, respectively) were determined by noting the rate at which isotope leaves the cell and crosses the membrane exposed to the bath. Our measured values for the flux of 36Cl and tritiated H2O approximate reported values for free-floating cells. 36Cl efflux, in addition, is inhibited by 4-acetamido-4'-isothiocyano-stilbene 2,2'-disulfonic acid (SITS) and furosemide, known inhibitors of the anion exchange mechanism responsible for the rapid anion fluxes of red blood cells. One can also demonstrate directly that > 89% of 36Cl efflux is "electrically silent" by analyzing the flux in the presence of an imposed transcellular voltage.

  8. Single cell ionization by a laser trap: a preliminary study in measuring radiation dose and charge in BT20 breast carcinoma cells

    PubMed Central

    Kelley, Michele; Gao, Ying; Erenso, Daniel

    2016-01-01

    In this work, a preliminary study in the application of a laser trap for ionization of living carcinoma cells is presented. The study was conducted using BT20 breast carcinoma cells cultured and harvested in our laboratory. Each cell, for a total of 50 cells, was trapped and ionized by a high intensity infrared laser at 1064 nm. The threshold radiation dose and the resultant charge from the ionization for each cell were determined. With the laser trap serving as a radiation source, the cell underwent dielectric breakdown of the membrane. When this process occurs, the cell becomes highly charged and its dielectric susceptibility changes. The charge creates an increasing electrostatic force while the changing dielectric susceptibility diminishes the strength of the trapping force. Consequently, at some instant of time the cell gets ejected from the trap. The time inside the trap while the cell is being ionized, the intensity of the radiation, and the post ionization trajectory of the cell were used to determine the threshold radiation dose and the charge for each cell. The measurement of the charge vs ionization radiation dose at single cell level could be useful in the accuracy of radiotherapy as the individual charges can collectively create a strong enough electrical interaction to cause dielectric breakdown in other cells in a tumor. PMID:27699110

  9. Single cell ionization by a laser trap: a preliminary study in measuring radiation dose and charge in BT20 breast carcinoma cells

    PubMed Central

    Kelley, Michele; Gao, Ying; Erenso, Daniel

    2016-01-01

    In this work, a preliminary study in the application of a laser trap for ionization of living carcinoma cells is presented. The study was conducted using BT20 breast carcinoma cells cultured and harvested in our laboratory. Each cell, for a total of 50 cells, was trapped and ionized by a high intensity infrared laser at 1064 nm. The threshold radiation dose and the resultant charge from the ionization for each cell were determined. With the laser trap serving as a radiation source, the cell underwent dielectric breakdown of the membrane. When this process occurs, the cell becomes highly charged and its dielectric susceptibility changes. The charge creates an increasing electrostatic force while the changing dielectric susceptibility diminishes the strength of the trapping force. Consequently, at some instant of time the cell gets ejected from the trap. The time inside the trap while the cell is being ionized, the intensity of the radiation, and the post ionization trajectory of the cell were used to determine the threshold radiation dose and the charge for each cell. The measurement of the charge vs ionization radiation dose at single cell level could be useful in the accuracy of radiotherapy as the individual charges can collectively create a strong enough electrical interaction to cause dielectric breakdown in other cells in a tumor.

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

    PubMed

    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.

  11. 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

  12. BENZO[A]PYRENE AND ITS K-REGION DIOL INDUCE DNA DAMAGE IN C3H10T1/2C18 CELLS AS MEASURED BY THE ALKALINE SINGLE CELL GEL (COMET) ASSAY

    EPA Science Inventory


    160. Benzo[a]pyrene and its K-region diol induce DNA damage in C3HlOTl/2Cl8 cells as measured by the alkaline single cell gel (Comet) assay

    In a continuing series of studies on the genotoxicity ofK-region dihydrodiols of polycyclic aromatic hydrocarbons, we have repo...

  13. Quantitative Measurement of Oxygen in Microgravity Combustion

    NASA Technical Reports Server (NTRS)

    Silver, Joel A.

    1997-01-01

    A low-gravity environment, in space or in ground-based facilities such as drop towers, provides a unique setting for studying combustion mechanisms. Understanding the physical phenomena controlling the ignition and spread of flames in microgravity has importance for space safety as well as for better characterization of dynamical and chemical combustion processes which are normally masked by buoyancy and other gravity-related effects. Due to restrictions associated with performing measurements in reduced gravity, diagnostic methods which have been applied to microgravity combustion studies have generally been limited to capture of flame emissions on film or video, laser Schlieren imaging and (intrusive) temperature measurements using thermocouples. Given the development of detailed theoretical models, more sophisticated diagnostic methods are needed to provide the kind of quantitative data necessary to characterize the properties of microgravity combustion processes as well as provide accurate feedback to improve the predictive capabilities of the models. When the demands of space flight are considered, the need for improved diagnostic systems which are rugged, compact, reliable, and operate at low power becomes apparent. The objective of this research is twofold. First, we want to develop a better understanding of the relative roles of diffusion and reaction of oxygen in microgravity combustion. As the primary oxidizer species, oxygen plays a major role in controlling the observed properties of flames, including flame front speed (in solid or liquid flames), extinguishment characteristics, flame size and flame temperature. The second objective is to develop better diagnostics based on diode laser absorption which can be of real value in both microgravity combustion research and as a sensor on-board Spacelab as either an air quality monitor or as part of a fire detection system. In our prior microgravity work, an eight line-of-sight fiber optic system measured

  14. Chemical Analysis of Single Cells

    NASA Astrophysics Data System (ADS)

    Borland, Laura M.; Kottegoda, Sumith; Phillips, K. Scott; Allbritton, Nancy L.

    2008-07-01

    Chemical analysis of single cells requires methods for quickly and quantitatively detecting a diverse array of analytes from extremely small volumes (femtoliters to nanoliters) with very high sensitivity and selectivity. Microelectrophoretic separations, using both traditional capillary electrophoresis and emerging microfluidic methods, are well suited for handling the unique size of single cells and limited numbers of intracellular molecules. Numerous analytes, ranging from small molecules such as amino acids and neurotransmitters to large proteins and subcellular organelles, have been quantified in single cells using microelectrophoretic separation techniques. Microseparation techniques, coupled to varying detection schemes including absorbance and fluorescence detection, electrochemical detection, and mass spectrometry, have allowed researchers to examine a number of processes inside single cells. This review also touches on a promising direction in single cell cytometry: the development of microfluidics for integrated cellular manipulation, chemical processing, and separation of cellular contents.

  15. Measuring Traces Of Oxygen By Resonant Electron Attachment

    NASA Technical Reports Server (NTRS)

    Man, Kin Fung; Boumsellek, Said; Chutjian, Ara

    1995-01-01

    Method of detecting trace amounts of oxygen based on dissociative attachment of electrons to oxygen molecules followed by measurement of resulting flux of negative oxygen ions in mass spectrometer. High sensitivity achieved in method by exploiting resonance in dissociative attachment of electrons to oxygen molecules: electron-attachment cross section rises to high peak at incident electron kinetic energy of 6.2 eV. Relative concentrations below 1 ppb detected. Devised to increase sensitivity of detection of oxygen in processing chambers in which oxygen regarded as contaminant; for example, chambers used in making semiconductor devices and in growing high-purity crystals.

  16. 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…

  17. 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

  18. Optoacoustic measurements of human placenta and umbilical blood oxygenation

    NASA Astrophysics Data System (ADS)

    Nanovskaya, T. N.; Petrov, I. Y.; Petrov, Y.; Patrikeeva, S. L.; Ahmed, M. S.; Hankins, G. D. V.; Prough, D. S.; Esenaliev, R. O.

    2016-03-01

    Adequate oxygenation is essential for normal embryogenesis and fetal growth. Perturbations in the intrauterine oxidative environment during pregnancy are associated with several pathophysiological disorders such as pregnancy loss, preeclampsia, and intrauterine growth restriction. We proposed to use optoacoustic technology for monitoring placental and fetal umbilical blood oxygenation. In this work, we studied optoacoustic monitoring of oxygenation in placenta and umbilical cord blood ex vivo using technique of placenta perfusion. We used a medical grade, nearinfrared, tunable, optoacoustic system developed and built for oxygenation monitoring in blood vessels and in tissues. First, we calibrated the system for cord blood oxygenation measurements by using a CO-Oximeter (gold standard). Then we performed validation in cord blood circulating through the catheters localized on the fetal side of an isolated placental lobule. Finally, the oxygenation measurements were performed in the perfused placental tissue. To increase or decrease blood oxygenation, we used infusion of a gas mixture of 95% O2 + 5% CO2 and 95% N2 + 5% CO2, respectively. In placental tissue, up to four cycles of changes in oxygenation were performed. The optoacoustically measured oxygenation in circulating cord blood and in placental lobule closely correlated with the actual oxygenation data measured by CO-Oximeter. We plan to further test the placental and cord blood oxygenation monitoring with optoacoustics in animal and clinical studies.

  19. Single Cell Electrical Characterization Techniques.

    PubMed

    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

  20. A novel method for multiparameter physiological phenotype characterization at the single-cell level

    NASA Astrophysics Data System (ADS)

    Kelbauskas, Laimonas; Ashili, Shashanka; Houkal, Jeff; Smith, Dean; Mohammadreza, Aida; Lee, Kristen; Kumar, Ashok; Anis, Yasser; Paulson, Tom; Youngbull, Cody; Tian, Yanqing; Johnson, Roger; Holl, Mark; Meldrum, Deirdre

    2011-02-01

    Non-genetic intercellular heterogeneity has been increasingly recognized as one of the key factors in a variety of core cellular processes including proliferation, stimulus response, carcinogenesis and drug resistance. Many diseases, including cancer, originate in a single or a few cells. Early detection and characterization of these abnormal cells can provide new insights into the pathogenesis and serve as a tool for better disease diagnosis and treatment. We report on a novel technology for multiparameter physiological phenotype characterization at the single-cell level. It is based on real-time measurements of concentrations of several metabolites by means of extracellular optical sensors in microchambers of sub-nL volume containing single cells. In its current configuration, the measurement platform features the capability to detect oxygen consumption rate and pH changes under normoxic and hypoxic conditions at the single-cell level. We have conceived, designed and developed a semi-automated method for single-cell manipulation and loading into microwells utilizing custom, high-precision fluid handling at the nanoliter scale. We present the results of a series of measurements of oxygen consumption rates (OCRs) of single human metaplastic esophageal epithelial cells. In addition, to assess the effects of cell-to-cell interactions, we have measured OCRs of two and three cells placed in a single well. The major advantages of the approach are a) multiplexed characterization of cell phenotype at the single-cell level, b) minimal invasiveness due to the distant positioning of sensors, and c) flexibility in terms of accommodating measurements of other metabolites or biomolecules of interest.

  1. Device for measuring the total concentration of oxygen in gases

    DOEpatents

    Isaacs, Hugh S.; Romano, Anthony J.

    1977-01-01

    This invention provides a CO equilibrium in a device for measuring the total concentration of oxygen impurities in a fluid stream. To this end, the CO equilibrium is produced in an electrochemical measuring cell by the interaction of a carbon element in the cell with the chemically combined and uncombined oxygen in the fluid stream at an elevated temperature.

  2. Single cell dynamic phenotyping

    PubMed Central

    Patsch, Katherin; Chiu, Chi-Li; Engeln, Mark; Agus, David B.; Mallick, Parag; Mumenthaler, Shannon M.; Ruderman, Daniel

    2016-01-01

    Live cell imaging has improved our ability to measure phenotypic heterogeneity. However, bottlenecks in imaging and image processing often make it difficult to differentiate interesting biological behavior from technical artifact. Thus there is a need for new methods that improve data quality without sacrificing throughput. Here we present a 3-step workflow to improve dynamic phenotype measurements of heterogeneous cell populations. We provide guidelines for image acquisition, phenotype tracking, and data filtering to remove erroneous cell tracks using the novel Tracking Aberration Measure (TrAM). Our workflow is broadly applicable across imaging platforms and analysis software. By applying this workflow to cancer cell assays, we reduced aberrant cell track prevalence from 17% to 2%. The cost of this improvement was removing 15% of the well-tracked cells. This enabled detection of significant motility differences between cell lines. Similarly, we avoided detecting a false change in translocation kinetics by eliminating the true cause: varied proportions of unresponsive cells. Finally, by systematically seeking heterogeneous behaviors, we detected subpopulations that otherwise could have been missed, including early apoptotic events and pre-mitotic cells. We provide optimized protocols for specific applications and step-by-step guidelines for adapting them to a variety of biological systems. PMID:27708391

  3. 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.

  4. Measurement of arterial plasma oxygenation in dynamic oxygen-enhanced MRI.

    PubMed

    Kershaw, Lucy E; Naish, Josephine H; McGrath, Deirdre M; Waterton, John C; Parker, Geoffrey J M

    2010-12-01

    Inhaled oxygen can be used as a contrast agent for magnetic resonance imaging, due to the T(1) shortening effect of the oxygen dissolved in blood and tissue water. In this study, blood T(1) was measured dynamically in 14 volunteers (seven smokers, seven never-smokers) as the inhaled gas was switched from medical air to 100% oxygen and back to medical air. These T(1) values were converted to changes in partial pressure of oxygen, which were found to be in agreement with literature values. There were differences in curve shape and curve height between the smoker and never-smoker groups, suggesting differences in lung function due to smoking-related damage. These curves could be used as an input function for modeling of oxygen uptake in tissues. The differences between groups highlight the importance of measuring such an input function for each individual rather than relying on an assumed measurement.

  5. Measurement of tumor oxygenation using new frequency domain phosphorometers.

    PubMed

    Wilson, David F; Vinogradov, Sergei A; Dugan, Benjamin W; Biruski, Dubravko; Waldron, Lee; Evans, Sydney A

    2002-05-01

    Oxygen dependent quenching of phosphorescence allows for non-invasive measurements of oxygen in tissue. We have designed and constructed a novel multi-frequency instrument for measurement of phosphorescence lifetimes and developed algorithms for determining the distribution of oxygen (oxygen histogram) in the microvasculature of tissue with good temporal resolution (Vinogradov et al., 2002, Compar. Biochem. A, these proceedings). This technology, in combination with a new water soluble near infra red phosphor (Oxyphor G2), was used to examine the oxygenation of subcutaneous Q7 tumors grown on the flank of Buffalo rats and their response to giving the rats oxygen or carbogen to breathe. Phosphorescence was measured using excitation at 635 nm and emission at >700 nm (the phosphorescence maximum is near 800 nm). The excitation and collection light guides were placed on the surface of the skin of the anesthetized animals separated by approximately 0.8 cm. A 6 x 6 or 7 x 7 grid (approx. 4 cm x 4 cm) was drawn on the flank and oxygen histograms were measured in each square, providing 'images' of the oxygen distribution in the tissue. This procedure determines the tissue oxygen distribution at each position in the grid. Regions of relative hypoxia (associated with the tumor) can be readily localized and the extent of hypoxia quantitatively evaluated.

  6. Measurement of oxygen transfer from air into organic solvents

    PubMed Central

    Ramesh, Hemalata; Hobisch, Mathias; Borisov, Sergey; Klimant, Ingo; Krühne, Ulrich; Woodley, John M

    2015-01-01

    Abstract BACKGROUND The use of non‐aqueous organic media is becoming increasingly important in many biotechnological applications in order to achieve process intensification. Such media can be used, for example, to directly extract poorly water‐soluble toxic products from fermentations. Likewise many biological reactions require the supply of oxygen, most normally from air. However, reliable online measurements of oxygen concentration in organic solvents (and hence oxygen transfer rates from air to the solvent) has to date proven impossible due to limitations in the current analytical methods. RESULTS For the first time, online oxygen measurements in non‐aqueous media using a novel optical sensor are demonstrated. The sensor was used to measure oxygen concentration in various organic solvents including toluene, THF, isooctane, DMF, heptane and hexane (which have all been shown suitable for several biological applications). Subsequently, the oxygen transfer rates from air into these organic solvents were measured. CONCLUSION The measurement of oxygen transfer rates from air into organic solvents using the dynamic method was established using the solvent resistant optical sensor. The feasibility of online oxygen measurements in organic solvents has also been demonstrated, paving the way for new opportunities in process control. © 2015 The Authors. Journal of Chemical Technology & Biotechnology published by JohnWiley & Sons Ltd on behalf of Society of Chemical Industry.

  7. Visible light optical coherence tomography measures retinal oxygen metabolic response to systemic oxygenation

    PubMed Central

    Yi, Ji; Liu, Wenzhong; Chen, Siyu; Backman, Vadim; Sheibani, Nader; Sorenson, Christine M.; Fawzi, Amani A.; Linsenmeier, Robert A.; Zhang, Hao F.

    2015-01-01

    The lack of capability to quantify oxygen metabolism noninvasively impedes both fundamental investigation and clinical diagnosis of a wide spectrum of diseases including all the major blinding diseases such as age-related macular degeneration, diabetic retinopathy, and glaucoma. Using visible light optical coherence tomography (vis-OCT), we demonstrated accurate and robust measurement of retinal oxygen metabolic rate (rMRO2) noninvasively in rat eyes. We continuously monitored the regulatory response of oxygen consumption to a progressive hypoxic challenge. We found that both oxygen delivery, and rMRO2 increased from the highly regulated retinal circulation (RC) under hypoxia, by 0.28 ± 0.08 μL min−1 (p < 0.001), and 0.20 ± 0.04 μL min−1 (p < 0.001) per 100 mmHg systemic pO2 reduction, respectively. The increased oxygen extraction compensated for the deficient oxygen supply from the poorly regulated choroidal circulation. Results from an oxygen diffusion model based on previous oxygen electrode measurements corroborated our in vivo observations. We believe that vis-OCT has the potential to reveal the fundamental role of oxygen metabolism in various retinal diseases. PMID:26658555

  8. 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

  9. ASRDI oxygen technology survey. Volume 6: Flow measurement instrumentation

    NASA Technical Reports Server (NTRS)

    Mann, D. B.

    1974-01-01

    A summary is provided of information available on liquid and gaseous oxygen flowmetering including an evaluation of commercial meters. The instrument types, physical principles of measurement, and performance characteristics are described. Problems concerning flow measurements of less than plus or minus two percent uncertainty are reviewed. Recommendations concerning work on flow reference systems, the use of surrogate fluids, and standard tests for oxygen flow measurements are also presented.

  10. ASRDI oxygen technology survey. Volume 8: Pressure measurement

    NASA Technical Reports Server (NTRS)

    Arvidson, J. M.; Brennan, J. A.

    1975-01-01

    Pressure transducers and their current uses with gaseous or liquid oxygen are reviewed. All transducer types such as strain gage, capacitance, potentiometric, piezoelectric, etc., are included. Topics covered include: cryogenic pressure measurement; material compatibility with gaseous and liquid oxygen; cleaning procedures; pressure tap connections; transducer types and descriptions; and calibration techniques.

  11. Visible light optical coherence tomography measure retinal oxygen metabolic response to systemic oxygenation (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Yi, Ji; Liu, Wenzhong; Chen, Siyu; Backman, Vadim; Sheibani, Nader; Sorenson, Christine M.; Fawzi, Amani A.; Linsenmeier, Robert A.; Zhang, Hao F.

    2016-03-01

    The lack of capability to quantify oxygen metabolism noninvasively impedes both fundamental investigation and clinical diagnosis of a wide spectrum of diseases including all the major blinding diseases such as age-related macular degeneration, diabetic retinopathy, and glaucoma. Using visible light optical coherence tomography (vis-OCT), we demonstrated accurate and robust measurement of retinal oxygen metabolic rate (rMRO2) noninvasively in rat eyes. The rMRO2 was calculated by concurrent measurement of blood flow and blood oxygen saturation (sO2). Blood flow was calculated by the principle of Doppler optical coherence tomography, where the phase shift between two closely spaced A-lines measures the axial velocity. The distinct optical absorption spectra of oxy- and deoxy-hemoglobin provided the contrast for sO2 measurement, combined with the spectroscopic analysis of vis-OCT signal within the blood vessels. We continuously monitored the regulatory response of oxygen consumption to a progressive hypoxic challenge. We found that both oxygen delivery, and rMRO2 increased from the highly regulated retinal circulation (RC) under hypoxia, by 0.28+/-0.08 μL/min (p<0.001), and 0.20+/-0.04 μL/min (p<0.001) per 100 mmHg systemic pO2 reduction, respectively. The increased oxygen extraction compensated for the deficient oxygen supply from the poorly regulated choroidal circulation (CC).

  12. Outcome measures for palliative oxygen therapy: relevance and practical utility.

    PubMed

    Antoniu, Sabina; Mihaltan, Florin

    2014-06-01

    Dyspnea is a common symptom in many advanced malignant and non-malignant diseases and often is refractory to the usual therapies. In such circumstances palliative care approaches are necessary and among them palliative care oxygen therapy can be applied although currently its effectiveness is rather uncertain. Palliative oxygen therapy can be given on either continuous basis or on demand. Often the continuous palliative oxygen therapy is seen as long-term oxygen therapy although their aims are rather different. Palliative oxygen therapy was evaluated in populations with mixed underlying diseases, with outcome measures not only the most appropriate for the setting and therefore these limitations might have influenced the overall perceived therapeutic benefit. Therefore an evaluation of this method in subsets defined based on the etiology and pathogenic mechanisms and with appropriate outcome measures would help to better define the criteria for its indication and would increase its acceptability.

  13. FIELD MEASUREMENT OF DISSOLVED OXYGEN: A COMPARISON OF TECHNIQUES

    EPA Science Inventory

    The measurement and interpretation of geochemical redox parameters are key components of ground water remedial investigations. Dissolved oxygen (DO) is perhaps the most robust geochemical parameter in redox characterization; however, recent work has indicated a need for proper da...

  14. HERSCHEL MEASUREMENTS OF MOLECULAR OXYGEN IN ORION

    SciTech Connect

    Goldsmith, Paul F.; Chen, Jo-Hsin; Li Di; Liseau, Rene; Black, John H.; Bell, Tom A.; Hollenbach, David; Kaufman, Michael J.; Lis, Dariusz C.; Melnick, Gary; Neufeld, David; Pagani, Laurent; Encrenaz, Pierre; Snell, Ronald; Benz, Arnold O.; Bruderer, Simon; Bergin, Edwin; Caselli, Paola; Caux, Emmanuel; Falgarone, Edith

    2011-08-20

    We report observations of three rotational transitions of molecular oxygen (O{sub 2}) in emission from the H{sub 2} Peak 1 position of vibrationally excited molecular hydrogen in Orion. We observed the 487 GHz, 774 GHz, and 1121 GHz lines using the Heterodyne Instrument for the Far Infrared on the Herschel Space Observatory, having velocities of 11 km s{sup -1} to 12 km s{sup -1} and widths of 3 km s{sup -1}. The beam-averaged column density is N(O{sub 2}) = 6.5 x 10{sup 16} cm{sup -2}, and assuming that the source has an equal beam-filling factor for all transitions (beam widths 44, 28, and 19''), the relative line intensities imply a kinetic temperature between 65 K and 120 K. The fractional abundance of O{sub 2} relative to H{sub 2} is (0.3-7.3) x 10{sup -6}. The unusual velocity suggests an association with a {approx}5'' diameter source, denoted Peak A, the Western Clump, or MF4. The mass of this source is {approx}10 M{sub sun} and the dust temperature is {>=}150 K. Our preferred explanation of the enhanced O{sub 2} abundance is that dust grains in this region are sufficiently warm (T {>=} 100 K) to desorb water ice and thus keep a significant fraction of elemental oxygen in the gas phase, with a significant fraction as O{sub 2}. For this small source, the line ratios require a temperature {>=}180 K. The inferred O{sub 2} column density {approx_equal}5 x 10{sup 18} cm{sup -2} can be produced in Peak A, having N(H{sub 2}) {approx_equal} 4 x 10{sup 24} cm{sup -2}. An alternative mechanism is a low-velocity (10-15 km s{sup -1}) C-shock, which can produce N(O{sub 2}) up to 10{sup 17} cm{sup -2}.

  15. Automated micropipette aspiration of single cells.

    PubMed

    Shojaei-Baghini, Ehsan; Zheng, Yi; Sun, Yu

    2013-06-01

    This paper presents a system for mechanically characterizing single cells using automated micropipette aspiration. Using vision-based control and position control, the system controls a micromanipulator, a motorized translation stage, and a custom-built pressure system to position a micropipette (4 μm opening) to approach a cell, form a seal, and aspirate the cell into the micropipette for quantifying the cell's elastic and viscoelastic parameters as well as viscosity. Image processing algorithms were developed to provide controllers with real-time visual feedback and to accurately measure cell deformation behavior on line. Experiments on both solid-like and liquid-like cells demonstrated that the system is capable of efficiently performing single-cell micropipette aspiration and has low operator skill requirements.

  16. Dissolved-oxygen quenching of in-situ fluorescence measurements

    NASA Astrophysics Data System (ADS)

    Chudyk, Wayne; Tonaszuck, David; Pohlig, Kenneth

    1993-04-01

    In-situ fluorescence measurements of aromatic organic ground water contaminants do not always agree with gas chromatographic methods. Dissolved oxygen quenching of fluorescence may be an interferant in field measurements. Two standard fluorescent aromatics, quinine sulfate and naphthalene, were evaluated in this study. Over the range of dissolved oxygen concentrations expected to be encountered in the field, no effects of oxygen quenching on fluorescence of these compounds was observed. Quenching of quinine sulfate fluorescence by sodium chloride was observed using this system. Sodium chloride quenching was shown to follow the Stern-Volmer relation.

  17. Surface pressure measurement by oxygen quenching of luminescence

    NASA Technical Reports Server (NTRS)

    Gouterman, Martin P. (Inventor); Kavandi, Janet L. (Inventor); Gallery, Jean (Inventor); Callis, James B. (Inventor)

    1994-01-01

    Methods and compositions for measuring the pressure of an oxygen-containing gas on an aerodynamic surface, by oxygen-quenching of luminescence of molecular sensors is disclosed. Objects are coated with luminescent films containing a first sensor and at least one of two additional sensors, each of the sensors having luminescences that have different dependencies on temperature and oxygen pressure. Methods and compositions are also provided for improving pressure measurements (qualitative or quantitive) on surfaces coated with a film having one or more types of sensor.

  18. Surface pressure measurement by oxygen quenching of luminescence

    NASA Technical Reports Server (NTRS)

    Gouterman, Martin P. (Inventor); Kavandi, Janet L. (Inventor); Gallery, Jean (Inventor); Callis, James B. (Inventor)

    1993-01-01

    Methods and compositions for measuring the pressure of an oxygen-containing gas on an aerodynamic surface, by oxygen-quenching of luminescence of molecular sensors is disclosed. Objects are coated with luminescent films containing a first sensor and at least one of two additional sensors, each of the sensors having luminescences that have different dependencies on temperature and oxygen pressure. Methods and compositions are also provided for improving pressure measurements (qualitative or quantitive) on surfaces coated with a film having one or more types of sensor.

  19. Validation of oxygen saturation measurements in a canine model of hemoglobin-based oxygen carrier infusion.

    PubMed

    Jahr, Jonathan S; Lurie, Fedor; Driessen, Bernd; Tang, Zuping; Louie, Richard F; Kost, Gerald

    2003-01-01

    This study was designed to validate oxygen saturation measurements from the NOVA CO-Oximeter (NOVA Biomedical Corporation, Waltham, MA), the i-STAT System (Sensor Devices, Waukesha, WI), and the Corning 170 blood gas analyzer (Bayer Corporation, East Walpole, MA) under conditions similar to the clinical application of a hemoglobin-based oxygen carrier (HBOC, hemoglobin glutamer-200 [bovine]; Oxyglobin, Biopure Corporation, Cambridge, MA). A canine model was used for both in vitro and in vivo experiments. In vivo experiments were conducted in a canine laboratory, and in vitro experiments were conducted in a tonometry laboratory. Study subjects were six mixed-breed dogs, each weighing approximately 30 kg. In the first set of experiments, the target blood po(2) levels were reached by tonometry. In the second set of experiments, quantitative measurements of total oxygen content with the LEXO2CON-K (HOSPEX Fiberoptics, Chestnut Hill, MA) were performed, immediately followed by measurements with the NOVA CO-Oximeter and the i-STAT system. HBOC was added in concentrations of 16.2, 32.5, 65, and 97.5 g/L. To analyze the clinical significance of the differences in the results obtained with the each investigated instrument, blood samples from dogs treated with HBOC after acute hemorrhagic shock were used. Oxygen saturation, oxygen content, and po(2) were measured. There was a strong correlation between the oxygen saturation values measured with the investigated instruments in samples after tonometry and known po(2). The total calculated oxygen content varied by 5% based on results generated by calculations using the investigated instruments. The results did not change with different oxygenation of the sample. The differences among methods were not significant when the HBOC concentration was 16.2 g/L. Higher concentrations of HBOC increased the difference between calculated and measured oxygen content; the i-STAT system demonstrated a greater deviation compared with the

  20. Ultra High Precision Laser Monitor for Oxygen Eddy Flux Measurements

    NASA Astrophysics Data System (ADS)

    Nelson, David; Herndon, Scott; McManus, Barry; Roscioli, Rob; Jervis, Dylan; Zahniser, Mark

    2016-04-01

    Atmospheric oxygen provides one of the most powerful tracers to study the carbon cycle through its close interaction with carbon dioxide. Keeling and co-workers demonstrated this at the global scale by using small variations in atmospheric oxygen content to disentangle oceanic and terrestrial carbon sinks. It would be very exciting to apply similar ideas at the ecosystem level to improve our understanding of biosphere-atmosphere exchange and our ability to predict the response of the biosphere and atmosphere to climate change. The eddy covariance technique is perhaps the most effective approach available to quantify the exchange of gases between these spheres. Therefore, eddy covariance flux measurements of oxygen would be extremely valuable. However, this requires a fast response (0.1 seconds), high relative precision (0.001% or 10 per meg) oxygen sensor. We report recent progress in developing such a sensor using a high resolution visible laser to probe the oxygen A-band electronic transition. We have demonstrated precision of 1 ppmv or 5 per meg for a 100 second measurement duration. This sensor will enable oxygen flux measurements using eddy covariance. In addition, we will incorporate a second laser in this instrument to simultaneously determine the fluxes of oxygen, carbon dioxide and water vapor within the same sampling cell. This will provide a direct, real time measurement of the ratio of the flux of oxygen to that of carbon dioxide. This ratio is expected to vary on short time scales and small spatial scales due to the differing stoichiometry of processes producing and consuming carbon dioxide. Thus measuring the variations in the ratio of oxygen and carbon dioxide fluxes will provide mechanistic information to improve our understanding of the crucial exchange of carbon between the atmosphere and biosphere.

  1. Single cell-resolution western blotting.

    PubMed

    Kang, Chi-Chih; Yamauchi, Kevin A; Vlassakis, Julea; Sinkala, Elly; Duncombe, Todd A; Herr, Amy E

    2016-08-01

    This protocol describes how to perform western blotting on individual cells to measure cell-to-cell variation in protein expression levels and protein state. Like conventional western blotting, single-cell western blotting (scWB) is particularly useful for protein targets that lack selective antibodies (e.g., isoforms) and in cases in which background signal from intact cells is confounding. scWB is performed on a microdevice that comprises an array of microwells molded in a thin layer of a polyacrylamide gel (PAG). The gel layer functions as both a molecular sieving matrix during PAGE and a blotting scaffold during immunoprobing. scWB involves five main stages: (i) gravity settling of cells into microwells; (ii) chemical lysis of cells in each microwell; (iii) PAGE of each single-cell lysate; (iv) exposure of the gel to UV light to blot (immobilize) proteins to the gel matrix; and (v) in-gel immunoprobing of immobilized proteins. Multiplexing can be achieved by probing with antibody cocktails and using antibody stripping/reprobing techniques, enabling detection of 10+ proteins in each cell. We also describe microdevice fabrication for both uniform and pore-gradient microgels. To extend in-gel immunoprobing to gels of small pore size, we describe an optional gel de-cross-linking protocol for more effective introduction of antibodies into the gel layer. Once the microdevice has been fabricated, the assay can be completed in 4-6 h by microfluidic novices and it generates high-selectivity, multiplexed data from single cells. The technique is relevant when direct measurement of proteins in single cells is needed, with applications spanning the fundamental biosciences to applied biomedicine. PMID:27466711

  2. Single Cell Transcriptomics: Methods and Applications

    PubMed Central

    Kanter, Itamar; Kalisky, Tomer

    2015-01-01

    Traditionally, gene expression measurements were performed on “bulk” samples containing populations of thousands of cells. Recent advances in genomic technology have made it possible to measure gene expression in hundreds of individual cells at a time. As a result, cellular properties that were previously masked in “bulk” measurements can now be observed directly. In this review, we will survey emerging technologies for single cell transcriptomics, and describe how they are used to study complex disease such as cancer, as well as other biological phenomena such as tissue regeneration, embryonic development, and immune response. PMID:25806353

  3. Quantitative measurement of damage caused by 1064-nm wavelength optical trapping of Escherichia coli cells using on-chip single cell cultivation system

    SciTech Connect

    Ayano, Satoru; Wakamoto, Yuichi; Yamashita, Shinobu; Yasuda, Kenji . E-mail: yasuda.bmi@tmd.ac.jp

    2006-11-24

    We quantitatively examined the possible damage to the growth and cell division ability of Escherichia coli caused by 1064-nm optical trapping. Using the synchronous behavior of two sister E. coli cells, the growth and interdivision times between those two cells, one of which was trapped by optical tweezers, the other was not irradiated, were compared using an on-chip single cell cultivation system. Cell growth stopped during the optical trapping period, even with the smallest irradiated power on the trapped cells. Moreover, the damage to the cell's growth and interdivision period was proportional to the total irradiated energy (work) on the cell, i.e., irradiation time multiplied by irradiation power. The division ability was more easily affected by a smaller energy, 0.36 J, which was 30% smaller than the energy that adversely affected growth, 0.54 J. The results indicate that the damage caused by optical trapping can be estimated from the total energy applied to cells, and furthermore, that the use of optical trapping for manipulating cells might cause damage to cell division and growth mechanisms, even at wavelengths under 1064 nm, if the total irradiation energy is excessive.

  4. Ultra High Precision Laser Monitor for Oxygen Eddy Flux Measurements

    NASA Astrophysics Data System (ADS)

    Zahniser, M. S.; Nelson, D. D.; Roscioli, J. R.; Herndon, S. C.; McManus, J. B.; Jervis, D.

    2015-12-01

    Atmospheric oxygen provides one of the most powerful tracers to study the carbon cycle through its close interaction with carbon dioxide. Keeling and co-workers demonstrated this at the global scale by using small variations in atmospheric oxygen content to disentangle oceanic and terrestrial carbon sinks. It would be very exciting to apply similar ideas at the ecosystem level to improve our understanding of biosphere-atmosphere exchange and our ability to predict the response of the biosphere and atmosphere to climate change. The eddy covariance technique is perhaps the most effective approach available to quantify the exchange of gases between these spheres. Therefore, eddy covariance flux measurements of oxygen would be extremely valuable. However, this requires a fast response (0.1 seconds), high relative precision (0.001% or 10 per meg) oxygen sensor. We report recent progress in developing such a sensor using a high resolution visible laser to probe the oxygen A-band electronic transition. This sensor will enable oxygen flux measurements using eddy covariance. In addition, we will incorporate a second laser in this instrument to simultaneously determine the fluxes of oxygen, carbon dioxide and water vapor within the same sampling cell. This will provide a direct, real time measurement of the ratio of the flux of oxygen to that of carbon dioxide. This ratio is expected to vary on short time scales and small spatial scales due to the differing stoichiometry of processes producing and consuming carbon dioxide. Thus measuring the variations in the ratio of oxygen and carbon dioxide fluxes will provide mechanistic information to improve our understanding of the crucial exchange of carbon between the atmosphere and biosphere.

  5. A tandem mass spectrometric method for singlet oxygen measurement.

    PubMed

    Karonen, Maarit; Mattila, Heta; Huang, Ping; Mamedov, Fikret; Styring, Stenbjörn; Tyystjärvi, Esa

    2014-01-01

    Singlet oxygen, a harmful reactive oxygen species, can be quantified with the substance 2,2,6,6-tetramethylpiperidine (TEMP) that reacts with singlet oxygen, forming a stable nitroxyl radical (TEMPO). TEMPO has earlier been quantified with electron paramagnetic resonance (EPR) spectroscopy. In this study, we designed an ultra-high-performance liquid chromatographic-tandem mass spectrometric (UHPLC-ESI-MS/MS) quantification method for TEMPO and showed that the method based on multiple reaction monitoring (MRM) can be used for the measurements of singlet oxygen from both nonbiological and biological samples. Results obtained with both UHPLC-ESI-MS/MS and EPR methods suggest that plant thylakoid membranes produce 3.7 × 10(-7) molecules of singlet oxygen per chlorophyll molecule in a second when illuminated with the photosynthetic photon flux density of 2000 μmol m(-2 ) s(-1). PMID:24849296

  6. 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

  7. Light-addressable measurements of cellular oxygen consumption rates in microwell arrays based on phase-based phosphorescence lifetime detection

    PubMed Central

    Huang, Shih-Hao; Hsu, Yu-Hsuan; Wu, Chih-Wei; Wu, Chang-Jer

    2012-01-01

    A digital light modulation system that utilizes a modified commercial digital micromirror device (DMD) projector, which is equipped with a UV light-emitting diode as a light modulation source, has been developed to spatially direct excited light toward a microwell array device to detect the oxygen consumption rate (OCR) of single cells via phase-based phosphorescence lifetime detection. The microwell array device is composed of a combination of two components: an array of glass microwells containing Pt(II) octaethylporphine (PtOEP) as the oxygen-sensitive luminescent layer and a microfluidic module with pneumatically actuated glass lids set above the microwells to controllably seal the microwells of interest. By controlling the illumination pattern on the DMD, the modulated excitation light can be spatially projected to only excite the sealed microwell for cellular OCR measurements. The OCR of baby hamster kidney-21 fibroblast cells cultivated on the PtOEP layer within a sealed microwell has been successfully measured at 104 ± 2.96 amol s−1 cell−1. Repeatable and consistent measurements indicate that the oxygen measurements did not adversely affect the physiological state of the measured cells. The OCR of the cells exhibited a good linear relationship with the diameter of the microwells, ranging from 400 to 1000 μm and containing approximately 480 to 1200 cells within a microwell. In addition, the OCR variation of single cells in situ infected by Dengue virus with a different multiplicity of infection was also successfully measured in real-time. This proposed platform provides the potential for a wide range of biological applications in cell-based biosensing, toxicology, and drug discovery. PMID:24348889

  8. Measuring oxygen reduction/evolution reactions on the nanoscale.

    PubMed

    Kumar, Amit; Ciucci, Francesco; Morozovska, Anna N; Kalinin, Sergei V; Jesse, Stephen

    2011-08-14

    The efficiency of fuel cells and metal-air batteries is significantly limited by the activation of oxygen reduction and evolution reactions. Despite the well-recognized role of oxygen reaction kinetics on the viability of energy technologies, the governing mechanisms remain elusive and until now have been addressable only by macroscopic studies. This lack of nanoscale understanding precludes optimization of material architecture. Here, we report direct measurements of oxygen reduction/evolution reactions and oxygen vacancy diffusion on oxygen-ion conductive solid surfaces with sub-10 nm resolution. In electrochemical strain microscopy, the biased scanning probe microscopy tip acts as a moving, electrocatalytically active probe exploring local electrochemical activity. The probe concentrates an electric field in a nanometre-scale volume of material, and bias-induced, picometre-level surface displacements provide information on local electrochemical processes. Systematic mapping of oxygen activity on bare and platinum-functionalized yttria-stabilized zirconia surfaces is demonstrated. This approach allows direct visualization of the oxygen reduction/evolution reaction activation process at the triple-phase boundary, and can be extended to a broad spectrum of oxygen-conductive and electrocatalytic materials.

  9. Potentials of single-cell biology in identification and validation of disease biomarkers.

    PubMed

    Niu, Furong; Wang, Diane C; Lu, Jiapei; Wu, Wei; Wang, Xiangdong

    2016-09-01

    Single-cell biology is considered a new approach to identify and validate disease-specific biomarkers. However, the concern raised by clinicians is how to apply single-cell measurements for clinical practice, translate the message of single-cell systems biology into clinical phenotype or explain alterations of single-cell gene sequencing and function in patient response to therapies. This study is to address the importance and necessity of single-cell gene sequencing in the identification and development of disease-specific biomarkers, the definition and significance of single-cell biology and single-cell systems biology in the understanding of single-cell full picture, the development and establishment of whole-cell models in the validation of targeted biological function and the figure and meaning of single-molecule imaging in single cell to trace intra-single-cell molecule expression, signal, interaction and location. We headline the important role of single-cell biology in the discovery and development of disease-specific biomarkers with a special emphasis on understanding single-cell biological functions, e.g. mechanical phenotypes, single-cell biology, heterogeneity and organization of genome function. We have reason to believe that such multi-dimensional, multi-layer, multi-crossing and stereoscopic single-cell biology definitely benefits the discovery and development of disease-specific biomarkers.

  10. Noncontact tissue oxygenation measurement using near-infrared spectroscopy

    NASA Astrophysics Data System (ADS)

    Niwayama, Masatsugu; Murata, Hideaki; Shinohara, Shigenobu

    2006-07-01

    Here, we present a noncontact tissue oxygenation monitor that uses near-infrared spectroscopy (NIRS). We examined changes in sensitivity of tissue oxygenation measurement due to changes in the distance between the optical probe and the skin surface using a Monte Carlo simulation and in vivo tests. We also examined the effects of skin and fat layer thickness. Photon migration was analyzed in a model consisting of the skin, fat, and muscle layers. The relationship between measurement sensitivity and the probe-tissue distance was obtained from the results of the simulation and was used for correction of measurements. A noncontact tissue oximeter was used to perform the in vivo tests and measure oxygen consumption of the forearm muscle. The value of corrected oxygen consumption was 0.12±0.03ml/(100gmin), which is consistent with previously reported values obtained using contact NIRS measurement and magnetic resonance spectroscopy. Quantitative measurement of oxygenation using noncontact NIRS is potentially useful for novel applications such as quantification of inflammation.

  11. Fetal oxygenation measurement using wireless near infrared spectroscopy

    NASA Astrophysics Data System (ADS)

    Macnab, Andrew; Shadgan, Babak; Janssen, Patricia; Rurak, Dan

    2012-03-01

    Background: Fetal well-being is determined in large part by how well the placenta is able to supply oxygen and nutrients, but current technology is unable to directly measure how well a placenta functions. Near-infrared spectroscopy (NIRS) utilizes optical methods to measure tissue oxygenation. This pilot project evaluated the feasibility of NIRS for fetal monitoring through the maternal abdominal wall using a sheep model. Methods: A miniature wireless 2-wavelength NIRS device was placed on the abdominal skin over the placenta of a pregnant ewe whose fetus had been chronically catheterized to allow arterial sampling for measurement of arterial oxygen saturation. The NIRS device has 3-paired light emitting diodes and a single photodiode detector; allowing measurement of an index of tissue oxygen saturation (TSI%). Fetal limb TSI% values were compared before and during fetal breathing movements. Correlation was made during these events between arterial values and placental TSI% monitored continuously in real time. Results: Serial measurements were obtained in a single experiment. The correlation between transcutaneous NIRS derived TSI% and direct arterial oxygen saturation was very high (R2=0.86). Measures of fetal limb TSI% were declined after episodes of fetal breathing (P<0.005). Conclusions: This correlation suggests that NIRS is sensitive enough to detect changes in fetal tissue oxygenation noninvasively through the maternal abdominal wall in real-time in a sheep model. NIRS data confirmed that fetal breathing movements decrease arterial oxygen saturation in fetal lambs. If validated by further study this optical methodology could be applied as means of monitoring fetal wellbeing in humans.

  12. 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. PMID:26124131

  13. 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

  14. Leaf water oxygen isotope measurement by direct equilibration.

    PubMed

    Song, Xin; Barbour, Margaret M

    2016-08-01

    The oxygen isotope composition of leaf water imparts a signal to a range of molecules in the atmosphere and biosphere, but has been notoriously difficult to measure in studies requiring a large number of samples as a consequence of the labour-intensive extraction step. We tested a method of direct equilibration of water in fresh leaf samples with CO2 , and subsequent oxygen isotope analysis on an optical spectrometer. The oxygen isotope composition of leaf water measured by the direct equilibration technique was strongly linearly related to that of cryogenically extracted leaf water in paired samples for a wide range of species with differing anatomy, with an R(2) of 0.95. The somewhat more enriched values produced by the direct equilibration method may reflect lack of full equilibration with unenriched water in the vascular bundles, but the strong relationship across a wide range of species suggests that this difference can be adequately corrected for using a simple linear relationship.

  15. Prolonged and Continuous Measurement of Kidney Oxygenation in Conscious Rats.

    PubMed

    Koeners, Maarten P; Ow, Connie P C; Russell, David M; Evans, Roger G; Malpas, Simon C

    2016-01-01

    A relative deficiency in kidney oxygenation, i.e., renal hypoxia, may contribute to the initiation and progression of acute and chronic kidney disease. A critical barrier to investigate this is the lack of methods allowing measurement of the partial pressure of oxygen in kidney tissue for long periods in vivo. We have developed, validated, and tested a novel telemetric method that can do this. Here we provide details on the calibration, implantation, implementation for data recording, and reuse of this telemetry-based technology for measurement of medullary tissue oxygen tension in conscious, unrestrained rats. This technique provides an important additional tool for investigating the impact of renal hypoxia in biology and pathophysiology.

  16. Development of microcoulometry for measuring oxygen content in copper oxides

    NASA Astrophysics Data System (ADS)

    Sato, Fumiaki; Fujihara, Masaaki; Komiyama, Naokazu; Kambe, Shiro; Ishii, Osamu

    2005-12-01

    A microcoulometry system was developed and employed for measuring the oxygen content in copper oxides using a reduced amount of material. To achieve this reduction in sample size, Ar flow rate, and coulometric current were optimized. When using 5.0 mg of YBa2Cu3O6.71, which is about one-tenth of the amount used in conventional coulometry, the oxygen content was determined to be 6.68±0.03 in the optimized conditions that Ar flow rate, and coulometric current were 0.1l/min and 1.0 mA, respectively. This method will be useful for measuring the oxygen content of oxide samples smaller than 10 mg, i.e., oxide materials produced in small quantities for electronic applications.

  17. Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

    PubMed Central

    Biddlestone, John; Druker, Jimena; Shmakova, Alena; Ferguson, Gus; Swedlow, Jason R.; Rocha, Sonia

    2014-01-01

    Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.  Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress. PMID:24895009

  18. Measurement of intracellular oxygen concentration during photodynamic therapy in vitro.

    PubMed

    Weston, Mark A; Patterson, Michael S

    2014-01-01

    A technique is introduced that monitors the depletion of intracellular ground state oxygen concentration ([(3)O(2)]) during photodynamic therapy of Mat-LyLu cell monolayers and cell suspensions. The photosensitizer Pd(II) meso-tetra(4-carboxyphenyl)porphine (PdT790) is used to manipulate and indicate intracellular [(3)O(2)] in both of the in vitro models. The Stern-Volmer relationship for PdT790 phosphorescence was characterized in suspensions by flowing nitrogen over the suspension while short pulses of 405 nm light were used to excite the sensitizer. The bleaching of sensitizer and the oxygen consumption rate were also measured during continuous exposure of the cell suspension to the 405 nm laser. Photodynamic therapy (PDT) was conducted in both cell suspensions and in cell monolayers under different treatment conditions while the phosphorescence signal was acquired. The intracellular [(3)O(2)] during PDT was calculated by using the measured Stern-Volmer relationship and correcting for sensitizer photobleaching. In addition, the amount of oxygen that was consumed during the treatments was calculated. It was found that even at large oxygen consumption rates, cells remain well oxygenated during PDT of cell suspensions. For monolayer treatments, it was found that intracellular [(3)O(2)] is rapidly depleted over the course of PDT.

  19. Nonlinear photoacoustic measurements of oxygen saturation levels in blood

    NASA Astrophysics Data System (ADS)

    Kamanzi, Albert

    Oxygen is necessary for metabolism. It is carried from lungs to the rest of the body by hemoglobin in blood. As each hemoglobin molecule can carry a maximum of four oxygen molecules, oxygen saturation (sO2) is the measure of percentage of oxygen content in blood. For a normal person sO2 is 95% - 100%. Point-of-care testing of sO2 in blood is important in medicine. It enables doctors and caregivers for monitoring a wide variety of chronic illnesses. On the other hand, mapping of sO2 values by performing a raster scan across the region of interest in vivo is also essential in clinical and research settings, such as to evaluate the therapeutic effects of a treatment, monitoring healing of wounds, etc. Several non-invasive methods have been developed for this purpose. In this thesis, I measured the nonlinear absorption coefficient (beta) of blood samples using photoacoustic Z-scan technique. Results depict linear dependency between beta and blood oxygenation levels.

  20. FIELD MEASUREMENT OF DISSOLVED OXYGEN: A COMPARISON OF METHODS

    EPA Science Inventory

    The ability to confidently measure the concentration of dissolved oxygen (D.O.) in ground water is a key aspect of remedial selection and assessment. Presented here is a comparison of the commonly practiced methods for determining D.O. concentrations in ground water, including c...

  1. Measuring oxygen pressures using triplet quenching of Pd-porphine

    NASA Astrophysics Data System (ADS)

    Sinaasappel, Michiel; Ince, C.; Sanderse, E. A.; Bruining, Hajo A.

    1994-02-01

    A non-invasive optical method for measuring free oxygen in vivo is described. The method, introduced by Wilson and co-workers, is based on the quenching of the triplet state of Pd- porphine by oxygen and is described by the Stern-Volmer relation. The quenching of the triplet state is determined by measurement of the phosphorescence decay following excitation by a pulse of light. Measuring decay times has the advantage that they are independent of the changing optical properties of tissue and concentration of the dye. We describe a setup to measure the phosphorescence decay and present the values of Kq and (tau) o as a function of pH and temperature. Furthermore, some experiments on a rat liver and gut are presented.

  2. ASRDI oxygen technology survey. Volume 4: Low temperature measurement

    NASA Technical Reports Server (NTRS)

    Sparks, L. L.

    1974-01-01

    Information is presented on temperature measurement between the triple point and critical point of liquid oxygen. The criterion selected is that all transducers which may reasonably be employed in the liquid oxygen (LO2) temperature range are considered. The temperature range for each transducer is the appropriate full range for the particular thermometer. The discussion of each thermometer or type of thermometer includes the following information: (1) useful temperature range, (2) general and particular methods of construction and the advantages of each type, (3) specifications (accuracy, reproducibility, response time, etc.), (4) associated instrumentation, (5) calibrations and procedures, and (6) analytical representations.

  3. 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

  4. Calibrated BOLD using direct measurement of changes in venous oxygenation.

    PubMed

    Driver, Ian D; Hall, Emma L; Wharton, Samuel J; Pritchard, Susan E; Francis, Susan T; Gowland, Penny A

    2012-11-15

    Calibration of the BOLD signal is potentially of great value in providing a closer measure of the underlying changes in brain function related to neuronal activity than the BOLD signal alone, but current approaches rely on an assumed relationship between cerebral blood volume (CBV) and cerebral blood flow (CBF). This is poorly characterised in humans and does not reflect the predominantly venous nature of BOLD contrast, whilst this relationship may vary across brain regions and depend on the structure of the local vascular bed. This work demonstrates a new approach to BOLD calibration which does not require an assumption about the relationship between cerebral blood volume and cerebral blood flow. This method involves repeating the same stimulus both at normoxia and hyperoxia, using hyperoxic BOLD contrast to estimate the relative changes in venous blood oxygenation and venous CBV. To do this the effect of hyperoxia on venous blood oxygenation has to be calculated, which requires an estimate of basal oxygen extraction fraction, and this can be estimated from the phase as an alternative to using a literature estimate. Additional measurement of the relative change in CBF, combined with the blood oxygenation change can be used to calculate the relative change in CMRO(2) due to the stimulus. CMRO(2) changes of 18 ± 8% in response to a motor task were measured without requiring the assumption of a CBV/CBF coupling relationship, and are in agreement with previous approaches.

  5. Redox-filled Carbon-Fiber Microelectrodes for Single-Cell Exocytosis

    PubMed Central

    Cox, Jonathan T.; Gunderson, Christopher G.; Zhang, Bo

    2014-01-01

    Carbon-fiber microelectrodes (CFEs) are the primary electroanalytical tool in single-cell exocytosis and in-vivo studies. Here we report a new study on the kinetic properties of electrolyte-filled CFEs in single-cell measurements and demonstrate that the addition of outer sphere redox species, such as Fe(CN)63− and Ru(NH3)63+, in the backfill electrolyte solution can greatly enhance the kinetic response of CFEs. We show that at 750 mV, a voltage normally applied for detection of dopamine, the presence of fast outer sphere redox species in the backfilling solution significantly enhances the kinetic response of CFEs toward fast dopamine detection at single PC12 cells. Moreover, we also demonstrate that the use of Fe(CN)63− in the backfilling solution has enabled direct measurement of dopamine at applied voltages as low as 200 mV. This kinetic enhancement is believed to be due to faster electron-transfer kinetics on the coupling pole as compared to the sluggish reduction of oxygen. We anticipate that such redox-filled CFE ultramicroelectrodes will find many useful applications in single cell exocytosis and in-vivo sensing. PMID:24833889

  6. Measuring oxygen uptake in fishes with bimodal respiration.

    PubMed

    Lefevre, S; Bayley, M; McKenzie, D J

    2016-01-01

    Respirometry is a robust method for measurement of oxygen uptake as a proxy for metabolic rate in fishes, and how species with bimodal respiration might meet their demands from water v. air has interested researchers for over a century. The challenges of measuring oxygen uptake from both water and air, preferably simultaneously, have been addressed in a variety of ways, which are briefly reviewed. These methods are not well-suited for the long-term measurements necessary to be certain of obtaining undisturbed patterns of respiratory partitioning, for example, to estimate traits such as standard metabolic rate. Such measurements require automated intermittent-closed respirometry that, for bimodal fishes, has only recently been developed. This paper describes two approaches in enough detail to be replicated by the interested researcher. These methods are for static respirometry. Measuring oxygen uptake by bimodal fishes during exercise poses specific challenges, which are described to aid the reader in designing experiments. The respiratory physiology and behaviour of air-breathing fishes is very complex and can easily be influenced by experimental conditions, and some general considerations are listed to facilitate the design of experiments. Air breathing is believed to have evolved in response to aquatic hypoxia and, probably, associated hypercapnia. The review ends by considering what realistic hypercapnia is, how hypercapnic tropical waters can become and how this might influence bimodal animals' gas exchange.

  7. Measuring oxygen uptake in fishes with bimodal respiration.

    PubMed

    Lefevre, S; Bayley, M; McKenzie, D J

    2016-01-01

    Respirometry is a robust method for measurement of oxygen uptake as a proxy for metabolic rate in fishes, and how species with bimodal respiration might meet their demands from water v. air has interested researchers for over a century. The challenges of measuring oxygen uptake from both water and air, preferably simultaneously, have been addressed in a variety of ways, which are briefly reviewed. These methods are not well-suited for the long-term measurements necessary to be certain of obtaining undisturbed patterns of respiratory partitioning, for example, to estimate traits such as standard metabolic rate. Such measurements require automated intermittent-closed respirometry that, for bimodal fishes, has only recently been developed. This paper describes two approaches in enough detail to be replicated by the interested researcher. These methods are for static respirometry. Measuring oxygen uptake by bimodal fishes during exercise poses specific challenges, which are described to aid the reader in designing experiments. The respiratory physiology and behaviour of air-breathing fishes is very complex and can easily be influenced by experimental conditions, and some general considerations are listed to facilitate the design of experiments. Air breathing is believed to have evolved in response to aquatic hypoxia and, probably, associated hypercapnia. The review ends by considering what realistic hypercapnia is, how hypercapnic tropical waters can become and how this might influence bimodal animals' gas exchange. PMID:26358224

  8. 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.

  9. Kinetics of virus production from single cells.

    PubMed

    Timm, Andrea; Yin, John

    2012-03-01

    The production of virus by infected cells is an essential process for the spread and persistence of viral diseases, the effectiveness of live-viral vaccines, and the manufacture of viruses for diverse applications. Yet despite its importance, methods to precisely measure virus production from cells are lacking. Most methods test infected-cell populations, masking how individual cells behave. Here we measured the kinetics of virus production from single cells. We combined simple steps of liquid-phase infection, serial dilution, centrifugation, and harvesting, without specialized equipment, to track the production of virus particles from BHK cells infected with vesicular stomatitis virus. Remarkably, cell-to-cell differences in latent times to virus release were within a factor of two, while production rates and virus yields spanned over 300-fold, highlighting an extreme diversity in virus production for cells from the same population. These findings have fundamental and technological implications for health and disease.

  10. Highly sensitive thermometer using a vacuum-packed Si resonator in a microfluidic chip for the thermal measurement of single cells.

    PubMed

    Inomata, Naoki; Toda, Masaya; Ono, Takahito

    2016-09-21

    A highly sensitive thermometer system for a living cell is proposed, fabricated, and evaluated. The system possesses a resonant thermal sensor surrounded by vacuum in a microfluidic chip. The measurement principle relies on resonant frequency tracking of the resonator in temperature variations due to the heat from a sample cell; the heat is conducted from the sample cell in the microfluidic channel via a heat guide connecting the resonator to a sample stage. This configuration can reduce heat loss from the resonator to the surroundings and damping in water. Two types of resonators are prepared, i.e., a cantilevered resonator and a double-supported resonator. The resonator sizes as a sensor are 30 × 50 × 1.5 μm in the cantilevered resonator, 30 × 75 × 0.40 μm in the double-supported one, respectively. The temperature and thermal resolutions of 79 μK and 1.90 nW, respectively, are achieved using the double-supported resonator. Two types of heat emissions from single brown fat cells are detected; one is continuous heat generation in the presence of chemical stimulation by a norepinephrine solution, and the other is pulsed without any stimulation. PMID:27526966

  11. Single-Cell Genomics for Virology

    PubMed Central

    Ciuffi, Angela; Rato, Sylvie; Telenti, Amalio

    2016-01-01

    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. PMID:27153082

  12. Single-Cell Genomics for Virology.

    PubMed

    Ciuffi, Angela; Rato, Sylvie; Telenti, Amalio

    2016-01-01

    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. PMID:27153082

  13. Redefining Signaling Pathways with an Expanding Single-Cell Toolbox.

    PubMed

    Gaudet, Suzanne; Miller-Jensen, Kathryn

    2016-06-01

    Genetically identical cells respond heterogeneously to uniform environmental stimuli. Consequently, investigating the signaling networks that control these cell responses using 'average' bulk cell measurements can obscure underlying mechanisms and misses information emerging from cell-to-cell variability. Here we review recent technological advances including live-cell fluorescence imaging-based approaches and microfluidic devices that enable measurements of signaling networks, dynamics, and responses in single cells. We discuss how these single-cell tools have uncovered novel mechanistic insights for canonical signaling pathways that control cell proliferation (ERK), DNA-damage responses (p53), and innate immune and stress responses (NF-κB). Future improvements in throughput and multiplexing, analytical pipelines, and in vivo applicability will all significantly expand the biological information gained from single-cell measurements of signaling pathways. PMID:26968612

  14. Redefining Signaling Pathways with an Expanding Single-Cell Toolbox.

    PubMed

    Gaudet, Suzanne; Miller-Jensen, Kathryn

    2016-06-01

    Genetically identical cells respond heterogeneously to uniform environmental stimuli. Consequently, investigating the signaling networks that control these cell responses using 'average' bulk cell measurements can obscure underlying mechanisms and misses information emerging from cell-to-cell variability. Here we review recent technological advances including live-cell fluorescence imaging-based approaches and microfluidic devices that enable measurements of signaling networks, dynamics, and responses in single cells. We discuss how these single-cell tools have uncovered novel mechanistic insights for canonical signaling pathways that control cell proliferation (ERK), DNA-damage responses (p53), and innate immune and stress responses (NF-κB). Future improvements in throughput and multiplexing, analytical pipelines, and in vivo applicability will all significantly expand the biological information gained from single-cell measurements of signaling pathways.

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

    PubMed Central

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

    2016-01-01

    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. PMID:27194954

  16. 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

  17. Device for measuring oxygen activity in liquid sodium

    DOEpatents

    Roy, P.; Young, R.S.

    1973-12-01

    A composite ceramic electrolyte in a configuration (such as a closed end tube or a plate) suitable to separate liquid sodium from a reference electrode with a high impedance voltmeter connected to measure EMF between the sodium and the reference electrode as a measure of oxygen activity in the sodium is described. The composite electrolyte consists of zirconiacalcia with a bonded layer of thoria-yttria. The device is used with a gaseous reference electrode on the zirconia-calcia side and liquid sodium on the thoria-yttria side of the electrolyte. (Official Gazette)

  18. Single cell protein as an occupational hazard.

    PubMed Central

    Ekenvall, L; Dölling, B; Göthe, C J; Ebbinghaus, L; von Stedingk, L V; Wasserman, J

    1983-01-01

    Single cell protein (SCP) intended for animal feed purposes was produced in a pilot plant. The SCP consisted of Methylomonas methanolica, a pseudomonas species which is an obligate methanol user. The SCP was cultured in fermenters and later dewatered and dried in a spray-drier. Seven of eight research workers had febrile reactions 6-12 hours after exposure to SCP dust. All workers had high titres of IgG and IgM antibodies against the pseudomonas species as measured with indirect ELISA and passive haemagglutination techniques. The mechanism behind the febrile reaction is judged to be a non-immunological reaction caused by endotoxins. By increasing the particle size of the SCP through using different drying procedures, a product which generated less dust was obtained. PMID:6830720

  19. Leaf water oxygen isotope measurement by direct equilibration.

    PubMed

    Song, Xin; Barbour, Margaret M

    2016-08-01

    The oxygen isotope composition of leaf water imparts a signal to a range of molecules in the atmosphere and biosphere, but has been notoriously difficult to measure in studies requiring a large number of samples as a consequence of the labour-intensive extraction step. We tested a method of direct equilibration of water in fresh leaf samples with CO2 , and subsequent oxygen isotope analysis on an optical spectrometer. The oxygen isotope composition of leaf water measured by the direct equilibration technique was strongly linearly related to that of cryogenically extracted leaf water in paired samples for a wide range of species with differing anatomy, with an R(2) of 0.95. The somewhat more enriched values produced by the direct equilibration method may reflect lack of full equilibration with unenriched water in the vascular bundles, but the strong relationship across a wide range of species suggests that this difference can be adequately corrected for using a simple linear relationship. PMID:27147584

  20. A new method for measuring the oxygen diffusion constant and oxygen consumption rate of arteriolar walls.

    PubMed

    Sasaki, Nobuhiko; Horinouchi, Hirohisa; Ushiyama, Akira; Minamitani, Haruyuki

    2012-01-01

    Oxygen transport is believed to primarily occur via capillaries and depends on the oxygen tension gradient between the vessels and tissues. As blood flows along branching arterioles, the O(2) saturation drops, indicating either consumption or diffusion. The blood flow rate, the O(2) concentration gradient, and Krogh's O(2) diffusion constant (K) of the vessel wall are parameters affecting O(2)delivery. We devised a method for evaluating K of arteriolar wall in vivo using phosphorescence quenching microscopy to measure the partial pressure of oxygen in two areas almost simultaneously. The K value of arteriolar wall (inner diameter, 63.5 ± 11.9 μm; wall thickness, 18.0 ± 1.2 μm) was found to be 6.0 ± 1.2 × 10(-11) (cm(2)/s)(ml O(2)·cm(-3) tissue·mmHg(-1)). The arteriolar wall O(2) consumption rate (M) was 1.5 ± 0.1 (ml O(2)·100 cm(-3) tissue·min(-1)), as calculated using Krogh's diffusion equation. These results suggest that the arteriolar wall consumes a considerable proportion of the O(2) that diffuses through it.

  1. Electrical impedance tomographic imaging of a single cell electroporation.

    PubMed

    Meir, Arie; Rubinsky, Boris

    2014-06-01

    A living cell placed in a high strength electric field, can undergo a process known as electroporation. It is believed that during electroporation nano-scale defects (pores) occur in the membrane of the cell, causing dramatic changes to the permeability of its membrane. Electroporation is an important technique in biotechnology and medicine and numerous methods are being developed to improve the understanding and use of the technology. We propose to extend the toolbox available for studying electroporation by generating impedance distribution images of the cell as it undergoes electroporation using Electrical Impedance Tomography (EIT). To investigate the feasibility of this concept, we develop a mathematical model of the process of electroporation in a single cell and of EIT of the process and show simulation results of a computer-based finite element model (FEM). Our work is an attempt to develop a new imaging tool for visualizing electroporation in a single cell, offering a different temporal and spatial resolution compared to the state of the art, which includes bulk measurements of electrical properties during single cell electroporation, patch clamp and voltage clamp measurement in single cells and optical imaging with colorimetric dyes during single cell electroporation. This paper is a preliminary theoretic feasibility study.

  2. Overview of single-cell elastic light scattering techniques.

    PubMed

    Kinnunen, Matti; Karmenyan, Artashes

    2015-05-01

    We present and discuss several modern optical methods based on elastic light scattering (ELS), along with their technical features and applications in biomedicine and life sciences. In particular, we review some ELS experiments at the single-cell level and explore new directions of applications. Due to recent developments in experimental systems (as shown in the literature), ELS lends itself to useful applications in the life sciences. Of the developed methods, we cover elastic scattering spectroscopy, optical tweezer-assisted measurement, goniometers, Fourier transform light scattering (FTLS), and microscopic methods. FTLS significantly extends the potential analysis of single cells by allowing monitoring of dynamical changes at the single-cell level. The main aim of our review is to demonstrate developments in the experimental investigation of ELS in single cells including issues related to theoretical “representations” and modeling of biological systems (cells, cellular systems, tissues, and so on). Goniometric measurements of ELS from optically trapped single cells are shown and the importance of the experimental verification of theoretical models of ELS in the context of biomedical applications is discussed.

  3. Thenar oxygen saturation and invasive oxygen delivery measurements in critically ill patients in early septic shock.

    PubMed

    Mesquida, Jaume; Gruartmoner, Guillem; Martínez, Maria Luisa; Masip, Jordi; Sabatier, Caroline; Espinal, Cristina; Artigas, Antonio; Baigorri, Francisco

    2011-05-01

    This prospective study was aimed to test the hypothesis that tissue hemoglobin oxygen saturation (StO₂) measured noninvasively using near-infrared spectroscopy is a reliable indicator of global oxygen delivery (DO₂) measured invasively using a pulmonary artery catheter (PAC) in patients with septic shock. The study setting was a 26-bed medical-surgical intensive care unit at a university hospital. Subjects were adult patients in septic shock who required PAC hemodynamic monitoring for resuscitation. Interventions included transient ischemic challenge on the forearm. After blood pressure normalization, hemodynamic and oximetric PAC variables and, simultaneously, steady-state StO₂ and its changes from ischemic challenge (deoxygenation and reoxygenation rates) were measured. Fifteen patients were studied. All the patients had a mean arterial pressure above 65 mmHg. The DO₂ index (iDO₂) range in the studied population was 215 to 674 mL O₂/min per m. The mean mixed venous oxygen saturation value was 61% ± 10%, mean cardiac index was 3.4 ± 0.9 L/min per m, and blood lactate level was 4.6 ± 2.7 mmol/L. Steady-state StO₂ significantly correlated with iDO₂, arterial and venous O₂ content, and O₂ extraction ratio. A StO₂ cutoff value of 75% predicted iDO₂ below 450, with a sensitivity of 0.9 and a specificity of 0.9. In patients in septic shock and normalized MAP, low StO₂ reflects extremely low iDO₂. Steady-state StO₂ does not correlate with moderately low iDO₂, indicating poor sensitivity of StO₂ to rule out hypoperfusion.

  4. Single-cell technologies for monitoring immune systems

    PubMed Central

    Chattopadhyay, Pratip K; Gierahn, Todd M; Roederer, Mario; Love, J Christopher

    2014-01-01

    The complex heterogeneity of cells, and their interconnectedness with each other, are major challenges to identifying clinically relevant measurements that reflect the state and capability of the immune system. Highly multiplexed, single-cell technologies may be critical for identifying correlates of disease or immunological interventions as well as for elucidating the underlying mechanisms of immunity. Here we review limitations of bulk measurements and explore advances in single-cell technologies that overcome these problems by expanding the depth and breadth of functional and phenotypic analysis in space and time. The geometric increases in complexity of data make formidable hurdles for exploring, analyzing and presenting results. We summarize recent approaches to making such computations tractable and discuss challenges for integrating heterogeneous data obtained using these single-cell technologies. PMID:24448570

  5. Oxygen measurements in brain stem slices exposed to normobaric hyperoxia and hyperbaric oxygen.

    PubMed

    Mulkey, D K; Henderson, R A; Olson, J E; Putnam, R W; Dean, J B

    2001-05-01

    We previously reported (J Appl Physiol 89: 807-822, 2000) that < or =10 min of hyperbaric oxygen (HBO(2); < or = 2,468 Torr) stimulates solitary complex neurons. To better define the hyperoxic stimulus, we measured PO(2) in the solitary complex of 300-microm-thick rat medullary slices, using polarographic carbon fiber microelectrodes, during perfusion with media having PO(2) values ranging from 156 to 2,468 Torr. Under control conditions, slices equilibrated with 95% O(2) at barometric pressure of 1 atmospheres absolute had minimum PO(2) values at their centers (291 +/- 20 Torr) that were approximately 10-fold greater than PO(2) values measured in the intact central nervous system (10-34 Torr). During HBO(2), PO(2) increased at the center of the slice from 616 +/- 16 to 1,517 +/- 15 Torr. Tissue oxygen consumption tended to decrease at medium PO(2) or = 1,675 Torr to levels not different from values measured at PO(2) found in all media in metabolically poisoned slices (2-deoxy-D-glucose and antimycin A). We conclude that control medium used in most brain slice studies is hyperoxic at normobaric pressure. During HBO(2), slice PO(2) increases to levels that appear to reduce metabolism. PMID:11299283

  6. Measuring Apoptosis at the Single Cell Level

    PubMed Central

    Bouchier-Hayes, Lisa; Muñoz-Pinedo, Cristina; Connell, Samuel; Green, Douglas R.

    2008-01-01

    The use of live cell microscopy has made a number of contributions to the study of apoptosis. Many of the tools and techniques are available that allow us to image the key events that occur during cell death including mitochondrial outer membrane permeabilization, mitochondrial transmembrane potential changes, translocation of Bcl-2 family members, caspase activation, phosphatidylserine flip and plasma membrane rupture. We discuss these techniques here and highlight the advantages and drawbacks of using such approaches to study apoptosis. PMID:18314052

  7. 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

  8. Electrochemical synthesis on single cells as templates.

    PubMed

    Tam, Jasper; Salgado, Shehan; Miltenburg, Mark; Maheshwari, Vivek

    2013-10-01

    The cell surface is made electrochemically active by interfacing with graphene sheets. The electrical and thermal properties of graphene allow the control of cell surface potential for electrochemical synthesis. Using this approach radially projecting ZnO nanorods are templated on the surface of single cells. This reported single cell photosensor has superior performance than similar devices made on planar surfaces.

  9. Gas temperature measurements in a microcathode sustained discharge in oxygen

    NASA Astrophysics Data System (ADS)

    Puech, V.; Lagrange, J. F.; Sadeghi, N.; Touzeau, M.; Bauville, G.; Lacour, B.

    2006-10-01

    Microcathode sustained discharges (MCSD) produced between a microhollow cathode discharge (MHCD) and a third electrode offer the possibility to produce high density plasmas at low E/N values. Such discharges in oxygen could be attractive for efficient production of singlet O2 if the gas temperature remains low. The temperature of a discharge in oxygen at 50 Torr and for currents up to 2 mA and E/N of 25 Td was measured through a spectroscopic investigation of the plasma emission. The spatial distribution of the O2(b^1σ) and O(5p) was measured. These species have a very different behaviour: O(5p) is mainly produced inside the hole of the 0.2 mm diameter MHCD and its density decreases by two orders of magnitude over a distance of 3 mm. On the other hand, the O2(b^1σ) production by the MHCD is very low, and this state is mainly produced in the MCSD, with a smooth density gradient in the interelectrode gap. The gas temperature was determined in the MCSD from the high resolution spectra of the atmospheric band at 760 nm, while the gas temperature inside the hole of the MHCD was measured through the rotational spectra of the 337 nm 2^nd positive band of nitrogen, introduced at low concentration in the discharge. In our experimental conditions, the gas temperature in the MHCD is lower than 650 K and less than 400 K in the MCSD.

  10. Immobilised activated sludge based biosensor for biochemical oxygen demand measurement.

    PubMed

    Liu, J; Björnsson, L; Mattiasson, B

    2000-02-01

    A biochemical oxygen demand (BOD) sensor, based on an immobilised mixed culture of microorganisms in combination with a dissolved oxygen electrode, has been developed for the purpose of on-line monitoring of the biological treatment process for waste and wastewater. The sensor was designed for easy replacement of the biomembrane, thereby making it suitable for short-term use. The drawbacks of activated sludge based sensor, such as short sensor lifetime, were thereby circumvented. The sensor BOD measurements were carried out in the kinetic mode using a flow injection system, resulting in 25 s for one measurement followed by 4-8 min recovery time. Based on the results of normalised sensor responses, the OECD synthetic wastewater was considered to be a more suitable calibration solution in comparison with the GGA solution. Good agreement was achieved between the results of the sensor BOD measurement and those obtained from BOD5 analysis of a wastewater sample from a food-processing factory. Reproducibility of responses using one sensor was below +/- 5.6%, standard deviation. Reproducibility of responses using different sensors was within acceptable bias limits, viz. +/- 15% standard deviation.

  11. Sound speed measurements in liquid oxygen-liquid nitrogen mixtures

    NASA Technical Reports Server (NTRS)

    Zuckerwar, A. J.; Mazel, D. S.

    1985-01-01

    The sound speed in liquid oxygen (LOX), liquid nitrogen (LN2), and five LOX-LN2 mixtures was measured by an ultrasonic pulse-echo technique at temperatures in the vicinity of -195.8C, the boiling point of N2 at a pressure of I atm. Under these conditions, the measurements yield the following relationship between sound speed in meters per second and LN2 content M in mole percent: c = 1009.05-1.8275M+0.0026507 M squared. The second speeds of 1009.05 m/sec plus or minus 0.25 percent for pure LOX and 852.8 m/sec plus or minus 0.32 percent for pure LN2 are compared with those reported by past investigators. Measurement of sound speed should prove an effective means for monitoring the contamination of LOX by Ln2.

  12. 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.

  13. 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

  14. Single Cell Transcriptome Amplification with MALBAC

    PubMed Central

    Tan, Longzhi; Tang, Fuchou; Xie, X. Sunney

    2015-01-01

    Recently, Multiple Annealing and Looping-Based Amplification Cycles (MALBAC) has been developed for whole genome amplification of an individual cell, relying on quasilinear instead of exponential amplification to achieve high coverage. Here we adapt MALBAC for single-cell transcriptome amplification, which gives consistently high detection efficiency, accuracy and reproducibility. With this newly developed technique, we successfully amplified and sequenced single cells from 3 germ layers from mouse embryos in the early gastrulation stage, and examined the epithelial-mesenchymal transition (EMT) program among cells in the mesoderm layer on a single-cell level. PMID:25822772

  15. Comparison of measured and calculated thermospheric molecular oxygen densities

    NASA Technical Reports Server (NTRS)

    Potter, W. E.; Kayser, D. C.; Brinton, H. C.; Brace, L. H.; Oppenheimer, M.

    1977-01-01

    The open source neutral mass spectrometers on the AE-C, -D, and -E satellites were equipped with a 'fly-through' mode of operation which has provided direct measurements of molecular oxygen densities over a large portion of the globe. A complementary set of O2 densities is derived by using AE ion measurements and a scheme based on the daytime ion chemistry of O2(+) in the thermosphere. A comparison of the two data sets reveals general agreement over northern latitudes during periods of relatively low Ap and F10.7. The simplifying assumptions made in the photochemical scheme require that caution be used in calculating O2, especially at high latitudes and altitudes below 200 km

  16. 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.

  17. Using oxygen species to measure marine production in Drake Passage

    NASA Astrophysics Data System (ADS)

    Castro Morales, Karel; Cassar, Nicolas; Bender, Michael; Kaiser, Jan

    2010-05-01

    Marine biological production is key to understanding the global carbon cycle, particularly the role of the Southern Ocean as a sink of CO2. Measurements of oxygen in the surface ocean allow quantifying marine biological productivity, since CO2 and O2 are linked via photosynthesis and respiration. Measurements of O2/Ar ratios and dissolved O2 isotopologues, together with wind-speed gas exchange parameterizations, give estimates of biological oxygen air-sea fluxes (Fbio) and gross photosynthetic production (G) in the mixed layer (zmix). In the absence of vertical mixing, Fbio can be used as a proxy for net community production (N). O2/Ar ratios and O2 concentrations were measured continuously in the uncontaminated seawater supply on board the RRS James Clark Ross along two sections across Drake Passage (DP). The DP1 section (southbound, 27 February-3 March 2007) represented mid-summer; DP2 represented early autumn (northbound, 12-15 April, 2007). The time difference between the two transects was 40 days. Weighted average gas exchange rates were calculated using the WOCE-NODC ocean mixed layer depth climatology and ECMWF wind speeds over 60 days prior to sample collection. The WOCE-NODC climatology shows a deepening of the zmix by on average 46 m within 40 days. The sea surface temperature decreased about 2.4 °C from DP1 to DP2. This reflects the seasonal transition from late summer to early autumn. In agreement with previous observations, we observed a strong north-south gradient of biological oxygen production in the DP. Our results also show high temporal variability over the course of 40 days. During late summer, the physical supersaturation contributes to about 3.6% of the total O2 supersaturation (?O2) for the Subantarctic and Polar Frontal Zones (SAZ and PFZ, respectively). In the other hand, the biological O2 supersaturation (?O2/Ar) showed mainly positive and homogeneous values (~1%) along the Antarctic Zone and Southern Antarctic Circumpolar Current Zone

  18. Continuous Dissolved Oxygen Measurements and Modelling Metabolism in Peatland Streams.

    PubMed

    Dick, Jonathan J; Soulsby, Chris; Birkel, Christian; Malcolm, Iain; Tetzlaff, Doerthe

    2016-01-01

    Stream water dissolved oxygen was monitored in a 3.2km2 moorland headwater catchment in the Scottish Highlands. The stream consists of three 1st order headwaters and a 2nd order main stem. The stream network is fringed by peat soils with no riparian trees, though dwarf shrubs provide shading in the lower catchment. Dissolved oxygen (DO) is regulated by the balance between atmospheric re-aeration and the metabolic processes of photosynthesis and respiration. DO was continuously measured for >1 year and the data used to calibrate a mass balance model, to estimate primary production, respiration and re-aeration for a 1st order site and in the 2nd order main stem. Results showed that the stream was always heterotrophic at both sites. Sites were most heterotrophic in the summer reflecting higher levels of stream metabolism. The 1st order stream appeared more heterotrophic which was consistent with the evident greater biomass of macrophytes in the 2nd order stream, with resulting higher primary productivity. Comparison between respiration, primary production, re-aeration and potential physical controls revealed only weak relationships. However, the most basic model parameters (e.g. the parameter linking light and photosynthesis) controlling ecosystem processes resulted in significant differences between the sites which seem related to the stream channel geometry. PMID:27556278

  19. Continuous Dissolved Oxygen Measurements and Modelling Metabolism in Peatland Streams

    PubMed Central

    Dick, Jonathan J.; Soulsby, Chris; Birkel, Christian; Malcolm, Iain; Tetzlaff, Doerthe

    2016-01-01

    Stream water dissolved oxygen was monitored in a 3.2km2 moorland headwater catchment in the Scottish Highlands. The stream consists of three 1st order headwaters and a 2nd order main stem. The stream network is fringed by peat soils with no riparian trees, though dwarf shrubs provide shading in the lower catchment. Dissolved oxygen (DO) is regulated by the balance between atmospheric re-aeration and the metabolic processes of photosynthesis and respiration. DO was continuously measured for >1 year and the data used to calibrate a mass balance model, to estimate primary production, respiration and re-aeration for a 1st order site and in the 2nd order main stem. Results showed that the stream was always heterotrophic at both sites. Sites were most heterotrophic in the summer reflecting higher levels of stream metabolism. The 1st order stream appeared more heterotrophic which was consistent with the evident greater biomass of macrophytes in the 2nd order stream, with resulting higher primary productivity. Comparison between respiration, primary production, re-aeration and potential physical controls revealed only weak relationships. However, the most basic model parameters (e.g. the parameter linking light and photosynthesis) controlling ecosystem processes resulted in significant differences between the sites which seem related to the stream channel geometry. PMID:27556278

  20. 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. PMID:27243002

  1. Efficient Synergistic Single-Cell Genome Assembly

    PubMed Central

    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. PMID:27243002

  2. Single-cell intracellular nano-pH probes†

    PubMed Central

    Özel, Rıfat Emrah; Lohith, Akshar; Mak, Wai Han; Pourmand, Nader

    2016-01-01

    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. PMID:27708772

  3. Single-cell intracellular nano-pH probes†

    PubMed Central

    Özel, Rıfat Emrah; Lohith, Akshar; Mak, Wai Han; Pourmand, Nader

    2016-01-01

    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.

  4. Estimating peak oxygen uptake based on postexercise measurements in swimming.

    PubMed

    Chaverri, Diego; Iglesias, Xavier; Schuller, Thorsten; Hoffmann, Uwe; Rodríguez, Ferran A

    2016-06-01

    To assess the validity of postexercise measurements in estimating peak oxygen uptake (V̇O2peak) in swimming, we compared oxygen uptake (V̇O2) measurements during supramaximal exercise with various commonly adopted methods, including a recently developed heart rate - V̇O2 modelling procedure. Thirty-one elite swimmers performed a 200-m maximal swim where V̇O2 was measured breath-by-breath using a portable gas analyzer connected to a respiratory snorkel, 1 min before, during, and 3 min postexercise. V̇O2peak(-20-0) was the average of the last 20 s of effort. The following postexercise measures were compared: (i) first 20-s average (V̇O2peak(0-20)); (ii) linear backward extrapolation (BE) of the first 20 s (BE(20)), 30 s, and 3 × 20-, 4 × 20-, and 3 or 4 × 20-s averages; (iii) semilogarithmic BE at 20 s (LOG(20)) and at the other same time intervals as in linear BE; and (iv) predicted V̇O2peak using mathematical modelling (pV̇O2(0-20)]. Repeated-measures ANOVA and post-hoc Bonferroni tests compared V̇O2peak (criterion) and each estimated value. Pearson's coefficient of determination (r(2)) was used to assess correlation. Exercise V̇O2peak(-20-0) (mean ± SD 3531 ± 738 mL·min(-1)) was not different (p > 0.30) from pV̇O2(0-20) (3571 ± 735 mL·min(-1)), BE(20) (3617 ± 708 mL·min(-1)), or LOG(20) (3627 ± 746 mL·min(-1)). pV̇O2(0-20) was very strongly correlated with exercise V̇O2peak (r(2) = 0.962; p < 0.001), and showed a low standard error of the estimate (146 mL·min(-1), 4.1%) and the lowest mean difference (40 mL·min(-1); 1.1%). We confirm that the new modelling procedure based on postexercise V̇O2 and heart rate measurements is a valid and accurate procedure for estimating V̇O2peak in swimmers and avoids the estimation bias produced by other methods.

  5. 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.

  6. Measurement and chemical kinetic model predictions of detonation cell size in methanol-oxygen mixtures

    NASA Astrophysics Data System (ADS)

    Eaton, R.; Zhang, B.; Bergthorson, J. M.; Ng, H. D.

    2012-03-01

    In this study, detonation cell sizes of methanol-oxygen mixtures are experimentally measured at different initial pressures and compositions. Good agreement is found between the experiment data and predictions based on the chemical length scales obtained from a detailed chemical kinetic model. To assess the detonation sensitivity in methanol-oxygen mixtures, the results are compared with those of hydrogen-oxygen and methane-oxygen mixtures. Based on the cell size comparison, it is shown that methanol-oxygen is more detonation sensitive than methane-oxygen but less sensitive than hydrogen-oxygen.

  7. Biochemical oxygen demand measurement by mediator method in flow system.

    PubMed

    Liu, Ling; Bai, Lu; Yu, Dengbin; Zhai, Junfeng; Dong, Shaojun

    2015-06-01

    Using mediator as electron acceptor for biochemical oxygen demand (BOD) measurement was developed in the last decade (BODMed). However, until now, no BOD(Med) in a flow system has been reported. This work for the first time describes a flow system of BOD(Med) method (BOD(Med)-FS) by using potassium ferricyanide as mediator and carbon fiber felt as substrate material for microbial immobilization. The system can determine the BOD value within 30 min and possesses a wider analytical linear range for measuring glucose-glutamic acid (GGA) standard solution from 2 up to 200 mg L(-1) without the need of dilution. The analytical performance of the BOD(Med)-FS is comparable or better than that of the previously reported BOD(Med) method, especially its superior long-term stability up to 2 months under continuous operation. Moreover, the BOD(Med)-FS has same determination accuracy with the conventional BOD5 method by measuring real samples from a local wastewater treatment plant (WWTP).

  8. 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

  9. Selective single cell isolation for genomics using microraft arrays.

    PubMed

    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

    2016-09-30

    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.

  10. Intrinsic oxygen fugacity measurements of some Allende Type B inclusions

    SciTech Connect

    Kozul, J.M.; Hewins, R.H. ); Ulmer, G.C. )

    1988-11-01

    Intrinsic oxygen fugacity (IOF) measurements for whole rock samples of two Type B Ca-, Al-rich inclusions (CAI) from the Allende meteorite show them to be 6-8 orders of magnitude more oxidized than the canonical solar nebular gas. Melilite separates from the two CAI give IOF measurements 8-9 orders of magnitude more oxidized than the solar nebular gas. Melilite crystallized in a reducing solar gas and later underwent a solid-state oxidation. Vacancies due to crystallization in a reducing solar gas and an open crystal structure were filled, probably through an O diffusion mechanism, during equilibration at temperatures higher than 700{degree}C with an oxidized gas. O-rich vapors released from an unaltered Vigarano inclusion during high-temperature volatilization may have caused oxidation of fassaite and possibly melilite in the inclusion and this may be a mechanism for oxidation in Allende inclusions. IOF measurements of fine-grained alteration/rim minerals in one Type B inclusion yield an oxidation state more oxidized than the whole rock CAI but more reduced than melilite from the same inclusion. Disequilibrium between melilite and alteration minerals with respect to fO{sub 2} may seem unlikely in light of their similar O isotope compositions but recent O isotope measurements of an unaltered Vigarano CAI indicate that melilite exchange O with the nebula without being altered. Allende melilites isotope exchange may involve CO but its oxidation requires a source of O which might also supply the Na, Fe, Cl, etc. of the alteration minerals. Whether the oxidation of melilite actually occurred with its alteration is unknown.

  11. 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.

  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. Technologies for Single-Cell Isolation.

    PubMed

    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

  14. Technologies for Single-Cell Isolation.

    PubMed

    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.

  15. Ultrasensitive detection of proteins and sugars at single-cell level

    PubMed Central

    Watabe, Satoshi; Morikawa, Mika; Kaneda, Mugiho; Nakaishi, Kazunari; Nakatsuma, Akira; Ninomiya, Masaki; Yoshimura, Teruki; Miura, Toshiaki; Ito, Etsuro

    2016-01-01

    ABSTRACT Each cell produces its own responses even if it appears identical to other cells. To analyze these individual cell characteristics, we need to measure trace amounts of molecules in a single cell. Nucleic acids in a single cell can be easily amplified by polymerase chain reaction, but single-cell measurement of proteins and sugars will require de novo techniques. In the present study, we outline the techniques we have developed toward this end. For proteins, our ultrasensitive enzyme-linked immunosorbent assay (ELISA) coupled with thionicotinamide-adenine dinucleotide cycling can detect proteins at subattomoles per assay. For sugars, fluorescence correlation spectroscopy coupled with glucose oxidase-catalyzed reaction allows us to measure glucose at tens of nM. Our methods thus offer versatile techniques for single-cell-level analyses, and they are hoped to strongly promote single-cell biology as well as to develop noninvasive tests in clinical medicine. PMID:27064305

  16. Quantitating intracellular oxygen tension in vivo by phosphorescence lifetime measurement.

    PubMed

    Hirakawa, Yosuke; Yoshihara, Toshitada; Kamiya, Mako; Mimura, Imari; Fujikura, Daichi; Masuda, Tsuyoshi; Kikuchi, Ryohei; Takahashi, Ippei; Urano, Yasuteru; Tobita, Seiji; Nangaku, Masaomi

    2015-01-01

    Hypoxia appears to have an important role in pathological conditions in many organs such as kidney; however, a method to quantify intracellular oxygen tension in vivo has not been well established. In this study, we established an optical method to quantify oxygen tension in mice kidneys using a cationic lipophilic phosphorescence probe, BTPDM1, which has an intracellular oxygen concentration-sensitive phosphorescence lifetime. Since this probe is distributed inside the tubular cells of the mice kidney, we succeeded in detecting acute renal hypoxic conditions and chronic kidney disease. This technique enabled us to estimate intracellular partial pressures of oxygen in vivo by extrapolating the calibration curve generated from cultured tubular cells. Since intracellular oxygen tension is directly related to cellular hypoxic reactions, such as the activation of hypoxia-inducible factors, our method will shed new light on hypoxia research in vivo. PMID:26644023

  17. Quantitating intracellular oxygen tension in vivo by phosphorescence lifetime measurement

    PubMed Central

    Hirakawa, Yosuke; Yoshihara, Toshitada; Kamiya, Mako; Mimura, Imari; Fujikura, Daichi; Masuda, Tsuyoshi; Kikuchi, Ryohei; Takahashi, Ippei; Urano, Yasuteru; Tobita, Seiji; Nangaku, Masaomi

    2015-01-01

    Hypoxia appears to have an important role in pathological conditions in many organs such as kidney; however, a method to quantify intracellular oxygen tension in vivo has not been well established. In this study, we established an optical method to quantify oxygen tension in mice kidneys using a cationic lipophilic phosphorescence probe, BTPDM1, which has an intracellular oxygen concentration-sensitive phosphorescence lifetime. Since this probe is distributed inside the tubular cells of the mice kidney, we succeeded in detecting acute renal hypoxic conditions and chronic kidney disease. This technique enabled us to estimate intracellular partial pressures of oxygen in vivo by extrapolating the calibration curve generated from cultured tubular cells. Since intracellular oxygen tension is directly related to cellular hypoxic reactions, such as the activation of hypoxia-inducible factors, our method will shed new light on hypoxia research in vivo. PMID:26644023

  18. Quantitating intracellular oxygen tension in vivo by phosphorescence lifetime measurement.

    PubMed

    Hirakawa, Yosuke; Yoshihara, Toshitada; Kamiya, Mako; Mimura, Imari; Fujikura, Daichi; Masuda, Tsuyoshi; Kikuchi, Ryohei; Takahashi, Ippei; Urano, Yasuteru; Tobita, Seiji; Nangaku, Masaomi

    2015-12-08

    Hypoxia appears to have an important role in pathological conditions in many organs such as kidney; however, a method to quantify intracellular oxygen tension in vivo has not been well established. In this study, we established an optical method to quantify oxygen tension in mice kidneys using a cationic lipophilic phosphorescence probe, BTPDM1, which has an intracellular oxygen concentration-sensitive phosphorescence lifetime. Since this probe is distributed inside the tubular cells of the mice kidney, we succeeded in detecting acute renal hypoxic conditions and chronic kidney disease. This technique enabled us to estimate intracellular partial pressures of oxygen in vivo by extrapolating the calibration curve generated from cultured tubular cells. Since intracellular oxygen tension is directly related to cellular hypoxic reactions, such as the activation of hypoxia-inducible factors, our method will shed new light on hypoxia research in vivo.

  19. Models at the Single Cell Level

    PubMed Central

    Cheong, Raymond; Paliwal, Saurabh

    2014-01-01

    Many cellular behaviors cannot be completely captured or appropriately described at the cell population level. Noise induced by stochastic chemical reactions, spatially polarized signaling networks and heterogeneous cell-cell communication are among the many phenomena that require fine-grained analysis. Accordingly, the mathematical models used to describe such systems must be capable of single cell or subcellular resolution. Here, we review techniques for modeling single cells, including models of stochastic chemical kinetics, spatially heterogeneous intracellular signaling, and spatial stochastic systems. We also briefly discuss applications of each type of model. PMID:20836009

  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. ASRDI oxygen technology survey. Volume 5: Density and liquid level measurement instrumentation for the cryogenic fluids oxygen, hydrogen, and nitrogen

    NASA Technical Reports Server (NTRS)

    Roder, H. M.

    1974-01-01

    Information is presented on instrumentation for density measurement, liquid level measurement, quantity gauging, and phase measurement. Coverage of existing information directly concerned with oxygen was given primary emphasis. A description of the physical principle of measurement for each instrumentation type is included. The basic materials of construction are listed if available from the source document for each instrument discussed. Cleaning requirements, procedures, and verification techniques are included.

  2. Design and optimization of a widely tunable semiconductor laser for blood oxygenation and blood flow measurements

    NASA Astrophysics Data System (ADS)

    Feng, Yafei; Deng, Haoyu; Song, Guangyi; He, Jian-Jun

    2014-11-01

    A method for measuring blood oxygenation and blood flow rate using a single widely tunable semiconductor laser is proposed and investigated. It is shown that a 700-nm-band tunable laser gives the highest sensitivity for blood oxygen measurement. The corresponding tunable laser is designed using the V-coupled cavity structure. The wavelength tuning range can reach 8 nm, which is sufficient for the blood oxygenation measurement in the 700-nm-band by using the Beer- Lambert law. In contrast to conventional blood oxygenation measurement method based on two LEDs, the laser can be used at the same time to measure the blood flow rate based on the Doppler principle.

  3. Antioxidative activity of lactobacilli measured by oxygen radical absorbance capacity.

    PubMed

    Saide, J A O; Gilliland, S E

    2005-04-01

    The reducing ability and antioxidative activity of some species of Lactobacillus were compared under in vitro conditions. Cultures of Lactobacillus delbrueckii ssp. lactis, Lactobacillus delbrueckii ssp. bulgaricus, Lactobacillus acidophilus, and Lactobacillus casei were grown at 37 degrees C in de Man, Rogosa, Sharpe (MRS) broth supplemented with 0.5% 2,3,5 triphenyl tetrazolium chloride (TTC) to evaluate reducing activity. Reduced TTC was extracted from the cultures with acetone, and the intensity of the red color measured colorimetrically at 485 nm was an indication of reducing activity. The lactobacilli varied significantly in relative ability to reduce TTC when grown in MRS broth for 15 h. The relative amounts of growth as indicated by pH values at 18 h appeared to influence the amount of reduction. Antioxidative activity was evaluated by the ability of the whole cells or the cell-free extracts from cultures to protect a protein from being attacked by free radicals. These analyses were performed using the oxygen radical absorbance capacity method. All cultures tested exhibited some degree of antioxidative activity. Among the treatments, the cell-free extracts from cells grown in MRS broth exhibited significantly higher values than did whole cells. There was no apparent relationship between the reducing and antioxidative activities of the cultures evaluated. The results from this study show that these cultures can provide a source of dietary antioxidants. Furthermore, selection of cultures that produce antioxidants as starters could provide yet another health or nutritional benefit from cultured or culture-containing dairy products.

  4. Method for physiologic phenotype characterization at the single-cell level in non-interacting and interacting cells

    PubMed Central

    Ashili, Shashanka P.; Houkal, Jeff; Smith, Dean; Mohammadreza, Aida; Lee, Kristen B.; Forrester, Jessica; Kumar, Ashok; Anis, Yasser H.; Paulson, Thomas G.; Youngbull, Cody A.; Tian, Yanqing; Holl, Mark R.; Johnson, Roger H.; Meldrum, Deirdre R.

    2012-01-01

    Abstract. Intercellular heterogeneity is a key factor in a variety of core cellular processes including proliferation, stimulus response, carcinogenesis, and drug resistance. However, cell-to-cell variability studies at the single-cell level have been hampered by the lack of enabling experimental techniques. We present a measurement platform that features the capability to quantify oxygen consumption rates of individual, non-interacting and interacting cells under normoxic and hypoxic conditions. It is based on real-time concentration measurements of metabolites of interest by means of extracellular optical sensors in cell-isolating microwells of subnanoliter volume. We present the results of a series of measurements of oxygen consumption rates (OCRs) of individual non-interacting and interacting human epithelial cells. We measured the effects of cell-to-cell interactions by using the system’s capability to isolate two and three cells in a single well. The major advantages of the approach are: 1. ratiometric, intensity-based characterization of the metabolic phenotype at the single-cell level, 2. minimal invasiveness due to the distant positioning of sensors, and 3. ability to study the effects of cell-cell interactions on cellular respiration rates. PMID:22502580

  5. Method for physiologic phenotype characterization at the single-cell level in non-interacting and interacting cells

    NASA Astrophysics Data System (ADS)

    Kelbauskas, Laimonas; Ashili, Shashanka P.; Houkal, Jeff; Smith, Dean; Mohammadreza, Aida; Lee, Kristen B.; Forrester, Jessica; Kumar, Ashok; Anis, Yasser H.; Paulson, Thomas G.; Youngbull, Cody A.; Tian, Yanqing; Holl, Mark R.; Johnson, Roger H.; Meldrum, Deirdre R.

    2012-03-01

    Intercellular heterogeneity is a key factor in a variety of core cellular processes including proliferation, stimulus response, carcinogenesis, and drug resistance. However, cell-to-cell variability studies at the single-cell level have been hampered by the lack of enabling experimental techniques. We present a measurement platform that features the capability to quantify oxygen consumption rates of individual, non-interacting and interacting cells under normoxic and hypoxic conditions. It is based on real-time concentration measurements of metabolites of interest by means of extracellular optical sensors in cell-isolating microwells of subnanoliter volume. We present the results of a series of measurements of oxygen consumption rates (OCRs) of individual non-interacting and interacting human epithelial cells. We measured the effects of cell-to-cell interactions by using the system's capability to isolate two and three cells in a single well. The major advantages of the approach are: 1. ratiometric, intensity-based characterization of the metabolic phenotype at the single-cell level, 2. minimal invasiveness due to the distant positioning of sensors, and 3. ability to study the effects of cell-cell interactions on cellular respiration rates.

  6. TOPAZ-2 single-cell TFE electric insulation properties study

    SciTech Connect

    Vasilchenko, A.V.; Izhvanov, O.L.

    1996-03-01

    TOPAZ-II single cell thermoinic fuel element (TFE) electric insulation parameters under testing with electric heating were measured. TFE electric design schematic, experimental procedure and measurements results are described. Collector resistance was measured in helium at 420{endash}890 K. Metal ceramic ceals insulation properties were measured in vacuum P=10{sup {minus}4} Pa and in cesium vapor P=10{sup {minus}1}{minus}260 Pa, at 420{endash}730 K. Results of separate TFE are compared with the data; that were measured during nuclear power system (NPS) Ya-21U test. Based upon this data NPS power losses were estimated. {copyright} {ital 1996 American Institute of Physics.}

  7. The Rate of Photorespiration as Measured by Means of Oxygen Uptake and Its Respiratory Quotient

    PubMed Central

    Samish, Yochai B.

    1971-01-01

    Oxygen recycling inside photosynthesizing leaves was found to amount to less than 0.3% of the oxygen consumed by photorespiration under natural conditions, provided the influence of buildup of oxygen released by photosynthesis into the external air was taken into consideration. When this is related to the amounts of photorespired CO2, which had been previously found to be reabsorbed by photosynthesis, it appears that previous respiratory quotients reported for photorespiration were underestimated. For the same reason the photosynthetic quotient was overestimated. Actually, quotients of photorespiration and of photosynthesis approach the more normal range of respiratory quotients int the dark. The oxygen recycling was calculated according to an electrical analogue to oxygen flow. The determination of photorespiration, when measured by oxygen uptake, can be more accurate than that by CO2 measurement. However, recycling of oxygen occurs in larger amounts at lower oxygen and higher CO2 concentrations, as well as under conditions of high resistance to transpiration. PMID:16657795

  8. 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

  9. 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.

  10. 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

  11. Exploring symbioses by single-cell genomics.

    PubMed

    Kamke, Janine; Bayer, Kristina; Woyke, Tanja; Hentschel, Ute

    2012-08-01

    Single-cell genomics has advanced the field of microbiology from the analysis of microbial metagenomes where information is "drowning in a sea of sequences," to recognizing each microbial cell as a separate and unique entity. Single-cell genomics employs Phi29 polymerase-mediated whole-genome amplification to yield microgram-range genomic DNA from single microbial cells. This method has now been applied to a handful of symbiotic systems, including bacterial symbionts of marine sponges, insects (grasshoppers, termites), and vertebrates (mouse, human). In each case, novel insights were obtained into the functional genomic repertoire of the bacterial partner, which, in turn, led to an improved understanding of the corresponding host. Single-cell genomics is particularly valuable when dealing with uncultivated microorganisms, as is still the case for many bacterial symbionts. In this review, we explore the power of single-cell genomics for symbiosis research and highlight recent insights into the symbiotic systems that were obtained by this approach. PMID:22983031

  12. CONTINUOUS, AUTOMATED AND SIMULTANEOUS MEASUREMENT OF OXYGEN UPTAKE AND CARBON DIOXIDE EVOLUTION IN BIOLOGICAL SYSTEMS

    EPA Science Inventory

    Commercial respirometers are capable of continuously and automatically measuring oxygen uptake in bioreactors. A method for continuously and automatically measuring carbon dioxide evolution can be retrofitted to commercial respirometers. Continuous and automatic measurements of...

  13. Measurement of oxygen fugacities under reducing conditions: non-Nernstian behavior of Y 2O 3-doped zirconia oxygen sensors

    NASA Astrophysics Data System (ADS)

    Mendybaev, R. A.; Beckett, J. R.; Stolper, E.; Grossman, L.

    1998-09-01

    A calibration procedure is presented for the use of a Y 2O 3-stabilized zirconia (YSZ) oxygen sensor in 1 atm gas-mixing furnaces in the temperature range 1200-1500°C and 0-8 orders of magnitude below the iron-wüstite (IW) buffer. Corrections to the Nernst equation were obtained by measuring apparent oxygen fugacities of gases in equilibrium with graphite (equilibrated with pure CO vapor), Cr + Cr 2O 3, and Ta + Ta 2O 5. Under reducing conditions, fO 2s calculated using the ideal form of the Nernst equation are erroneously high, by <0.1 log units at IW but by nearly three log units for Ta-Ta 2O 5 at 1000°C. The deviations between measured emfs and those calculated assuming Nernstian behavior of the electrolyte in the oxygen sensor reflect mixed ionic-electronic conduction. Measured emfs under reducing conditions are readily corrected for this effect via experimentally determined values of P θ, the oxygen fugacity at which electronic conduction constitutes half of the total conductivity. For the oxygen sensors used in this study, logP θ(±0.20,1σ)=3.70(±0.72)- (32.95±1.15)×10 3/T(K). Even under conditions more reducing than a gas of solar composition ( fO 2 = 10 -18 at 1200°C), YSZ oxygen sensors can be used to determine absolute values of the oxygen fugacity to within ±0.2 log units.

  14. Resolution of Conflicting Signals at the Single-Cell Level in the Regulation of Cyanobacterial Photosynthesis and Nitrogen Fixation

    PubMed Central

    Mohr, Wiebke; Vagner, Tomas; Kuypers, Marcel M. M.; Ackermann, Martin; LaRoche, Julie

    2013-01-01

    Unicellular, diazotrophic cyanobacteria temporally separate dinitrogen (N2) fixation and photosynthesis to prevent inactivation of the nitrogenase by oxygen. This temporal segregation is regulated by a circadian clock with oscillating activities of N2 fixation in the dark and photosynthesis in the light. On the population level, this separation is not always complete, since the two processes can overlap during transitions from dark to light. How do single cells avoid inactivation of nitrogenase during these periods? One possibility is that phenotypic heterogeneity in populations leads to segregation of the two processes. Here, we measured N2 fixation and photosynthesis of individual cells using nanometer-scale secondary ion mass spectrometry (nanoSIMS) to assess both processes in a culture of the unicellular, diazotrophic cyanobacterium Crocosphaera watsonii during a dark-light and a continuous light phase. We compared single-cell rates with bulk rates and gene expression profiles. During the regular dark and light phases, C. watsonii exhibited the temporal segregation of N2 fixation and photosynthesis commonly observed. However, N2 fixation and photosynthesis were concurrently measurable at the population level during the subjective dark phase in which cells were kept in the light rather than returned to the expected dark phase. At the single-cell level, though, cells discriminated against either one of the two processes. Cells that showed high levels of photosynthesis had low nitrogen fixing activities, and vice versa. These results suggest that, under ambiguous environmental signals, single cells discriminate against either photosynthesis or nitrogen fixation, and thereby might reduce costs associated with running incompatible processes in the same cell. PMID:23805199

  15. Intrinsic artefacts in optical oxygen sensors--how reliable are our measurements?

    PubMed

    Lehner, Philipp; Staudinger, Christoph; Borisov, Sergey M; Regensburger, Johannes; Klimant, Ingo

    2015-03-01

    Optical oxygen sensing is of broad interest in many areas of research, such as medicine, food processing, and micro- and marine biology. The operation principle of optical oxygen sensors is well established and these sensors are routinely employed in lab and field experiments. Ultratrace oxygen sensors, which enable measurements in the sub-nanomolar region (dissolved oxygen), are becoming increasingly important. Such sensors prominently exhibit phenomena that complicate calibration and measurements. However, these phenomena are not constrained to ultratrace sensors; rather, these effects are inherent to the way optical oxygen sensors work and may influence any optical oxygen measurement when certain conditions are met. This scenario is especially true for applications that deal with high-excitation light intensities, such as microscopy and microfluidic applications. Herein, we present various effects that we could observe in our studies with ultratrace oxygen sensors and discuss the reasons for their appearance, the mechanism by which they influence measurements, and how to best reduce their impact. The phenomena discussed are oxygen photoconsumption in the sensor material; depletion of the dye ground state by high-excitation photon-flux values, which can compromise both intensity and ratiometric-based measurements; triplet-triplet annihilation; and singlet-oxygen accumulation, which affects measurements at very low oxygen concentrations.

  16. A simplified headspace biochemical oxygen demand test protocol based on oxygen measurements using a fiber optic probe.

    PubMed

    Min, Booki; Kohler, David; Logan, Bruce E

    2004-01-01

    Batch respirometric tests have many advantages over the conventional biochemical oxygen demand (BOD) method for analysis of wastewaters, including the use of nondiluted samples, a more rapid exertion of oxygen demand, and reduced sample preparation time. The headspace biochemical oxygen demand (HBOD) test can be used to obtain oxygen demands in 2 or 3 days that can predict 5-day biochemical oxygen demand (BOD5) results. The main disadvantage of the HBOD and other respirometric tests has been the lack of a simple and direct method to measure oxygen concentrations in the gas phase. The recent commercial production of a new type of fiber optic oxygen probe, however, provides a method to eliminate this disadvantage. This fiber optic probe, referred to here as the HBOD probe, was tested to see if it could be used in HBOD tests. Gas-phase oxygen measurements made with the HBOD probe took only a few seconds and were not significantly different from those made using a gas chromatograph (t test: n = 15, R2 = 0.9995, p < 0.001). In field tests using the HBOD probe procedure, the probe greatly reduced sample analysis time compared with previous HBOD and BOD protocols and produced more precise results than the BOD test for wastewater samples from two treatment plants (University Area Joint Authority [UAJA] Wastewater Treatment Plant in University Park, Pennsylvania, and The Pennsylvania State University [PSU] Wastewater Treatment Plant in University Park). Headspace biochemical oxygen demand measurements on UAJA primary clarifier effluent were 59.9 +/- 2.4% after 2 days (HBOD2) and 73.0 +/- 3.1% after 3 days (HBOD) of BOD, values, indicating that BOD5 values could be predicted by multiplying HBOD2 values by 1.67 +/- 0.07 or HBOD3 by 1.37 +/- 0.06. Similarly, tests using PSU wastewater samples could be used to provide BOD5 estimates by multiplying the HBOD2 by 1.24 +/- 0.04 or by multiplying the HBOD3 by 0.97 +/- 0.03. These results indicate that the HBOD fiber optic probe can

  17. Massive and parallel expression profiling using microarrayed single-cell sequencing

    PubMed Central

    Vickovic, Sanja; Ståhl, Patrik L.; Salmén, Fredrik; Giatrellis, Sarantis; Westholm, Jakub Orzechowski; Mollbrink, Annelie; Navarro, José Fernández; Custodio, Joaquin; Bienko, Magda; Sutton, Lesley-Ann; Rosenquist, Richard; Frisén, Jonas; Lundeberg, Joakim

    2016-01-01

    Single-cell transcriptome analysis overcomes problems inherently associated with averaging gene expression measurements in bulk analysis. However, single-cell analysis is currently challenging in terms of cost, throughput and robustness. Here, we present a method enabling massive microarray-based barcoding of expression patterns in single cells, termed MASC-seq. This technology enables both imaging and high-throughput single-cell analysis, characterizing thousands of single-cell transcriptomes per day at a low cost (0.13 USD/cell), which is two orders of magnitude less than commercially available systems. Our novel approach provides data in a rapid and simple way. Therefore, MASC-seq has the potential to accelerate the study of subtle clonal dynamics and help provide critical insights into disease development and other biological processes. PMID:27739429

  18. 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.

  19. Measurement and Control of Oxygen Partial Pressure in an Electrostatic Levitator

    NASA Technical Reports Server (NTRS)

    SanSoucie, Michael P.; Rogers, Jan R.

    2014-01-01

    Recently the NASA Marshall Space Flight Center electrostatic levitation (ESL) laboratory has been upgraded to include an oxygen control system. This system allows the oxygen partial pressure within the vacuum chamber to be measured and controlled, at elevated temperatures, theoretically in the range from 10(exp -36) to 10(exp 0) bar. The role of active surface agents in liquid metals is fairly well known; however, published surface tension data typically has large scatter, which has been hypothesized to be caused by the presence of oxygen. The surface tension of metals is affected by even a small amount of adsorption of oxygen. It has even been shown that oxygen partial pressures may need to be as low as 10(exp -24) bar to avoid oxidation. While electrostatic levitation is done under high vacuum, oxide films or dissolved oxygen may have significant effects on materials properties, such as surface tension and viscosity. Therefore, the ability to measure and control the oxygen partial pressure within the chamber is highly desirable. The oxygen control system installed at MSFC contains a potentiometric sensor, which measures the oxygen partial pressure, and an oxygen ion pump. In the pump, a pulse-width modulated electric current is applied to yttrium-stabilized zirconia, resulting in oxygen transfer into or out of the system. Also part of the system is a control unit, which consists of temperature controllers for the sensor and pump, PID-based current loop for the ion pump, and a control algorithm. This system can be used to study the effects of oxygen on the thermophysical properties of metals, ceramics, glasses, and alloys. It can also be used to provide more accurate measurements by processing the samples at very low oxygen partial pressures. The oxygen control system will be explained in more detail and an overview of its use and limitations in an electrostatic levitator will be described. Some preliminary measurements have been made, and the results to date will

  20. Measuring Spatial and Temporal Heterogeneity of Dissolved Oxygen in Streambed Sediments Using Pressure Sensitive Paint (PSP)

    NASA Astrophysics Data System (ADS)

    Huynh, K. T.; Salus, A.; Xie, M.; Roche, K. R.; Packman, A. I.

    2014-12-01

    Pressure sensitive paints (PSP) have been largely used in aerodynamic applications to measure pressure distributions on complex bodies such as aircraft. One common family of PSPs employ fluorescent pigments that are quenched in the presence of oxygen, yielding an inverse relationship between fluorescence intensity and oxygen concentration that is used to measure pressure in aerodynamic applications through the partial pressure of oxygen. These PSPs offer unexplored potential for visualizing dissolved oxygen (DO) concentration distributions on surfaces underwater. PSP was used to measure dissolved oxygen concentrations in streambed sediments in a laboratory flume. Two PSP-coated 2.5 cm diameter spheres were emplaced in a bed of similar material, and imaged under varying DO concentrations. Calibration curves relating fluorescence intensity to dissolved oxygen concentration were developed on a pixel-by-pixel basis, enabling spatial patterns of oxygen to be resolved in the sediment bed. This method of measuring dissolved oxygen concentration is advantageous because of its fast response time and ability to measure heterogeneous oxygen distributions in sediments. Future work will explore the combined effects of stream flow and biofilm growth on oxygen distributions in streambed sediments.

  1. High-Dimensional Single-Cell Cancer Biology

    PubMed Central

    Doxie, Deon B.

    2014-01-01

    Cancer cells are distinguished from each other and from healthy cells by features that drive clonal evolution and therapy resistance. New advances in high-dimensional flow cytometry make it possible to systematically measure mechanisms of tumor initiation, progression, and therapy resistance on millions of cells from human tumors. Here we describe flow cytometry techniques that enable a ‘single-cell systems biology’ view of cancer. High-dimensional techniques like mass cytometry enable multiplexed single-cell analysis of cell identity, clinical biomarkers, signaling network phospho-proteins, transcription factors, and functional readouts of proliferation, cell cycle status, and apoptosis. This capability pairs well with a signaling profiles approach that dissects mechanism by systematically perturbing and measuring many nodes in a signaling network. Single-cell approaches enable study of cellular heterogeneity of primary tissues and turn cell subsets into experimental controls or opportunities for new discovery. Rare populations of stem cells or therapy resistant cancer cells can be identified and compared to other types of cells within the same sample. In the long term, these techniques will enable tracking of minimal residual disease (MRD) and disease progression. By better understanding biological systems that control development and cell-cell interactions in healthy and diseased contexts, we can learn to program cells to become therapeutic agents or target malignant signaling events to specifically kill cancer cells. Single-cell approaches that provide deep insight into cell signaling and fate decisions will be critical to optimizing the next generation of cancer treatments combining targeted approaches and immunotherapy. PMID:24671264

  2. Measurement of oxygen consumption during muscle flaccidity exercise by near-infrared spectroscopy

    NASA Astrophysics Data System (ADS)

    Fukuda, K.; Fukawa, Y.

    2013-03-01

    Quantitative measurement oxygen consumption in the muscles is important to evaluate the effect of the exercise. Near-infrared spectroscopy (NIRS) is a noninvasive method for measuring muscle oxygenation. However, measurement results are affected by blood volume change due to changes in the blood pressure. In order to evaluate changes in blood volume and to improve measurement accuracy, we proposed a calculation method of three-wavelength measurement with considering the scattering factor and the measurement with monitoring blood flow for measuring the temporal change of the oxygen concentration more precisely. We applied three-wavelength light source (680nm, 808nm and 830nm) for the continued wave measurement. Two detectors (targeted detector and the reference detector) were placed near the target muscle and apart from it. We measured the blood flow by controlling the intravascular pressure and the oxygen consumption with the handgrip exercise in the forearm. The measured results show that the scattering factor contains the artifact at the surface and the blood flow in the artery and the vein in the same phase. The artifact and the blood flow in the same phase are reduced from the oxygenated and the deoxygenated hemoglobin densities. Thus our proposed method is effective for reducing the influence of the artifact and the blood flow in the same phase from the oxygen consumption measurement. Further, it is shown that the oxygen consumption is measured more accurately by subtracting the blood flow measured by the reference detector.

  3. Simulation of oxygen saturation measurement in a single blood vein.

    PubMed

    Duadi, Hamootal; Nitzan, Meir; Fixler, Dror

    2016-09-15

    The value of oxygen saturation in venous blood, SvO2, has important clinical significance since it is related to the tissue oxygen utilization, which is related to the blood flow to the tissue and to its metabolism rate. However, existing pulse oximetry techniques are not suitable for blood in veins. In the current study we examine the feasibility of difference oximetry to assess SvO2 by using two near-infrared wavelengths and collecting the backscattered light from two photodetectors located at different distances from the light source. PMID:27628385

  4. Measurement in a marine environment using low cost sensors of temperature and dissolved oxygen

    USGS Publications Warehouse

    Godshall, F.A.; Cory, R.L.; Phinney, D.E.

    1974-01-01

    Continuous records of physical parameters of the marine environment are difficult as well as expensive to obtain. This paper describes preliminary results of an investigative program with the purpose of developing low cost time integrating measurement and averaging devices for water temperature and dissolved oxygen. Measurements were made in an estuarine area of the Chesapeake Bay over two week periods. With chemical thermometers average water temperature for the two week period was found to be equal to average water temperature measured with thermocouples plus or minus 1.0 C. The slow diffusion of oxygen through the semipermiable sides of plastic bottles permitted the use of water filled bottles to obtain averaged oxygen measurements. Oxygen measurements for two week averaging times using 500 ml polyethylene bottles were found to vary from conventionally measured and averaged dissolved oxygen by about 1.8 mg/l. ?? 1974 Estuarine Research Federation.

  5. Techniques for Measuring Low Earth Orbital Atomic Oxygen Erosion of Polymers

    NASA Technical Reports Server (NTRS)

    deGroh, Kim K.; Banks, Bruce A.; Demko, Rikako

    2002-01-01

    Polymers such as polyimide Kapton and Teflon FEP (fluorinated ethylene propylene) are commonly used spacecraft materials due to their desirable properties such as flexibility, low density, and in the case of FEP, a low solar absorptance and high thermal emittance. Polymers on the exterior of spacecraft in the low Earth orbit (LEO) environment are exposed to energetic atomic oxygen. Atomic oxygen reaction with polymers causes erosion, which is a threat to spacecraft durability. It is therefore important to understand the atomic oxygen erosion yield (E, the volume loss per incident oxygen atom) of polymers being considered in spacecraft design. The most common technique for determining E is through mass loss measurements. For limited duration exposure experiments, such as shuttle experiments, where the atomic oxygen fluence is often so low that mass loss measurements can not produce acceptable uncertainties, recession measurements based on atomic force microscopy analyses can be used. Equally necessary to knowing the mass loss or recession depth for determining the erosion yield of polymers is the knowledge of the atomic oxygen fluence that the polymers were exposed to in space. This paper discusses the procedures and relevant issues for mass loss and recession depth measurements for passive atomic oxygen erosion yield characterization of polymers, along with techniques for active atomic oxygen fluence and erosion characterization. One active atomic oxygen erosion technique discussed is a new technique based on optical measurements. Details including the use of both semi-transparent and opaque polymers for active erosion measurement are reviewed.

  6. A system using solid ceramic oxygen electrolyte cells to measure oxygen fugacities in gas-mixing systems

    NASA Technical Reports Server (NTRS)

    Williams, R. J.; Mullins, O.

    1976-01-01

    Details are given for the construction and operation of a 101.3 kN/sq m (1 atmosphere) redox control system. A solid ceramic oxygen electrolyte cell is used to monitor the oxygen fugacity in the furnace. The system consists of a vertical quench, gas mixing furnace with heads designed for mounting the electrolyte cell and with facilities for inserting and removing the samples. The system also contains the high input impedance electronics necessary for measurements, a simplified version of a gas mixing apparatus, and devices for experiments under controlled rates of change relative to temperature and redox state. The calibration and maintenance of the system are discussed.

  7. JSC systems using solid ceramic oxygen electrolyte cells to measure oxygen fugacites in gas-mixing systems

    NASA Technical Reports Server (NTRS)

    Williams, R. J.; Mullins, O.

    1981-01-01

    Details are given for the construction and operation of a 101.3 KN/sq meter (1 atmosphere) redox control system. A solid ceramic oxygen electrolyte cell is used to monitor the oxygen fugacity in the furnace. The system consists of a vertical quench gas mixing furnace with heads designed for mounting the electrolyte cell and with facilities for inserting and removing the samples, a simplified version of a gas mixing apparatus, and devices for experiments under controlled rates of change of temperature. A thermogravimetric analysis system employing these techniques of redox control and measurement is also described. The calibration and maintenance of the system are discussed.

  8. A Simple Experiment To Measure the Content of Oxygen in the Air Using Heated Steel Wool

    ERIC Educational Resources Information Center

    Vera, Francisco; Rivera, Rodrigo; Nunez, Cesar

    2011-01-01

    The typical experiment to measure the oxygen content in the atmosphere uses the rusting of steel wool inside a closed volume of air. Two key aspects of this experiment that make possible a successful measurement of the content of oxygen in the air are the use of a closed atmosphere and the use of a chemical reaction that involves the oxidation of…

  9. 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

  10. Digital microfluidic immunocytochemistry in single cells

    PubMed Central

    Ng, Alphonsus H. C.; Dean Chamberlain, M.; 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

  11. A stochastic transcriptional switch model for single cell imaging data

    PubMed Central

    Hey, Kirsty L.; Momiji, Hiroshi; Featherstone, Karen; Davis, Julian R.E.; White, Michael R.H.; Rand, David A.; Finkenstädt, Bärbel

    2015-01-01

    Gene expression is made up of inherently stochastic processes within single cells and can be modeled through stochastic reaction networks (SRNs). In particular, SRNs capture the features of intrinsic variability arising from intracellular biochemical processes. We extend current models for gene expression to allow the transcriptional process within an SRN to follow a random step or switch function which may be estimated using reversible jump Markov chain Monte Carlo (MCMC). This stochastic switch model provides a generic framework to capture many different dynamic features observed in single cell gene expression. Inference for such SRNs is challenging due to the intractability of the transition densities. We derive a model-specific birth–death approximation and study its use for inference in comparison with the linear noise approximation where both approximations are considered within the unifying framework of state-space models. The methodology is applied to synthetic as well as experimental single cell imaging data measuring expression of the human prolactin gene in pituitary cells. PMID:25819987

  12. 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

  13. Mie scatter corrections in single cell infrared microspectroscopy.

    PubMed

    Konevskikh, Tatiana; Lukacs, Rozalia; Blümel, Reinhold; Ponossov, Arkadi; Kohler, Achim

    2016-06-23

    Strong Mie scattering signatures hamper the chemical interpretation and multivariate analysis of the infrared microscopy spectra of single cells and tissues. During recent years, several numerical Mie scatter correction algorithms for the infrared spectroscopy of single cells have been published. In the paper at hand, we critically reviewed existing algorithms for the correction of Mie scattering and suggest improvements. We developed an iterative algorithm based on Extended Multiplicative Scatter Correction (EMSC), for the retrieval of pure absorbance spectra from highly distorted infrared spectra of single cells. The new algorithm uses the van de Hulst approximation formula for the extinction efficiency employing a complex refractive index. The iterative algorithm involves the establishment of an EMSC meta-model. While existing iterative algorithms for the correction of resonant Mie scattering employ three independent parameters for establishing a meta-model, we could decrease the number of parameters from three to two independent parameters, which reduced the calculation time for the Mie scattering curves for the iterative EMSC meta-model by a factor of 10. Moreover, by employing the Hilbert transform for evaluating the Kramers-Kronig relations based on a FFT algorithm in Matlab, we further improved the speed of the algorithm by a factor of 100. For testing the algorithm we simulate distorted apparent absorbance spectra by utilizing the exact theory for the scattering of infrared light at absorbing spheres, taking into account the high numerical aperture of infrared microscopes employed for the analysis of single cells and tissues. In addition, the algorithm was applied to measured absorbance spectra of single lung cancer cells. PMID:27034998

  14. Mie scatter corrections in single cell infrared microspectroscopy.

    PubMed

    Konevskikh, Tatiana; Lukacs, Rozalia; Blümel, Reinhold; Ponossov, Arkadi; Kohler, Achim

    2016-06-23

    Strong Mie scattering signatures hamper the chemical interpretation and multivariate analysis of the infrared microscopy spectra of single cells and tissues. During recent years, several numerical Mie scatter correction algorithms for the infrared spectroscopy of single cells have been published. In the paper at hand, we critically reviewed existing algorithms for the correction of Mie scattering and suggest improvements. We developed an iterative algorithm based on Extended Multiplicative Scatter Correction (EMSC), for the retrieval of pure absorbance spectra from highly distorted infrared spectra of single cells. The new algorithm uses the van de Hulst approximation formula for the extinction efficiency employing a complex refractive index. The iterative algorithm involves the establishment of an EMSC meta-model. While existing iterative algorithms for the correction of resonant Mie scattering employ three independent parameters for establishing a meta-model, we could decrease the number of parameters from three to two independent parameters, which reduced the calculation time for the Mie scattering curves for the iterative EMSC meta-model by a factor of 10. Moreover, by employing the Hilbert transform for evaluating the Kramers-Kronig relations based on a FFT algorithm in Matlab, we further improved the speed of the algorithm by a factor of 100. For testing the algorithm we simulate distorted apparent absorbance spectra by utilizing the exact theory for the scattering of infrared light at absorbing spheres, taking into account the high numerical aperture of infrared microscopes employed for the analysis of single cells and tissues. In addition, the algorithm was applied to measured absorbance spectra of single lung cancer cells.

  15. Single-cell photonic nanocavity probes

    PubMed Central

    Shambat, Gary; Kothapalli, Sri-Rajasekhar; Provine, J; Sarmiento, Tomas; Harris, James; Gambhir, Sanjiv Sam; Vučković, Jelena

    2013-01-01

    Single-cell interrogation at the nanoscale can take on many forms—namely, pillars1, tubes2–5, and wires6–8 as probes for gentle insertion into a cell’s interior, allowing for studies on single-cell cargo delivery4, electrochemistry3 or electrophysiology1, and optical signaling9–10. However, optical probes such as nanowires9 or tapered fibers10 are passive in nature and have only served as conduits to guide light into and out of a cell. Here we demonstrate a nanobeam photonic crystal (PC) cavity as an advanced cellular nanoprobe, active in nature, and configurable to provide a multitude of actions for both intracellular sensing and control. Our semiconductor nanocavity probes emit photoluminescence (PL) from embedded quantum dots (QD) and sustain high quality resonant photonic modes inside cells. The beams can be loaded in cells and tracked for days at a time, with cells undergoing regular division with the beams. Furthermore, we present in vitro label-free protein sensing with our probes as a path towards real-time biomarker detection in single cells. PMID:23387382

  16. Optical manipulation and microfluidics for studies of single cell dynamics

    NASA Astrophysics Data System (ADS)

    Eriksson, E.; Scrimgeour, J.; Granéli, A.; Ramser, K.; Wellander, R.; Enger, J.; Hanstorp, D.; Goksör, M.

    2007-08-01

    Most research on optical manipulation aims towards investigation and development of the system itself. In this paper we show how optical manipulation, imaging and microfluidics can be combined for investigations of single cells. Microfluidic systems have been fabricated and are used, in combination with optical tweezers, to enable environmental changes for single cells. The environment within the microfluidic system has been modelled to ensure control of the process. Three biological model systems have been studied with different combinations of optical manipulation, imaging techniques and microfluidics. In Saccharomyces cerevisiae, environmentally induced size modulations and spatial localization of proteins have been studied to elucidate various signalling pathways. In a similar manner the oxygenation cycle of single red blood cells was triggered and mapped using Raman spectroscopy. In the third experiment the forces between the endoplasmic reticulum and chloroplasts were studied in Pisum sativum and Arabidopsis thaliana. By combining different techniques we make advanced biological research possible, revealing information on a cellular level that is impossible to obtain with traditional techniques.

  17. Measurements of oxygen pressure in a copper flash smelting furnace by an EMF method

    NASA Astrophysics Data System (ADS)

    Kemori, Nobumasa; Shibata, Yukio; Tomono, Mutsuo

    1986-01-01

    Oxygen pressures in a copper flash smelting furnace were measured by means of the following galvanic cell: Fe, FeO/ZrO2 + MgO/ barO in slag or matte. Measured oxygen pressures were normalized to 1523 K with respect to the reaction: 4 FeO(l) + O2(g) = 4 FeO1.5(l). Vertical and horizontal variations of normalized oxygen pressures in the reaction shaft and in the settler were studied. The equilibrium relation between normalized oxygen pressure and the ratio of ferric to ferrous oxide content in the furnace slag was confirmed, and the activity coefficient ratio of these oxides was determined.

  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.; 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. 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.

  20. Vacuum Ultraviolet Absorption Measurements of Atomic Oxygen in a Shock Tube

    NASA Technical Reports Server (NTRS)

    Meyer, Scott Andrew

    1995-01-01

    The absorption of vacuum ultraviolet light by atomic oxygen has been measured in the Electric Arc-driven Shock Tube (EAST) Facility at NASA-Ames Research Center. This investigation demonstrates the instrumentation required to determine atomic oxygen concentrations from absorption measurements in impulse facilities. A shock wave dissociates molecular oxygen, producing a high temperature sample of atomic oxygen in the shock tube. A probe beam is generated with a Raman-shifted ArF excimer laser. By suitable tuning of the laser, absorption is measured over a range of wavelengths in the region of the atomic line at 130.49 nm. The line shape function is determined from measurements at atomic oxygen densities of 3 x 10(exp 17) and 9 x 10(exp 17) cm(exp -3). The broadening coefficient for resonance interactions is deduced from this data, and this value is in accord with available theoretical models.

  1. Vacuum Ultraviolet Absorption Measurements of Atomic Oxygen in a Shock Tube

    NASA Technical Reports Server (NTRS)

    Meyer, Scott Andrew

    1995-01-01

    The absorption of vacuum ultraviolet light by atomic oxygen has been measured in the Electric Arc-driven Shock Tube (EAST) Facility at NASA-Ames Research Center. This investigation demonstrates the instrumentation required to determine atomic oxygen concentrations from absorption measurements in impulse facilities. A shock wave dissociates molecular oxygen, producing a high temperature sample of atomic oxygen in the shock tube. A probe beam is generated with a Raman-shifted ArF excimer laser. By suitable tuning of the laser, absorption is measured over a range of wavelengths in the region of the atomic line at 130.49 nm. The line shape function is determined from measurements at atomic oxygen densities of 3x10(exp 17) and 9x10(exp 17) cm(exp -3). The broadening coefficient for resonance interactions is deduced from this data, and this value is in accord with available theoretical models.

  2. Vacuum Ultraviolet Absorption Measurements of Atomic Oxygen in a Shock Tube

    NASA Technical Reports Server (NTRS)

    Meyer, Scott Andrew

    1995-01-01

    The absorption of vacuum ultraviolet light by atomic oxygen has been measured in the Electric Arc-driven Shock Tube (EAST) Facility at NASA-Ames Research Center. This investigation demonstrates the instrumentation required to determine atomic oxygen concentrations from absorption measurements in impulse facilities. A shock wave dissociates molecular oxygen, producing a high temperature sample of atomic oxygen in the shock tube. A probe beam is generated with a Raman-shifted ArF excimer laser. By suitable tuning of the laser, absorption is measured over a range of wavelengths in the region of the atomic line at 130.49 nm. The line shape function is determined from measurements at atomic oxygen densities of 3 x 10(exp 17) and 9 x 10(exp 17)/cu cm. The broadening coefficient for resonance interactions is deduced from this data, and this value is in accord with available theoretical models.

  3. Precision of cerebral oxygenation and hemoglobin concentration measurements in neonates measured by near-infrared spectroscopy

    NASA Astrophysics Data System (ADS)

    Arri, Sandra Jasminder; Muehlemann, Thomas; Biallas, Martin; Bucher, Hans Ulrich; Wolf, Martin

    2011-04-01

    Background and aim: One source of error with near-infrared spectroscopy (NIRS) is the assumption that the measured tissue is optically homogeneous. This is not always the case. Our aim is to assess the impact of tissue homogeneity (TH) on the precision of NIRS measurements in neonates. Methods: On 36 term and 27 preterm neonates at least five 1-min measurements are performed on each subject using the OxiplexTS. The sensor position is slightly changed before each measurement while assessing TH. The precision for cerebral tissue oxygenation saturation (StO2) and total hemoglobin concentration (tHb) are calculated by repeated measures analysis of variance. Results: The mean StO2 is not significantly different between term and preterm infants. The mean tHb is significantly lower in preterm infants (p < 0.01). With increasing TH, the precision of StO2 increase from 5.6 to 4.6% for preterm and from 11.0 to 2.0% for term infants; the precision of tHb increases from 10.1 to 7.5μM for preterm and from 16.4 to 3.5μM for term infants. The precision for StO2 is higher in term than in preterm infants. The precision for tHb shows no significant difference between the two groups. Conclusions: The precision of NIRS measurements correlates with tissue homogeneity.

  4. Oxygen Pickup Ions Measured by MAVEN Outside the Martian Bow Shock

    NASA Astrophysics Data System (ADS)

    Rahmati, A.; Cravens, T.; Larson, D. E.; Lillis, R. J.; Dunn, P.; Halekas, J. S.; Connerney, J. E. P.; Eparvier, F. G.; Thiemann, E.; Mitchell, D. L.; Jakosky, B. M.

    2015-12-01

    The MAVEN (Mars Atmosphere and Volatile EvolutioN) spacecraft entered orbit around Mars on September 21, 2014 and has since been detecting energetic oxygen pickup ions by its SEP (Solar Energetic Particles) and SWIA (Solar Wind Ion Analyzer) instruments. The oxygen pickup ions detected outside the Martian bowshock and in the upstream solar wind are associated with the extended hot oxygen exosphere of Mars, which is created mainly by the dissociative recombination of molecular oxygen ions with electrons in the ionosphere. We use analytic solutions to the equations of motion of pickup ions moving in the undisturbed upstream solar wind magnetic and motional electric fields and calculate the flux of oxygen pickup ions at the location of MAVEN. Our model calculates the ionization rate of oxygen atoms in the exosphere based on the hot oxygen densities predicted by Rahmati et al. (2014), and the sources of ionization include photo-ionization, charge exchange, and electron impact ionization. The photo-ionization frequency is calculated using the FISM (Flare Irradiance Spectral Model) solar flux model, based on MAVEN EUVM (Extreme Ultra-Violet Monitor) measurements. The frequency of charge exchange between a solar wind proton and an oxygen atom is calculated using MAVEN SWIA solar wind proton flux measurements, and the electron impact ionization frequency is calculated based on MAVEN SWEA (Solar Wind Electron Analyzer) solar wind electron flux measurements. The solar wind magnetic field used in the model is from the measurements taken by MAVEN MAG (magnetometer) in the upstream solar wind. The good agreement between our predicted pickup oxygen fluxes and the MAVEN SEP and SWIA measured ones confirms detection of oxygen pickup ions and these model-data comparisons can be used to constrain models of hot oxygen densities and photochemical escape flux.

  5. Investigating evolutionary perspective of carcinogenesis with single-cell transcriptome analysis

    PubMed Central

    Zhang, Xi; Zhang, Cheng; Li, Zhongjun; Zhong, Jiangjian; Weiner, Leslie P.; Zhong, Jiang F.

    2013-01-01

    We developed phase-switch microfluidic devices for molecular profiling of a large number of single cells. Whole genome microarrays and RNA-sequencing are commonly used to determine the expression levels of genes in cell lysates (a physical mix of millions of cells) for inferring gene functions. However, cellular heterogeneity becomes an inherent noise in the measurement of gene expression. The unique molecular characteristics of individual cells, as well as the temporal and quantitative information of gene expression in cells, are lost when averaged among all cells in cell lysates. Our single-cell technology overcomes this limitation and enables us to obtain a large number of single-cell transcriptomes from a population of cells. A collection of single-cell molecular profiles allows us to study carcinogenesis from an evolutionary perspective by treating cancer as a diverse population of cells with abnormal molecular characteristics. Because a cancer cell population contains cells at various stages of development toward drug resistance, clustering similar single-cell molecular profiles could reveal how drug-resistant sub-clones evolve during cancer treatment. Here, we discuss how single-cell transcriptome analysis technology could enable the study of carcinogenesis from an evolutionary perspective and the development of drug-resistance in leukemia. The single-cell transcriptome analysis reported here could have a direct and significant impact on current cancer treatments and future personalized cancer therapies. PMID:23706768

  6. 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

  7. 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. PMID:22025223

  8. Thermoresponsive Micropatterned Substrates for Single Cell Studies

    PubMed Central

    Mandal, Kalpana; Balland, Martial; Bureau, Lionel

    2012-01-01

    We describe the design of micropatterned surfaces for single cell studies, based on thermoresponsive polymer brushes. We show that brushes made of poly(N-isopropylacrylamide) grafted at high surface density display excellent protein and cell anti-adhesive properties. Such brushes are readily patterned at the micron scale via deep UV photolithography. A proper choice of the adhesive pattern shapes, combined with the temperature-dependent swelling properties of PNIPAM, allow us to use the polymer brush as a microactuator which induces cell detachment when the temperature is reduced below C. PMID:22701519

  9. System for measuring oxygen consumption rates of mammalian cells in static culture under hypoxic conditions.

    PubMed

    Kagawa, Yuki; Miyahara, Hirotaka; Ota, Yuri; Tsuneda, Satoshi

    2016-01-01

    Estimating the oxygen consumption rates (OCRs) of mammalian cells in hypoxic environments is essential for designing and developing a three-dimensional (3-D) cell culture system. However, OCR measurements under hypoxic conditions are infrequently reported in the literature. Here, we developed a system for measuring OCRs at low oxygen levels. The system injects nitrogen gas into the environment and measures the oxygen concentration by an optical oxygen microsensor that consumes no oxygen. The developed system was applied to HepG2 cells in static culture. Specifically, we measured the spatial profiles of the local dissolved oxygen concentration in the medium, then estimated the OCRs of the cells. The OCRs, and also the pericellular oxygen concentrations, decreased nonlinearly as the oxygen partial pressure in the environment decreased from 19% to 1%. The OCRs also depended on the culture period and the matrix used for coating the dish surface. Using this system, we can precisely estimate the OCRs of various cell types under environments that mimic 3-D culture conditions, contributing crucial data for an efficient 3-D culture system design. PMID:26558344

  10. An Inexpensive Electrode and Cell for Measurement of Oxygen Uptake in Chemical and Biochemical Systems.

    ERIC Educational Resources Information Center

    Brunet, Juan E.; And Others

    1983-01-01

    The continuous measurement of oxygen consumption in an enzymatic reaction is a frequent experimental fact and extremely important in the enzymatic activity of oxygenase. An electrochemical system, based on a polarographic method, has been developed to monitor the oxygen uptake. The system developed and electrode used are described. (JN)

  11. Single Cell Adhesion Assay Using Computer Controlled Micropipette

    PubMed Central

    Salánki, Rita; Hős, Csaba; Orgovan, Norbert; Péter, Beatrix; Sándor, Noémi; Bajtay, Zsuzsa; Erdei, Anna; Horvath, Robert; Szabó, Bálint

    2014-01-01

    Cell adhesion is a fundamental phenomenon vital for all multicellular organisms. Recognition of and adhesion to specific macromolecules is a crucial task of leukocytes to initiate the immune response. To gain statistically reliable information of cell adhesion, large numbers of cells should be measured. However, direct measurement of the adhesion force of single cells is still challenging and today’s techniques typically have an extremely low throughput (5–10 cells per day). Here, we introduce a computer controlled micropipette mounted onto a normal inverted microscope for probing single cell interactions with specific macromolecules. We calculated the estimated hydrodynamic lifting force acting on target cells by the numerical simulation of the flow at the micropipette tip. The adhesion force of surface attached cells could be accurately probed by repeating the pick-up process with increasing vacuum applied in the pipette positioned above the cell under investigation. Using the introduced methodology hundreds of cells adhered to specific macromolecules were measured one by one in a relatively short period of time (∼30 min). We blocked nonspecific cell adhesion by the protein non-adhesive PLL-g-PEG polymer. We found that human primary monocytes are less adherent to fibrinogen than their in vitro differentiated descendants: macrophages and dendritic cells, the latter producing the highest average adhesion force. Validation of the here introduced method was achieved by the hydrostatic step-pressure micropipette manipulation technique. Additionally the result was reinforced in standard microfluidic shear stress channels. Nevertheless, automated micropipette gave higher sensitivity and less side-effect than the shear stress channel. Using our technique, the probed single cells can be easily picked up and further investigated by other techniques; a definite advantage of the computer controlled micropipette. Our experiments revealed the existence of a sub

  12. Single cell adhesion assay using computer controlled micropipette.

    PubMed

    Salánki, Rita; Hős, Csaba; Orgovan, Norbert; Péter, Beatrix; Sándor, Noémi; Bajtay, Zsuzsa; Erdei, Anna; Horvath, Robert; Szabó, Bálint

    2014-01-01

    Cell adhesion is a fundamental phenomenon vital for all multicellular organisms. Recognition of and adhesion to specific macromolecules is a crucial task of leukocytes to initiate the immune response. To gain statistically reliable information of cell adhesion, large numbers of cells should be measured. However, direct measurement of the adhesion force of single cells is still challenging and today's techniques typically have an extremely low throughput (5-10 cells per day). Here, we introduce a computer controlled micropipette mounted onto a normal inverted microscope for probing single cell interactions with specific macromolecules. We calculated the estimated hydrodynamic lifting force acting on target cells by the numerical simulation of the flow at the micropipette tip. The adhesion force of surface attached cells could be accurately probed by repeating the pick-up process with increasing vacuum applied in the pipette positioned above the cell under investigation. Using the introduced methodology hundreds of cells adhered to specific macromolecules were measured one by one in a relatively short period of time (∼30 min). We blocked nonspecific cell adhesion by the protein non-adhesive PLL-g-PEG polymer. We found that human primary monocytes are less adherent to fibrinogen than their in vitro differentiated descendants: macrophages and dendritic cells, the latter producing the highest average adhesion force. Validation of the here introduced method was achieved by the hydrostatic step-pressure micropipette manipulation technique. Additionally the result was reinforced in standard microfluidic shear stress channels. Nevertheless, automated micropipette gave higher sensitivity and less side-effect than the shear stress channel. Using our technique, the probed single cells can be easily picked up and further investigated by other techniques; a definite advantage of the computer controlled micropipette. Our experiments revealed the existence of a sub-population of

  13. Airborne Lidar Measurements of Atmospheric Pressure Made Using the Oxygen A-Band

    NASA Technical Reports Server (NTRS)

    Riris, Haris; Rodriquez, Michael; Allan, Graham R.; Hasselbrack, William E.; Stephen, Mark A.; Abshire, James B.

    2011-01-01

    We report on airborne measurements of atmospheric pressure using a fiber-laser based lidar operating in the oxygen A-band near 765 nm and the integrated path differential absorption measurement technique. Our lidar uses fiber optic technology and non-linear optics to generate tunable laser radiation at 765 nm, which overlaps an absorption line pair in the Oxygen A-band. We use a pulsed time resolved technique, which rapidly steps the laser wavelength across the absorption line pair, a 20 cm telescope and photon counting detector to measure Oxygen concentrations.

  14. Making an honest measurement scale out of the oxygen isotope delta-values.

    PubMed

    Gat, Joel R; DeBievre, Paul

    2002-01-01

    The differential measurement of the abundance of oxygen isotopes based on reference materials, such as VSMOW for the case of water, was used because the precision of the absolute mass-spectrometric determination of the abundance fell short of the differences to be measured. Since then these measurements have been much improved, so that a calibration scheme of the oxygen isotope abundance in water, carbonates, silica, phosphates, sulfates, nitrates and organic materials is suggested, based on an accredited primary standard of oxygen in air and using standard fluorination and O(2) to CO(2) conversion techniques. PMID:12442297

  15. In situ global method for measurement of oxygen demand and mass transfer

    SciTech Connect

    Klasson, K.T.; Lundbaeck, K.M.O.; Clausen, E.C.; Gaddy, J.L.

    1997-05-01

    Two aerobic microorganisms, Saccharomycopsis lipolytica and Brevibacterium lactofermentum, have been used in a study of mass transfer and oxygen uptake from a global perspective using a closed gas system. Oxygen concentrations in the gas and liquid were followed using oxygen electrodes, and the results allowed for easy calculation of in situ oxygen transport. The cell yields on oxygen for S. lipolytica and B. lactofermentum were 1.01 and 1.53 g/g respectively. The mass transfer coefficient was estimated as 10 h{sup {minus}1} at 500 rpm for both fermentations. The advantages with this method are noticeable since the use of model systems may be avoided, and the in situ measurements of oxygen demand assure reliable data for scale-up.

  16. New instrumentation for optical measuring of oxygen in gas or dissolved in liquids

    NASA Astrophysics Data System (ADS)

    Trettnak, W.; Gruber, W.; Reininger, F.; O'Leary, P.; Klimant, I.

    The optical oxygen sensor is a novel device for the determination of oxygen in gases or dissolved in liquids. It is based on the measurement principle of fluorescence quenching, which is completely different from that of polarographic oxygen sensors (today the most widespread devices for oxygen detection). The new instrument offers features and advantages, which render it not only a realistic alternative, but, for specific applications, make it superior to existing electrochemical methods. The system is based on low-cost semiconductor devices (light-emitting diodes, photodiodes, low-cost analogue and digital components) and new LED-compatible oxygen-sensitive membranes. The flow cell of the instrument may be thermostatted and the sensor can be calibrated by a simple two-point calibration procedure. The optical oxygen sensor is particularly suitable for measuring dissolved oxygen in respirometry, since no oxygen is consumed by the device and the signal is independent of sample flowrate or stirring speed. Typical fields of application are monitoring of oxygen in ground and drinking water, in process controll in bioreactors and in breath gas and blood gas analysis.

  17. The Oxygen Ratio: A Fuel-Independent Measure of Mixture Stoichiometry

    SciTech Connect

    Mueller, C J; Musculus, M P; Pickett, L M; Pitz, W J; Westbrook, C K

    2003-12-19

    The pollutant-formation characteristics and other properties of a combustion reaction typically depend strongly on the proximity of the mixture to its stoichiometric condition, i.e., the ''mixture stoichiometry.'' A quantitative, widely applicable measure of this mixture property is therefore a critical independent variable in the study of combustion systems. Such a parameter enables the clear separation of mixture stoichiometry effects from other effects (e.g., fuel molecular structure, product temperature, diluent concentration, pressure). The parameter most often used to quantify mixture stoichiometry is the equivalence ratio. Unfortunately, the equivalence ratio fails to properly account for oxygen in oxygenates, i.e., compounds that have oxygen chemically bound within the fuel molecule. This manuscript introduces the oxygen ratio, a parameter that properly characterizes mixture stoichiometry for a broader class of reactants than does the equivalence ratio, including oxygenates. A detailed definition of the oxygen ratio is provided and used to show its relationship to the equivalence ratio. The definition is also used to quantify errors involved when the equivalence ratio is used as a measure of mixture stoichiometry with oxygenates. Proper usage of the oxygen ratio is discussed and the oxygen ratio is used to interpret results in a practical example.

  18. 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

  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.

  20. Self-Folding Single Cell Grippers

    PubMed Central

    2015-01-01

    Given the heterogeneous nature of cultures, tumors, and tissues, the ability to capture, contain, and analyze single cells is important for genomics, proteomics, diagnostics, therapeutics, and surgery. Moreover, for surgical applications in small conduits in the body such as in the cardiovascular system, there is a need for tiny tools that approach the size of the single red blood cells that traverse the blood vessels and capillaries. We describe the fabrication of arrayed or untethered single cell grippers composed of biocompatible and bioresorbable silicon monoxide and silicon dioxide. The energy required to actuate these grippers is derived from the release of residual stress in 3–27 nm thick films, did not require any wires, tethers, or batteries, and resulted in folding angles over 100° with folding radii as small as 765 nm. We developed and applied a finite element model to predict these folding angles. Finally, we demonstrated the capture of live mouse fibroblast cells in an array of grippers and individual red blood cells in untethered grippers which could be released from the substrate to illustrate the potential utility for in vivo operations. PMID:24937214

  1. Direct measurement of local dissolved oxygen concentration spatial profiles in a cell culture environment.

    PubMed

    Kagawa, Yuki; Matsuura, Katsuhisa; Shimizu, Tatsuya; Tsuneda, Satoshi

    2015-06-01

    Controlling local dissolved oxygen concentration (DO) in media is critical for cell or tissue cultures. Various biomaterials and culture methods have been developed to modulate DO. Direct measurement of local DO in cultures has not been validated as a method to test DO modulation. In the present study we developed a DO measurement system equipped with a Clark-type oxygen microelectrode manipulated with 1 μm precision in three-dimensional space to explore potential applications for tissue engineering. By determining the microelectrode tip position precisely against the bottom plane of culture dishes with rat or human cardiac cells in static monolayer culture, we successfully obtained spatial distributions of DO in the medium. Theoretical quantitative predictions fit the obtained data well. Based on analyses of the variance between samples, we found the data reflected "local" oxygen consumption in the vicinity of the microelectrode and the detection of temporal changes in oxygen consumption rates of cultured cells was limited by the diffusion rate of oxygen in the medium. This oxygen measuring system monitors local oxygen consumption and production with high spatial resolution, and can potentially be used with recently developed oxygen modulating biomaterials to design microenvironments and non-invasively monitor local DO dynamics during culture.

  2. Measurement and interpretation of low levels of dissolved oxygen in ground water

    USGS Publications Warehouse

    White, A.F.; Peterson, M.L.; Solbau, R.D.

    1990-01-01

    A Rhodazine-D colorimetric technique was adapted to measure low-level dissolved oxygen concentrations in ground water. Prepared samples containing between 0 and 8.0 ??moles L-1 dissolved oxygen in equilibrium with known gas mixtures produced linear spectrophotometric absorbance with a lower detection limit of 0.2 ??moles L-1. Excellent reproducibility was found for solutions ranging in composition from deionized water to sea water with chemical interferences detected only for easily reduced metal species such as ferric ion, cupric ion, and hexavalent chromium. Such effects were correctable based on parallel reaction stoichiometries relative to oxygen. The technique, coupled with a downhole wire line tool, permitted low-level monitoring of dissolved oxygen in wells at the selenium-contaminated Kesterson Reservoir in California. Results indicated a close association between low but measurable dissolved oxygen concentrations and mobility of oxidized forms of selenium. -from Authors

  3. Image-guided optical measurement of blood oxygen saturation within capillary vessels (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Akons, Kfir; Zeidan, Adel; Yeheskely-Hayon, Daniella; Minai, Limor; Yelin, Dvir

    2016-03-01

    Values of blood oxygenation levels are useful for assessing heart and lung conditions, and are frequently monitored during routine patient care. Independent measurement of the oxygen saturation in capillary blood, which is significantly different from that of arterial blood, is important for diagnosing tissue hypoxia and for increasing the accuracy of existing techniques that measure arterial oxygen saturation. Here, we developed a simple, non-invasive technique for measuring the reflected spectra from individual capillary vessels within a human lip, allowing local measurement of the blood oxygen saturation. The optical setup includes a spatially incoherent broadband light that was focused onto a specific vessel below the lip surface. Backscattered light was imaged by a camera for identifying a target vessel and pointing the illumination beam to its cross section. Scattered light from the vessel was then collected by a single-mode fiber and analyzed by a fast spectrometer. Spectra acquired from small capillary vessels within a volunteer lip showed the characteristic oxyhemoglobin absorption bands in real time and with a high signal-to-noise ratio. Measuring capillary oxygen saturation using this technique would potentially be more accurate compared to existing pulse oximetry techniques due to its insensitivity to the patient's skin color, pulse rate, motion, and medical condition. It could be used as a standalone endoscopic technique for measuring tissue hypoxia or in conjunction with conventional pulse oximetry for a more accurate measurement of oxygen transport in the body.

  4. Detecting Bacterial Surface Organelles on Single Cells Using Optical Tweezers.

    PubMed

    Zakrisson, Johan; Singh, Bhupender; Svenmarker, Pontus; Wiklund, Krister; Zhang, Hanqing; Hakobyan, Shoghik; Ramstedt, Madeleine; Andersson, Magnus

    2016-05-10

    Bacterial cells display a diverse array of surface organelles that are important for a range of processes such as intercellular communication, motility and adhesion leading to biofilm formation, infections, and bacterial spread. More specifically, attachment to host cells by Gram-negative bacteria are mediated by adhesion pili, which are nanometers wide and micrometers long fibrous organelles. Since these pili are significantly thinner than the wavelength of visible light, they cannot be detected using standard light microscopy techniques. At present, there is no fast and simple method available to investigate if a single cell expresses pili while keeping the cell alive for further studies. In this study, we present a method to determine the presence of pili on a single bacterium. The protocol involves imaging the bacterium to measure its size, followed by predicting the fluid drag based on its size using an analytical model, and thereafter oscillating the sample while a single bacterium is trapped by an optical tweezer to measure its effective fluid drag. Comparison between the predicted and the measured fluid drag thereby indicate the presence of pili. Herein, we verify the method using polymer coated silica microspheres and Escherichia coli bacteria expressing adhesion pili. Our protocol can in real time and within seconds assist single cell studies by distinguishing between piliated and nonpiliated bacteria.

  5. Microchip platforms for multiplex single-cell functional proteomics with applications to immunology and cancer research

    PubMed Central

    2013-01-01

    Single-cell functional proteomics assays can connect genomic information to biological function through quantitative and multiplex protein measurements. Tools for single-cell proteomics have developed rapidly over the past 5 years and are providing approaches for directly elucidating phosphoprotein signaling networks in cancer cells or for capturing high-resolution snapshots of immune system function in patients with various disease conditions. We discuss advances in single-cell proteomics platforms, with an emphasis on microchip methods. These methods can provide a direct correlation of morphological, functional and molecular signatures at the single-cell level. We also provide examples of how those platforms are being applied to both fundamental biology and clinical studies, focusing on immune-system monitoring and phosphoprotein signaling networks in cancer. PMID:23998271

  6. [Design of Non-Invasive Blood Oxygen Measurement Based on AFE4490].

    PubMed

    Zhu, Jinsong; Wu, Shouhao; Guo, Wenxiu; Zheng, Hui; Tang, Dong

    2015-09-01

    From the perspective of portable monitoring devices,we use an analog front-end AFE4490 design a module of Non-invasive blood oxygen measurement, used to collect human pulse wave signal and peak (valley) value detection and then use the principles of non-invasive oximetry calculated oxygen saturation (SPO2). This design of noninvasive oximetry module has the characteristics of small size, low power consumption, and the results of test show that the measurement of oxygen saturation are correct. PMID:26904876

  7. 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

  8. 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.

  9. 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.

  10. JGI Genomic Single-cell Assembly Workflow

    SciTech Connect

    Trong, S.

    2011-09-16

    JIGSAW is a software package disigned to quality control and assemble genomic DNA sequences from single-cell bacterial and archaeal genomes. Amplification of singel-cell genomes using multiple displacement amplification technology presents challenges that magnify the amount of contaminants in the sample and produce non uniform depth of sequence coverage. these factors pose problems whan assembling the genomic data using currently availible short read assembles. The software addresses these problems by removing contaminants and normalizing the sequence read coverage prior to assemble. A hybrid assembly approach using two different open source genome assembly tools is then applied to piece together the DNA fragments. Additional reporting of QC metrics for the input sample and the genome assembly is provided for further analysis.

  11. JGI Genomic Single-cell Assembly Workflow

    2011-09-16

    JIGSAW is a software package disigned to quality control and assemble genomic DNA sequences from single-cell bacterial and archaeal genomes. Amplification of singel-cell genomes using multiple displacement amplification technology presents challenges that magnify the amount of contaminants in the sample and produce non uniform depth of sequence coverage. these factors pose problems whan assembling the genomic data using currently availible short read assembles. The software addresses these problems by removing contaminants and normalizing the sequencemore » read coverage prior to assemble. A hybrid assembly approach using two different open source genome assembly tools is then applied to piece together the DNA fragments. Additional reporting of QC metrics for the input sample and the genome assembly is provided for further analysis.« less

  12. Skeletal Muscle Oxygenation Measured by EPR Oximetry Using a Highly Sensitive Polymer-Encapsulated Paramagnetic Sensor.

    PubMed

    Hou, H; Khan, N; Nagane, M; Gohain, S; Chen, E Y; Jarvis, L A; Schaner, P E; Williams, B B; Flood, A B; Swartz, H M; Kuppusamy, P

    2016-01-01

    We have incorporated LiNc-BuO, an oxygen-sensing paramagnetic material, in polydimethylsiloxane (PDMS), which is an oxygen-permeable, biocompatible, and stable polymer. We fabricated implantable and retrievable oxygen-sensing chips (40 % LiNc-BuO in PDMS) using a 20-G Teflon tubing to mold the chips into variable shapes and sizes for in vivo studies in rats. In vitro EPR measurements were used to test the chip's oxygen response. Oxygen induced linear and reproducible line broadening with increasing partial pressure (pO2). The oxygen response was similar to that of bare (unencapsulated) crystals and did not change significantly on sterilization by autoclaving. The chips were implanted in rat femoris muscle and EPR oximetry was performed repeatedly (weekly) for 12 weeks post-implantation. The measurements showed good reliability and reproducibility over the period of testing. These results demonstrated that the new formulation of OxyChip with 40 % LiNc-BuO will enable the applicability of EPR oximetry for long-term measurement of oxygen concentration in tissues and has the potential for clinical applications. PMID:27526163

  13. Measuring hemoglobin amount and oxygen saturation of skin with advancing age

    NASA Astrophysics Data System (ADS)

    Watanabe, Shumpei; Yamamoto, Satoshi; Yamauchi, Midori; Tsumura, Norimichi; Ogawa-Ochiai, Keiko; Akiba, Tetsuo

    2012-03-01

    We measured the oxygen saturation of skin at various ages using our previously proposed method that can rapidly simulate skin spectral reflectance with high accuracy. Oxygen saturation is commonly measured by a pulse oximeter to evaluate oxygen delivery for monitoring the functions of heart and lungs at a specific time. On the other hand, oxygen saturation of skin is expected to assess peripheral conditions. Our previously proposed method, the optical path-length matrix method (OPLM), is based on a Monte Carlo for multi-layered media (MCML), but can simulate skin spectral reflectance 27,000 times faster than MCML. In this study, we implemented an iterative simulation of OPLM with a nonlinear optimization technique such that this method can also be used for estimating hemoglobin concentration and oxygen saturation from the measured skin spectral reflectance. In the experiments, the skin reflectance spectra of 72 outpatients aged between 20 and 86 years were measured by a spectrophotometer. Three points were measured for each subject: the forearm, the thenar eminence, and the intermediate phalanx. The result showed that the oxygen saturation of skin remained constant at each point as the age varied.

  14. Single-Cell Analysis of Growth and Cell Division of the Anaerobe Desulfovibrio vulgaris Hildenborough.

    PubMed

    Fievet, Anouchka; Ducret, Adrien; Mignot, Tâm; Valette, Odile; Robert, Lydia; Pardoux, Romain; Dolla, Alain R; Aubert, Corinne

    2015-01-01

    Recent years have seen significant progress in understanding basic bacterial cell cycle properties such as cell growth and cell division. While characterization and regulation of bacterial cell cycle is quite well-documented in the case of fast growing aerobic model organisms, no data has been so far reported for anaerobic bacteria. This lack of information in anaerobic microorganisms can mainly be explained by the absence of molecular and cellular tools such as single cell microscopy and fluorescent probes usable for anaerobes and essential to study cellular events and/or subcellular localization of the actors involved in cell cycle. In this study, single-cell microscopy has been adapted to study for the first time, in real time, the cell cycle of a bacterial anaerobe, Desulfovibrio vulgaris Hildenborough (DvH). This single-cell analysis provides mechanistic insights into the cell division cycle of DvH, which seems to be governed by the recently discussed so-called incremental model that generates remarkably homogeneous cell sizes. Furthermore, cell division was reversibly blocked during oxygen exposure. This may constitute a strategy for anaerobic cells to cope with transient exposure to oxygen that they may encounter in their natural environment, thereby contributing to their aerotolerance. This study lays the foundation for the first molecular, single-cell assay that will address factors that cannot otherwise be resolved in bulk assays and that will allow visualization of a wide range of molecular mechanisms within living anaerobic cells. PMID:26696987

  15. Single-Cell Analysis of Growth and Cell Division of the Anaerobe Desulfovibrio vulgaris Hildenborough

    PubMed Central

    Fievet, Anouchka; Ducret, Adrien; Mignot, Tâm; Valette, Odile; Robert, Lydia; Pardoux, Romain; Dolla, Alain R.; Aubert, Corinne

    2015-01-01

    Recent years have seen significant progress in understanding basic bacterial cell cycle properties such as cell growth and cell division. While characterization and regulation of bacterial cell cycle is quite well-documented in the case of fast growing aerobic model organisms, no data has been so far reported for anaerobic bacteria. This lack of information in anaerobic microorganisms can mainly be explained by the absence of molecular and cellular tools such as single cell microscopy and fluorescent probes usable for anaerobes and essential to study cellular events and/or subcellular localization of the actors involved in cell cycle. In this study, single-cell microscopy has been adapted to study for the first time, in real time, the cell cycle of a bacterial anaerobe, Desulfovibrio vulgaris Hildenborough (DvH). This single-cell analysis provides mechanistic insights into the cell division cycle of DvH, which seems to be governed by the recently discussed so-called incremental model that generates remarkably homogeneous cell sizes. Furthermore, cell division was reversibly blocked during oxygen exposure. This may constitute a strategy for anaerobic cells to cope with transient exposure to oxygen that they may encounter in their natural environment, thereby contributing to their aerotolerance. This study lays the foundation for the first molecular, single-cell assay that will address factors that cannot otherwise be resolved in bulk assays and that will allow visualization of a wide range of molecular mechanisms within living anaerobic cells. PMID:26696987

  16. New Active Optical Technique Developed for Measuring Low-Earth-Orbit Atomic Oxygen Erosion of Polymers

    NASA Technical Reports Server (NTRS)

    Banks, Bruce A.; deGroh, Kim K.; Demko, Rikako

    2003-01-01

    Polymers such as polyimide Kapton (DuPont) and Teflon FEP (DuPont, fluorinated ethylene propylene) are commonly used spacecraft materials because of desirable properties such as flexibility, low density, and in the case of FEP, a low solar absorptance and high thermal emittance. Polymers on the exterior of spacecraft in the low-Earth-orbit (LEO) environment are exposed to energetic atomic oxygen. Atomic oxygen reaction with polymers causes erosion, which is a threat to spacecraft performance and durability. It is, therefore, important to understand the atomic oxygen erosion yield E (the volume loss per incident oxygen atom) of polymers being considered in spacecraft design. The most common technique for determining E is a passive technique based on mass-loss measurements of samples exposed to LEO atomic oxygen during a space flight experiment. There are certain disadvantages to this technique. First, because it is passive, data are not obtained until after the flight is completed. Also, obtaining the preflight and postflight mass measurements is complicated by the fact that many polymers absorb water and, therefore, the mass change due to water absorption can affect the E data. This is particularly true for experiments that receive low atomic oxygen exposures or for samples that have a very low E. An active atomic oxygen erosion technique based on optical measurements has been developed that has certain advantages over the mass-loss technique. This in situ technique can simultaneously provide the erosion yield data on orbit and the atomic oxygen exposure fluence, which is needed for erosion yield determination. In the optical technique, either sunlight or artificial light can be used to measure the erosion of semitransparent or opaque polymers as a result of atomic oxygen attack. The technique is simple and adaptable to a rather wide range of polymers, providing that they have a sufficiently high optical absorption coefficient. If one covers a photodiode with a

  17. [Measurement of multi-wavelength pulse oxygen saturation based on dynamic spectroscopy].

    PubMed

    Wang, Xiao-Fei; Zhao, Wen-Jun

    2014-05-01

    The present paper puts forward multi-wavelength pulse oxygen saturation measurement based on dynamic spectroscopy to do the non-invasive determination of oxygen saturation. Compared to conventional ways, the new method makes full use of more wavelengths light and improves the measurement accuracy. During the experiment, the in-vivo measurements were carried out on 60 patients and their spectroscopic data were collected by the high sensitivity type fiber optic spectrometer. Singletrial estimation method was used to extract the dynamic spectroscopy at the wavelengths of 606. 44 approximately 987. 55 nm. Oxygen saturation obtained from arterial blood gas analysis is regarded as the true value. Synergy interval partial least square (siPLS) was used to establish the calibration model of subjects' oxygen saturation values against dynamic spectroscopy data. The relative error of prediction is +/-0. 017 6, but the relative error of the subjects in the same set measured by the patient monitor which was two-wavelength measure system is +/-0. 116 4. Measurement results show that the use of the high sensitivity type fiber optic spectrometer to collect multi-wavelength spectroscopic data and dynamic spectroscopy method to process data can do better in improving the accuracy of the oxygen saturation measurement.

  18. [Measurement of multi-wavelength pulse oxygen saturation based on dynamic spectroscopy].

    PubMed

    Wang, Xiao-Fei; Zhao, Wen-Jun

    2014-05-01

    The present paper puts forward multi-wavelength pulse oxygen saturation measurement based on dynamic spectroscopy to do the non-invasive determination of oxygen saturation. Compared to conventional ways, the new method makes full use of more wavelengths light and improves the measurement accuracy. During the experiment, the in-vivo measurements were carried out on 60 patients and their spectroscopic data were collected by the high sensitivity type fiber optic spectrometer. Singletrial estimation method was used to extract the dynamic spectroscopy at the wavelengths of 606. 44 approximately 987. 55 nm. Oxygen saturation obtained from arterial blood gas analysis is regarded as the true value. Synergy interval partial least square (siPLS) was used to establish the calibration model of subjects' oxygen saturation values against dynamic spectroscopy data. The relative error of prediction is +/-0. 017 6, but the relative error of the subjects in the same set measured by the patient monitor which was two-wavelength measure system is +/-0. 116 4. Measurement results show that the use of the high sensitivity type fiber optic spectrometer to collect multi-wavelength spectroscopic data and dynamic spectroscopy method to process data can do better in improving the accuracy of the oxygen saturation measurement. PMID:25095431

  19. Development of 200-channel mapping system for tissue oxygenation measured by near-infrared spectroscopy

    NASA Astrophysics Data System (ADS)

    Niwayama, Masatsugu; Kohata, Daisuke; Shao, Jun; Kudo, Nobuki; Hamaoka, Takatumi; Katsumura, Toshihito; Yamamoto, Katsuyuki

    2000-07-01

    Near-infrared spectroscopy (NIRS) is a very useful technique for noninvasive measurement of tissue oxygenation. Among various methods of NIRS, continuous wave near-infrared spectroscopy (CW- NIRS) is especially suitable for real-time measurement and for practical use. CW-NIRS has recently been applied in vivo reflectance imaging of muscle oxygenation and brain activity. However, conventional mapping systems do not have a sufficient mapping area at present. Moreover, they do not enable quantitative measurement of tissue oxygenation because conventional NIRS is based on the inappropriate assumption that tissue is homogeneous. In this study, we developed a 200-channel mapping system that enables measurement of changes in oxygenation and blood volume and that covers a wider area (30 cm x 20 cm) than do conventional systems. The spatial resolution (source- detector separation) of this system is 15 mm. As for the effcts of tissue inhomogeneity on muscle oxygenation measurement, subcutaneous adipose tissue greatly reduces measurement sensitivity. Therefore, we also used a correction method for influence of the subcutaneous fat layer so that we could obtain quantitative changes in concentrations of oxy- and deoxy- hemoglobin. We conducted exercise tests and measured the changed in hemoglobin concentration in the thigh using the new system. The working muscles in the exercises could be imaged, and the heterogeneity of the muscles was shown. These results demonstrated the new 200-channel mapping system enables observation of the distribution of muscle metabolism and localization of muscle function.

  20. The efficiency of the CO2-concentrating mechanism during single-cell C4 photosynthesis.

    PubMed

    King, Jenny L; Edwards, Gerald E; Cousins, Asaph B

    2012-03-01

    The photosynthetic efficiency of the CO(2)-concentrating mechanism in two forms of single-cell C(4) photosynthesis in the family Chenopodiaceae was characterized. The Bienertioid-type single-cell C(4) uses peripheral and central cytoplasmic compartments (Bienertia sinuspersici), while the Borszczowioid single-cell C(4) uses distal and proximal compartments of the cell (Suaeda aralocaspica). C(4) photosynthesis within a single-cell raises questions about the efficiency of this type of CO(2) -concentrating mechanism compared with the Kranz-type. We used measurements of leaf CO(2) isotope exchange (Δ(13) C) to compare the efficiency of the single-cell and Kranz-type forms of C(4) photosynthesis under various temperature and light conditions. Comparisons were made between the single-cell C(4) and a sister Kranz form, S. eltonica[NAD malic enzyme (NAD ME) type], and with Flaveria bidentis[NADP malic enzyme (NADP-ME) type with Kranz Atriplicoid anatomy]. There were similar levels of Δ(13) C discrimination and CO(2) leakiness (Φ) in the single-cell species compared with the Kranz-type. Increasing leaf temperature (25 to 30 °C) and light intensity caused a decrease in Δ(13) C and Φ across all C(4) types. Notably, B. sinuspersici had higher Δ(13) C and Φ than S. aralocaspica under lower light. These results demonstrate that rates of photosynthesis and efficiency of the CO(2) -concentrating mechanisms in single-cell C(4) plants are similar to those in Kranz-type.

  1. Preliminary results of oxygen isotope ratio measurement with a particle-gamma coincidence method

    NASA Astrophysics Data System (ADS)

    Borysiuk, Maciek; Kristiansson, Per; Ros, Linus; Abdel, Nassem S.; Elfman, Mikael; Nilsson, Charlotta; Pallon, Jan

    2015-04-01

    The possibility to study variations in the oxygen isotopic ratio with photon tagged nuclear reaction analysis (pNRA) is evaluated in the current work. The experiment described in the article was performed at Lund Ion Beam Analysis Facility (LIBAF) with a 2 MeV deuteron beam. Isotopic fractionation of light elements such as carbon, oxygen and nitrogen is the basis of many analytical tools in hydrology, geology, paleobiology and paleogeology. IBA methods provide one possible tool for measurement of isotopic content. During this experimental run we focused on measurement of the oxygen isotopic ratio. The measurement of stable isotopes of oxygen has a number of applications; the particular one driving the current investigation belongs to the field of astrogeology and specifically evaluation of fossil extraterrestrial material. There are three stable isotopes of oxygen: 16O, 17O and 18O. We procured samples highly enriched with all three isotopes. Isotopes 16O and 18O were easily detected in the enriched samples, but no significant signal from 17O was detected in the same samples. The measured yield was too low to detect 18O in a sample with natural abundances of oxygen isotopes, at least in the current experimental setup, but the spectral line from the reaction with 16O was clearly visible.

  2. Atomic Oxygen Density Measurements in a Low Pressure Textile Processing Plasma

    NASA Astrophysics Data System (ADS)

    Gomez, S.; Steen, P. G.; Graham, W. G.

    1999-10-01

    There is increasing interest in plasma processing of textile materials. Here the effect of textile materials on low pressure oxygen plasmas has been investigated. In particular laser induced fluorescence (LIF) measurements of the atomic oxygen density with and without textile samples are presented. Polypropylene and polyester samples were placed on the lower electrode of an inductively coupled Gaseous Electronic Conference (GEC) reactor. This had to be operated at low power and hence in the capacitive mode to avoid toasting the material. Operation with a bare stainless steel electrode and one loaded with the sample materials is contrasted by comparing spatially resolved LIF measurements of atomic oxygen under a wide range of pressures and powers, from a few Pa to 133 Pa, and from 10 to 300 W. Atomic oxygen densities with samples present are around one third lower than those without samples, and in both cases the atomic oxygen density increases linearly with gas pressure. Previous optical emission spectroscopy (OES) measurements indicate that plasma interaction with the substrate commences a few seconds after plasma turn on. Similar trends are observed with time resolved LIF measurements of the atomic oxygen.

  3. Phosphorescent nanoparticles for quantitative measurements of oxygen profiles in vitro and in vivo

    PubMed Central

    Choi, Nak Won; Verbridge, Scott S.; Williams, Rebecca M.; Chen, Jin; Kim, Ju-Young; Schmehl, Russel; Farnum, Cornelia E.; Zipfel, Warren R.; Fischbach, Claudia; Stroock, Abraham D.

    2012-01-01

    We present the development and characterization of nanoparticles loaded with a custom phosphor; we exploit these nanoparticles to perform quantitative measurements of the concentration of oxygen within three-dimensional (3-D) tissue cultures in vitro and blood vessels in vivo. We synthesized a customized ruthenium (Ru)-phosphor and incorporated it into polymeric nanoparticles via self-assembly. We demonstrate that the encapsulated phosphor is non-toxic with and without illumination. We evaluated two distinct modes of employing the phosphorescent nanoparticles for the measurement of concentrations of oxygen: 1) in vitro, in a 3-D microfluidic tumor model via ratiometric measurements of intensity with an oxygen-insensitive fluorophore as a reference, and 2) in vivo, in mouse vasculature using measurements of phosphorescence lifetime. With both methods, we demonstrated micrometer-scale resolution and absolute calibration to the dissolved oxygen concentration. Based on the ease and customizability of the synthesis of the nanoparticles and the flexibility of their application, these oxygen-sensing polymeric nanoparticles will find a natural home in a range of biological applications, benefiting studies of physiological as well as pathological processes in which oxygen availability and concentration play a critical role. PMID:22240511

  4. Detecting Antigen-Specific T Cell Responses: From Bulk Populations to Single Cells.

    PubMed

    Phetsouphanh, Chansavath; Zaunders, John James; Kelleher, Anthony Dominic

    2015-01-01

    A new generation of sensitive T cell-based assays facilitates the direct quantitation and characterization of antigen-specific T cell responses. Single-cell analyses have focused on measuring the quality and breadth of a response. Accumulating data from these studies demonstrate that there is considerable, previously-unrecognized, heterogeneity. Standard assays, such as the ICS, are often insufficient for characterization of rare subsets of cells. Enhanced flow cytometry with imaging capabilities enables the determination of cell morphology, as well as the spatial localization of the protein molecules within a single cell. Advances in both microfluidics and digital PCR have improved the efficiency of single-cell sorting and allowed multiplexed gene detection at the single-cell level. Delving further into the transcriptome of single-cells using RNA-seq is likely to reveal the fine-specificity of cellular events such as alternative splicing (i.e., splice variants) and allele-specific expression, and will also define the roles of new genes. Finally, detailed analysis of clonally related antigen-specific T cells using single-cell TCR RNA-seq will provide information on pathways of differentiation of memory T cells. With these state of the art technologies the transcriptomics and genomics of Ag-specific T cells can be more definitively elucidated. PMID:26274954

  5. Analysis and methodology for measuring oxygen concentration in liquid sodium with a plugging meter

    SciTech Connect

    Nollet, B. K.; Hvasta, M.; Anderson, M.

    2012-07-01

    Oxygen concentration in liquid sodium is a critical measurement in assessing the potential for corrosion damage in sodium-cooled fast reactors (SFRs). There has been little recent work on sodium reactors and oxygen detection. Thus, the technical expertise dealing with oxygen measurements within sodium is no longer readily available in the U.S. Two methods of oxygen detection that have been investigated are the plugging meter and the galvanic cell. One of the overall goals of the Univ. of Wisconsin's sodium research program is to develop an affordable, reliable galvanic cell oxygen sensor. Accordingly, attention must first be dedicated to a well-known standard known as a plugging meter. Therefore, a sodium loop has been constructed on campus in effort to develop the plugging meter technique and gain experience working with liquid metal. The loop contains both a galvanic cell test section and a plugging meter test section. Consistent plugging results have been achieved below 20 [wppm], and a detailed process for achieving effective plugging has been developed. This paper will focus both on an accurate methodology to obtain oxygen concentrations from a plugging meter, and on how to easily control the oxygen concentration of sodium in a test loop. Details of the design, materials, manufacturing, and operation will be presented. Data interpretation will also be discussed, since a modern discussion of plugging data interpretation does not currently exist. (authors)

  6. Fluorescence lifetime imaging to quantify sub-cellular oxygen measurements in live macrophage during bacterial invasion

    NASA Astrophysics Data System (ADS)

    Dragavon, Joe; Amiri, Megdouda; Marteyn, Benoit; Sansonetti, Philipe; Shorte, Spencer

    2011-03-01

    Fluorophore concentration, the surrounding microenvironment, and photobleaching greatly influence the fluorescence intensity of a fluorophore, increasing the difficulty to directly observe micro-environmental factors such as pH and oxygen. However, the fluorescence lifetime of a fluorophore is essentially independent of both the fluorophore concentration and photobleaching, providing a viable alternative to intensity measurements. The development of fluorescence lifetime imaging (FLI) allows for the direct measurement of the microenvironment surrounding a fluorophore. Pt-porphyrin is a fluorophore whose optical properties include a very stable triplet excited state. This energy level overlaps strongly with the ground triplet state of oxygen, making the phosphorescent lifetime directly proportional to the surrounding oxygen concentration. Initial experiments using this fluorophore involved the use of individual microwells coated with the porphyrin. Cells were allowed to enter the micro-wells before being sealed to create a diffusionally isolated volume. The decrease in the extracellular oxygen concentration was observed using FLI. However, this isolation technique provides only the consumption rate but cannot indicate the subcellular oxygen distribution. To improve upon this, live macrophages are loaded with the porphyrin and the fluorescence lifetime determined using a Lambert Instruments Lifa-X FLI system. Initial results indicate that an increase in subcellular oxygen is observed upon initial exposure to invasive bacteria. A substantial decrease in oxygen is observed after about 1 hour of exposure. The cells remain in this deoxygenated state until the bacteria are removed or cell death occurs.

  7. Predicting stochastic gene expression dynamics in single cells.

    PubMed

    Mettetal, Jerome T; Muzzey, Dale; Pedraza, Juan M; Ozbudak, Ertugrul M; van Oudenaarden, Alexander

    2006-05-01

    Fluctuations in protein numbers (noise) due to inherent stochastic effects in single cells can have large effects on the dynamic behavior of gene regulatory networks. Although deterministic models can predict the average network behavior, they fail to incorporate the stochasticity characteristic of gene expression, thereby limiting their relevance when single cell behaviors deviate from the population average. Recently, stochastic models have been used to predict distributions of steady-state protein levels within a population but not to predict the dynamic, presteady-state distributions. In the present work, we experimentally examine a system whose dynamics are heavily influenced by stochastic effects. We measure population distributions of protein numbers as a function of time in the Escherichia coli lactose uptake network (lac operon). We then introduce a dynamic stochastic model and show that prediction of dynamic distributions requires only a few noise parameters in addition to the rates that characterize a deterministic model. Whereas the deterministic model cannot fully capture the observed behavior, our stochastic model correctly predicts the experimental dynamics without any fit parameters. Our results provide a proof of principle for the possibility of faithfully predicting dynamic population distributions from deterministic models supplemented by a stochastic component that captures the major noise sources. PMID:16648266

  8. Block-Cell-Printing for live single-cell printing

    PubMed Central

    Zhang, Kai; Chou, Chao-Kai; Xia, Xiaofeng; Hung, Mien-Chie; Qin, Lidong

    2014-01-01

    A unique live-cell printing technique, termed “Block-Cell-Printing” (BloC-Printing), allows for convenient, precise, multiplexed, and high-throughput printing of functional single-cell arrays. Adapted from woodblock printing techniques, the approach employs microfluidic arrays of hook-shaped traps to hold cells at designated positions and directly transfer the anchored cells onto various substrates. BloC-Printing has a minimum turnaround time of 0.5 h, a maximum resolution of 5 µm, close to 100% cell viability, the ability to handle multiple cell types, and efficiently construct protrusion-connected single-cell arrays. The approach enables the large-scale formation of heterotypic cell pairs with controlled morphology and allows for material transport through gap junction intercellular communication. When six types of breast cancer cells are allowed to extend membrane protrusions in the BloC-Printing device for 3 h, multiple biophysical characteristics of cells—including the protrusion percentage, extension rate, and cell length—are easily quantified and found to correlate well with their migration levels. In light of this discovery, BloC-Printing may serve as a rapid and high-throughput cell protrusion characterization tool to measure the invasion and migration capability of cancer cells. Furthermore, primary neurons are also compatible with BloC-Printing. PMID:24516129

  9. Limitations of fitting angular scattering from single cells (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Fan, Xing; Cannaday, Ashley E.; Berger, Andrew J.

    2016-04-01

    The literature contains several reports of Mie-like fits to angular-domain elastic scattering measurements from multiple cells or isolated mitochondria. In these studies, the sampling volume typically contains hundreds or thousands of mitochondria, allowing for the size distribution of mitochondria to be modeled as a smooth function, (e.g. Gaussian or log-normal) with a small number of free parameters. In the case of a single-cell volume containing significantly fewer mitochondria, the true size distribution will no longer be as smooth. Increasing the number of free parameters can lead to unstable fits, however, as the forward-directed angular scattering pattern from such a population illuminated with 785 nm light is a monotonically decaying radial function with few distinct features. Using simulations, we have investigated the limitations of modeling single-cell mitochondrial scattering using smooth population distributions of Mie scatterers. In different instances, the fidelity of the estimated size information can be limited by the number of organelles, the angular detection range, or the non-ideality of the data (both speckle and shot noise). We will describe the conditions under which each of these effects dominates. We will also discuss whether mean and standard deviation are the best sizes to report from such Mie modeling, or if there are other size parameters that have greater fidelity to the true, non-smooth size distributions.

  10. Cell tracing dyes significantly change single cell mechanics

    PubMed Central

    Lulevich, Valentin; Shih, Yi-Ping; Lo, Su Hao; Liu, Gang-yu

    2009-01-01

    Cell tracing dyes are very frequently utilized in cellular biology research because they provide highly sensitive fluorescent tags that do not compromise cellular functions such as growth and proliferation. In many investigations concerning cellular adhesion and mechanics, fluorescent dyes have been employed with the assumption of little impact on the results. Using the single-cell compression technique developed by our team, the single-cell mechanics of MDA-MB-468 and MLC-SV40 cells were investigated as a function of dye uptake. Cell tracing dyes increase living cell stiffness 3-6 times and cell-to-probe adhesion up to 7 times. These results suggest a more significant effect than toxins, such as Thrombin. A simple analytical model was derived to enable the extraction of the Young’s moduli of the cell membrane and cytoskeleton from the force-deformation profiles measured for individual cells. The increase in Young’s modulus of the membrane is 3-7 times, which is more significant than that of the cytoskeleton (1.1-3.4 times). We propose that changes in cell mechanics upon the addition of fluorescent tracing dye are primarily due to incorporation of amphiphilic dye molecules into the cellular plasma membrane, which increases the lateral interaction among phospholipid chains and thus enhances their rigidity and adhesion. PMID:19366241

  11. 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

  12. Single-cell force spectroscopy of pili-mediated adhesion

    NASA Astrophysics Data System (ADS)

    Sullan, Ruby May A.; Beaussart, Audrey; Tripathi, Prachi; Derclaye, Sylvie; El-Kirat-Chatel, Sofiane; Li, James K.; Schneider, Yves-Jacques; Vanderleyden, Jos; Lebeer, Sarah; Dufrêne, Yves F.

    2013-12-01

    Although bacterial pili are known to mediate cell adhesion to a variety of substrates, the molecular interactions behind this process are poorly understood. We report the direct measurement of the forces guiding pili-mediated adhesion, focusing on the medically important probiotic bacterium Lactobacillus rhamnosus GG (LGG). Using non-invasive single-cell force spectroscopy (SCFS), we quantify the adhesion forces between individual bacteria and biotic (mucin, intestinal cells) or abiotic (hydrophobic monolayers) surfaces. On hydrophobic surfaces, bacterial pili strengthen adhesion through remarkable nanospring properties, which - presumably - enable the bacteria to resist high shear forces under physiological conditions. On mucin, nanosprings are more frequent and adhesion forces larger, reflecting the influence of specific pili-mucin bonds. Interestingly, these mechanical responses are no longer observed on human intestinal Caco-2 cells. Rather, force curves exhibit constant force plateaus with extended ruptures reflecting the extraction of membrane nanotethers. These single-cell analyses provide novel insights into the molecular mechanisms by which piliated bacteria colonize surfaces (nanosprings, nanotethers), and offer exciting avenues in nanomedicine for understanding and controlling the adhesion of microbial cells (probiotics, pathogens).

  13. Chip Based Single Cell Analysis for Nanotoxicity Assessment

    PubMed Central

    Shah, Pratikkumar; Kaushik, Ajeet; Zhu, Xuena; Zhang, Chengxiao; Li, Chen-Zhong

    2014-01-01

    Nanomaterials, because of their tunable properties and performances, have been utilized extensively in everyday life related consumable products and technology. On exposure, beyond physiological range, nanomaterials cause health risks via affecting the function of organisms, genomic systems, and even central nervous system. Thus, new analytical approaches for nanotoxicity assessment to verify the feasibility of nanomaterials for future use are in demand. The conventional analytical techniques, such as spectrophotometric assay-based techniques usually require a lengthy and time-consuming process and many times produces false positives, and many times cannot be implemented at a single cell level measurement for studying cell behavior without interference of their surrounding environment. Hence, there is demand of a precise, accurate, sensitive assessment for toxicity using single cell. Recently, due to advantages of automation of fluids and minimization of human error, the integration of cell-on-a-chip (CoC) with microfluidic system is in practice for nanotoxicity assessments. This review explains nanotoxicity and assessment approaches with advantages/limitations and new approaches to overcome the confines of traditional techniques. Recent advances in nanotoxicity assessment using CoC integrated with microfluidic system are also discussed in this review, which may be of use for nanotoxicity assessment and diagnostics. PMID:24567949

  14. Novel needle-electrochemical microsensor for in-vitro and in-vivo measurements of oxygen

    NASA Astrophysics Data System (ADS)

    Xu, Weiya; Ma, Wentao; Li, Kaiyang; Hu, Jiming; Li, Hongyi; Cao, Lianxin; Song, Yu; Zhao, Lan

    2001-09-01

    Electrochemical microsensors have been applied in the field of biomedicine for many years. The aim of this work was to develop a novel oxygen sensor to monitor the partial pressure of oxygen in tissues and acupuncture points. The functions of microsensor were evaluated through in vitro experiments. In vivo in tissues and acupuncture points. The data from oxygen microsensor were compared with the data from blood gas analyzer. The measurements depend on the physiological changes of experimental animal. The further development of this new sensor is to be a tool for meridian research.

  15. An optical multifrequency phase-modulation method using microbeads for measuring intracellular oxygen concentrations in plants.

    PubMed

    Schmälzlin, Elmar; van Dongen, Joost T; Klimant, Ingo; Marmodée, Bettina; Steup, Martin; Fisahn, Joachim; Geigenberger, Peter; Löhmannsröben, Hans-Gerd

    2005-08-01

    A technique has been developed to measure absolute intracellular oxygen concentrations in green plants. Oxygen-sensitive phosphorescent microbeads were injected into the cells and an optical multifrequency phase-modulation technique was used to discriminate the sensor signal from the strong autofluorescence of the plant tissue. The method was established using photosynthesis-competent cells of the giant algae Chara corallina L., and was validated by application to various cell types of other plant species.

  16. Rebreathing method for the simultaneous measurement of oxygen consumption and effective pulmonary blood flow during exercise.

    PubMed

    Bush, A; Busst, C M; Johnson, S; Denison, D M

    1988-04-01

    This paper describes a rebreathing method for the simultaneous measurement of oxygen consumption (VO2) and effective pulmonary blood flow (QP. eff) at rest and during exercise. Subjects rebreathed a test gas consisting of 35% oxygen, 3.5% chlorodifluoromethane (freon-22), and 10% argon in nitrogen for 30 seconds or until the respired oxygen tension fell to below 13.3 kPa. Sixty normal subjects were studied on a motorized treadmill, the Bruce protocol being used. The rebreathing manoeuvre was performed at three minute intervals, and was initially practised sitting down. Measurements were then made with the subjects standing at rest, and subsequently during the last minute of each stage of the Bruce exercise protocol until the subjects were exhausted. Heart rate was recorded from the electrocardiogram. Oxygen uptake plotted against calculated power (watts) showed a discontinuity between resting and exercise values, probably because power output during treadmill exercise is underestimated. The arbitrary addition of 30 watts to the exercise power output abolished this discontinuity. There was good agreement between rebreathing estimates of oxygen consumption and values measured during a second exercise test by the conventional open circuit argon dilution method. Coefficients of variation of oxygen consumption and effective pulmonary blood flow measured by rebreathing were usually less than 10% even during maximal exertion. At rest mean (SD) effective pulmonary blood flow corrected for body surface area was 2.2 (0.46) l/min/m2. Effective pulmonary blood flow rose linearly with oxygen consumption. At rest the arteriovenous oxygen content difference for pulmonary blood (VO2/QP eff) was 9.1 (1.6) ml/dl, rising to a maximum of 16.4 (1.8) ml/dl. The stroke volume index was 27.5 (6.8) ml/m2, rising to a maximum of 46.5 (7.1) ml/m2 during exertion.

  17. Rebreathing method for the simultaneous measurement of oxygen consumption and effective pulmonary blood flow during exercise.

    PubMed Central

    Bush, A; Busst, C M; Johnson, S; Denison, D M

    1988-01-01

    This paper describes a rebreathing method for the simultaneous measurement of oxygen consumption (VO2) and effective pulmonary blood flow (QP. eff) at rest and during exercise. Subjects rebreathed a test gas consisting of 35% oxygen, 3.5% chlorodifluoromethane (freon-22), and 10% argon in nitrogen for 30 seconds or until the respired oxygen tension fell to below 13.3 kPa. Sixty normal subjects were studied on a motorized treadmill, the Bruce protocol being used. The rebreathing manoeuvre was performed at three minute intervals, and was initially practised sitting down. Measurements were then made with the subjects standing at rest, and subsequently during the last minute of each stage of the Bruce exercise protocol until the subjects were exhausted. Heart rate was recorded from the electrocardiogram. Oxygen uptake plotted against calculated power (watts) showed a discontinuity between resting and exercise values, probably because power output during treadmill exercise is underestimated. The arbitrary addition of 30 watts to the exercise power output abolished this discontinuity. There was good agreement between rebreathing estimates of oxygen consumption and values measured during a second exercise test by the conventional open circuit argon dilution method. Coefficients of variation of oxygen consumption and effective pulmonary blood flow measured by rebreathing were usually less than 10% even during maximal exertion. At rest mean (SD) effective pulmonary blood flow corrected for body surface area was 2.2 (0.46) l/min/m2. Effective pulmonary blood flow rose linearly with oxygen consumption. At rest the arteriovenous oxygen content difference for pulmonary blood (VO2/QP eff) was 9.1 (1.6) ml/dl, rising to a maximum of 16.4 (1.8) ml/dl. The stroke volume index was 27.5 (6.8) ml/m2, rising to a maximum of 46.5 (7.1) ml/m2 during exertion. PMID:3406913

  18. LUMOS - A Sensitive and Reliable Optode System for Measuring Dissolved Oxygen in the Nanomolar Range

    PubMed Central

    Lehner, Philipp; Larndorfer, Christoph; Garcia-Robledo, Emilio; Larsen, Morten; Borisov, Sergey M.; Revsbech, Niels-Peter; Glud, Ronnie N.; Canfield, Donald E.; Klimant, Ingo

    2015-01-01

    Most commercially available optical oxygen sensors target the measuring range of 300 to 2 μmol L-1. However these are not suitable for investigating the nanomolar range which is relevant for many important environmental situations. We therefore developed a miniaturized phase fluorimeter based measurement system called the LUMOS (Luminescence Measuring Oxygen Sensor). It consists of a readout device and specialized “sensing chemistry” that relies on commercially available components. The sensor material is based on palladium(II)-5,10,15,20-tetrakis-(2,3,4,5,6-pentafluorphenyl)-porphyrin embedded in a Hyflon AD 60 polymer matrix and has a KSV of 6.25 x 10-3 ppmv-1. The applicable measurement range is from 1000 nM down to a detection limit of 0.5 nM. A second sensor material based on the platinum(II) analogue of the porphyrin is spectrally compatible with the readout device and has a measurement range of 20 μM down to 10 nM. The LUMOS device is a dedicated system optimized for a high signal to noise ratio, but in principle any phase flourimeter can be adapted to act as a readout device for the highly sensitive and robust sensing chemistry. Vise versa, the LUMOS fluorimeter can be used for read out of less sensitive optical oxygen sensors based on the same or similar indicator dyes, for example for monitoring oxygen at physiological conditions. The presented sensor system exhibits lower noise, higher resolution and higher sensitivity than the electrochemical STOX sensor previously used to measure nanomolar oxygen concentrations. Oxygen contamination in common sample containers has been investigated and microbial or enzymatic oxygen consumption at nanomolar concentrations is presented. PMID:26029920

  19. Atomic oxygen and O2(a^1δg) density measurements in a Micro-Cathode Sustained Discharge in oxygen and rare gases/oxygen mixtures.

    NASA Astrophysics Data System (ADS)

    Magne, L.; Bauville, G.; Jeanney, P.; Lacour, B.; Puech, V.

    2006-10-01

    This work presents first experimental investigations of atomic oxygen density and O2(a^1δg) production in a Micro-Cathode Sustained Discharge (MCSD) in pure O2 and in argon (or helium)/O2 mixtures for a total pressure up to 130 Torr. A micro-hollow cathode discharge (MHCD), 200 micron in diameter, is used as plasma cathode for a discharge between the MHCD and a third electrode placed 8 mm away. In pure oxygen, the absolute atom density was measured by Two-photon Absorption Laser Induced Fluorescence (TALIF). It will be shown that, for a current of 1 mA and a pressure of 50 Torr, an atomic density of 3 10^15 cm-3 is obtained near the micro-hollow cathode, and it decreases to 5 10^14 cm-3 near the third electrode. If the MCSD is switched off while the MHCD is still on, the atom density decreases by an order of magnitude. 2D cartography of the atom distributions will be presented for different operating conditions. The density of the O2(a^1δg) metastable state was evaluated from the intensity of the 1.27 μm transition measured with a calibrated InGaAs detector. It will be shown that O2(a^1δg) densities up to 10^16 cm-3 have been obtained for 10% O2 in an argon/oxygen mixture at 50 Torr. Work is in progress to determine conditions for generating higher O2(a^1δg) densities.

  20. ACE EPAM and Van Allen Probes RBSPICE measurements of interplanetary oxygen injection to the inner magnetosphere

    NASA Astrophysics Data System (ADS)

    Patterson, J. D.; Manweiler, J. W.; Gerrard, A. J.; Lanzerotti, L. J.

    2015-12-01

    On March 17, 2015, a significant oxygen-rich interplanetary event was measure by the Advanced Composition Explorer (ACE) Electron Proton Alpha Monitor (EPAM) instrument. At the same time the Van Allen Probes Radiation Belt Storm Probes Ion Composition Experiment (RBSPICE) instrument recorded significant enhancements of oxygen in the inner magnetosphere. We present a detailed analysis of this event utilizing a new method of exploiting the EPAM Pulse Height Analyzer (PHA) data to precisely resolve helium and oxygen spectra within the 0.5 to 5 MeV/nuc range. We also present the flux, partial particle pressures, and pitch angle distributions of the ion measurements from RBSPICE. During this event, both EPAM and RBSPICE measured O:He ratios greater than 10:1. The pitch angle distributions from RBSPICE-B show a strong beam of oxygen at an L ~ 5.8 early on March 17th during orbit. The timing between the observations of the oxygen peak at ACE and the beam observed at RBSPICE-B is consistent with the travel-time required for energetic particle transport from L1 to Earth and access to the magnetosphere. We assert that the oxygen seen by RBSPICE during the initial phase of this event is the result of direct injection from the interplanetary medium of energetic ions. This poster contains the observations and detailed calculations to support this assertion.

  1. LIF measurements of oxygen concentration gradients along flat and wavy air-water interfaces

    NASA Astrophysics Data System (ADS)

    Woodrow, Philip T., Jr.; Duke, Steve R.

    Instantaneous spatially-varying measurements of concentration gradients occurring during aeration for flat, stagnant air-water interfaces and for interfaces with mechanically-generated waves are presented. Measurements were obtained in a laboratory wave tank using a laser-induced fluorescence (LIF) technique that images planar oxygen concentration fields near air-water interfaces. Pulsed nitrogen laser light focused to a thin sheet induces the fluorescence of pyrene butyric acid (in micromolar concentration) in deoxygenated water. The PBA fluorescence is quenched by dissolved oxygen. A high-resolution CCD camera images in two dimensions the intensities of the fluorescence field, providing spatial measurements of oxygen concentration with magnification of 7 μm per pixel. The concentration fields, gradients, and boundary layer thicknesses along the flat and wavy air-water interfaces are quantified and compared to previous measurements associated with sheared gas-liquid interfaces and with wind-generated waves.

  2. Oxygen Isotope Measurements of a Rare Murchison Type A CAI and Its Rim

    NASA Technical Reports Server (NTRS)

    Matzel, J. E. P.; Simon, J. I.; Hutcheon, I. D.; Jacobsen, B.; Simon, S. B.; Grossman, L.

    2013-01-01

    Ca-, Al-rich inclusions (CAIs) from CV chondrites commonly show oxygen isotope heterogeneity among different mineral phases within individual inclusions reflecting the complex history of CAIs in both the solar nebula and/or parent bodies. The degree of isotopic exchange is typically mineral-specific, yielding O-16-rich spinel, hibonite and pyroxene and O-16-depleted melilite and anorthite. Recent work demonstrated large and systematic variations in oxygen isotope composition within the margin and Wark-Lovering rim of an Allende Type A CAI. These variations suggest that some CV CAIs formed from several oxygen reservoirs and may reflect transport between distinct regions of the solar nebula or varying gas composition near the proto-Sun. Oxygen isotope compositions of CAIs from other, less-altered chondrites show less intra-CAI variability and 16O-rich compositions. The record of intra-CAI oxygen isotope variability in CM chondrites, which commonly show evidence for low-temperature aqueous alteration, is less clear, in part because the most common CAIs found in CM chondrites are mineralogically simple (hibonite +/- spinel or spinel +/- pyroxene) and are composed of minerals less susceptible to O-isotopic exchange. No measurements of the oxygen isotope compositions of rims on CAIs in CM chondrites have been reported. Here, we present oxygen isotope data from a rare, Type A CAI from the Murchison meteorite, MUM-1. The data were collected from melilite, hibonite, perovskite and spinel in a traverse into the interior of the CAI and from pyroxene, melilite, anorthite, and spinel in the Wark-Lovering rim. Our objectives were to (1) document any evidence for intra-CAI oxygen isotope variability; (2) determine the isotopic composition of the rim minerals and compare their composition(s) to the CAI interior; and (3) compare the MUM-1 data to oxygen isotope zoning profiles measured from CAIs in other chondrites.

  3. Emergent Collective Chemotaxis without Single-Cell Gradient Sensing

    NASA Astrophysics Data System (ADS)

    Camley, Brian A.; Zimmermann, Juliane; Levine, Herbert; Rappel, Wouter-Jan

    2016-03-01

    Many eukaryotic cells chemotax, sensing and following chemical gradients. However, experiments show that even under conditions when single cells cannot chemotax, small clusters may still follow a gradient. This behavior is observed in neural crest cells, in lymphocytes, and during border cell migration in Drosophila, but its origin remains puzzling. Here, we propose a new mechanism underlying this "collective guidance," and study a model based on this mechanism both analytically and computationally. Our approach posits that contact inhibition of locomotion, where cells polarize away from cell-cell contact, is regulated by the chemoattractant. Individual cells must measure the mean attractant value, but need not measure its gradient, to give rise to directional motility for a cell cluster. We present analytic formulas for how the cluster velocity and chemotactic index depend on the number and organization of cells in the cluster. The presence of strong orientation effects provides a simple test for our theory of collective guidance.

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

    PubMed

    Efroni, Idan; Birnbaum, Kenneth D

    2016-01-01

    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. PMID:27048384

  5. Comparison of atomic oxygen measurements by incoherent scatter and satellite-borne mass spectrometer techniques

    NASA Technical Reports Server (NTRS)

    Hedin, A. E.; Alcayde, D.

    1974-01-01

    Atomic oxygen densities determined by the incoherent scatter technique are compared to densities deduced from satellite-borne mass spectrometer measurements and are found to agree within experimental error. The diurnal variations inferred from the incoherent scatter measurements do show, however, some departure from diurnal variations found by modeling the mass spectrometer results. Some implications of these departures are briefly discussed.

  6. Brief inhalation method to measure cerebral oxygen extraction fraction with PET: Accuracy determination under pathologic conditions

    SciTech Connect

    Altman, D.I.; Lich, L.L.; Powers, W.J. )

    1991-09-01

    The initial validation of the brief inhalation method to measure cerebral oxygen extraction fraction (OEF) with positron emission tomography (PET) was performed in non-human primates with predominantly normal cerebral oxygen metabolism (CMRO2). Sensitivity analysis by computer simulation, however, indicated that this method may be subject to increasing error as CMRO2 decreases. Accuracy of the method under pathologic conditions of reduced CMRO2 has not been determined. Since reduced CMRO2 values are observed frequently in newborn infants and in regions of ischemia and infarction in adults, we determined the accuracy of the brief inhalation method in non-human primates by comparing OEF measured with PET to OEF measured by arteriovenous oxygen difference (A-VO2) under pathologic conditions of reduced CMRO2 (0.27-2.68 ml 100g-1 min-1). A regression equation of OEF (PET) = 1.07 {times} OEF (A-VO2) + 0.017 (r = 0.99, n = 12) was obtained. The absolute error in oxygen extraction measured with PET was small (mean 0.03 {plus minus} 0.04, range -0.03 to 0.12) and was independent of cerebral blood flow, cerebral blood volume, CMRO2, or OEF. The percent error was higher (19 {plus minus} 37), particularly when OEF is below 0.15. These data indicate that the brief inhalation method can be used for measurement of cerebral oxygen extraction and cerebral oxygen metabolism under pathologic conditions of reduced cerebral oxygen metabolism, with these limitations borne in mind.

  7. Position-specific measurement of oxygen isotope ratios in cellulose: Isotopic exchange during heterotrophic cellulose synthesis

    NASA Astrophysics Data System (ADS)

    Waterhouse, John S.; Cheng, Shuying; Juchelka, Dieter; Loader, Neil J.; McCarroll, Danny; Switsur, V. Roy; Gautam, Lata

    2013-07-01

    We describe the first reported method for the measurement of oxygen isotope ratios at each position in the glucose units of the cellulose molecule. The overall process comprises a series of synthetic organic sequences, by which α-cellulose is hydrolysed to glucose, and oxygen atoms at specific positions in the glucose molecule are removed in samples of benzoic acid for measurement of δ18O. Values of δ18O at specific positions in cellulose are calculated from these δ18O values and the overall δ18O value of the cellulose. We apply the method to determine the degree to which oxygen atoms at each position undergo isotopic exchange with water during heterotrophic cellulose synthesis, such as occurs in the cambium of trees. To do this we extract α-cellulose from wheat seedlings germinated in the dark in aqueous media of differing oxygen isotope ratios. Results indicate that oxygen atoms at positions 5 and 6 (O-5 and O-6 respectively) undergo around 80% exchange with medium water, O-3 undergoes around 50% exchange, and O-2 and O-4 do not undergo isotopic exchange. The results have important implications for extracting palaeoclimatic records from oxygen isotope time series obtained from tree ring cellulose. As O-5 and O-6 undergo significant exchange with medium water during heterotrophic cellulose synthesis, oxygen isotopes at these positions in tree ring cellulose should carry a predominantly trunk (source) water signal. On the other hand, O-2 and O-4 should retain the isotopic signature of leaf water in tree ring cellulose. Our method therefore potentially enables the separate reconstruction of past temperature and humidity data from oxygen isotope ratios of tree ring cellulose - something that has hitherto not been possible. The measured degrees of isotopic exchange are to some extent unexpected and cannot be fully explained using current biochemical mechanisms, suggesting that knowledge of these processes is incomplete.

  8. The Kety-Schmidt Technique for Quantitative Perfusion and Oxygen Metabolism Measurements in the MR Environment

    PubMed Central

    Lee, John J.; Powers, William J.; Faulkner, Chad B.; Boyle, Patrick J.; Derdeyn, Colin P.

    2013-01-01

    The Kety-Schmidt technique provides quantitative measurement of whole brain cerebral blood flow (CBF). CBF is measured as the area between the arterial and venous washout curves of a diffusible tracer. Oxygen extraction and metabolism may be calculated from arterial and venous samples. In this report we present a method for performing these measurements in an MR environment. This technique could be useful for validation of MR methods of hemodynamic and metabolic measurements in humans. PMID:22997166

  9. Live single-cell laser tag

    PubMed Central

    Binan, Loïc; Mazzaferri, Javier; Choquet, Karine; Lorenzo, Louis-Etienne; Wang, Yu Chang; Affar, El Bachir; De Koninck, Yves; Ragoussis, Jiannis; Kleinman, Claudia L.; Costantino, Santiago

    2016-01-01

    The ability to conduct image-based, non-invasive cell tagging, independent of genetic engineering, is key to cell biology applications. Here we introduce cell labelling via photobleaching (CLaP), a method that enables instant, specific tagging of individual cells based on a wide array of criteria such as shape, behaviour or positional information. CLaP uses laser illumination to crosslink biotin onto the plasma membrane, coupled with streptavidin conjugates to label individual cells for genomic, cell-tracking, flow cytometry or ultra-microscopy applications. We show that the incorporated mark is stable, non-toxic, retained for several days, and transferred by cell division but not to adjacent cells in culture. To demonstrate the potential of CLaP for genomic applications, we combine CLaP with microfluidics-based single-cell capture followed by transcriptome-wide next-generation sequencing. Finally, we show that CLaP can also be exploited for inducing transient cell adhesion to substrates for microengineering cultures with spatially patterned cell types. PMID:27198043

  10. Nitrogen assimilation by single cells in hot springs

    NASA Astrophysics Data System (ADS)

    Poret-peterson, A. T.; Romaniello, S. J.; Bose, M.; Williams, P.; Elser, J. J.; Shock, E.; Anbar, A. D.; Hartnett, H. E.

    2012-12-01

    Microorganisms drive biogeochemical cycles and require nutrients, such as ammonium and nitrate, to function. As a result, following nutrient flows provides opportunities to study how microbial activity influences ecosystem-level processes. Most past measurements of microbial nutrient uptake rely on bulk measurements, which are informative but provide little information about heterogeneity among community members involved in elemental transformations, nor about possible effects of physiological state or taxonomic identity. Since microbial communities tend to be phylogenetically and physiologically diverse, it is reasonable to expect that community members will respond differently to nutrient addition. Here, we examine nitrogen assimilation (via addition of 15N-labeled ammonium or nitrate) in Yellowstone hot spring microbial communities. Using the NanoSIMS, we imaged cells at a very high spatial resolution (nanometer scale) necessary to determine 15N enrichments in single micron-sized cells. We compare the N isotopic enrichments observed in single cells to that determined in bulk sediments by standard isotope ratio mass spectrometry. NanoSIMS imaging of 56 individual cells from sediments of an acidic hot spring (pH 4.7, T=67oC) incubated with 15N-ammonium shows that about two-thirds of the cells (38) exhibited 15N-enrichment. Most cells had 15N enrichments from 0.39 to 0.91 atom %, while some cells were much more significantly enriched. Bulk analyses of sediments show that ammonium assimilation and nitrate assimilation readily occurred at this spring. These findings show that microbes in this hot spring may differentially take up ammonium, which may arise from a number of factors including differences in cellular N requirements, growth rates, and the ability to transport ammonium. This work represents some of the first single-cell isotopic measurements from an extreme environment. Efforts are underway to image sediment samples from other hot springs and to pair Nano

  11. 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.

  12. 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

  13. 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.

  14. Self-Digitization Microfluidic Chip for Absolute Quantification of mRNA in Single Cells

    PubMed Central

    2015-01-01

    Quantification of mRNA in single cells provides direct insight into how intercellular heterogeneity plays a role in disease progression and outcomes. Quantitative polymerase chain reaction (qPCR), the current gold standard for evaluating gene expression, is insufficient for providing absolute measurement of single-cell mRNA transcript abundance. Challenges include difficulties in handling small sample volumes and the high variability in measurements. Microfluidic digital PCR provides far better sensitivity for minute quantities of genetic material, but the typical format of this assay does not allow for counting of the absolute number of mRNA transcripts samples taken from single cells. Furthermore, a large fraction of the sample is often lost during sample handling in microfluidic digital PCR. Here, we report the absolute quantification of single-cell mRNA transcripts by digital, one-step reverse transcription PCR in a simple microfluidic array device called the self-digitization (SD) chip. By performing the reverse transcription step in digitized volumes, we find that the assay exhibits a linear signal across a wide range of total RNA concentrations and agrees well with standard curve qPCR. The SD chip is found to digitize a high percentage (86.7%) of the sample for single-cell experiments. Moreover, quantification of transferrin receptor mRNA in single cells agrees well with single-molecule fluorescence in situ hybridization experiments. The SD platform for absolute quantification of single-cell mRNA can be optimized for other genes and may be useful as an independent control method for the validation of mRNA quantification techniques. PMID:25390242

  15. Self-digitization microfluidic chip for absolute quantification of mRNA in single cells.

    PubMed

    Thompson, Alison M; Gansen, Alexander; Paguirigan, Amy L; Kreutz, Jason E; Radich, Jerald P; Chiu, Daniel T

    2014-12-16

    Quantification of mRNA in single cells provides direct insight into how intercellular heterogeneity plays a role in disease progression and outcomes. Quantitative polymerase chain reaction (qPCR), the current gold standard for evaluating gene expression, is insufficient for providing absolute measurement of single-cell mRNA transcript abundance. Challenges include difficulties in handling small sample volumes and the high variability in measurements. Microfluidic digital PCR provides far better sensitivity for minute quantities of genetic material, but the typical format of this assay does not allow for counting of the absolute number of mRNA transcripts samples taken from single cells. Furthermore, a large fraction of the sample is often lost during sample handling in microfluidic digital PCR. Here, we report the absolute quantification of single-cell mRNA transcripts by digital, one-step reverse transcription PCR in a simple microfluidic array device called the self-digitization (SD) chip. By performing the reverse transcription step in digitized volumes, we find that the assay exhibits a linear signal across a wide range of total RNA concentrations and agrees well with standard curve qPCR. The SD chip is found to digitize a high percentage (86.7%) of the sample for single-cell experiments. Moreover, quantification of transferrin receptor mRNA in single cells agrees well with single-molecule fluorescence in situ hybridization experiments. The SD platform for absolute quantification of single-cell mRNA can be optimized for other genes and may be useful as an independent control method for the validation of mRNA quantification techniques.

  16. DESIGN AND PERFORMANCE OBJECTIVES OF THE SINGLE CELL TEST SYSTEM FOR SO2 DEPOLARIZED ELECTROLYZER DEVELOPMENT

    SciTech Connect

    Steimke, J

    2007-01-15

    The single cell test system development for the SRNL sulfur dioxide-depolarized electrolyzer has been completed. Operating experience and improved operating procedures were developed during test operations in FY06 and the first quarter of FY07. Eight different cell configurations, using various MEA designs, have been tested. The single cell test electrolyzer has been modified to overcome difficulties experienced during testing, including modifications to the inlet connection to eliminate minute acid leaks that caused short circuits. The test facility was modified by adding a water bath for cell heating, thus permitting operation over a wider range of flowrates and cell temperatures. Modifications were also identified to permit continuous water flushing of the cathode to remove sulfur, thus extending operating time between required shutdowns. This is also expected to permit a means of independently measuring the rate of sulfur formation, and the corresponding SO{sub 2} flux through the membrane. This report contains a discussion of the design issues being addressed by the single cell test program, a test matrix being conducted to address these issues, and a summary of the performance objectives for the single cell test system. The current primary objective of single cell test system is to characterize and qualify electrolyzer configurations for the following 100-hour longevity tests. Although the single cell test system development is considered complete, SRNL will continue to utilize the test facility and the single cell electrolyzer to measure the operability and performance of various cell design configurations, including new MEA's produced by the component development tasks.

  17. 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.

  18. Laser induced fluorescence measurements of dissolved oxygen concentration fields near air bubble surfaces

    NASA Astrophysics Data System (ADS)

    Roy, Sabita; Duke, Steve R.

    2000-09-01

    This article describes a laser-induced fluorescence (LIF) technique for measuring dissolved oxygen concentration gradients in water near the surface of an air bubble. Air bubbles are created at the tip of a needle in a rectangular bubble column filled with water that contains pyrenebutyric acid (PBA). The fluorescence of the PBA is induced by a planar pulse of nitrogen laser light. Oxygen transferring from the air bubble to the deoxygenated water quenches the fluorescence of the PBA. Images of the instantaneous and two-dimensional fluorescence field are obtained by a UV-intensified charge-coupled device (CCD) camera. Quenching of fluorescence intensity is determined at each pixel in the CCD image to measure dissolved oxygen concentration. Two-dimensional concentration fields are presented for a series of measurements of oxygen transfer from 1.6 mm bubbles suspended on the tip of a needle in a quiescent fluid. The images show the spatially varying concentration profiles, gradients, and boundary layer thicknesses at positions around the bubble surfaces. These direct and local measurements of concentration behavior within the mass transfer boundary layer show the potential of this LIF technique for the development of general and mechanistic models for oxygen transport across the air-water interface.

  19. The Choroidal Eye Oximeter - An instrument for measuring oxygen saturation of choroidal blood in vivo

    NASA Technical Reports Server (NTRS)

    Laing, R. A.; Danisch, L. A.; Young, L. R.

    1975-01-01

    The Choroidal Eye Oximeter is an electro-optical instrument that noninvasively measures the oxygen saturation of choroidal blood in the back of the human eye by a spectrophotometric method. Since choroidal blood is characteristic of blood which is supplied to the brain, the Choroidal Eye Oximeter can be used to monitor the amount of oxygen which is supplied to the brain under varying external conditions. The instrument consists of two basic systems: the optical system and the electronic system. The optical system produces a suitable bi-chromatic beam of light, reflects this beam from the fundus of the subject's eye, and onto a low-noise photodetector. The electronic system amplifies the weak composite signal from the photodetector, computes the average oxygen saturation from the area of the fundus that was sampled, and displays the value of the computed oxygen saturation on a panel meter.

  20. Development of micro-coulometry for measuring oxygen content in copper oxides

    NASA Astrophysics Data System (ADS)

    Sato, Fumiaki; Fujihara, Masaaki; Kambe, Shiro; Ishii, Osamu

    2006-10-01

    A micro-coulometry system was newly developed and employed for measuring the oxygen content in copper oxides using a reduced amount of material. To achieve this reduction in sample size, Ar flow rate, and coulometric current were optimized. When using 5.0 mg of YBa2Cu3O6.71, which is about 1/10 the amount used in conventional coulometry, the oxygen content was successfully determined to be 6.68 ± 0.03, Ar flow rate, and coulometric current were 0.1 l/min, and 1.0 mA, respectively. It was found to be necessary to continue to pass Ar through the solution before coulometry more than ten minutes and during the coulometric measurement. This method will be useful for measuring the oxygen content of oxide samples smaller than 10 mg, i.e. oxide materials produced in small quantities for electronic applications.

  1. Flexible Sheet-Type Sensor for Noninvasive Measurement of Cellular Oxygen Metabolism on a Culture Dish

    PubMed Central

    Akagi, Takanori; Shiono, Hirofumi; Ichiki, Takanori

    2015-01-01

    A novel flexible sensor was developed for the noninvasive oxygen metabolism measurement of cultivated cells and tissues. This device is composed of a transparent double-layered polymer sheet of ethylene-vinyl alcohol (EVOH) and poly(dimethylsiloxane) (PDMS) having an array of microhole structures of 90 μm diameter and 50 μm depth on its surface. All the microhole structures were equipped with a 1-μm-thick optical chemical sensing layer of platinum porphyrin-fluoropolymer on their bottom. The three-dimensional microstructures of the sensor were fabricated by a newly developed simple and low-cost production method named self-aligned hot embossing. The device was designed to be attached slightly above the cells cultivated on a dish to form a temporarily closed microspace over the target cells during measurement. Since the change in oxygen concentration is relatively fast in the microcompartmentalized culture medium, a rapid evaluation of the oxygen consumption rate is possible by measuring the phosphorescence lifetime of the platinum porphyrin-fluoropolymer. The combined use of the device and an automated optical measurement system enabled the high-throughput sensing of cellular oxygen consumption (100 points/min). We monitored the oxygen metabolism of the human breast cancer cell line MCF7 on a Petri dish and evaluated the oxygen consumption rate to be 0.72 ± 0.12 fmol/min/cell. Furthermore, to demonstrate the utility of the developed sensing system, we demonstrated the mapping of the oxygen consumption rate of rat brain slices and succeeded in visualizing a clear difference among the layer structures of the hippocampus, i.e., the cornu ammonis (CA1 and CA3) and dentate gyrus (DG). PMID:26624889

  2. Measuring and modeling oxygen diffusion in niobium-vanadium and niobium-palladium alloys

    NASA Astrophysics Data System (ADS)

    Hennessey, Theresa P.

    Niobium alloys are under consideration for high-temperature aerospace applications, but they have poor oxidation resistance and need high-temperature coatings for protection in severe environments. Our approach to creating an oxidation-resistant Nb alloy is to identify substitutional solute elements that lower the diffusivity of oxygen in Nb. In theory, this will induce a transition from internal to external oxidation and promote the formation of a desirable, protective oxide scale. The objective of this particular project is to compare the oxygen diffusivity in Nb alloys that contain either trap or repulsive sites. In Nb, oxygen atoms diffuse via an interstitial mechanism, and they can interact with substitutional solute atoms in different ways. The interstitial sites adjacent to a substitutional atom constitute a "zone of influence". If the sites in this zone have a lower energy than the normal sites, they are called "trap" sites. If these sites have a higher energy, they are called "repulsive" sites. Oxygen diffusion is inhibited in both cases: trap sites hold the oxygen and keep it from diffusing further, while repulsive sites block the path of the oxygen. Two new mathematical models for interstitial diffusion in these systems were derived from probability and statistical thermodynamic theory. The models were verified using a new random-walk computer simulation of oxygen diffusion through Nb alloys. These models were also tested experimentally by measuring oxygen diffusivity in Nb-V and Nb-Pd alloys. These results showed that V atoms create trap sites for oxygen atoms, confirming previous work. However, there was not enough data to prove definitively that Pd atoms create repulsive sites, as expected by theory.

  3. Calibration-free measurement of the oxygen saturation in human retinal vessels

    NASA Astrophysics Data System (ADS)

    Schweitzer, Dietrich; Leistritz, Lutz; Hammer, Martin; Scibor, Mateusz; Bartsch, Ulrich; Strobel, Juergen

    1995-05-01

    The detection of alterations in the microcirculation requires both the measurement of the blood-flow and the measurement of the oxygen saturation in whole blood. The basis for the non-invasive estimation of the oxygen saturation is the difference between extinction-spectra of hemoglobin and of oxyhemoglobin. Further in whole blood the scattering at the erythrocytes has to be taken into account. In principle spectral measurements at three neighboring wavelengths are sufficient for the calculation of the oxygen saturation, the concentration- thickness-geometry product and the scattering intensity. Caused by the maximal permissible exposure, the signal/noise ratio is very low in fundus reflectometry. But if the wavelength- range from 520 nm to 620 nm is evaluated, the requirement at the signal/noise ratio is reduced. This reduction corresponds to the square root of the number of discrete wavelengths at which the ocular fundus reflectance is measured. So the oxygen saturation can be calculated with an error lower than +/- 4%. For this purpose the extinction spectrum of whole blood is approximated by a model, including besides the above mentioned unknowns the spectral dependency of the scattering. The experimental arrangement for the measurement of the oxygen saturation is an imaging ophthalmo-spectrometer which allows reflectance measurements with a good spectral (< 3 nm) and local (> 3 micrometers ) resolution simultaneously at a vessel and in its neighborhood. The extinction of blood is calculated as the logarithm of the ratio of the reflectance of the neighborhood and of the vessel. In this calculation the influences of the ocular media, of the background and of eye movements are eliminated. The sensitivity of the detector system has to be very high in order to detect the light which is reflected at a dark background and travels through the blood. The new method was tested by a comparison the oxygen saturation of the blood in an arteriole and a venule in the brain

  4. Simultaneous Noninvasive Determination of Regional Myocardial Perfusion and Oxygen Content in Rabbits: Toward Direct Measurement of Myocardial Oxygen Consumption at MR Imaging1

    PubMed Central

    Reeder, Scott B.; Holmes, A. Alexander; McVeigh, Elliot R.; Forder, John R.

    2007-01-01

    PURPOSE To determine whether myocardial arterial perfusion and oxygen concentration can be quantified simultaneously from the same images by using spin labeling and the blood oxygenation level-dependent (BOLD) effect with fast spin-echo (SE) imaging. MATERIALS AND METHODS A T2-weighted fast SE pulse sequence was written to image isolated, arrested, blood-perfused rabbit hearts (n = 6) at 4.7 T. Perfusion images with intensity in units of milliliters per minute per gram that covered the entire left ventricle with 0.39 × 0.39 × 3.00-mm resolution were obtained in less than 15 minutes with a 32-fold reduction in imaging time from that of a previous study. Estimates of oxygen concentration were made from the same images acquired for calculation of perfusion images. RESULTS Estimates of regional myocardial oxygen content could be made from the perfusion images; this demonstrated the feasibility of three-dimensional calculation of regional oxygen consumption, which requires concomitant measurement of both oxygen content and flow. Fast SE imaging was shown to bas sensitive to hemoglobin desaturation as standard SE imaging. Perfusion abnormalities and oxygen deficits were easily identified and verified qualitatively with gadopentetate dimeglumine on both perfusion and BOLD images obtained after coronary arterial ligation. CONCLUSION T2-weighted fast SE imaging combined with perfusion-sensitive spin labeling can be used to measure myocardial arterial perfusion and oxygen concentration. This provides the groundwork for calculation of regional myocardial oxygen consumption. PMID:10478241

  5. Measuring reactive oxygen and nitrogen species with fluorescent probes: challenges and limitations

    PubMed Central

    Kalyanaraman, Balaraman; Darley-Usmar, Victor; Davies, Kelvin J.A.; Dennery, Phyllis A.; Forman, Henry Jay; Grisham, Matthew B.; Mann, Giovanni E.; Moore, Kevin; Roberts, L. Jackson; Ischiropoulos, Harry

    2013-01-01

    The purpose of this position paper is to present a critical analysis of the challenges and limitations of the most widely used fluorescent probes for detecting and measuring reactive oxygen and nitrogen species. Where feasible, we have made recommendations for the use of alternate probes and appropriate analytical techniques that measure the specific products formed from the reactions between fluorescent probes and reactive oxygen and nitrogen species. We have proposed guidelines that will help present and future researchers with regard to the optimal use of selected fluorescent probes and interpretation of results. PMID:22027063

  6. 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. PMID:20728993

  7. 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.

  8. Sample Targeting During Single-Particle Single-Cell Irradiation

    NASA Astrophysics Data System (ADS)

    Bigelow, A. W.; Randers-Pehrson, G.; Michel, K. A.; Brenner, D. J.; Dymnikov, A. D.

    2003-08-01

    An apertured microbeam is used for single-particle single-cell irradiation to study radiobiological effects at the Radiological Research Accelerator Facility (RARAF), Center for Radiological Research, Columbia University. The present sample targeting system involves imaging techniques and a stepping motor stage to sequentially position a cell nucleus above a vertical ion beam. An interest expressed by the biology research community in targeting subnuclear components has spurred the development of microbeam II, a next-generation facility to include a focused ion beam and a more precise sample manipulator, a voice coil stage. Sample positioning precision will rely on a feedback circuit incorporating linear variable differential transformer (LVDT) position measurements. In addition, post-lens electrostatic deflection is a contender for a point-and-shoot system that could speed up the cell irradiation process for cells within an image frame. Crucial to this development is that ion beam blow up must be minimal during deflection.

  9. Sample Targeting During Single-Particle Single-Cell Irradiation

    SciTech Connect

    Bigelow, A.W.; Randers-Pehrson, G.; Michel, K.A.; Brenner, D.J.; Dymnikov, A.D.

    2003-08-26

    An apertured microbeam is used for single-particle single-cell irradiation to study radiobiological effects at the Radiological Research Accelerator Facility (RARAF), Center for Radiological Research, Columbia University. The present sample targeting system involves imaging techniques and a stepping motor stage to sequentially position a cell nucleus above a vertical ion beam. An interest expressed by the biology research community in targeting subnuclear components has spurred the development of microbeam II, a next-generation facility to include a focused ion beam and a more precise sample manipulator, a voice coil stage. Sample positioning precision will rely on a feedback circuit incorporating linear variable differential transformer (LVDT) position measurements. In addition, post-lens electrostatic deflection is a contender for a point-and-shoot system that could speed up the cell irradiation process for cells within an image frame. Crucial to this development is that ion beam blow up must be minimal during deflection.

  10. Limits of sensing temporal concentration changes by single cells.

    PubMed

    Mora, Thierry; Wingreen, Ned S

    2010-06-18

    Berg and Purcell [Biophys. J. 20, 193 (1977)] calculated how the accuracy of concentration sensing by single-celled organisms is limited by noise from the small number of counted molecules. Here we generalize their results to the sensing of concentration ramps, which is often the biologically relevant situation (e.g., during bacterial chemotaxis). We calculate lower bounds on the uncertainty of ramp sensing by three measurement devices: a single receptor, an absorbing sphere, and a monitoring sphere. We contrast two strategies, simple linear regression of the input signal versus maximum likelihood estimation, and show that the latter can be twice as accurate as the former. Finally, we consider biological implementations of these two strategies, and identify possible signatures that maximum likelihood estimation is implemented by real biological systems.

  11. Limits of Sensing Temporal Concentration Changes by Single Cells

    NASA Astrophysics Data System (ADS)

    Mora, Thierry; Wingreen, Ned S.

    2010-06-01

    Berg and Purcell [Biophys. J. 20, 193 (1977)BIOJAU0006-349510.1016/S0006-3495(77)85544-6] calculated how the accuracy of concentration sensing by single-celled organisms is limited by noise from the small number of counted molecules. Here we generalize their results to the sensing of concentration ramps, which is often the biologically relevant situation (e.g., during bacterial chemotaxis). We calculate lower bounds on the uncertainty of ramp sensing by three measurement devices: a single receptor, an absorbing sphere, and a monitoring sphere. We contrast two strategies, simple linear regression of the input signal versus maximum likelihood estimation, and show that the latter can be twice as accurate as the former. Finally, we consider biological implementations of these two strategies, and identify possible signatures that maximum likelihood estimation is implemented by real biological systems.

  12. Microscopic Local Measurement of Blood Flow and Oxygen Tension in Brain Microcirculation

    NASA Astrophysics Data System (ADS)

    Minamitani, Haruyuki; Takahashi, Ryota; Tsukada, Kousuke

    A multi-photonic imaging system was proposed for measuring blood flow velocity, vessel diameter and blood oxygen tension pO2 simultaneously with high spatio-temporal resolution in the parenchymatous organ microcirculation, such as pial tissue, by using a closed cranial window and two light sources. FITC-stained erythrocytes was used to visualize the microcirculation, and the fluorescent image was recorded by a high-speed video camera for measuring blood flow velocity. Oxygen tension pO2 was measured by oxygen-dependent quenching of phosphorescent molecules, Pd-TCPP, in the microvessels after irradiation of second harmonic light of Nd:YAG pulse laser (532nm). Animal experiments were performed for investigation of blood flow dynamics and oxygen diffusion phenomenon during acute cerebral ischemia using photochemical thrombus formation in the closed cranial window of male Wistar rats. Experimental results showed specific and significant blood flow and oxygen diffusion phenomena related to the abnormal organ tissues, from those the proposed technique would contribute to the trasnlational research for the clinical medicine, concerned in the ischemic dysfunction, angiogenisis, tumorgenisis and so on.

  13. Kinetics of single cells: observation and modeling of a stochastic process.

    PubMed

    Pin, Carmen; Baranyi, József

    2006-03-01

    The successive generation times for single cells of Escherichia coli K-12 were measured as described by A. Elfwing, Y. LeMarc, J. Baranyi, and A. Ballagi (Appl. Environ. Microbiol. 70:675-678, 2004), and the histograms they generated were used as empirical distributions to simulate growth of the population as the result of the multiplication of its single cells. This way, a stochastic birth model in which the underlying distributions were measured experimentally was simulated. To validate the model, analogous bacterial growth curves were generated by the use of different inoculum levels. The agreement with the simulation was very good, proving that the growth of the population can be predicted accurately if the distribution of the first few division times for the single cells within that population is known. Two questions were investigated by the simulation. (i) To what extent can we say that the distribution of the detection time, i.e., the time by which a single-cell-generated subpopulation reaches a detectable level, can be identified with that of the lag time of the original single cell? (ii) For low inocula, how does the inoculum size affect the lag time of the population?

  14. Dual transcript and protein quantification in a massive single cell array.

    PubMed

    Park, Seung-Min; Lee, Jae Young; Hong, Soongweon; Lee, Sang Hun; Dimov, Ivan K; Lee, Hojae; Suh, Susie; Pan, Qiong; Li, Keyu; Wu, Anna M; Mumenthaler, Shannon M; Mallick, Parag; Lee, Luke P

    2016-10-01

    Recently, single-cell molecular analysis has been leveraged to achieve unprecedented levels of biological investigation. However, a lack of simple, high-throughput single-cell methods has hindered in-depth population-wide studies with single-cell resolution. We report a microwell-based cytometric method for simultaneous measurements of gene and protein expression dynamics in thousands of single cells. We quantified the regulatory effects of transcriptional and translational inhibitors on cMET mRNA and cMET protein in cell populations. We studied the dynamic responses of individual cells to drug treatments, by measuring cMET overexpression levels in individual non-small cell lung cancer (NSCLC) cells with induced drug resistance. Across NSCLC cell lines with a given protein expression, distinct patterns of transcript-protein correlation emerged. We believe this platform is applicable for interrogating the dynamics of gene expression, protein expression, and translational kinetics at the single-cell level - a paradigm shift in life science and medicine toward discovering vital cell regulatory mechanisms. PMID:27546183

  15. Single-cell detection of mRNA expression using nanofountain-probe electroporated molecular beacons.

    PubMed

    Giraldo-Vela, Juan P; Kang, Wonmo; McNaughton, Rebecca L; Zhang, Xuemei; Wile, Brian M; Tsourkas, Andrew; Bao, Gang; Espinosa, Horacio D

    2015-05-01

    New techniques for single-cell analysis enable new discoveries in gene expression and systems biology. Time-dependent measurements on individual cells are necessary, yet the common single-cell analysis techniques used today require lysing the cell, suspending the cell, or long incubation times for transfection, thereby interfering with the ability to track an individual cell over time. Here a method for detecting mRNA expression in live single cells using molecular beacons that are transfected into single cells by means of nanofountain probe electroporation (NFP-E) is presented. Molecular beacons are oligonucleotides that emit fluorescence upon binding to an mRNA target, rendering them useful for spatial and temporal studies of live cells. The NFP-E is used to transfect a DNA-based beacon that detects glyceraldehyde 3-phosphate dehydrogenase and an RNA-based beacon that detects a sequence cloned in the green fluorescence protein mRNA. It is shown that imaging analysis of transfection and mRNA detection can be performed within seconds after electroporation and without disturbing adhered cells. In addition, it is shown that time-dependent detection of mRNA expression is feasible by transfecting the same single cell at different time points. This technique will be particularly useful for studies of cell differentiation, where several measurements of mRNA expression are required over time.

  16. Oxidative damage induced by copper in mouse primary hepatocytes by single-cell analysis.

    PubMed

    Jing, Mingyang; Liu, Yang; Song, Wei; Yan, Yunxing; Yan, Wenbao; Liu, Rutao

    2016-01-01

    Copper can disturb the intracellular redox balance, induce oxidative stress, and subsequently cause irreversible damage, leading to a variety of diseases. In the present study, mouse primary hepatocytes were chosen to elucidate the in vitro oxidative damage of short-term copper exposure (10-200 μM) by single-cell analysis. We evaluated the toxicity of copper by reactive oxygen species (ROS), glutathione (GSH), and oxidative DNA damage at the single-cell level. Oxidative damage induced by copper was verified by the morphological changes, persistent elevations of excessive ROS and malondialdehyde (MDA), a decrease in GSH level, and the oxidative DNA damage. Furthermore, the average ROS generation, GSH consumption, and the indicators in DNA damage did not significantly change at relatively low concentrations (10 or 50 μM), but we can find the alterations of parameters in some single cells clearly. Emphasis on the analysis of single cells is conducive to gain a better understanding on the toxicity of copper. This study will also complement studies on the environmental risk assessment of copper pollution.

  17. Limitations to the Measurement of Oxygen Concentrations by HRTEM Imposed by Surface Roughness

    SciTech Connect

    Lupini, Andrew R; Chisholm, Matthew F; van Benthem, Klaus; Allen, L. J.; Oxley, Mark P; Findlay, Scott D.; Varela del Arco, Maria; Pennycook, Stephen J

    2005-01-01

    In an article published in Microscopy and Microanalysis recently (Jia et al., 2004), it was claimed that aberration-corrected high resolution transmission electron microscopy (HRTEM) allows the quantitative measurement of oxygen concentrations in ceramic materials with atomic resolution. Similar claims have recently appeared elsewhere, based on images obtained through aberration correction (Jia et al., 2003; Jia & Urban, 2004) or very high voltages (Zhang et al., 2003). Seeing oxygen columns is a significant achievement of great importance (Spence, 2003) that will doubtlessly allow some exciting new science; however, other models could provide a better explanation for some of the experimental data than variations in the oxygen concentration. Quantification of the oxygen concentrations was attempted by comparing experimental images with simulations in which the fractional occupancy in individual oxygen columns was reduced. The results were interpreted as representing nonstoichiometry within the bulk and at grain boundaries. This is plausible because previous studies have shown that grain boundaries can be nonstoichiometric (Kim et al., 2001), and it is indeed possible that oxygen vacancies are present at boundaries or in the bulk. However, is this the only possible interpretation? We show that for the thicknesses considered a better match to the images is obtained using a simple model of surface damage in which atoms are removed from the surface, which would usually be interpreted as surface damage or local thickness variation (from ion milling, for example).

  18. Measurement of atomic oxygen and related airglows in the lower thermosphere

    NASA Technical Reports Server (NTRS)

    Thomas, R. J.; Young, R. A.

    1981-01-01

    Instruments on board a sounding rocket were used to make simultaneous observations of atomic oxygen density and airglow emissions between 80 and 120 km. Atomic oxygen was measured with a resonance lamp and was found to have a peak density of 6 x 10 to the 11th at 94 km. Similar structure is seen in the oxygen density profile on both uplegs and downlegs. The following airglow emissions were measured by using vertical-viewing photometers: Herzberg I bands near 300 nm; O(1S) green line at 557.7 nm; background at 566 nm; O2(1 Delta g) bands at 1.27 microns; and OH (X 2 pi) Meinel bands near 1.7 microns.

  19. Quantifying the effect of electric current on cell adhesion studied by single-cell force spectroscopy.

    PubMed

    Jaatinen, Leena; Young, Eleanore; Hyttinen, Jari; Vörös, János; Zambelli, Tomaso; Demkó, László

    2016-03-20

    This study presents the effect of external electric current on the cell adhesive and mechanical properties of the C2C12 mouse myoblast cell line. Changes in cell morphology, viability, cytoskeleton, and focal adhesion structure were studied by standard staining protocols, while single-cell force spectroscopy based on the fluidic force microscopy technology provided a rapid, serial quantification and detailed analysis of cell adhesion and its dynamics. The setup allowed measurements of adhesion forces up to the μN range, and total detachment distances over 40 μm. Force-distance curves have been fitted with a simple elastic model including a cell detachment protocol in order to estimate the Young's modulus of the cells, as well as to reveal changes in the dynamic properties as functions of the applied current dose. While the cell spreading area decreased monotonously with increasing current doses, small current doses resulted only in differences related to cell elasticity. Current doses above 11 As/m(2), however, initiated more drastic changes in cell morphology, viability, cellular structure, as well as in properties related to cell adhesion. The observed differences, eventually leading to cell death toward higher doses, might originate from both the decrease in pH and the generation of reactive oxygen species.

  20. Measurement of atomic oxygen in the middle atmosphere using solid electrolyte sensors and catalytic probes

    NASA Astrophysics Data System (ADS)

    Eberhart, M.; Löhle, S.; Steinbeck, A.; Binder, T.; Fasoulas, S.

    2015-09-01

    The middle- and upper-atmospheric energy budget is largely dominated by reactions involving atomic oxygen (O). Modeling of these processes requires detailed knowledge about the distribution of this oxygen species. Understanding the mutual contributions of atomic oxygen and wave motions to the atmospheric heating is the main goal of the rocket project WADIS (WAve propagation and DISsipation in the middle atmosphere). It includes, amongst others, our instruments for the measurement of atomic oxygen that have both been developed with the aim of resolving density variations on small vertical scales along the trajectory. In this paper the instrument based on catalytic effects (PHLUX: Pyrometric Heat Flux Experiment) is introduced briefly. The experiment employing solid electrolyte sensors (FIPEX: Flux φ(Phi) Probe Experiment) is presented in detail. These sensors were laboratory calibrated using a microwave plasma as a source of atomic oxygen in combination with mass spectrometer reference measurements. The spectrometer was in turn calibrated for O with a method based on methane. In order to get insight into the horizontal variability, the rocket payload had instrument decks at both ends. Each housed several sensor heads measuring during both the up- and downleg of the trajectory. The WADIS project comprises two rocket flights during different geophysical conditions. Results from WADIS-1 are presented, which was successfully launched in June 2013 from the Andøya Space Center, Norway. FIPEX data were sampled at 100 Hz and yield atomic oxygen density profiles with a vertical resolution better than 9 m. This allows density variations to be studied on very small spatial scales. Numerical simulations of the flow field around the rocket were done at several points of the trajectory to assess the influence of aerodynamic effects on the measurement results. Density profiles peak at 3 × 1010 cm-3 at altitudes of 93.6 and 96 km for the up- and downleg, respectively.

  1. Single-Cell Approaches: Pandora's Box of Developmental Mechanisms.

    PubMed

    Hadjantonakis, Anna-Katerina; Arias, Alfonso Martinez

    2016-09-26

    Single-cell approaches are providing a new lexicon of developmental cell biology by revealing heterogeneities in seemingly uniform cellular populations. By bridging scales, single-cell approaches should, in principle, galvanize our understanding of how individual cells adopt distinct fates as they build complex tissues. PMID:27676428

  2. 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.

  3. Direct Measurement of Oxygen Incorporation into Thin Film Oxides at Room Temperature Upon Ultraviolet Phton Irradiation

    SciTech Connect

    Tsuchiya, Masaru; Shutthanandan, Vaithiyalingam; Engelhard, Mark H.; Ramanathan, Shriram

    2008-12-31

    Minute changes in oxygen concentration in complex oxides even of the order of ~0.001% can significantly influence functional properties ranging from onset of superconductivity to colossal dielectric constant and ferroic response. In this letter, we report on direct experimental measurement of enhanced oxygen incorporation into ultra-thin oxide films at room temperature under gentle UV photon exposure. Oxygen concentration changes in nanoscale yttria-doped-zirconia (YDZ) films grown on Ge substrate were quantified using the 16O(d,p)17O nuclear reaction. The oxygen concentration was consistently ~ 3 % larger in UV irradiated YDZ films compared to as-grown YDZ films and can be kinetically controlled. Possible incorporation mechanisms are discussed. This suggests a novel approach to modulate oxygen concentration in complex oxides. There is tremendous interest in the science and applications of ultra-thin oxide films, such as electrolyte membranes for solid oxide fuel cells 1, high-dielectric constant (high-κ) oxides for metal-oxide-semiconductor (MOS) devices 2 and multi-ferroics 3. In addition, thin film oxides also serve as model systems to investigate space charge effects on carrier transport and strongly correlated phenomena such as phase transitions. An overarching problem of central importance is the controlled synthesis of oxide films and how they impact functional properties. Particularly, the role of oxygen vacancies or non-stoichiometry has been found to be crucial in this regard. Examples include large magneto resistance effect and metal-to-insulator transition introduced by reducing oxygen stoichiometry of poly crystalline La0.67Ba0.33MnOz 3, nonsuperconducting-to-superconducting transformation by minute amount of oxygen incorporation upon annealing YBa2Cu3O7-δ(YBCO) films 4, and blue light emission at room temperature in oxygen deficient SrTiO3 (STO) 5, 6. These studies revealed

  4. Electrochemical Technology for Oxygen Removal and Measurement in the CELSS Test Facility, Engineering Development Unit

    NASA Technical Reports Server (NTRS)

    Drews, Michael E.; Covington, Al (Technical Monitor)

    1994-01-01

    , the amount of oxygen that is removed from the EDU is directly proportional to the cell input current via Faraday's constant, potentially allowing for a mol/electron measurement of photosynthetic rate. The currently operative oxygen removal system has maintained reduced oxygen set points within the EDU, and preparation is underway to verify of the accuracy of electrochemical measurement of oxygen production and hence, photosynthesis. This paper examines the working principles of the electrochemical cell, outlines the overall design of the oxygen removal system and its integration with other EDU subsystems, and summarizes test results obtained over crop growth cycles in the CTF-EDU.

  5. An irradiation system for photodynamic therapy with a fiber-optic sensor for measuring tissue oxygen

    NASA Astrophysics Data System (ADS)

    Quintanar, L.; Fabila, D.; Stolik, S.; de la Rosa, J. M.

    2013-11-01

    Photodynamic Therapy is a well known treatment based on the interaction of light of specific wavelength with a photosensitizing drug. In the presence of oxygen molecules, the illumination of the photosensitizer can activate the production of reactive oxygen species, which leads to the death of target cells within the treated tissue. In order to obtain the best therapy response, the tissue oxygen concentration should be measured to adjust the therapy parameters before and during the treatment. In this work, an irradiation system for 5-Aminolevulinic Acid Photodynamic Therapy is presented. It allows the application of visible light radiation of 630 nm using as a light source a high-brightness light emitting diode with an optical-power automatic control considering a light depth-distribution model. A module to measure the tissue oxygen saturation has been implemented into the system. It is based on two light emitting diodes of 660 nm and 940 nm as light sources, a photodiode as a detector and a new handheld fiber optic reflectance pulse oximetry sensor for estimating the blood oxygen saturation within the tissue. The pulse oximetry sensor was modeled through multilayered Monte Carlo simulations to study the behavior of the sensor with changes in skin thickness and melanin content.

  6. Instrument for stable high temperature Seebeck coefficient and resistivity measurements under controlled oxygen partial pressure

    DOE PAGESBeta

    Ihlefeld, Jon F.; Brown-Shaklee, Harlan James; Sharma, Peter Anand

    2015-04-28

    The transport properties of ceramic materials strongly depend on oxygen activity, which is tuned by changing the partial oxygen pressure (pO2) prior to and during measurement. Within, we describe an instrument for highly stable measurements of Seebeck coefficient and electrical resistivity at temperatures up to 1300 K with controlled oxygen partial pressure. An all platinum construction is used to avoid potential materials instabilities that can cause measurement drift. Two independent heaters are employed to establish a small temperature gradient for Seebeck measurements, while keeping the average temperature constant and avoiding errors associated with pO2-induced drifts in thermocouple readings. Oxygen equilibriummore » is monitored using both an O2 sensor and the transient behavior of the resistance as a proxy. A pO2 range of 10-25–100 atm can be established with appropriate gas mixtures. Seebeck measurements were calibrated against a high purity platinum wire, Pt/Pt–Rh thermocouple wire, and a Bi2Te3 Seebeck coefficient Standard Reference Material. To demonstrate the utility of this instrument for oxide materials we present measurements as a function of pO2 on a 1 % Nb-doped SrTiO3 single crystal, and show systematic changes in properties consistent with oxygen vacancy defect chemistry. Thus, an approximately 11% increase in power factor over a pO2 range of 10-19–10-8 atm at 973 K for the donor-doped single crystals is observed.« less

  7. Instrument for stable high temperature Seebeck coefficient and resistivity measurements under controlled oxygen partial pressure

    SciTech Connect

    Ihlefeld, Jon F.; Brown-Shaklee, Harlan James; Sharma, Peter Anand

    2015-04-28

    The transport properties of ceramic materials strongly depend on oxygen activity, which is tuned by changing the partial oxygen pressure (pO2) prior to and during measurement. Within, we describe an instrument for highly stable measurements of Seebeck coefficient and electrical resistivity at temperatures up to 1300 K with controlled oxygen partial pressure. An all platinum construction is used to avoid potential materials instabilities that can cause measurement drift. Two independent heaters are employed to establish a small temperature gradient for Seebeck measurements, while keeping the average temperature constant and avoiding errors associated with pO2-induced drifts in thermocouple readings. Oxygen equilibrium is monitored using both an O2 sensor and the transient behavior of the resistance as a proxy. A pO2 range of 10-25–100 atm can be established with appropriate gas mixtures. Seebeck measurements were calibrated against a high purity platinum wire, Pt/Pt–Rh thermocouple wire, and a Bi2Te3 Seebeck coefficient Standard Reference Material. To demonstrate the utility of this instrument for oxide materials we present measurements as a function of pO2 on a 1 % Nb-doped SrTiO3 single crystal, and show systematic changes in properties consistent with oxygen vacancy defect chemistry. Thus, an approximately 11% increase in power factor over a pO2 range of 10-19–10-8 atm at 973 K for the donor-doped single crystals is observed.

  8. RELATIONSHIPS BETWEEN NEAR-BOTTOM DISSOLVED OXYGEN AND SEDIMENT PROFILE CAMERA MEASUREMENTS

    EPA Science Inventory

    The United States Environmental Protection Agency (U.S. EPA) and other environmental authorities regulate concentrations of dissolved oxygen (DO) as a measure of nutrient-related eutrophication in estuarine and coastal waters. However, in situ DO concentrations are extremely var...

  9. Atmospheric Airborne Pressure Measurements using the Oxygen A Band for the ASCENDS Mission

    NASA Astrophysics Data System (ADS)

    Riris, H.; Rodriguez, M.

    2014-12-01

    We report on an airborne demonstration of atmospheric oxygen optical depth measurements with an Integrated Path Differential Absorption (IPDA) lidar using a fiber-based laser system and a photon counting detector. Accurate knowledge of atmospheric temperature and pressure is required for NASA's Active Sensing of CO2 Emissions over Nights, Days and Seasons (ASCENDS) space mission, and climate modeling studies. The lidar uses a doubled Erbium Doped Fiber amplifier and single photon counting detector to measure oxygen absorption at 765 nm. Our approach uses a sequence of laser pulses at increasing wavelengths that sample a pair of absorption lines in the Oxygen A-band at 764.7 nm. The O2 lines were selected after careful spectroscopic analysis to minimize the O2 line temperature dependence and the availability of the transmitter and receiver technology to maximize transmitter power, doubling efficiency, and detector sensitivity. We compare our 2013 and 2014 Oxygen IPDA lidar measurements and evaluate the impact of receiver dynamic range, transmitter stability and signal to noise ratio on the differential optical depth measurements.

  10. 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.

  11. New Approach to Investigate the Cytotoxicity of Nanomaterials Using Single Cell Mechanics

    PubMed Central

    2015-01-01

    Current in vitro methods to assess nanomaterial cytotoxicity involve various assays to monitor specific cellular dysfunction, such as metabolic imbalance or inflammation. Although high throughput, fast, and animal-free, these in vitro methods suffer from unreliability and lack of relevance to in vivo situations. New approaches, especially with the potential to reliably relate to in vivo studies directly, are in critical need. This work introduces a new approach, single cell mechanics, derived from atomic force microscopy-based single cell compression. The single cell based approach is intrinsically advantageous in terms of being able to directly correlate to in vivo investigations. Its reliability and potential to measure cytotoxicity is evaluated using known systems: zinc oxide (ZnO) and silicon dioxide (SiO2) nanoparticles (NP) on human aortic endothelial cells (HAECs). This investigation clearly indicates the reliability of single cell compression. For example, ZnO NPs cause significant changes in force vs relative deformation profiles, whereas SiO2 NPs do not. New insights into NPs–cell interactions pertaining to cytotoxicity are also revealed from this single cell mechanics approach, in addition to a qualitative cytotoxicity conclusion. The advantages and disadvantages of this approach are also compared with conventional cytotoxicity assays. PMID:24417356

  12. Mixture models for single-cell assays with applications to vaccine studies.

    PubMed

    Finak, Greg; McDavid, Andrew; Chattopadhyay, Pratip; Dominguez, Maria; De Rosa, Steve; Roederer, Mario; Gottardo, Raphael

    2014-01-01

    Blood and tissue are composed of many functionally distinct cell subsets. In immunological studies, these can be measured accurately only using single-cell assays. The characterization of these small cell subsets is crucial to decipher system-level biological changes. For this reason, an increasing number of studies rely on assays that provide single-cell measurements of multiple genes and proteins from bulk cell samples. A common problem in the analysis of such data is to identify biomarkers (or combinations of biomarkers) that are differentially expressed between two biological conditions (e.g. before/after stimulation), where expression is defined as the proportion of cells expressing that biomarker (or biomarker combination) in the cell subset(s) of interest. Here, we present a Bayesian hierarchical framework based on a beta-binomial mixture model for testing for differential biomarker expression using single-cell assays. Our model allows the inference to be subject specific, as is typically required when assessing vaccine responses, while borrowing strength across subjects through common prior distributions. We propose two approaches for parameter estimation: an empirical-Bayes approach using an Expectation-Maximization algorithm and a fully Bayesian one based on a Markov chain Monte Carlo algorithm. We compare our method against classical approaches for single-cell assays including Fisher's exact test, a likelihood ratio test, and basic log-fold changes. Using several experimental assays measuring proteins or genes at single-cell level and simulations, we show that our method has higher sensitivity and specificity than alternative methods. Additional simulations show that our framework is also robust to model misspecification. Finally, we demonstrate how our approach can be extended to testing multivariate differential expression across multiple biomarker combinations using a Dirichlet-multinomial model and illustrate this approach using single-cell gene

  13. A Fiber Optic Catalytic Sensor for Neutral Atom Measurements in Oxygen Plasma

    PubMed Central

    Zaplotnik, Rok; Vesel, Alenka; Mozetic, Miran

    2012-01-01

    The presented sensor for neutral oxygen atom measurement in oxygen plasma is a catalytic probe which uses fiber optics and infrared detection system to measure the gray body radiation of the catalyst. The density of neutral atoms can be determined from the temperature curve of the probe, because the catalyst is heated predominantly by the dissipation of energy caused by the heterogeneous surface recombination of neutral atoms. The advantages of this sensor are that it is simple, reliable, easy to use, noninvasive, quantitative and can be used in plasma discharge regions. By using different catalyst materials the sensor can also be applied for detection of neutral atoms in other plasmas. Sensor design, operation, example measurements and new measurement procedure for systematic characterization are presented. PMID:22666005

  14. Measurement of cell respiration and oxygenation in standard multichannel biochips using phosphorescent O2-sensitive probes.

    PubMed

    Kondrashina, Alina V; Papkovsky, Dmitri B; Dmitriev, Ruslan I

    2013-09-01

    Measurement of cell oxygenation and oxygen consumption is useful for studies of cell bioenergetics, metabolism, mitochondrial function, drug toxicity and common pathophysiological conditions. Here we present a new platform for such applications which uses commercial multichannel biochips (μ-slides, Ibidi) and phosphorescent O2 sensitive probes. This platform was evaluated with both extracellular and intracellular O2 probes, several different cell types and treatments including mitochondrial uncoupling and inhibition, depletion of extracellular Ca(2+) and inhibition of V-ATPase and histone deacetylases. The results show that compared to the standard microwell plates currently used, the μ-slide platform provides facile O2 measurements with both suspension and adherent cells, higher sensitivity and reproducibility, and faster measurement time. It also allows re-perfusion and multiple treatments of cells and multi-parametric analyses in conjunction with other probes. Optical measurements are conducted on standard fluorescence readers and microscopes.

  15. Magnetic susceptibility measurement of solid oxygen at pressures up to 3.3 GPa

    SciTech Connect

    Mito, M. Yamaguchi, S.; Tsuruda, H.; Deguchi, H.; Ishizuka, M.

    2014-01-07

    The magnetic susceptibility of solid oxygen had long been observed only in the restricted pressure region below 0.8 GPa. We succeeded in extending the pressure region up to 3.3 GPa by clamping condensed oxygen in the sample chamber of a miniature diamond anvil cell and measuring the dc magnetic susceptibility using a superconducting quantum interference device magnetometer. In this experiment, the well-known α–β and β–γ transitions are observed in the phase diagram, suggesting consistency with the previous results of X-ray and Raman studies. In addition, a new magnetic anomaly is observed in the β phase.

  16. Multiplexed, targeted profiling of single-cell proteomes and transcriptomes in a single reaction.

    PubMed

    Genshaft, Alex S; Li, Shuqiang; Gallant, Caroline J; Darmanis, Spyros; Prakadan, Sanjay M; Ziegler, Carly G K; Lundberg, Martin; Fredriksson, Simon; Hong, Joyce; Regev, Aviv; Livak, Kenneth J; Landegren, Ulf; Shalek, Alex K

    2016-01-01

    We present a scalable, integrated strategy for coupled protein and RNA detection from single cells. Our approach leverages the DNA polymerase activity of reverse transcriptase to simultaneously perform proximity extension assays and complementary DNA synthesis in the same reaction. Using the Fluidigm C1™ system, we profile the transcriptomic and proteomic response of a human breast adenocarcinoma cell line to a chemical perturbation, benchmarking against in situ hybridizations and immunofluorescence staining, as well as recombinant proteins, ERCC Spike-Ins, and population lysate dilutions. Through supervised and unsupervised analyses, we demonstrate synergies enabled by simultaneous measurement of single-cell protein and RNA abundances. Collectively, our generalizable approach highlights the potential for molecular metadata to inform highly-multiplexed single-cell analyses. PMID:27640647

  17. The workflow of single-cell expression profiling using quantitative real-time PCR

    PubMed Central

    Ståhlberg, Anders; Kubista, Mikael

    2014-01-01

    Biological material is heterogeneous and when exposed to stimuli the various cells present respond differently. Much of the complexity can be eliminated by disintegrating the sample, studying the cells one by one. Single-cell profiling reveals responses that go unnoticed when classical samples are studied. New cell types and cell subtypes may be found and relevant pathways and expression networks can be identified. The most powerful technique for single-cell expression profiling is currently quantitative reverse transcription real-time PCR (RT-qPCR). A robust RT-qPCR workflow for highly sensitive and specific measurements in high-throughput and a reasonable degree of multiplexing has been developed for targeting mRNAs, but also microRNAs, non-coding RNAs and most recently also proteins. We review the current state of the art of single-cell expression profiling and present also the improvements and developments expected in the next 5 years. PMID:24649819

  18. Microfabricated Collector-Generator Electrode Sensor for Measuring Absolute pH and Oxygen Concentrations.

    PubMed

    Dengler, Adam K; Wightman, R Mark; McCarty, Gregory S

    2015-10-20

    Fast-scan cyclic voltammetry (FSCV) has attracted attention for studying in vivo neurotransmission due to its subsecond temporal resolution, selectivity, and sensitivity. Traditional FSCV measurements use background subtraction to isolate changes in the local electrochemical environment, providing detailed information on fluctuations in the concentration of electroactive species. This background subtraction removes information about constant or slowly changing concentrations. However, determination of background concentrations is still important for understanding functioning brain tissue. For example, neural activity is known to consume oxygen and produce carbon dioxide which affects local levels of oxygen and pH. Here, we present a microfabricated microelectrode array which uses FSCV to detect the absolute levels of oxygen and pH in vitro. The sensor is a collector-generator electrode array with carbon microelectrodes spaced 5 μm apart. In this work, a periodic potential step is applied at the generator producing transient local changes in the electrochemical environment. The collector electrode continuously performs FSCV enabling these induced changes in concentration to be recorded with the sensitivity and selectivity of FSCV. A negative potential step applied at the generator produces a transient local pH shift at the collector. The generator-induced pH signal is detected using FSCV at the collector and correlated to absolute solution pH by postcalibration of the anodic peak position. In addition, in oxygenated solutions a negative potential step at the generator produces hydrogen peroxide by reducing oxygen. Hydrogen peroxide is detected with FSCV at the collector electrode, and the magnitude of the oxidative peak is proportional to absolute oxygen concentrations. Oxygen interference on the pH signal is minimal and can be accounted for with a postcalibration.

  19. Measurements of tissue oxygen tension in vascularised jejunal autografts in pigs.

    PubMed

    Nielsen, I M; Riis, A; Jahn, H; Gottrup, F

    1995-12-01

    Tissue oxygen measurements were evaluated as a monitor of the jejunal flap in seven female landrace pigs. A small polarographic sensor (diameter 0.55 mm) was used in which interstitial tissue oxygen tension was measured continuously in a jejunal flap and a muscle flap (rectus abdominis) during arterial and venous occlusion. Mean (SEM) tissue oxygen tension in the two types of flap were 44(9) mmHg (jejunal flap) and 47(8) mmHg (rectus flap). After arterial occlusion for 30 minutes the values dropped to 17(4) mmHg for the jejunal flap and 12(2) mmHg for the muscle flap. The decline became significant after five minutes. During venous occlusion (30 minutes) the values fell to 20(4) mmHg and 14(1) mmHg. The arterial occlusion was undetectable by the naked eye, but the enteric tissue after venous occlusion became severely congested and blue-black in colour. The condition returned to normal after release of the clamp. We conclude that direct measurement of tissue oxygen tension in a jejunal flap is a reliable method of detecting impaired perfusion. This method may in the future be used to monitor vascularised jejunal autografts. PMID:8771255

  20. Noninvasive Measurement of Microvascular and Interstitial Oxygen Profiles in a Human Tumor in SCID Mice

    NASA Astrophysics Data System (ADS)

    Torres Filho, Ivo P.; Leunig, Michael; Yuan, Fan; Intaglietta, Marcos; Jain, Rakesh K.

    1994-03-01

    Simultaneous measurements of intravascular and interstitial oxygen partial pressure (Po_2) in any tissue have not previously been reported, despite the importance of oxygen in health and in disease. This is due to the limitations of current techniques, both invasive and noninvasive. We have optically measured microscopic profiles of Po_2 with high spatial resolution in subcutaneous tissue and transplanted tumors in mice by combining an oxygen-dependent phosphorescence quenching method and a transparent tissue preparation. The strengths of our approach include the ability to follow Po_2 in the same location for several weeks and to relate these measurements to local blood flow and vascular architecture. Our results show that (i) Po_2 values in blood vessels in well-vascularized regions of a human colon adenocarcinoma xenograft are comparable to those in surrounding arterioles and venules, (ii) carbogen (95% O_2/5% CO_2) breathing increases microvascular Po_2 in tumors, and (iii) in unanesthetized and anesthetized mice Po_2 drops to hypoxic values at <200 μm from isolated vessels but drops by <5 mmHg (1 mmHg = 133 Pa) in highly vascularized tumor regions. Our method should permit noninvasive evaluations of oxygen-modifying agents and offer further mechanistic information about tumor pathophysiology in tissue preparations where the surface of the tissue can be observed.

  1. Limitations of quantitative photoacoustic measurements of blood oxygenation in small vessels.

    PubMed

    Sivaramakrishnan, Mathangi; Maslov, Konstantin; Zhang, Hao F; Stoica, George; Wang, Lihong V

    2007-03-01

    We investigate the feasibility of obtaining accurate quantitative information, such as local blood oxygenation level (sO2), with a spatial resolution of about 50 microm from spectral photoacoustic (PA) measurements. The optical wavelength dependence of the peak values of the PA signals is utilized to obtain the local blood oxygenation level. In our in vitro experimental models, the PA signal amplitude is found to be linearly proportional to the blood optical absorption coefficient when using ultrasonic transducers with central frequencies high enough such that the ultrasonic wavelengths are shorter than the light penetration depth into the blood vessels. For an optical wavelength in the 578-596 nm region, with a transducer central frequency that is above 25 MHz, the sensitivity and accuracy of sO2 inversion is shown to be better than 4%. The effect of the transducer focal position on the accuracy of quantifying blood oxygenation is found to be negligible. In vivo oxygenation measurements of rat skin microvasculature yield results consistent with those from in vitro studies, although factors specific to in vivo measurements, such as the spectral dependence of tissue optical attenuation, dramatically affect the accuracy of sO2 quantification in vivo.

  2. Embedded silver PDMS electrodes for single cell electrical impedance spectroscopy

    NASA Astrophysics Data System (ADS)

    Wei, Yuan; Xu, Zhensong; Cachia, Mark A.; Nguyen, John; Zheng, Yi; Wang, Chen; Sun, Yu

    2016-09-01

    This paper presents a microfluidic device with wide channels and embedded AgPDMS electrodes for measuring the electrical properties of single cells. The work demonstrates the feasibility of using a large channel design and embedded electrodes for impedance spectroscopy to circumvent issues such as channel clogging and limited device re-usability. AgPDMS electrodes were formed on channel sidewalls for impedance detection and cell electrical properties measurement. Equivalent circuit models were used to interpret multi-frequency impedance data to quantify each cell’s cytoplasm conductivity and specific membrane capacitance. T24 cells were tested to validate the microfluidic system and modeling results. Comparisons were then made by measuring two leukemia cell lines (AML-2 and HL-60) which were found to have different cytoplasm conductivity values (0.29  ±  0.15 S m-1 versus 0.47  ±  0.20 S m-1) and specific membrane capacitance values (41  ±  25 mF m-2 versus 55  ±  26 mF m-2) when the cells were flown through the wide channel and measured by the AgPDMS electrodes.

  3. Embedded silver PDMS electrodes for single cell electrical impedance spectroscopy

    NASA Astrophysics Data System (ADS)

    Wei, Yuan; Xu, Zhensong; Cachia, Mark A.; Nguyen, John; Zheng, Yi; Wang, Chen; Sun, Yu

    2016-09-01

    This paper presents a microfluidic device with wide channels and embedded AgPDMS electrodes for measuring the electrical properties of single cells. The work demonstrates the feasibility of using a large channel design and embedded electrodes for impedance spectroscopy to circumvent issues such as channel clogging and limited device re-usability. AgPDMS electrodes were formed on channel sidewalls for impedance detection and cell electrical properties measurement. Equivalent circuit models were used to interpret multi-frequency impedance data to quantify each cell’s cytoplasm conductivity and specific membrane capacitance. T24 cells were tested to validate the microfluidic system and modeling results. Comparisons were then made by measuring two leukemia cell lines (AML-2 and HL-60) which were found to have different cytoplasm conductivity values (0.29  ±  0.15 S m‑1 versus 0.47  ±  0.20 S m‑1) and specific membrane capacitance values (41  ±  25 mF m‑2 versus 55  ±  26 mF m‑2) when the cells were flown through the wide channel and measured by the AgPDMS electrodes.

  4. Synchronizing stochastic circadian oscillators in single cells of Neurospora crassa

    PubMed Central

    Deng, Zhaojie; Arsenault, Sam; Caranica, Cristian; Griffith, James; Zhu, Taotao; Al-Omari, Ahmad; Schüttler, Heinz-Bernd; Arnold, Jonathan; Mao, Leidong

    2016-01-01

    The synchronization of stochastic coupled oscillators is a central problem in physics and an emerging problem in biology, particularly in the context of circadian rhythms. Most measurements on the biological clock are made at the macroscopic level of millions of cells. Here measurements are made on the oscillators in single cells of the model fungal system, Neurospora crassa, with droplet microfluidics and the use of a fluorescent recorder hooked up to a promoter on a clock controlled gene-2 (ccg-2). The oscillators of individual cells are stochastic with a period near 21 hours (h), and using a stochastic clock network ensemble fitted by Markov Chain Monte Carlo implemented on general-purpose graphical processing units (or GPGPUs) we estimated that >94% of the variation in ccg-2 expression was stochastic (as opposed to experimental error). To overcome this stochasticity at the macroscopic level, cells must synchronize their oscillators. Using a classic measure of similarity in cell trajectories within droplets, the intraclass correlation (ICC), the synchronization surface ICC is measured on >25,000 cells as a function of the number of neighboring cells within a droplet and of time. The synchronization surface provides evidence that cells communicate, and synchronization varies with genotype. PMID:27786253

  5. Distinguishing phosphate from fertilizers and wastewater treatment plant effluents in Western Canada using oxygen isotope measurements

    NASA Astrophysics Data System (ADS)

    Fau, Veronique; Nightingale, Michael; Tamburini, Frederica; Mayer, Bernhard

    2014-05-01

    The successful application of oxygen isotope ratios as a tracer for phosphate in aquatic ecosystems requires that different sources of phosphate are isotopically distinct. The objective of this study was to determine whether the oxygen isotope ratios of phosphate from fertilizers and effluents from wastewater treatment plants in Western Canada are isotopically distinct. Therefore, we carried out oxygen isotope analyses on phosphate in effluent from five different wastewater treatment plants (WWTP) in the Bow River watershed of Alberta, Canada. Samples were collected directly from the final effluent (post-UV) in Banff and Canmore upstream of Calgary, and from effluents of Calgary's WWTPs at Bonnybrook, Fish Creek and Pine Creek. We also carried out oxygen isotope analyses on a variety of phosphate-containing fertilizers that are widely used in Western Canada. Historically, most of the phosphate contained in manufactured fertilizers sold in Alberta came from two distinct deposits: 1) a weathered Pliocene igneous carbonatite located in eastern Canada, and 2) the Permian Phosphoria Formation in the western USA. Phosphate (PO43-) contained in the water or the fertilizer was concentrated and quantitatively converted to pure silver phosphate (Ag3PO4). The silver phosphate was then reduced with carbon in an oxygen free environment using a TC/EA pyrolysis reactor linked to a mass spectrometer where 18O/16O ratios of CO were measured in continuous flow mode. Preparation of samples for δ18OPO4 analyses was conducted using the Magnesium Induced Coprecipitation (MAGIC) method. Expected oxygen isotope ratios for phosphate in equilibrium with water (δ18Oeq) were calculated using the Longinelli and Nuti equation: T (° C) = 111.4 - 4.3 (δ18Oeq - δ18Owater). Measured δ18O values of phosphate for fertilizer samples varied from 8 to 25 oÈ®n average, fertilizer samples of sedimentary origin had higher δ18O values (15.8) than those of igneous origin (11.5). Phosphate isotopic

  6. Monte Carlo method for calculating oxygen abundances and their uncertainties from strong-line flux measurements

    NASA Astrophysics Data System (ADS)

    Bianco, F. B.; Modjaz, M.; Oh, S. M.; Fierroz, D.; Liu, Y. Q.; Kewley, L.; Graur, O.

    2016-07-01

    We present the open-source Python code pyMCZ that determines oxygen abundance and its distribution from strong emission lines in the standard metallicity calibrators, based on the original IDL code of Kewley and Dopita (2002) with updates from Kewley and Ellison (2008), and expanded to include more recently developed calibrators. The standard strong-line diagnostics have been used to estimate the oxygen abundance in the interstellar medium through various emission line ratios (referred to as indicators) in many areas of astrophysics, including galaxy evolution and supernova host galaxy studies. We introduce a Python implementation of these methods that, through Monte Carlo sampling, better characterizes the statistical oxygen abundance confidence region including the effect due to the propagation of observational uncertainties. These uncertainties are likely to dominate the error budget in the case of distant galaxies, hosts of cosmic explosions. Given line flux measurements and their uncertainties, our code produces synthetic distributions for the oxygen abundance in up to 15 metallicity calibrators simultaneously, as well as for E(B- V) , and estimates their median values and their 68% confidence regions. We provide the option of outputting the full Monte Carlo distributions, and their Kernel Density estimates. We test our code on emission line measurements from a sample of nearby supernova host galaxies (z < 0.15) and compare our metallicity results with those from previous methods. We show that our metallicity estimates are consistent with previous methods but yield smaller statistical uncertainties. It should be noted that systematic uncertainties are not taken into account. We also offer visualization tools to assess the spread of the oxygen abundance in the different calibrators, as well as the shape of the estimated oxygen abundance distribution in each calibrator, and develop robust metrics for determining the appropriate Monte Carlo sample size. The code

  7. Airborne Lidar Measurements of Atmospheric Pressure Made Using the Oxygen A-Band

    NASA Technical Reports Server (NTRS)

    Riris, Haris; Rodriquez, Michael D.; Allan, Graham R.; Hasselbrack, William E.; Mao, Jianping; Stephen, Mark A.; Abshire, James B.

    2012-01-01

    Accurate measurements of greenhouse gas mixing ratios on a global scale are currently needed to gain a better understanding of climate change and its possible impact on our planet. In order to remotely measure greenhouse gas concentrations in the atmosphere with regard to dry air, the air number density in the atmosphere is also needed in deriving the greenhouse gas concentrations. Since oxygen is stable and uniformly mixed in the atmosphere at 20.95%, the measurement of an oxygen absorption in the atmosphere can be used to infer the dry air density and used to calculate the dry air mixing ratio of a greenhouse gas, such as carbon dioxide or methane. OUT technique of measuring Oxygen uses integrated path differential absorption (IPDA) with an Erbium Doped Fiber Amplifier (EDF A) laser system and single photon counting module (SPCM). It measures the absorbance of several on- and off-line wavelengths tuned to an O2 absorption line in the A-band at 764.7 nm. The choice of wavelengths allows us to maximize the pressure sensitivity using the trough between two absorptions in the Oxygen A-band. Our retrieval algorithm uses ancillary meteorological and aircraft altitude information to fit the experimentally obtained lidar O2 line shapes to a model atmosphere and derives the pressure from the profiles of the two lines. We have demonstrated O2 measurements from the ground and from an airborne platform. In this paper we will report on our airborne measurements during our 2011 campaign for the ASCENDS program.

  8. Single cell deposition and patterning with a robotic system.

    PubMed

    Lu, Zhe; Moraes, Christopher; Ye, George; Simmons, Craig A; Sun, Yu

    2010-01-01

    Integrating single-cell manipulation techniques in traditional and emerging biological culture systems is challenging. Microfabricated devices for single cell studies in particular often require cells to be spatially positioned at specific culture sites on the device surface. This paper presents a robotic micromanipulation system for pick-and-place positioning of single cells. By integrating computer vision and motion control algorithms, the system visually tracks a cell in real time and controls multiple positioning devices simultaneously to accurately pick up a single cell, transfer it to a desired substrate, and deposit it at a specified location. A traditional glass micropipette is used, and whole- and partial-cell aspiration techniques are investigated to manipulate single cells. Partially aspirating cells resulted in an operation speed of 15 seconds per cell and a 95% success rate. In contrast, the whole-cell aspiration method required 30 seconds per cell and achieved a success rate of 80%. The broad applicability of this robotic manipulation technique is demonstrated using multiple cell types on traditional substrates and on open-top microfabricated devices, without requiring modifications to device designs. Furthermore, we used this serial deposition process in conjunction with an established parallel cell manipulation technique to improve the efficiency of single cell capture from ∼80% to 100%. Using a robotic micromanipulation system to position single cells on a substrate is demonstrated as an effective stand-alone or bolstering technology for single-cell studies, eliminating some of the drawbacks associated with standard single-cell handling and manipulation techniques.

  9. Correlation of oxygenation and perfusion sensitive MRI with invasive micro probe measurements in healthy mice brain.

    PubMed

    Sedlacik, Jan; Reitz, Matthias; Bolar, Divya S; Adalsteinsson, Elfar; Schmidt, Nils O; Fiehler, Jens

    2015-03-01

    The non-invasive assessment of (patho-)physiological parameters such as, perfusion and oxygenation, is of great importance for the characterization of pathologies e.g., tumors, which may be helpful to better predict treatment response and potential outcome. To better understand the influence of physiological parameters on the investigated oxygenation and perfusion sensitive MRI methods, MRI measurements were correlated with subsequent invasive micro probe measurements during free breathing conditions of air, air+10% CO2 and 100% O2 in healthy mice brain. MRI parameters were the irreversible (R2), reversible (R2') and effective (R2*) transverse relaxation rates, venous blood oxygenation level assessed by quantitative blood oxygenation level dependent (qBOLD) method and cerebral blood flow (CBF) assessed by arterial spin labeling (ASL) using a 7 T small animal MRI scanner. One to two days after MRI, tissue perfusion and pO2 were measured by Laser-Doppler flowmetry and fluorescence quenching micro probes, respectively. The tissue pO2 values were converted to blood oxygen saturation by using the Hill equation. The animals were anesthetized by intra peritoneal injection of ketamine-xylazine-acepromazine (10-2-0.3 mg/ml · kg). Results for normal/hypercapnia/hyperoxia conditions were: R2[s(∧)-1] = 20.7/20.4/20.1, R2*[s(∧)-1] = 31.6/29.6/25.9, R2'[s-(∧)1] = 10.9/9.2/5.7, qBOLD venous blood oxygenation level = 0.43/0.51/0.56, CBF[ml · min(∧)-1 · 100 g(∧)-1] = 70.6/105.5/81.8, Laser-Doppler flowmetry[a.u.] = 89.2/120.2/90.6 and pO2[mmHg] = 6.3/32.3/46.7. All parameters were statistically significantly different with P < 0.001 between all breathing conditions. All MRI and the corresponding micro probe measurements were also statistically significantly (P ≤ 0.03) correlated with each other. However, converting the tissue pO2 to blood oxygen saturation = 0.02/0.34/0.63, showed only very limited agreement with the qBOLD venous blood oxygenation level. We found

  10. 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

  11. Single-cell Transcriptome Study as Big Data.

    PubMed

    Yu, Pingjian; Lin, Wei

    2016-02-01

    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

  12. Near-infrared spectroscopy measurement of blood oxygenation content and its application in sports practice

    NASA Astrophysics Data System (ADS)

    Xu, Guodong; Gong, Hui; Ge, Xinfa; Luo, Qingming

    2003-12-01

    To research the change characteristics of blood oxygenation content in skeletal muscle, the change regularity between blood oxygenation content and exercise intensity as well as HbO2 and blood lactate acid while taking incremental exercises, we took an in vivo, real-time and continuous measurement on the blood oxygenation content of eight sportsmen when they did incremental exercises of five degrees on a power bicycle using a portable tissue oximeter which is based on the principle of near-infrared spectroscopy(NIRS), simultaneously, we detected the blood lactate acid of subjects after each degree of incremental physical load instantly using a blood lactate analysis equipment. The results showed that the content of HbO2 descended regularly while that of Hb ascended; blood volume decreased; and the density of lactate increased as the intensity of exercises was heightened. The statistics analyses showed that the relationship between HbO2 and blood lactate is rather close (correlation coefficient r=-0.918). With this discovery, a theoretical basis in measuring the relative change of blood oxygenation content non-invasively was evidenced, and a novel technology for assessing the physical situation of sportsman, grasping sports density and evaluating the training effect could be imported.

  13. Atomic oxygen dosimetry measurements made on STS-46 by CONCAP 2

    NASA Technical Reports Server (NTRS)

    Gregory, J. C.; Miller, G. P.; Pettigrew, P. J.; Raikar, G. N.; Cross, Jon B.; Lan, E.; Renschler, C. L.; Sutherland, W. T.

    1995-01-01

    With increasing flight duration and the possibility of a permanent facility in space, long-term monitoring of material degradation due to atomic oxygen is increasing in importance. Reliance on models to determine the fluence of atomic oxygen is not only necessarily complex but also imprecise due to the strong dependence of oxygen concentration on day/night, latitude and solar activity. Mass-spectroscopy, the traditional method for determining the gas phase species densities at low pressure, is not only expensive but is limited in the area that it can monitor. Our group has developed a simple and inexpensive dosimeter to measure the atomic oxygen fluence via the change in resistance as the sensor element is gradually oxidized. The sensors consisted of thin-film circuit elements deposited on a suitable substrate. Four-point resistance measurements were used to monitor the change in resistance. Results obtained using silver and carbon dosimeters flown on STS-46 (CONCAP 2-01) will be discussed.

  14. Real-time frequency-domain fiber optic sensor for intra-arterial blood oxygen measurements

    NASA Astrophysics Data System (ADS)

    Alcala, J. R.; Scott, Ian L.; Parker, Jennifer W.; Atwater, Beauford W.; Yu, Clement; Fischer, Russell; Bellingrath, K.

    1993-05-01

    A real time frequency domain phosphorimeter capable of measuring precise and accurate excited state lifetimes for determining oxygen is described. This frequency domain instrument does not make use of cross correlation techniques traditionally used in frequency domain fluorometers. Instead, the electrical signal from the detector is filtered to contain only the first several harmonics. This filtered signal is then sampled and averaged over a few thousand cycles. The absolute phase and absolute modulation of each sampled harmonic of the excitation and of the luminescence is computed by employing fast Fourier transform algorithms. The phase delay and the modulation ratio is then calculated at each harmonic frequency. A least squares fit is performed in the frequency domain to obtain the lifetimes of discrete exponentials. Oxygen concentrations are computed from these lifetimes. Prototypes based on these techniques were built employing commercially available components. Results from measurements in saline solution and in the arterial blood of dogs show that oxygen concentrations can be determined reproducibly. The system drift is less than 1% in over 100 hours of continuous operation. The performance of fiber optic sensors was evaluated in dogs over a period of 10 hours. The sensors tracked changes in arterial oxygen tension over the course of the experiment without instabilities. The overall response of the system was about 90 seconds. The update time was 3 seconds.

  15. Spatially monitoring oxygen level in 3D microfabricated cell culture systems using optical oxygen sensing beads.

    PubMed

    Wang, Lin; Acosta, Miguel A; Leach, Jennie B; Carrier, Rebecca L

    2013-04-21

    Capability of measuring and monitoring local oxygen concentration at the single cell level (tens of microns scale) is often desirable but difficult to achieve in cell culture. In this study, biocompatible oxygen sensing beads were prepared and tested for their potential for real-time monitoring and mapping of local oxygen concentration in 3D micro-patterned cell culture systems. Each oxygen sensing bead is composed of a silica core loaded with both an oxygen sensitive Ru(Ph2phen3)Cl2 dye and oxygen insensitive Nile blue reference dye, and a poly-dimethylsiloxane (PDMS) shell rendering biocompatibility. Human intestinal epithelial Caco-2 cells were cultivated on a series of PDMS and type I collagen based substrates patterned with micro-well arrays for 3 or 7 days, and then brought into contact with oxygen sensing beads. Using an image analysis algorithm to convert florescence intensity of beads to partial oxygen pressure in the culture system, tens of microns-size oxygen sensing beads enabled the spatial measurement of local oxygen concentration in the microfabricated system. Results generally indicated lower oxygen level inside wells than on top of wells, and local oxygen level dependence on structural features of cell culture surfaces. Interestingly, chemical composition of cell culture substrates also appeared to affect oxygen level, with type-I collagen based cell culture systems having lower oxygen concentration compared to PDMS based cell culture systems. In general, results suggest that oxygen sensing beads can be utilized to achieve real-time and local monitoring of micro-environment oxygen level in 3D microfabricated cell culture systems.

  16. Quantitative photoacoustic blood oxygenation measurement of whole porcine blood samples using a multi-wavelength semiconductor laser system

    NASA Astrophysics Data System (ADS)

    Friedrich, Claus-Stefan; Mienkina, Martin P.; Brenner, Carsten; Gerhardt, Nils C.; Jörger, Manfred; Strauß, Andreas; Beckmann, Martin F.; Schmitz, Georg; Hofmann, Martin R.

    2011-07-01

    We present a photoacoustic measurement system based on semiconductor lasers for blood oxygenation measurements. It permits to use four different optical wavelengths (650nm, 808nm, 850nm, 905nm) to generate photoacoustic signals. As the optical extinction coefficient of oxygenated hemoglobin and deoxygenated hemoglobin is different at specific wavelengths, a blood oxygenation measurement by a multi-wavelength photoacoustic laser system is feasible. Especially at 650nm, the clear difference between the extinction coefficients of the two hemoglobin derivates permits to determine the blood oxygenation in combination with other near infrared wavelengths. A linear model based on tabulated values of extinction coefficients for fully oxygenated and fully deoxygenated hemoglobin is presented. We used heparin stabilized whole porcine blood samples to model the optical behavior of human blood, as the optical absorption behavior of porcine hemoglobin does not differ significantly from human hemoglobin. To determine the real oxygen saturation values of the blood samples, we measured the partial oxygen pressure with an IRMA Trupoint Blood Analysis System. The oxygen saturation values were calculated from a dissociation curve for porcine blood. The results of the photoacoustic measurement are in qualitatively good agreement with the predicted linear model. Further, we analyze the abilities and the limitations of quantitative oxygenation measurements.

  17. Continuous tissue oxygen tension measurement as a monitor of free-flap viability.

    PubMed

    Hirigoyen, M B; Blackwell, K E; Zhang, W X; Silver, L; Weinberg, H; Urken, M L

    1997-03-01

    Early recognition of vascular compromise within microvascular free-tissue transfers is essential if reexploration is to prove successful. Tissue oxygen tension is increasingly recognized to be a sensitive and reliable index of tissue perfusion, and preliminary studies suggest that it may be of value in the assessment of free-flap viability. We describe our investigation into the application of an implantable microcatheter oxygen sensor in the monitoring of free flaps used in head and neck and extremity reconstruction. In a preliminary study using the rabbit model, we sought to evaluate the response of oxygen tension as an index of tissue perfusion in myocutaneous (n = 20) and osteomyocutaneous flaps (n = 5) under conditions of arterial and venous occlusion. A clinical study was then undertaken to evaluate the role of this method in the monitoring of surface and buried free flaps. In 30 heterogeneous free-tissue transfers, sensors placed intraoperatively were used to provide continuous information about flap oxygen tension (mean monitoring period 3.2 +/- 0.8 days). The data generated were correlated with changes in clinical parameters and routine flap observations. Results for experimental and clinical data have confirmed the efficacy of continuous tissue oxygen measurements using this device as a method that provides an objective, recordable index of free-tissue transfer viability in a variety of circumstances and vascular events. Tissue oxygen tension is a suitable index by which to evaluate flap viability with the probe placed in muscle or bone but is unreliable when used for the monitoring of revascularized cutaneous flaps. PMID:9047197

  18. Measurements and Analysis of Oxygen Bubble Distributions in LiCl-KCl Molten Salt

    SciTech Connect

    Ryan W. Bezzant; Supathorn Phongikaroon; Michael F. Simpson

    2013-03-01

    Transparent system experimental studies have been performed to provide measurement and analysis of oxygen bubble distributions and mass transfer coefficients at different sparging rates ranging from 0.05 to 0.20 L/min in LiCl-KCl molten salt at 500 degrees C using a high-speed digital camera and an oxygen sensor. The results reveal that bubble sizes and rise velocities increased with an increase in oxygen sparging rate. The bubbles observed were ellipsoidal in shape, and an equivalent diameter based on the ellipsoid volume was calculated. The average equivalent bubble diameters at 500 degrees C and these oxygen sparging rates range from 2.63 to 4.07 mm. Results show that the bubble equivalent diameters at each respective sparging rate are normally distributed. A Fanning friction factor correlation was produced to predict a bubble’s rise velocity based on its equivalent diameter. The oxygen mass transfer coefficients for four sparging rates were calculated using the oxygenation model. These calculated values were within the order of magnitude of 10-2 cm/sec and followed a decreasing trend corresponding to an increasing bubble size and sparging rate. The diffusivities were calculated based on two different types of mechanisms, one based on physics of the bubbles and the other on systematic properties. The results reveal that diffusivity values calculated from bubble physics are 1.65 to 8.40 x 10-5 cm2/sec, which are within the range suggested by literature for gases in liquids of a similar viscosity.

  19. Determining Physical Mechanisms of Gene Expression Regulation from Single Cell Gene Expression Data

    PubMed Central

    Moignard, Victoria; Göttgens, Berthold; Adryan, Boris

    2016-01-01

    Many genes are expressed in bursts, which can contribute to cell-to-cell heterogeneity. It is now possible to measure this heterogeneity with high throughput single cell gene expression assays (single cell qPCR and RNA-seq). These experimental approaches generate gene expression distributions which can be used to estimate the kinetic parameters of gene expression bursting, namely the rate that genes turn on, the rate that genes turn off, and the rate of transcription. We construct a complete pipeline for the analysis of single cell qPCR data that uses the mathematics behind bursty expression to develop more accurate and robust algorithms for analyzing the origin of heterogeneity in experimental samples, specifically an algorithm for clustering cells by their bursting behavior (Simulated Annealing for Bursty Expression Clustering, SABEC) and a statistical tool for comparing the kinetic parameters of bursty expression across populations of cells (Estimation of Parameter changes in Kinetics, EPiK). We applied these methods to hematopoiesis, including a new single cell dataset in which transcription factors (TFs) involved in the earliest branchpoint of blood differentiation were individually up- and down-regulated. We could identify two unique sub-populations within a seemingly homogenous group of hematopoietic stem cells. In addition, we could predict regulatory mechanisms controlling the expression levels of eighteen key hematopoietic transcription factors throughout differentiation. Detailed information about gene regulatory mechanisms can therefore be obtained simply from high throughput single cell gene expression data, which should be widely applicable given the rapid expansion of single cell genomics. PMID:27551778

  20. Reduced-Gravity Measurements of the Effect of Oxygen on Properties of Zirconium

    NASA Technical Reports Server (NTRS)

    Zhao, J.; Lee, J.; Wunderlich, R.; Fecht, H.-J.; Schneider, S.; SanSoucie, M.; Rogers, J.; Hyers, R.

    2016-01-01

    The influence of oxygen on the thermophysical properties of zirconium is being investigated using MSL-EML (Material Science Laboratory - Electromagnetic Levitator) on ISS (International Space Station) in collaboration with NASA, ESA (European Space Agency), and DLR (German Aerospace Center). Zirconium samples with different oxygen concentrations will be put into multiple melt cycles, during which the density, viscosity, surface tension, heat capacity, and electric conductivity will be measured at various undercooled temperatures. The facility check-up of MSL-EML and the first set of melting experiments have been successfully performed in 2015. The first zirconium sample will be tested near the end of 2015. As part of ground support activities, the thermophysical properties of zirconium and ZrO were measured using a ground-based electrostatic levitator located at the NASA Marshall Space Flight Center. The influence of oxygen on the measured surface tension was evaluated. The results of this research will serve as reference data for those measured in ISS.

  1. Non-destructive measurement of carbonic anhydrase activity and the oxygen isotope composition of soil water

    NASA Astrophysics Data System (ADS)

    Jones, Sam; Sauze, Joana; Ogée, Jérôme; Wohl, Steven; Bosc, Alexandre; Wingate, Lisa

    2016-04-01

    oxygen isotope composition of ambient CO2. This non-destructive approach was tested through laboratory incubations of air-dried soils that were re-wetted with water of known isotopic composition. Performance was assessed by comparing estimates of the soil water oxygen isotope composition derived from open chamber flux measurements with those measured in the irrigation water and soil water extracted following incubations. The influence of soil pH and bovine carbonic anhydrase additions on these estimates was also investigated. Coherent values were found between the soil water composition estimates obtained from the dual steady state approach and those measured for irrigation waters. Estimates of carbonic anhydrase activity made using this approach also reflected well artificial increases to the concentration of carbonic anhydrase and indicated that this activity was sensitive to soil pH.

  2. The measurement of oxygen fugacities in flowing gas mixtures at temperatures below 1200°C

    NASA Astrophysics Data System (ADS)

    Beckett, J. R.; Mendybaev, R. A.

    1997-10-01

    We measured oxygen fugacities in H 2CO 2 and COCO 2 gas mixtures in the temperature interval 700-1350°C using an yttria-stabilized zirconia (YSZ) oxygen sensor. At high temperatures in excess of 1200°C, measured emfs are consistent with expectations based on the gas composition. At lower temperatures in H 2CO 2 gas mixtures, the oxygen fugacity ( fO 2) obtained assuming Nernstian behavior of the oxygen sensor is as much as two log units more reducing (˜900°C) to one log unit more oxidizing (˜700°C) than expected by assuming equilibrium speciation. The deviations in H 2CO 2 gas mixtures arise from two sources: (1) poor contact between the electrode and the zirconia electrolyte, leading to apparent fO 2 values that are higher than expected and (2) disequilibrium in the vapor, leading to lower than expected fO 2 values in the temperature range ˜700-1200°C (for experiments near the iron-wustite (IW) buffer) and higher than expected fO 2 at lower temperatures. The first problem can be alleviated by spring-loading and lightly sintering a Pt mesh internal electrode against the electrolyte and the second by forcing the entire gas stream to equilibrate by passing it through a Pt catalyst. With these measures, experiments employing H 2CO 2 gas mixtures can be conducted routinely in the temperature range 700-1200°C and the fO 2 determined with an accuracy comparable to that obtained at higher temperatures (2σ < ±0.1 log units). Above ˜770°C, apparent oxygen fugacities measured using an oxygen sensor in COCO 2 gas mixtures near IW are consistent with equilibration in the vapor regardless of whether or not a Pt catalyst is present. At lower temperatures, however, the measured values are more oxidizing than the expected equilibrium values. Under more reducing conditions, the deviations begin to occur at even higher temperatures, ˜930°C for IW-3. The anomalously high fO 2 values are probably related to the condensation of graphite, which removes C

  3. Atmospheric Airborne Pressure Measurements Using the Oxygen A Band for the ASCENDS Mission

    NASA Astrophysics Data System (ADS)

    Rodriguez, M.; Riris, H.; Abshire, J. B.; Allan, G. R.; Stephen, M.; Hasselbrack, W.; Mao, J.

    2012-12-01

    We report on airborne atmospheric pressure measurements using fiber-based laser technology and the oxygen A-band at 765 nm. Remote atmospheric temperature and pressure measurements are needed for NASA's Active Sensing of CO2 Emissions Over Nights, Days, and Seasons (ASCENDS) mission. ASCENDS will measure atmospheric CO2 dry mixing ratios on a global scale. Remote atmospheric pressure measurements are necessary to normalize ASCENDS CO2 measurements. Our work, funded by the ESTO IIP program, uses erbium doped fiber optic amplifiers and non-linear optics technology to tune laser radiation over the Oxygen A-band between 764.5 nm and 765 nm. Surface reflections are fiber-coupled from a receiver telescope to photon counting detectors. Our pulsed, time gated approach resolves ground reflections from cloud returns. This system successfully recorded O2 absorption spectra during two airborne campaigns aboard a NASA DC-8. Airborne data has been analyzed and fitted to HITRAN reference spectra based upon aircraft meteorological data. Our algorithm linearly scales the HITRAN reference until measurement errors are minimized. Atmospheric pressure changes are estimated by comparing the differential optical depth of the optimum scaled HITRAN spectra to the differential optical depth of the nominal HITRAN spectra. On flights over gradually sloping terrain, these results compare favorably with ground-based observations and predictions from computer models. Measurement uncertainty is commensurate with photon counting noise. We plan to reduce measurement uncertainty in future campaigns by improving transmitter pulse energy and increasing wavelength sweep frequency.

  4. 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.

  5. Droplet microfluidics--a tool for single-cell analysis.

    PubMed

    Joensson, Haakan N; Andersson Svahn, Helene

    2012-12-01

    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.

  6. Computational and analytical challenges in single-cell transcriptomics.

    PubMed

    Stegle, Oliver; Teichmann, Sarah A; Marioni, John C

    2015-03-01

    The development of high-throughput RNA sequencing (RNA-seq) at the single-cell level has already led to profound new discoveries in biology, ranging from the identification of novel cell types to the study of global patterns of stochastic gene expression. Alongside the technological breakthroughs that have facilitated the large-scale generation of single-cell transcriptomic data, it is important to consider the specific computational and analytical challenges that still have to be overcome. Although some tools for analysing RNA-seq data from bulk cell populations can be readily applied to single-cell RNA-seq data, many new computational strategies are required to fully exploit this data type and to enable a comprehensive yet detailed study of gene expression at the single-cell level.

  7. Direct Measurements of Oxygen Gradients in Spheroid Culture System Using Electron Parametric Resonance Oximetry

    PubMed Central

    Langan, Laura M.; Dodd, Nicholas J. F.; Owen, Stewart F.; Purcell, Wendy M.; Jackson, Simon K.; Jha, Awadhesh N.

    2016-01-01

    Advanced in vitro culture from tissues of different origin includes three-dimensional (3D) organoid micro structures that may mimic conditions in vivo. One example of simple 3D culture is spheroids; ball shaped structures typically used as liver and tumour models. Oxygen is critically important in physiological processes, but is difficult to quantify in 3D culture: and the question arises, how small does a spheroid have to be to have minimal micro-environment formation? This question is of particular importance in the growing field of 3D based models for toxicological assessment. Here, we describe a simple non-invasive approach modified for the quantitative measurement and subsequent evaluation of oxygen gradients in spheroids developed from a non-malignant fish cell line (i.e. RTG-2 cells) using Electron Paramagnetic Resonance (EPR) oximetry. Sonication of the paramagnetic probe Lithium phthalocyanine (LiPc) allows for incorporation of probe particulates into spheroid during its formation. Spectra signal strength after incorporation of probe into spheroid indicated that a volume of 20 μl of probe (stock solution: 0.10 mg/mL) is sufficient to provide a strong spectra across a range of spheroid sizes. The addition of non-toxic probes (that do not produce or consume oxygen) report on oxygen diffusion throughout the spheroid as a function of size. We provide evidence supporting the use of this model over a range of initial cell seeding densities and spheroid sizes with the production of oxygen distribution as a function of these parameters. In our spheroid model, lower cell seeding densities (∼2,500 cells/spheroid) and absolute size (118±32 μm) allow control of factors such as pre-existing stresses (e.g. ∼ 2% normoxic/hypoxic interface) for more accurate measurement of treatment response. The applied methodology provides an elegant, widely applicable approach to directly characterize spheroid (and other organoid) cultures in biomedical and toxicological

  8. Understanding Health and Disease with Multidimensional Single-Cell Methods

    PubMed Central

    Candia, Julián; Banavar, Jayanth R.; Losert, Wolfgang

    2014-01-01

    Current efforts in the biomedical sciences and related interdisciplinary fields are focused on gaining a molecular understanding of health and disease, which is a problem of daunting complexity that spans many orders of magnitude in characteristic length scales, from small molecules that regulate cell function to cell ensembles that form tissues and organs working together as an organism. In order to uncover the molecular nature of the emergent properties of a cell, it is essential to measure multiple cell components simultaneously in the same cell. In turn, cell heterogeneity requires multiple cells to be measured in order to understand health and disease in the organism. This review summarizes current efforts towards a data-driven framework that leverages single-cell technologies to build robust signatures of healthy and diseased phenotypes. While some approaches focus on multicolor flow cytometry data and other methods are designed to analyze high-content image-based screens, we emphasize the so-called Supercell/SVM paradigm (recently developed by the authors of this review and collaborators) as a unified framework that captures mesoscopic-scale emergence to build reliable phenotypes. Beyond their specific contributions to basic and translational biomedical research, these efforts illustrate, from a larger perspective, the powerful synergy that might be achieved from bringing together methods and ideas from statistical physics, data mining, and mathematics to solve the most pressing problems currently facing the life sciences. PMID:24451406

  9. Local Measurement of Flap Oxygen Saturation: An Application of Visible Light Spectroscopy.

    PubMed

    Nasseri, Nassim; Kleiser, Stefan; Reidt, Sascha; Wolf, Martin

    2016-01-01

    The aim was to develop and test a new device (OxyVLS) to measure tissue oxygen saturation by visible light spectroscopy independently of the optical pathlength and scattering. Its local applicability provides the possibility of real time application in flap reconstruction surgery. We tested OxyVLS in a liquid phantom with optical properties similar to human tissue. Our results were in good agreement with a conventional near infrared spectroscopy device.

  10. Validation of a New NIRS Method for Measuring Muscle Oxygenation During Rhythmic Handgrip Exercise

    NASA Technical Reports Server (NTRS)

    Hagan, R. Donald; Soller, Babs R.; Soyemi, Olusola; Landry, Michelle; Shear, Michael; Wu, Jacqueline

    2006-01-01

    Near infrared spectroscopy (NIRS) is commonly used to measure muscle oxygenation during exercise and recovery. Current NIRS algorithms do not account for variation in water content and optical pathlength during exercise. The current effort attempts to validate a newly developed NIRS algorithm during rhythmic handgrip exercise and recovery. Six female subjects, aver age 28 +/- 6 yrs, participated in the study. A venous catheter was placed in the retrograde direction in the antecubital space. A NIRS sensor with 30 mm source-detector separation was placed on the flexor digitorum profundus. Subjects performed two 5-min bouts of rhythmic handgrip exercise (2 s contraction/1 s relaxation) at 15% and 30% of maximal voluntary contraction. Venous blood was sampled before each bout, during the last minute of exercise, and after 5 minutes of recovery. Venous oxygen saturation (SvO2) was measured with a I-stat CG-4+ cartridge. Spectra were collected between 700-900 nm. A modified Beer's Law formula was used to calculate the absolute concentration of oxyhemoglobin (HbO2), deoxyhemoglobin (Hb) and water, as well as effective pathlength for each spectrum. Muscle oxygen saturation (SmO2) was calculated from the HbO2 and Hb results. The correlation between SvO2 and SmO2 was determined. Optical pathlength and water varied significantly during each exercise bout, with pathlength increasing approximately 20% and water increasing about 2%. R2 between blood and muscle SO2 was found to be 0.74, the figure shows the relationship over SvO2 values between 22% and 82%. The NIRS measurement was, on average, 6% lower than the blood measurement. It was concluded that pathlength changes during exercise because muscle contraction causes variation in optical scattering. Water concentration also changes, but only slightly. A new NIRS algorithm which accounts for exercise-induced variation in water and pathlength provided an accurate assessment of muscle oxygen saturation before, during and after

  11. 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

  12. Biased Allelic Expression in Human Primary Fibroblast Single Cells

    PubMed Central

    Borel, Christelle; Ferreira, Pedro G.; Santoni, Federico; Delaneau, Olivier; Fort, Alexandre; Popadin, Konstantin Y.; Garieri, Marco; Falconnet, Emilie; Ribaux, Pascale; Guipponi, Michel; Padioleau, Ismael; Carninci, Piero; Dermitzakis, Emmanouil T.; Antonarakis, Stylianos E.

    2015-01-01

    The study of gene expression in mammalian single cells via genomic technologies now provides the possibility to investigate the patterns of allelic gene expression. We used single-cell RNA sequencing to detect the allele-specific mRNA level in 203 single human primary fibroblasts over 133,633 unique heterozygous single-nucleotide variants (hetSNVs). We observed that at the snapshot of analyses, each cell contained mostly transcripts from one allele from the majority of genes; indeed, 76.4% of the hetSNVs displayed stochastic monoallelic expression in single cells. Remarkably, adjacent hetSNVs exhibited a haplotype-consistent allelic ratio; in contrast, distant sites located in two different genes were independent of the haplotype structure. Moreover, the allele-specific expression in single cells correlated with the abundance of the cellular transcript. We observed that genes expressing both alleles in the majority of the single cells at a given time point were rare and enriched with highly expressed genes. The relative abundance of each allele in a cell was controlled by some regulatory mechanisms given that we observed related single-cell allelic profiles according to genes. Overall, these results have direct implications in cellular phenotypic variability. PMID:25557783

  13. Microfluidic single-cell analysis of intracellular compounds

    PubMed Central

    Chao, Tzu-Chiao; Ros, Alexandra

    2008-01-01

    Biological analyses traditionally probe cell ensembles in the range of 103–106 cells, thereby completely averaging over relevant individual cell responses, such as differences in cell proliferation, responses to external stimuli or disease onset. In past years, this fact has been realized and increasing interest has evolved for single-cell analytical methods, which could give exciting new insights into genomics, proteomics, transcriptomics and systems biology. Microfluidic or lab-on-a-chip devices are the method of choice for single-cell analytical tools as they allow the integration of a variety of necessary process steps involved in single-cell analysis, such as selection, navigation, positioning or lysis of single cells as well as separation and detection of cellular analytes. Along with this advantageous integration, microfluidic devices confine single cells in compartments near their intrinsic volume, thus minimizing dilution effects and increasing detection sensitivity. This review overviews the developments and achievements of microfluidic single-cell analysis of intracellular compounds in the past few years, from proof-of-principle devices to applications demonstrating a high biological relevance. PMID:18682362

  14. Vapor Pressures and Thermodynamics of Oxygen-Containing Polycyclic Aromatic Hydrocarbons Measured Using Knudsen Effusion

    PubMed Central

    Goldfarb, Jillian L.

    2013-01-01

    Polycyclic aromatic hydrocarbons (PAHs) and their oxygenated derivatives (OPAHs) are ubiquitous environmental pollutants resulting from the incomplete combustion of coal and fossil fuels. Their vapor pressures are key thermodynamic data essential for modeling fate and transport within the environment. The present study involved nine PAHs containing oxygen heteroatoms, including aldehyde, carboxyl and nitro groups, specifically: 2-nitrofluorene; 9-fluorenecarboxylic acid; 2-fluorenecarboxaldehyde; 2-anthracenecarboxylic acid; 9-anthracenecarboxylic acid; 9-anthraldehyde; 1-nitropyrene; 1-pyrenecarboxaldehyde and 1-bromo-2-naphthoic acid. The vapor pressures of these compounds, with molecular weights ranging from 194 to 251 grams per mole, were measured using the isothermal Knudsen effusion technique in the temperature range of 329 to 421. The corresponding enthalpies of sublimation, calculated via the Clausius-Clapeyron equation, are compared to parent, non-oxygenated PAH compound data to determine the effect of the addition of these oxygen-containing heteroatoms. As expected, the addition of –CHO,–COOH, and –NO2 groups onto these PAHs increases the enthalpy of sublimation and decreases the vapor pressure as compared to the parent PAH; the position of substitution also plays a significant role in determining the vapor pressure of these OPAHs. PMID:18220445

  15. [Measurement of reactive oxygen species in a biological system and its perspectives].

    PubMed

    Todoki, K; Lee, C; Okabe, E

    1996-12-01

    In recent years, reactive oxygen species have been implicated in the pathogenesis of a wide variety of disorders. Although the existence of reactive oxygen intermediates in drug metabolism can be inferred from end product analysis or from the effects of antioxidants or enzymes such as superoxide dismutase, only the technique of electron spin resonance (ESR) allows the direct detection of these highly reactive species. However, some free radical species cannot be detected by ESR due to their extremely short half-lives, which result in low steady-state concentrations of the radicals or to short radical relaxation times, which lead to a very broad line. These facts made recent development of spin-trapping and chemiluminescence techniques are widely used to detect free radicals. The goal of this paper is to introduce the various assays available for measurement of reactive oxygen species in biological models. This paper will focus on two topics: (1) the spin-trapping/ESR technique in vitro and vivo and (2) the chemiluminescence-optical biosensor application of this technique, a very sensitive method that has the advantage of being able to provide continuous, online, nondestructive monitoring of reactive oxygen species.

  16. Atomic oxygen variations in MLT observed from SCIAMACHY sun-synchronous nightglow measurements

    NASA Astrophysics Data System (ADS)

    Lednyts'kyy, Olexandr; Von Savigny, Christian

    Atomic oxygen (O) is the most abundant chemically active trace gas in the Earth's mesosphere and lower thermosphere region, and plays a critical role in O_3 and OH(*) formation. The near-global night-time limb measurements of the O((1) S-(1) D) emission at 557.7 nm were performed with the sun-synchronous SCIAMACHY grating spectrometer on board of ENVISAT-1 satellite from August 2002 to April 2012 at approximately 22:00 LT. Assuming horizontal homogeneity of atmospheric layers as well as absence of absorption and scattering, the emission transport problem was analysed on the base of linear forward inversion employing regularized total least squares minimization. A photochemical model based on the generally accepted 2-step Barth transfer scheme was used for deriving atomic oxygen concentration ([O]) profiles from volume emission rate profiles. An error budget was established to quantify the maximum uncertainty, assuming independent contribution of errors for each considered model parameter. [O] profiles allow monitoring of the energy transport because of the long photochemical lifetime of ground state atomic oxygen (about some months at the 100 km altitude). A wavelet analysis was performed for the time series of [O] profiles with five degree latitude steps in daily resolution. Semiannual and significant annual oscillations, and a pronounced 11-year solar cycle were identified, considering the existence of quasi biennial oscillations. Further studies presume accounting the correlation of solar activity with atomic oxygen concentration at high altitudes.

  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. Reproducibility of cerebral tissue oxygen saturation measurements by near-infrared spectroscopy in newborn infants

    NASA Astrophysics Data System (ADS)

    Jenny, Carmen; Biallas, Martin; Trajkovic, Ivo; Fauchère, Jean-Claude; Bucher, Hans Ulrich; Wolf, Martin

    2011-09-01

    Early detection of cerebral hypoxemia is an important aim in neonatology. A relevant parameter to assess brain oxygenation may be the cerebral tissue oxygen saturation (StO2) measured by near-infrared spectroscopy (NIRS). So far the reproducibility of StO2 measurements was too low for clinical application, probably due to inhomogeneities. The aim of this study was to test a novel sensor geometry which reduces the influence of inhomogeneities. Thirty clinically stable newborn infants, with a gestational age of median 33.9 (range 26.9 to 41.9) weeks, birth weight of 2220 (820 to 4230) g, postnatal age of 5 (1 to 71) days were studied. At least four StO2 measurements of 1 min duration were carried out using NIRS on the lateral head. The sensor was repositioned between measurements. Reproducibility was calculated by a linear mixed effects model. The mean StO2 was 79.99 +/- 4.47% with a reproducibility of 2.76% and a between-infant variability of 4.20%. Thus, the error of measurement only accounts for 30.1% of the variability. The novel sensor geometry leads to considerably more precise measurements compared to previous studies with, e.g., ~5% reproducibility for the NIRO 300. The novel StO2 values hence have a higher clinical relevance.

  19. Turgor pressure: direct manometric measurement in single cells of Nitella.

    PubMed

    Green, P B; Stanton, F W

    1967-03-31

    A small capillary, fused at one end, serves as a micromanometer when the open end is inserted into a large Nitella cell. The cell's ability to compress the gas reveals its turgor pressure directly-save for a small correction due to capillarity. The method gives a lower limit to turgor pressure for the same cell in the normal state. The common method, incipient plasmolysis, gives an upper limit. On Nitella axillaris cells the two methods limit the turgor pressure at 5.1 to 5.7 atmospheres. The manometric method is also applicable to growing cells, where osmotic equilibrium is not present.

  20. Atmospheric Airborne Pressure Measurements Using the Oxygen A Band for the ASCENDS Mission

    NASA Technical Reports Server (NTRS)

    Riris, Haris; Rodriguez, Mike; Stephen, Mark; Hasselbrack, William; Allan, Graham; Mao, Jianping; Kawa, Stephen R.; Weaver, Clark J.

    2010-01-01

    We report on airborne atmospheric pressure measurements using new fiber-based laser technology and the oxygen A-band at 765 nm. Remote measurements of atmospheric temperature and pressure are required for a number of NASA Earth science missions and specifically for the Active Sensing of CO2 Emissions Over Nights, Days, and Seasons (ASCENDS) mission. Accurate measurements of tropospheric CO2 on a global scale are very important in order to better understand its sources and sinks and to improve predictions on any future climate change. The ultimate goal of a CO2 remote sensing mission, such as ASCENDS, is to derive the CO2 concentration in the atmosphere in terms of mole fraction in unit of parts-per-million (ppmv) with regard to dry air. Therefore, both CO2 and the dry air number of molecules in the atmosphere are needed in deriving this quantity. O2 is a stable molecule and uniformly mixed in the atmosphere. Measuring the O2 absorption in the atmosphere can thus be used to infer the dry air number of molecules and then used to calculate CO2 concentration. With the knowledge of atmospheric water vapor, we can then estimate the total surface pressure needed for CO2 retrievals. Our work, funded by the ESTO IIP program, uses fiber optic technology and non-linear optics to generate 765 nm laser radiation coincident with the Oxygen A-band. Our pulsed, time gated technique uses several on- and off-line wavelengths tuned to the O2 absorption line. The choice of wavelengths allows us to measure the pressure by using two adjacent O2 absorptions in the Oxygen A-band. Our retrieval algorithm fits the O2 lineshapes and derives the pressure. Our measurements compare favorably with a local weather monitor mounted outside our laboratory and a local weather station.

  1. Atmospheric Airborne Pressure Measurements Using the Oxygen A Band for the ASCENDS Mission

    NASA Technical Reports Server (NTRS)

    Riris, Haris; Rodriguez, Mike; Stephen, Mark; Hasselbrack, William; Allan, Graham; Mao, Jiamping,; Kawa, Stephan R.; Weaver, Clark J.

    2011-01-01

    We report on airborne atmospheric pressure measurements using new fiber-based laser technology and the oxygen A-band at 765 nm. Remote measurements of atmospheric temperature and pressure are required for a number of NASA Earth science missions and specifically for the Active Sensing of CO2 Emissions Over Nights, Days, and Seasons (ASCENDS) mission. Accurate measurements of tropospheric CO2 on a global scale are very important in order to better understand its sources and sinks and to improve predictions on any future climate change. The ultimate goal of a CO2 remote sensing mission, such as ASCENDS, is to derive the CO2 concentration in the atmosphere in terms of mole fraction in unit of parts-per-million (ppmv) with regard to dry air. Therefore, both CO2 and the dry air number of molecules in the atmosphere are needed in deriving this quantity. O2 is a stable molecule and uniformly mixed in the atmosphere. Measuring the O2 absorption in the atmosphere can thus be used to infer the dry air number of molecules and then used to calculate CO2 concentration. With the knowledge of atmospheric water vapor, we can then estimate the total surface pressure needed for CO2 retrievals. Our work, funded by the ESTO IIP program, uses fiber optic technology and non-linear optics to generate 765 nm laser radiation coincident with the Oxygen A-band. Our pulsed, time gated technique uses several on- and off-line wavelengths tuned to the O2 absorption line. The choice of wavelengths allows us to measure the pressure by using two adjacent O2 absorptions in the Oxygen A-band. Our retrieval algorithm fits the O2 lineshapes and derives the pressure. Our measurements compare favorably with a local weather monitor mounted outside our laboratory and a local weather station.

  2. [A nondestructive method to measure the oxygen binding and releasing properties of biodegradable polymers microcapsules intented for blood substitutes].

    PubMed

    Zhang, Xiaolan; Yuan, Yuan; Wang, Kai; Shan, Xiaoqian; Zhao, Jian; Sheng, Yan; Liu, Changsheng

    2008-10-01

    P50 is an important parameter reflecting the binding and releasing oxygen properties of blood substitutes. In this study, based on the strong penetrating property of near infrared light and the mechanism involved in the pulsatile oxygen meter in clinic as well as on the ability for penetrating biodegradable polymers and detecting bovine hemoglobin encapsulated within the microcapsules, we have made an airproof and equilibrium apparatus to measure oxygen saturation and oxygen partial pressure. Subsequently, we have obtained the oxygen dissociation curve and P50 of the microcapsules loaded bovine hemoglobin in the light of oxyHemoglobin and deoxyHemoglobin with different spectrum in the near infrared region. The above-mentioned apparatus and method are not destructive to the microcapsules, and the process is simple and nondestructive. So it is practical to take in-situ measurements of the oxygen binding and releasing property of biodegradable polymer microcapsules intented for the blood substitute.

  3. A simplified CARS measurement system for rapid determination of temperature and oxygen concentration

    NASA Technical Reports Server (NTRS)

    Fujii, Shoichi

    1987-01-01

    A new spectroscopic concept for the rapid determination of temperature and oxygen concentration by CARS (Coherent Anti-Stokes Raman Spectroscopy) was described. The ratio of two spectral regions in the broadband Q-branch spectrum was detected by photomultipliers in a monochromator, which ratio depends on temperature and species concentration. The comparison of the measured data with theory was made using a flat flame burner and an electric furnace, with reasonable results. Various optical techniques for alignment were introduced including a highly efficient, stable dye oscillator. The combination of the spectroscopic concept and the optical techniques will make the CARS measurement system rapid in data processing and simple in optical parts.

  4. Measurement of Oxygen A Band Line Parameters by Using Modulation Spectroscopy with Higher Harmonic Detection

    NASA Technical Reports Server (NTRS)

    Dharamsi, Amin

    1999-01-01

    Wavelength modulation spectroscopy is used to demonstrate that extremely weak absorption lines can be measured even when these lines suffer from interference from the wings of adjacent stronger lines. It is shown that the use of detection at several harmonics allows such interference to be examined clearly and conveniently. The results of experimental measurements on a weak magnetic dipole driven, spin-forbidden line in the oxygen A band, which experiences interference from the wings of a pair of adjacent lines towards the blue and red regions of line center, are presented. A comparison of the experimental results to theory is given.

  5. pyMCZ: Oxygen abundances calculations and uncertainties from strong-line flux measurements

    NASA Astrophysics Data System (ADS)

    Bianco, Federica B.; Modjaz, Maryam; Oh, Seung Man; Fierroz, David; Liu, Yuqian; Kewley, Lisa; Graur, Or

    2015-05-01

    pyMCZ calculates metallicity according to a number of strong line metallicity diagnostics from spectroscopy line measurements and obtain uncertainties from the line flux errors in a Monte Carlo framework. Given line flux measurements and their uncertainties, pyMCZ produces synthetic distributions for the oxygen abundance in up to 13 metallicity scales simultaneously, as well as for E(B-V), and estimates their median values and their 68% confidence regions. The code can output the full MC distributions and their kernel density estimates.

  6. Measurement of oxygen uptake in newborn infants during assisted and spontaneous ventilation.

    PubMed

    Rozé, J C; Chambille, B; Dehan, M; Gaultier, C

    1994-10-01

    Measurements of oxygen uptake (VO2) and CO2 output (VCO2) are useful in critically ill patients. However, VO2 is not routinely measured in intensive care during mechanical ventilation (MV) especially in premature newborns. The present study describes a new method of measuring VO2 and VCO2 using a double open circuit which accounts for gas leaks around the uncuffed tracheal tube. The accuracy of the method was assessed with N2 and CO2 infusion. In case of leaks, VO2 and VCO2 measurement was significantly underestimated by the simple circuit method. This underestimation was not present with double circuit method. Five preterm newborns were studied. VO2 and VCO2 using the double open circuit were compared with the classic simple circuit. During MV, the mean underestimation assessed by the difference between simple and double circuit measurement was -12% (range from 0 to -29%) for VO2 and -14% (range 0 to -26%) for VCO2.

  7. The measurement of sequential changes in cerebral blood flow and oxygen metabolism by positron computed tomography with continuous inhalation of oxygen-15 labeled gases

    SciTech Connect

    Tanada, S.; Yonekura, Y.; Senda, M.; Nishimura, K.; Tamaki, N.; Saji, H.; Fujita, T.; Kobayashi, A.; Taki, W.; Ishikawa, M.

    1984-01-01

    The use of continuous inhalation of oxygen-15 labeled gases is a widely accepted method to measure regional cerebral blood flow (CBF) and oxygen metabolism (CMRO/sub 2/) with positron computed tomography (PCT). The purpose of this study is to evaluate the feasibility to measure sequential changes in CBF and CMRO/sub 2/ by PCT. The functional images of CBF, oxygen extraction fraction (OEF), and CMRO/sub 2/ were obtained using continuous inhalation of oxygen-15 labeled carbon dioxide and oxygen. The effects of spinal drainage in CBF and CMRO/sub 2/ were studied in patients with hydrocephalus following subarachnoid hemorrhage due to the rupture of intracranial aneurysm. Following the measurement in control state, 20 ml of cerebrospinal fluid (CSF) were withdrawn gradually through lumbar puncture, and sequential PCT scans were performed. CBF and CMRO/sub 2/ were markedly depressed in the case with hydrocephalus. The drainage of CSF significantly improved OEF and CMRO/sub 2/, whereas CBF remained depressed. In patients with chronic cerebrovascular disease, the changes in CBF were studied with inhalation of 5% carbon dioxide (CO/sub 2/). CO/sub 2/ loading demonstrated the increase in CBF, while poor regional increase was observed in ''moyamoya'' disease, which permitted the assessment of vascular response to the elevation of plasma CO/sub 2/. The authors preliminary work indicated the potential usefulness of sequential PCT to study the changes in CBF and CMRO/sub 2/ with various interventions.

  8. Measurement of time-resolved oxygen concentration changes in photosynthetic systems by nitroxide-based EPR oximetry.

    PubMed

    Strzalka, K; Walczak, T; Sarna, T; Swartz, H M

    1990-09-01

    The application of recent developments of EPR oximetry to photosynthetic systems is described and used to study rapid processes in isolated thylakoid membranes from spinach and in intact photoautotrophic soybean cells. Using the peak heights of 15N perdeuterated Tempone and two microwave power levels oxygen evolution and consumption were measured. The method measured time-resolved oxygen concentration changes in the micromolar range. Oxygen evolution was linearly proportionate to the chlorophyl concentration of thylakoid membrane over the range studied (0-2 mg/ml). Oxygen evolution associated with single turnover light pulses was consistent with the four state model. The time (t1/2) to reach equilibrium of oxygen concentrations after a single turnover pulse was 0.4-0.5 ms, indicating that the evolution of oxygen coupled to the S4-S0 transition may be shorter than reported previously. The time for equilibrium of oxygen after single turnover pulses in soybean cells was relatively long (400 ms), which suggests that there are significant barriers to the free diffusion of oxygen in this system. The method also was used to study oxygen consumption by the electron transport chain of photosystem I and photosystem II. We conclude that EPR oximetry can provide quantitative and time-resolved data on oxygen concentrations with a sensitivity that is useful for studies of such systems. PMID:2168161

  9. Single-cell dynamics reveals sustained growth during diauxic shifts.

    PubMed

    Boulineau, Sarah; Tostevin, Filipe; Kiviet, Daniel J; ten Wolde, Pieter Rein; Nghe, Philippe; Tans, Sander J

    2013-01-01

    Stochasticity in gene regulation has been characterized extensively, but how it affects cellular growth and fitness is less clear. We study the growth of E. coli cells as they shift from glucose to lactose metabolism, which is characterized by an obligatory growth arrest in bulk experiments that is termed the lag phase. Here, we follow the growth dynamics of individual cells at minute-resolution using a single-cell assay in a microfluidic device during this shift, while also monitoring lac expression. Mirroring the bulk results, the majority of cells displays a growth arrest upon glucose exhaustion, and resume when triggered by stochastic lac expression events. However, a significant fraction of cells maintains a high rate of elongation and displays no detectable growth lag during the shift. This ability to suppress the growth lag should provide important selective advantages when nutrients are scarce. Trajectories of individual cells display a highly non-linear relation between lac expression and growth, with only a fraction of fully induced levels being sufficient for achieving near maximal growth. A stochastic molecular model together with measured dependencies between nutrient concentration, lac expression level, and growth accurately reproduces the observed switching distributions. The results show that a growth arrest is not obligatory in the classic diauxic shift, and underscore that regulatory stochasticity ought to be considered in terms of its impact on growth and survival. PMID:23637881

  10. Mass spectrometry imaging and profiling of single cells

    PubMed Central

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

    2012-01-01

    Mass spectrometry imaging and profiling of individual cells and subcellular structures provide unique analytical capabilities for biological and biomedical research, including determination of the biochemical heterogeneity of cellular populations and intracellular localization of pharmaceuticals. Two mass spectrometry technologies—secondary ion mass spectrometry (SIMS) and matrix assisted laser desorption ionization mass spectrometry (MALDI MS)—are most often used in micro-bioanalytical investigations. Recent advances in ion probe technologies have increased the dynamic range and sensitivity of analyte detection by SIMS, allowing two- and three-dimensional localization of analytes in a variety of cells. SIMS operating in the mass spectrometry imaging (MSI) mode can routinely reach spatial resolutions at the submicron level; therefore, it is frequently used in studies of the chemical composition of subcellular structures. MALDI MS offers a large mass range and high sensitivity of analyte detection. It has been successfully applied in a variety of single-cell and organelle profiling studies. Innovative instrumentation such as scanning microprobe MALDI and mass microscope spectrometers enable new subcellular MSI measurements. Other approaches for MS-based chemical imaging and profiling include those based on near-field laser ablation and inductively-coupled plasma MS analysis, which offer complementary capabilities for subcellular chemical imaging and profiling. PMID:22498881

  11. Reconstructing dynamic molecular states from single-cell time series.

    PubMed

    Huang, Lirong; Pauleve, Loic; Zechner, Christoph; Unger, Michael; Hansen, Anders S; Koeppl, Heinz

    2016-09-01

    The notion of state for a system is prevalent in the quantitative sciences and refers to the minimal system summary sufficient to describe the time evolution of the system in a self-consistent manner. This is a prerequisite for a principled understanding of the inner workings of a system. Owing to the complexity of intracellular processes, experimental techniques that can retrieve a sufficient summary are beyond our reach. For the case of stochastic biomolecular reaction networks, we show how to convert the partial state information accessible by experimental techniques into a full system state using mathematical analysis together with a computational model. This is intimately related to the notion of conditional Markov processes and we introduce the posterior master equation and derive novel approximations to the corresponding infinite-dimensional posterior moment dynamics. We exemplify this state reconstruction approach using both in silico data and single-cell data from two gene expression systems in Saccharomyces cerevisiae, where we reconstruct the dynamic promoter and mRNA states from noisy protein abundance measurements. PMID:27605167

  12. Simulation of neutron interactions at the single-cell level.

    PubMed

    Alard, Jean-Pierre; Bodez, Véronique; Tchirkov, Andrei; Nénot, Marie-Laure; Arnold, Jack; Crespin, Sylvain; Rapp, Maryse; Verrelle, Pierre; Dionet, Claude

    2002-11-01

    We recently reported that the exposure of cancer cells to 14 MeV neutrons at a very low dose rate (0.8 mGy min(-1)) produced a marked increase in cell killing at 5 cGy, followed by a plateau in survival and chromosomal damage. Simulation of the energy deposition events in irradiated cells may help to explain these unusual cell responses. We describe here a Monte Carlo simulation code, Energy Deposition in Cells Irradiated by Neutrons (EDCIN). The procedure considered the experimental setup and a hemispheric cell model. The simulation data fitted the dosimetric measurements performed using tissue-equivalent ionization chambers, Geiger-Müller counters, fission chambers, and silicon diodes. The simulation showed that 80% of the energy deposited in a single cell came from the interactions of neutrons outside the cell and only 20% came from neutron interactions inside the cell. Thus the "external" interactions that result in the production of recoil protons and secondary electrons may induce most of the biological damage, which may be repaired efficiently at low dose rate. The repair process may be triggered from a threshold level of damage, which would explain an initial increase cell death due to unrepaired sublethal damage, and then may compensate for induced damage, resulting in the plateaus. PMID:12385643

  13. Probing single-cell mechanics with picosecond ultrasonics.

    PubMed

    Dehoux, Thomas; Abi Ghanem, Maroun; Zouani, Omar F; Ducousso, Mathieu; Chigarev, Nikolay; Rossignol, Clément; Tsapis, Nicolas; Durrieu, Marie-Christine; Audoin, Bertrand

    2015-02-01

    The mechanical properties of cells play a key role in several fundamental biological processes, such as migration, proliferation, differentiation and tissue morphogenesis. The complexity of the inner cell composition and the intricate meshwork formed by transmembrane cell-substrate interactions demands a non-invasive technique to probe cell mechanics and cell adhesion at a subcell scale. In this paper we review the use of laser-generated GHz acoustic waves--a technique called picosecond ultrasonics (PU)--to probe the mechanical properties of single cells. We first describe applications to vegetal cells and biomimetic systems. We show how these systems can be used as simple models to understand more complex animal cells. We then present an opto-acoustic bio-transducer designed for in vivo measurements in physiological conditions. We illustrate the use of this transducer through the simultaneous probing of the density and compressibility of Allium cepa cells. Finally, we demonstrate that this technique can quantify animal-cell adhesion on metallic surfaces by analyzing the acoustic pulses reflected off the cell-metal interface. This innovative approach allows investigating quantitatively cell mechanics without fluorescent labels or mechanical contact to the cell.

  14. Direct measurement of singlet oxygen by using a photomultiplier tube-based detection system.

    PubMed

    Kim, In-Wook; Park, Jae Myung; Roh, Yoon Jin; Kim, Ju Hee; Choi, Myung-Gyu; Hasan, Tayyaba

    2016-06-01

    The effective dosimetry for photodynamic therapy (PDT) can be specified by direct measurement of singlet oxygen ((1)O2) production. The purpose of this study was to investigate the feasibility of a newly developed photomultiplier tube (PMT)-based singlet oxygen detection (SOD) system. The lowest and highest (1)O2 concentrations detectable by the PMT-SOD system were 15nM and 10μM, respectively. Dose-dependent quenching, by NaN3, of the fluorogenic reaction was observed, which was negatively correlated with the (1)O2 level measured by the PMT-SOD system. The lifetime of (1)O2, as measured by the PMT-SOD system, was found to be lengthened when H2O was replaced with deuterium oxide. (1)O2 photon counts were significantly and dose-dependently correlated with intracellular fluorescence intensity after photosensitizer treatments. In vitro cell viability test and in vivo xenografted-tumor mass shrinkage showed a positive association between PDT-induced cytotoxicity and (1)O2 production concomitantly measured by the PMT-SOD system. It was concluded that the PMT-SOD system is capable of measuring (1)O2 production directly and accurately, demonstrating that this system can be useful in the determination of dosimetry for PDT. PMID:26995671

  15. Twilight rocket measurements of high-latitude atomic oxygen density during the DYANA campaign

    NASA Astrophysics Data System (ADS)

    Ulwick, J. C.; Ratkowski, A. J.; Makhlouf, U.

    1994-12-01

    During the DYANA (DYnamics Adapted Network for the Atmosphere) campaign, a rocket containing a resonance fluorescence experiment for measurement of atomic oxygen concentrations was launced at twilight-dawn from ESRANGE, Kiruna, Sweden in March 1990. The measured atomic oxygen concetration rose very sharply near 80 km to about 10(exp 11) atoms/cu cm, achieved a peak value of 3 x 10(exp 11) atoms/cu cm between 90 to 105 km, and decreased with increasing rocket altitude to about 155 km where the instrument's noise level was reached. In addition, ground-based, near-infrared radiometric and spectral measurements of the mesospheric and lower thermospheric hydroxyl (OH) airglow emissions were also obtained during the night up to the time of rocket launch. We have used least-squares spectral fitting procedures to obtain the OH Meinel (3-1) band intensities and accurate (+/-2.5K) rotational temperatures from individual scans. The band intensities show considerable structure throughout the night, dropping sharply by about a factor of 3 during sunrise when the rocket was launched. The rotational temperatures were consistently around 225 K throughout the night and during the rocket flight. During the MAP/WINE campaign in February 1984, similar measurements using identical rocket-borne and ground-based techniques were made at night from ESRANGE. In this paper, the DYANA and MAP/WINE measurements are inter-compared and discussed and further compared with model calculations.

  16. Assessment of the uncertainty budget for the amperometric measurement of dissolved oxygen.

    PubMed

    Fisicaro, Paola; Adriaens, Annemie; Ferrara, Enzo; Prenesti, Enrico

    2007-07-30

    This work aimed at identifying the main sources of uncertainty for the measurement of dissolved oxygen concentration in aqueous solutions. The experimental apparatus consists of an amperometric cell based on the Clark-type sensor. The corresponding uncertainty budget was assessed, this being a fundamental step for the validation of a measurement method. The principle of the measurement, as well as the procedure for the set-up and the characterisation of the cell, are described. The measurement equation was defined as a combination of Faraday's and Fick's laws, and a method was worked out for the empirical determination of the diffusivity parameter. In this connection, the solutions of oxygen were standardised by way of the Winkler's titration, as suggested by the ISO Guide 5813 and 5814. With this approach we aimed at contributing to the development of a potential primary method of measurement. A discussion of all the contributions to the overall uncertainty is reported, allowing operators to locate the largest ones and plan specific improvements.

  17. [Laser Tuning Performance Testing and Optimization in TDLAS Oxygen Measuring Systems].

    PubMed

    He, Jun-feng; Hu, Jun; Kan, Rui-feng; Xu, Zhen-yu; Wang, Tao

    2015-03-01

    TDLAS (tunable diode laser absorption spectroscopy) technology, with its unmatched advantages such as high selectivity molecular spectra, fast response, high sensitivity, non-contact measuring, become the preferred scheme for combustion process diagnosis, and can be effectively used for oxygen measuring. DFB (distributed feedback) laser diode with its small size, low power consumption, long service life, narrow linewidth, tunable wavelength has become the main choice of the TDLAS system. Performance of laser tuning characteristics is a key factor restricting TDLAS's measuring performance. According to TDLAS oxygen measuring system's working requirements, a simple experimental method was used to test and analyze tuning characteristics such as wavelength current, power current and wavelength temperature of a 764 nm DFB laser diode in the system. Nonlinear distortion of tuning curves was obvious, which affects oxygen measuring accuracy. The laser spectra's characteristics such as narrow linewidth, high side mode suppression ratio and wide wavelength tuning range are obvious, while its wavelength-current tuning curve with a tuning rate of about 0.023 nm x mA(-1) is not strictly linear. The higher the temperature the greater the threshold current, the PI curve is not strictly linear either. Temperature tuning curve is of good linearity, temperature-wave-length tuning rate keeps constant of about 0.056 nm/DEG C. Temperature tuning nonlinearity can be improved by high temperature control accuracy, and current power nonlinearity can be improved by setting the reference light path. In order to solve the wavelength current tuning nonlinear problems, the method of DA controlling injection current was considered to compensate for non-linear wavelength current tuning according to DFB laser diode tuning mechanism and polynomial fitting of test results. In view of different type of lasers, this method needs only one polynomial fitting process before the system's initial work. The

  18. Measurements of elastohydrodynamic film thickness, wear and tempering behavior of high pressure oxygen turbopump bearings

    NASA Technical Reports Server (NTRS)

    Dufrane, K. F.; Merriman, T. L.; Kannel, J. W.; Stockwell, R. D.; Hauser, D.; Vanecho, J. A.

    1984-01-01

    The reusable design of the Space Shuttle requires a target life of 7.5 hours for the turbopumps of the Space Shuttle main engine (SSME). This large increase from the few hundred seconds required in single-use rockets has caused various problems with the bearings of the turbopumps. The berings of the high pressure oxygen turbopump (HPOTP) were of particular concern because of wear, spalling, and cage failures at service time well below the required 7.5 hours. Lubrication and wear data were developed for the bearings. Since the HPOTP bearings operate in liquid oxygen, conventional liquid lubricants cannot be applied. Therefore, solid lubricant coatings and lubricant transfer from the polytetrafluorethylene (FTFE) cage were the primary lubrication approaches for the bearings. Measurements were made using liquid nitrogen in a rolling disk machine to determine whether usable elastohydrodynamic films could be generated to assist in the bearing lubrication.

  19. Two-photon absorption laser induced fluorescence measurement of atomic oxygen density in an atmospheric pressure air plasma jet

    NASA Astrophysics Data System (ADS)

    Conway, J.; Gogna, G. S.; Gaman, C.; Turner, M. M.; Daniels, S.

    2016-08-01

    Atomic oxygen number density [O] is measured in an air atmospheric pressure plasma jet (APPJ) using two-photon absorption laser induced fluorescence (TALIF). Gas flow is fixed at 8 slpm, the RF power coupled into the plasma jet varied between 5 W and 20 W, and the resulting changes in atomic oxygen density measured. Photolysis of molecular oxygen is employed to allow in situ calibration of the TALIF system. During calibration, O2 photo-dissociation and two-photon excitation of the resulting oxygen atoms are achieved within the same laser pulse. The atomic oxygen density produced by photolysis is time varying and spatially non-uniform which needs to be corrected for to calibrate the TALIF system for measurement of atomic oxygen density in plasma. Knowledge of the laser pulse intensity I 0(t), wavelength, and focal spot size allows correction factors to be determined using a rate equation model. Atomic oxygen is used for calibration and measurement, so the laser intensity can be increased outside the TALIF quadratic laser power dependence region without affecting the calibration reliability as the laser power dependence will still be the same for both. The atomic O density results obtained are not directly benchmarked against other known density measurement techniques. The results show that the plasma jet atomic oxygen content increases as the RF power coupled into the plasma increases.

  20. SINCERA: A Pipeline for Single-Cell RNA-Seq Profiling Analysis.

    PubMed

    Guo, Minzhe; Wang, Hui; Potter, S Steven; Whitsett, Jeffrey A; Xu, Yan

    2015-11-01

    A major challenge in developmental biology is to understand the genetic and cellular processes/programs driving organ formation and differentiation of the diverse cell types that comprise the embryo. While recent studies using single cell transcriptome analysis illustrate the power to measure and understand cellular heterogeneity in complex biological systems, processing large amounts of RNA-seq data from heterogeneous cell populations creates the need for readily accessible tools for the analysis of single-cell RNA-seq (scRNA-seq) profiles. The present study presents a generally applicable analytic pipeline (SINCERA: a computational pipeline for SINgle CEll RNA-seq profiling Analysis) for processing scRNA-seq data from a whole organ or sorted cells. The pipeline supports the analysis for: 1) the distinction and identification of major cell types; 2) the identification of cell type specific gene signatures; and 3) the determination of driving forces of given cell types. We applied this pipeline to the RNA-seq analysis of single cells isolated from embryonic mouse lung at E16.5. Through the pipeline analysis, we distinguished major cell types of fetal mouse lung, including epithelial, endothelial, smooth muscle, pericyte, and fibroblast-like cell types, and identified cell type specific gene signatures, bioprocesses, and key regulators. SINCERA is implemented in R, licensed under the GNU General Public License v3, and freely available from CCHMC PBGE website, https://research.cchmc.org/pbge/sincera.html.

  1. SINCERA: A Pipeline for Single-Cell RNA-Seq Profiling Analysis

    PubMed Central

    Guo, Minzhe; Wang, Hui; Potter, S. Steven; Whitsett, Jeffrey A.; Xu, Yan

    2015-01-01

    A major challenge in developmental biology is to understand the genetic and cellular processes/programs driving organ formation and differentiation of the diverse cell types that comprise the embryo. While recent studies using single cell transcriptome analysis illustrate the power to measure and understand cellular heterogeneity in complex biological systems, processing large amounts of RNA-seq data from heterogeneous cell populations creates the need for readily accessible tools for the analysis of single-cell RNA-seq (scRNA-seq) profiles. The present study presents a generally applicable analytic pipeline (SINCERA: a computational pipeline for SINgle CEll RNA-seq profiling Analysis) for processing scRNA-seq data from a whole organ or sorted cells. The pipeline supports the analysis for: 1) the distinction and identification of major cell types; 2) the identification of cell type specific gene signatures; and 3) the determination of driving forces of given cell types. We applied this pipeline to the RNA-seq analysis of single cells isolated from embryonic mouse lung at E16.5. Through the pipeline analysis, we distinguished major cell types of fetal mouse lung, including epithelial, endothelial, smooth muscle, pericyte, and fibroblast-like cell types, and identified cell type specific gene signatures, bioprocesses, and key regulators. SINCERA is implemented in R, licensed under the GNU General Public License v3, and freely available from CCHMC PBGE website, https://research.cchmc.org/pbge/sincera.html. PMID:26600239

  2. Measuring gas exchange with step changes in inspired oxygen: an analysis of the assumption of oxygen steady state in patients suffering from COPD.

    PubMed

    Thomsen, Lars P; Weinreich, Ulla M; Karbing, Dan S; Wagner, Peter D; Rees, Stephen E

    2014-12-01

    Bedside estimation of pulmonary gas exchange efficiency may be possible from step changes in FIO2 and subsequent measurement of arterial oxygenation at steady state conditions. However, a steady state may not be achieved quickly after a change in FIO2, especially in patients with lung disease such as COPD, rendering this approach cumbersome. This paper investigates whether breath by breath measurement of respiratory gas and arterial oxygen levels as FIO2 is changed can be used as a much more rapid alternative to collecting data from steady state conditions for measuring pulmonary gas exchange efficiency. Fourteen patients with COPD were studied using 4-5 step changes in FIO2 in the range of 0.15-0.35. Values of expired respiratory gas and arterial oxygenation were used to calculate and compare the parameters of a mathematical model of pulmonary gas exchange in two cases: from data at steady state; and from breath by breath data prior to achievement of a steady state. For each patient, the breath by breath data were corrected for the delay in arterial oxygen saturation changes following each change in FIO2. Calculated model parameters were shown to be similar for the two data sets, with Bland-Altman bias and limits of agreement of -0.4 and -3.0 to 2.2 % for calculation of pulmonary shunt and 0.17 and -0.47 to 0.81 kPa for alveolar to end-capillary PO2, a measure of oxygen abnormality due to shunting plus regions of low [Formula: see text] A/[Formula: see text] ratio. This study shows that steady state oxygen levels may not be necessary when estimating pulmonary gas exchange using changes in FIO2. As such this technique may be applicable in patients with lung disease such as COPD.

  3. Carbon nanoelectrodes for single-cell probing.

    PubMed

    Anderson, Sean E; Bau, Haim H

    2015-05-01

    Carbon nanoelectrodes with tip diameters ranging from tens to hundreds of nanometers are fabricated by pyrolitic deposition of carbon films along the entire inner surfaces of pulled-glass pipettes. The pulled end of each glass pipette is then etched to expose a desired length (typically, a few micrometers) of carbon pipe. The carbon film provides an electrically conductive path from the nanoscopic carbon tip to the distal, macroscopic end of the pipette, bridging between the nanoscale tip and the macroscale handle, without a need for assembly. We used our nanoelectrodes to penetrate into individual cells and cell nuclei and measured the variations in the electrode impedance upon cell and nucleus penetration as well as the electrode impedance as a function of cell penetration depth. Theoretical predictions based on a simple circuit model were in good agreement with experimental data.

  4. Carbon Nanoelectrodes for Single-Cell Probing

    PubMed Central

    Anderson, Sean E.; Bau, Haim H.

    2015-01-01

    Carbon nanoelectrodes with tip diameters ranging from tens to hundreds of nm are fabricated by pyrolitic deposition of carbon films along the entire inner surfaces of pulled-glass pipettes. The pulled end of each glass pipette is then etched to expose a desired length (typically, a few µm) of carbon pipe. The carbon film provides an electrically conductive path from the nanoscopic carbon tip to the distal, macroscopic end of the pipette, bridging between the nanoscale tip and the macroscale handle, without a need for assembly. We used our nanoelectrodes to penetrate into individual cells and cell nuclei and measured the variations in the electrode impedance upon cell and nucleus penetration as well as the electrode impedance as a function of cell penetration depth. Theoretical predictions based on a simple circuit model were in good agreement with experimental data. PMID:25876625

  5. Carbon nanoelectrodes for single-cell probing

    NASA Astrophysics Data System (ADS)

    Anderson, Sean E.; Bau, Haim H.

    2015-05-01

    Carbon nanoelectrodes with tip diameters ranging from tens to hundreds of nanometers are fabricated by pyrolitic deposition of carbon films along the entire inner surfaces of pulled-glass pipettes. The pulled end of each glass pipette is then etched to expose a desired length (typically, a few micrometers) of carbon pipe. The carbon film provides an electrically conductive path from the nanoscopic carbon tip to the distal, macroscopic end of the pipette, bridging between the nanoscale tip and the macroscale handle, without a need for assembly. We used our nanoelectrodes to penetrate into individual cells and cell nuclei and measured the variations in the electrode impedance upon cell and nucleus penetration as well as the electrode impedance as a function of cell penetration depth. Theoretical predictions based on a simple circuit model were in good agreement with experimental data.

  6. Accurate, in vivo NIR measurement of skeletal muscle oxygenation through fat

    NASA Astrophysics Data System (ADS)

    Jin, Chunguang; Zou, Fengmei; Ellerby, Gwenn E. C.; Scott, Peter; Peshlov, Boyan; Soller, Babs R.

    2010-02-01

    Noninvasive near infrared (NIR) spectroscopic measurement of muscle oxygenation requires the penetration of light through overlying skin and fat layers. We have previously demonstrated a dual-light source design and orthogonalization algorithm that corrects for inference from skin absorption and fat scattering. To achieve accurate muscle oxygen saturation (SmO2) measurement, one must select the appropriate source-detector distance (SD) to completely penetrate the fat layer. Methods: Six healthy subjects were supine for 15min to normalize tissue oxygenation across the body. NIR spectra were collected from the calf, shoulder, lower and upper thigh muscles with long SD distances of 30mm, 35mm, 40mm and 45mm. Spectral preprocessing with the short SD (3mm) spectrum preceded SmO2 calculation with a Taylor series expansion method. Three-way ANOVA was used to compare SmO2 values over varying fat thickness, subjects and SD distances. Results: Overlying fat layers varied in thickness from 4.9mm to 19.6mm across all subjects. SmO2 measured at the four locations were comparable for each subject (p=0.133), regardless of fat thickness and SD distance. SmO2 (mean+/-std dev) measured at calf, shoulder, low and high thigh were 62+/-3%, 59+/-8%, 61+/-2%, 61+/-4% respectively for SD distance of 30mm. In these subjects no significant influence of SD was observed (p=0.948). Conclusions: The results indicate that for our sensor design a 30mm SD is sufficient to penetrate through a 19mm fat layer and that orthogonalization with short SD effectively removed spectral interference from fat to result in a reproducible determination of SmO2.

  7. Somatosensory evoked changes in cerebral oxygen consumption measured non-invasively in premature neonates

    PubMed Central

    Roche-Labarbe, Nadege; Fenoglio, Angela; Radakrishnan, Harsha; Kocienski-Filip, Marcia; Carp, Stefan A.; Dubb, Jay; Boas, David A.; Grant, P. Ellen; Franceschini, Maria Angela

    2013-01-01

    The hemodynamic functional response is used as a reliable marker of neuronal activity in countless studies of brain function and cognition. In newborns and infants, however, conflicting results have appeared in the literature concerning the typical response, and there is little information on brain metabolism and functional activation. Measurement of all hemodynamic components and oxygen metabolism is critical for understanding neurovascular coupling in the developing brain. To this end, we combined multiple near infrared spectroscopy techniques to measure oxy- and deoxy-hemoglobin concentrations, cerebral blood volume (CBV), and relative cerebral blood flow (CBF) in the somatosensory cortex of 6 preterm neonates during passive tactile stimulation of the hand. By combining these measures we estimated relative changes in the cerebral metabolic rate of oxygen consumption (rCMRO2). CBF starts increasing immediately after stimulus onset, and returns to baseline before blood volume. This is consistent with the model of pre-capillary arteriole active dilation driving the CBF response, with a subsequent CBV increase influenced by capillaries and veins dilating passively to accommodate the extra blood. rCMRO2 estimated using the steady-state formulation shows a biphasic pattern: an increase immediately after stimulus onset, followed by a post-stimulus undershoot due to blood flow returning faster to baseline than oxygenation. However, assuming a longer mean transit time from the arterial to the venous compartment, due to the immature vascular system of premature infants, reduces the post-stimulus undershoot and increases the flow/consumption ratio to values closer to adult values reported in the literature. We are the first to report changes in local rCBF and rCMRO2 during functional activation in preterm infants. The ability to measure these variables in addition to hemoglobin concentration changes is critical for understanding neurovascular coupling in the developing

  8. Automated single cell isolation from suspension with computer vision

    PubMed Central

    Ungai-Salánki, Rita; Gerecsei, Tamás; Fürjes, Péter; Orgovan, Norbert; Sándor, Noémi; Holczer, Eszter; Horvath, Robert; Szabó, Bálint

    2016-01-01

    Current robots can manipulate only surface-attached cells seriously limiting the fields of their application for single cell handling. We developed a computer vision-based robot applying a motorized microscope and micropipette to recognize and gently isolate intact individual cells for subsequent analysis, e.g., DNA/RNA sequencing in 1–2 nanoliters from a thin (~100 μm) layer of cell suspension. It can retrieve rare cells, needs minimal sample preparation, and can be applied for virtually any tissue cell type. Combination of 1 μm positioning precision, adaptive cell targeting and below 1 nl liquid handling precision resulted in an unprecedented accuracy and efficiency in robotic single cell isolation. Single cells were injected either into the wells of a miniature plate with a sorting speed of 3 cells/min or into standard PCR tubes with 2 cells/min. We could isolate labeled cells also from dense cultures containing ~1,000 times more unlabeled cells by the successive application of the sorting process. We compared the efficiency of our method to that of single cell entrapment in microwells and subsequent sorting with the automated micropipette: the recovery rate of single cells was greatly improved. PMID:26856740

  9. Automated single cell isolation from suspension with computer vision.

    PubMed

    Ungai-Salánki, Rita; Gerecsei, Tamás; Fürjes, Péter; Orgovan, Norbert; Sándor, Noémi; Holczer, Eszter; Horvath, Robert; Szabó, Bálint

    2016-02-09

    Current robots can manipulate only surface-attached cells seriously limiting the fields of their application for single cell handling. We developed a computer vision-based robot applying a motorized microscope and micropipette to recognize and gently isolate intact individual cells for subsequent analysis, e.g., DNA/RNA sequencing in 1-2 nanoliters from a thin (~100 μm) layer of cell suspension. It can retrieve rare cells, needs minimal sample preparation, and can be applied for virtually any tissue cell type. Combination of 1 μm positioning precision, adaptive cell targeting and below 1 nl liquid handling precision resulted in an unprecedented accuracy and efficiency in robotic single cell isolation. Single cells were injected either into the wells of a miniature plate with a sorting speed of 3 cells/min or into standard PCR tubes with 2 cells/min. We could isolate labeled cells also from dense cultures containing ~1,000 times more unlabeled cells by the successive application of the sorting process. We compared the efficiency of our method to that of single cell entrapment in microwells and subsequent sorting with the automated micropipette: the recovery rate of single cells was greatly improved.

  10. Single-cell printer: automated, on demand, and label free.

    PubMed

    Gross, Andre; Schöndube, Jonas; Niekrawitz, Sonja; Streule, Wolfgang; Riegger, Lutz; Zengerle, Roland; Koltay, Peter

    2013-12-01

    Within the past years, single-cell analysis has developed into a key topic in cell biology to study cellular functions that are not accessible by investigation of larger cell populations. Engineering approaches aiming to access single cells to extract information about their physiology, phenotype, and genotype at the single-cell level are going manifold ways, meanwhile allowing separation, sorting, culturing, and analysis of individual cells. Based on our earlier research toward inkjet-like printing of single cells, this article presents further characterization results obtained with a fully automated prototype instrument for printing of single living cells in a noncontact inkjet-like manner. The presented technology is based on a transparent microfluidic drop-on-demand dispenser chip coupled with a camera-assisted automatic detection system. Cells inside the chip are detected and classified with this detection system before they are expelled from the nozzle confined in microdroplets, thus enabling a "one cell per droplet" printing mode. To demonstrate the prototype instrument's suitability for biological and biomedical applications, basic experiments such as printing of single-bead and cell arrays as well as deposition and culture of single cells in microwell plates are presented. Printing efficiencies greater than 80% and viability rates about 90% were achieved.

  11. [Research on accurate measurement of oxygen content in coal using laser-induced breakdown spectroscopy in air environment].

    PubMed

    Yin, Wang-bao; Zhang, Lei; Wang, Le; Dong, Lei; Ma, Wei-guang; Jia, Suo-tang

    2012-01-01

    A technique about accurate measurement of oxygen content in coal in air environment using laser-induced breakdown spectroscopy (LIBS) is introduced in the present paper. Coal samples were excited by the laser, and plasma spectra were obtained. Combining internal standard method, temperature correction method and multi-line methods, the oxygen content of coal samples was precisely measured. The measurement precision is not less than 1.37% for oxygen content in coal analysis, so is satisfied for the requirement of coal-fired power plants in coal analysis. This method can be used in surveying, environmental protection, medicine, materials, archaeological and food safety, biochemical and metallurgy application.

  12. [Research on accurate measurement of oxygen content in coal using laser-induced breakdown spectroscopy in air environment].

    PubMed

    Yin, Wang-bao; Zhang, Lei; Wang, Le; Dong, Lei; Ma, Wei-guang; Jia, Suo-tang

    2012-01-01

    A technique about accurate measurement of oxygen content in coal in air environment using laser-induced breakdown spectroscopy (LIBS) is introduced in the present paper. Coal samples were excited by the laser, and plasma spectra were obtained. Combining internal standard method, temperature correction method and multi-line methods, the oxygen content of coal samples was precisely measured. The measurement precision is not less than 1.37% for oxygen content in coal analysis, so is satisfied for the requirement of coal-fired power plants in coal analysis. This method can be used in surveying, environmental protection, medicine, materials, archaeological and food safety, biochemical and metallurgy application. PMID:22497159

  13. Single Cell Mass Cytometry for Analysis of Immune System Functional States

    PubMed Central

    Bjornson, Zach B.; Nolan, Garry P.; Fantl, Wendy J.

    2013-01-01

    Single cell mass cytometry facilitates high-dimensional, quantitative analysis of the effects of bioactive molecules on cell populations at single-cell resolution. Datasets are generated with antibody panels (upwards of 40) in which each antibody is conjugated to a polymer chelated with a stable metal isotope, usually in the Lanthanide series of the periodic table. Isotope labelled antibodies recognize surface markers to delineate cell types and intracellular signaling molecules to provide a measure of the network state—and thereby demarcating multiple cell state functions such as apoptosis, DNA damage and cell cycle. By measuring all these parameters simultaneously, the signaling state of an individual cell can be measured at its network state. This review will cover the basics of mass cytometry as well as outline steps already taken to allow it to stand aside traditional fluorescence based cytometry in the immunologist’s analytical arsenal in their study of immune states during infection. PMID:23999316

  14. A comprehensive strategy for the analysis of acoustic compressibility and optical deformability on single cells

    NASA Astrophysics Data System (ADS)

    Yang, Tie; Bragheri, Francesca; Nava, Giovanni; Chiodi, Ilaria; Mondello, Chiara; Osellame, Roberto; Berg-Sørensen, Kirstine; Cristiani, Ilaria; Minzioni, Paolo

    2016-04-01

    We realized an integrated microfluidic chip that allows measuring both optical deformability and acoustic compressibility on single cells, by optical stretching and acoustophoresis experiments respectively. Additionally, we propose a measurement protocol that allows evaluating the experimental apparatus parameters before performing the cell-characterization experiments, including a non-destructive method to characterize the optical force distribution inside the microchannel. The chip was used to study important cell-mechanics parameters in two human breast cancer cell lines, MCF7 and MDA-MB231. Results indicate that MDA-MB231 has both higher acoustic compressibility and higher optical deformability than MCF7, but statistical analysis shows that optical deformability and acoustic compressibility are not correlated parameters. This result suggests the possibility to use them to analyze the response of different cellular structures. We also demonstrate that it is possible to perform both measurements on a single cell, and that the order of the two experiments does not affect the retrieved values.

  15. Atomic oxygen in the mesosphere and lower thermosphere derived from SABER: Algorithm theoretical basis and measurement uncertainty

    NASA Astrophysics Data System (ADS)

    Mlynczak, Martin G.; Hunt, Linda A.; Mast, Jeffrey C.; Thomas Marshall, B.; Russell, James M.; Smith, Anne K.; Siskind, David E.; Yee, Jeng-Hwa; Mertens, Christopher J.; Martin-Torres, F. Javier; Thompson, R. Earl; Drob, Douglas P.; Gordley, Larry L.

    2013-06-01

    Atomic oxygen (O) is a fundamental component in chemical aeronomy of Earth's mesosphere and lower thermosphere region extending from approximately 50 km to over 100 km in altitude. Atomic oxygen is notoriously difficult to measure, especially with remote sensing techniques from orbiting satellite sensors. It is typically inferred from measurements of the ozone concentration in the day or from measurements of the Meinel band emission of the hydroxyl radical (OH) at night. The Sounding of the Atmosphere using Broadband Emission Radiometry (SABER) instrument on the NASA Thermosphere-Ionosphere-Mesosphere Energetics and Dynamics (TIMED) satellite measures OH emission and ozone for the purpose of determining the O-atom concentration. In this paper, we present the algorithms used in the derivation of day and night atomic oxygen from these measurements. We find excellent consistency between the day and night O-atom concentrations from daily to annual time scales. We also examine in detail the collisional relaxation of the highly vibrationally excited OH molecule at night measured by SABER. Large rate coefficients for collisional removal of vibrationally excited OH molecules by atomic oxygen are consistent with the SABER observations if the deactivation of OH(9) proceeds solely by collisional quenching. An uncertainty analysis of the derived atomic oxygen is also given. Uncertainty in the rate coefficient for recombination of O and molecular oxygen is shown to be the largest source of uncertainty in the derivation of atomic oxygen day or night.

  16. Vapor pressures and thermodynamics of oxygen-containing polycyclic aromatic hydrocarbons measured using Knudsen effusion

    SciTech Connect

    Goldfarb, J.L.; Suuberg, E.M.

    2008-06-15

    Polycyclic aromatic hydrocarbons (PAHs) and their oxygenated derivatives (OPAHs) are ubiquitous environmental pollutants resulting from the incomplete combustion of coal and fossil fuels. Their vapor pressures are key thermodynamic data essential for modeling fate and transport within the environment. The present study involved nine PAHs containing oxygen heteroatoms, including aldehyde, carboxyl, and nitro groups, specifically 2-nitrofluorene, 9-fluorenecarboxylic acid, 2-fluorenecarboxaldehyde, 2-anthracenecarboxylic acid, 9-anthracenecarboxylic acid, 9-anthraldehyde, 1-nitropyrene, 1-pyrenecarboxaldehyde, and 1-bromo-2-naphthoic acid. The vapor pressures of these compounds, with molecular weights ranging from 194 to 251 g/mol, were measured using the isothermal Knudsen effusion technique in the temperature range of 329 to 421 K. The corresponding enthalpies of sublimation, calculated via the Clausius-Clapeyron equation, are compared to parent, nonoxygenated PAH compound data to determine the effect of the addition of these oxygen-containing heteroatoms. As expected, the addition of -CHO, -COOH, and -NO{sub 2} groups onto these PAHs increases the enthalpy of sublimation and decreases the vapor pressure as compared to the parent PAH; the position of substitution also plays a significant role in determining the vapor pressure of these OPAHs.

  17. Vapor pressures and thermodynamics of oxygen-containing polycyclic aromatic hydrocarbons measured using Knudsen effusion.

    PubMed

    Goldfarb, Jillian L; Suuberg, Eric M

    2008-06-01

    Polycyclic aromatic hydrocarbons (PAHs) and their oxygenated derivatives (OPAHs) are ubiquitous environmental pollutants resulting from the incomplete combustion of coal and fossil fuels. Their vapor pressures are key thermodynamic data essential for modeling fate and transport within the environment. The present study involved nine PAHs containing oxygen heteroatoms, including aldehyde, carboxyl, and nitro groups, specifically 2-nitrofluorene, 9-fluorenecarboxylic acid, 2-fluorenecarboxaldehyde, 2-anthracenecarboxylic acid, 9-anthracenecarboxylic acid, 9-anthraldehyde, 1-nitropyrene, 1-pyrenecarboxaldehyde, and 1-bromo-2-naphthoic acid. The vapor pressures of these compounds, with molecular weights ranging from 194 to 251 g/mol, were measured using the isothermal Knudsen effusion technique in the temperature range of 329 to 421 K. The corresponding enthalpies of sublimation, calculated via the Clausius-Clapeyron equation, are compared to parent, nonoxygenated PAH compound data to determine the effect of the addition of these oxygen-containing heteroatoms. As expected, the addition of -CHO, -COOH, and -NO(2) groups onto these PAHs increases the enthalpy of sublimation and decreases the vapor pressure as compared to the parent PAH; the position of substitution also plays a significant role in determining the vapor pressure of these OPAHs. PMID:18220445

  18. Measurement of the oxygen partial pressure and thermodynamic modeling of the U-Nd-O system

    NASA Astrophysics Data System (ADS)

    Lee, Seung Min; Knight, Travis W.; McMurray, Jacob W.; Besmann, Theodore M.

    2016-05-01

    Fission products greatly impact the properties of fuel necessitating a thorough understanding of the thermochemical properties of oxide fuels with fission products. However, thermochemical data for the U-Nd-O system is insufficient even though neodymium is a major fission product. As neodymium will likely be present as a solute in UO2, this research focuses on the study of (U1-yNdy)O2±x. Experimental measurements and analyses of the oxygen partial pressure (pO2)-temperature-oxygen to metal ratio (O/M ratio) relationships were performed using a thermogravimetric analyzer (TGA) and an oxygen analyzer. Thermodynamic computational modeling was performed using the CALPHAD (CALculation of PHAse Diagrams) method with the FactSage software. The Gibbs energy of the (U1-yNdy)O2±x solid solution was described by the compound energy formalism (CEF), which is based on earlier thermodynamic modeling data of the binary U-O system from Guéneau et al.. The thermodynamic and phase diagram data of the U-Nd-O system produced in this work show good agreement with the experimental data.

  19. Determination of Sediment Oxygen Demand in the Ziya River Watershed, China: Based on Laboratory Core Incubation and Microelectrode Measurements.

    PubMed

    Rong, Nan; Shan, Baoqing; Wang, Chao

    2016-02-19

    A study coupling sedimentcore incubation and microelectrode measurement was performed to explore the sediment oxygen demand (SOD) at 16 stations in the Ziya River Watershed, a severely polluted and anoxic river system in the north of China. Total oxygen flux values in the range 0.19-1.41 g/(m²·d) with an average of 0.62 g/(m²·d) were obtained by core incubations, and diffusive oxygen flux values in the range 0.15-1.38 g/(m²·d) with an average of 0.51 g/(m²·d) were determined by microelectrodes. Total oxygen flux obviously correlated with diffusive oxygen flux (R² = 0.842). The microelectrode method produced smaller results than the incubation method in 15 of 16 sites, and the diffusive oxygen flux was smaller than the total oxygen flux. Although the two sets of SOD values had significant difference accepted by the two methods via the Wilcoxon signed-rank test (p < 0.05), the microelectrode method was shown to produce results that were similar to those from the core incubation method. The microelectrode method, therefore, could be used as an alternative method for traditional core incubation method, or as a method to verify SOD rates measured by other methods. We consider that high potential sediment oxygen demand would occur in the Ziya River Watershed when the dissolved oxygen (DO) recovered in the overlying water.

  20. Measurement of changes in blood oxygenation using Multispectral Optoacoustic Tomography (MSOT) allows assessment of tumor development

    NASA Astrophysics Data System (ADS)

    Tomaszewski, Michal R.; Quiros-Gonzalez, Isabel; Joseph, James; Bohndiek, Sarah E.

    2016-03-01

    The ability to evaluate tumor oxygenation in the clinic could indicate prognosis and enable treatment monitoring, since oxygen deficient cancer cells are more resistant to chemotherapy and radiotherapy. MultiSpectral Optoacoustic Tomography (MSOT) is a hybrid technique combining the high contrast of optical imaging with the spatial resolution and penetration depth similar to ultrasound. We aim to demonstrate that MSOT can be used to monitor the development of tumor vasculature. To establish the relationship between MSOT derived imaging biomarkers and biological changes during tumor development, we performed MSOT on nude mice (n=10) bearing subcutaneous xenograft U87 glioblastoma tumors using a small animal optoacoustic tomography system. The mice were maintained under inhalation anesthesia during imaging and respired oxygen content was modified between 21% and 100%. The measurements from early (week 4) and late (week 7) stages of tumor development were compared. To further explore the functionality of the blood vessels, we examined the evolution of changes in the abundance of oxy- and deoxyhemoglobin in the tumors in response to a gas challenge. We found that the kinetics of the change in oxygen saturation (SO2) were significantly different between small tumors and the healthy blood vessels in nearby normal tissue (p=0.0054). Furthermore, we showed that there was a significant difference in the kinetics of the gas challenge between small and large tumors (p=0.0015). We also found that the tumor SO2 was significantly correlated (p=0.0057) with the tumor necrotic fraction as assessed by H&E staining in histology. In the future, this approach may be of use in the clinic as a method for tumor staging and assessment of treatment response.

  1. Tools for Single-Cell Kinetic Analysis of Virus-Host Interactions.

    PubMed

    Warrick, Jay W; Timm, Andrea; Swick, Adam; Yin, John

    2016-01-01

    Measures of cellular gene expression or behavior, when performed on individual cells, inevitably reveal a diversity of behaviors and outcomes that can correlate with normal or diseased states. For virus infections, the potential diversity of outcomes are pushed to an extreme, where measures of infection reflect features of the specific infecting virus particle, the individual host cell, as well as interactions between viral and cellular components. Single-cell measures, while revealing, still often rely on specialized fluid handling capabilities, employ end-point measures, and remain labor-intensive to perform. To address these limitations, we consider a new microwell-based device that uses simple pipette-based fluid handling to isolate individual cells. Our design allows different experimental conditions to be implemented in a single device, permitting easier and more standardized protocols. Further, we utilize a recently reported dual-color fluorescent reporter system that provides dynamic readouts of viral and cellular gene expression during single-cell infections by vesicular stomatitis virus. In addition, we develop and show how free, open-source software can enable streamlined data management and batch image analysis. Here we validate the integration of the device and software using the reporter system to demonstrate unique single-cell dynamic measures of cellular responses to viral infection. PMID:26752057

  2. Tools for Single-Cell Kinetic Analysis of Virus-Host Interactions

    PubMed Central

    Swick, Adam; Yin, John

    2016-01-01

    Measures of cellular gene expression or behavior, when performed on individual cells, inevitably reveal a diversity of behaviors and outcomes that can correlate with normal or diseased states. For virus infections, the potential diversity of outcomes are pushed to an extreme, where measures of infection reflect features of the specific infecting virus particle, the individual host cell, as well as interactions between viral and cellular components. Single-cell measures, while revealing, still often rely on specialized fluid handling capabilities, employ end-point measures, and remain labor-intensive to perform. To address these limitations, we consider a new microwell-based device that uses simple pipette-based fluid handling to isolate individual cells. Our design allows different experimental conditions to be implemented in a single device, permitting easier and more standardized protocols. Further, we utilize a recently reported dual-color fluorescent reporter system that provides dynamic readouts of viral and cellular gene expression during single-cell infections by vesicular stomatitis virus. In addition, we develop and show how free, open-source software can enable streamlined data management and batch image analysis. Here we validate the integration of the device and software using the reporter system to demonstrate unique single-cell dynamic measures of cellular responses to viral infection. PMID:26752057

  3. What Population Reveals about Individual Cell Identity: Single-Cell Parameter Estimation of Models of Gene Expression in Yeast

    PubMed Central

    Versari, Cristian; Cinquemani, Eugenio; Ferrari-Trecate, Giancarlo; Hersen, Pascal; Batt, Gregory

    2016-01-01

    Significant cell-to-cell heterogeneity is ubiquitously observed in isogenic cell populations. Consequently, parameters of models of intracellular processes, usually fitted to population-averaged data, should rather be fitted to individual cells to obtain a population of models of similar but non-identical individuals. Here, we propose a quantitative modeling framework that attributes specific parameter values to single cells for a standard model of gene expression. We combine high quality single-cell measurements of the response of yeast cells to repeated hyperosmotic shocks and state-of-the-art statistical inference approaches for mixed-effects models to infer multidimensional parameter distributions describing the population, and then derive specific parameters for individual cells. The analysis of single-cell parameters shows that single-cell identity (e.g. gene expression dynamics, cell size, growth rate, mother-daughter relationships) is, at least partially, captured by the parameter values of gene expression models (e.g. rates of transcription, translation and degradation). Our approach shows how to use the rich information contained into longitudinal single-cell data to infer parameters that can faithfully represent single-cell identity. PMID:26859137

  4. 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.

  5. 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

  6. Disentangling neural cell diversity using single-cell transcriptomics.

    PubMed

    Poulin, Jean-Francois; Tasic, Bosiljka; Hjerling-Leffler, Jens; Trimarchi, Jeffrey M; Awatramani, Rajeshwar

    2016-08-26

    Cellular specialization is particularly prominent in mammalian nervous systems, which are composed of millions to billions of neurons that appear in thousands of different 'flavors' and contribute to a variety of functions. Even in a single brain region, individual neurons differ greatly in their morphology, connectivity and electrophysiological properties. Systematic classification of all mammalian neurons is a key goal towards deconstructing the nervous system into its basic components. With the recent advances in single-cell gene expression profiling technologies, it is now possible to undertake the enormous task of disentangling neuronal heterogeneity. High-throughput single-cell RNA sequencing and multiplexed quantitative RT-PCR have become more accessible, and these technologies enable systematic categorization of individual neurons into groups with similar molecular properties. Here we provide a conceptual and practical guide to classification of neural cell types using single-cell gene expression profiling technologies. PMID:27571192

  7. 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

  8. Disentangling neural cell diversity using single-cell transcriptomics.

    PubMed

    Poulin, Jean-Francois; Tasic, Bosiljka; Hjerling-Leffler, Jens; Trimarchi, Jeffrey M; Awatramani, Rajeshwar

    2016-08-26

    Cellular specialization is particularly prominent in mammalian nervous systems, which are composed of millions to billions of neurons that appear in thousands of different 'flavors' and contribute to a variety of functions. Even in a single brain region, individual neurons differ greatly in their morphology, connectivity and electrophysiological properties. Systematic classification of all mammalian neurons is a key goal towards deconstructing the nervous system into its basic components. With the recent advances in single-cell gene expression profiling technologies, it is now possible to undertake the enormous task of disentangling neuronal heterogeneity. High-throughput single-cell RNA sequencing and multiplexed quantitative RT-PCR have become more accessible, and these technologies enable systematic categorization of individual neurons into groups with similar molecular properties. Here we provide a conceptual and practical guide to classification of neural cell types using single-cell gene expression profiling technologies.

  9. Defining heterogeneity within bacterial populations via single cell approaches.

    PubMed

    Davis, Kimberly M; Isberg, Ralph R

    2016-08-01

    Bacterial populations are heterogeneous, which in many cases can provide a selective advantage during changes in environmental conditions. In some instances, heterogeneity exists at the genetic level, in which significant allelic variation occurs within a population seeded by a single cell. In other cases, heterogeneity exists due to phenotypic differences within a clonal, genetically identical population. A variety of mechanisms can drive this latter strategy. Stochastic fluctuations can drive differential gene expression, but heterogeneity in gene expression can also be driven by environmental changes sensed by individual cells residing in distinct locales. Utilizing multiple single cell approaches, workers have started to uncover the extent of heterogeneity within bacterial populations. This review will first describe several examples of phenotypic and genetic heterogeneity, and then discuss many single cell approaches that have recently been applied to define heterogeneity within bacterial populations. PMID:27273675

  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. 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

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

    PubMed

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

    2011-02-01

    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 host 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.

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

    NASA Astrophysics Data System (ADS)

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

    2011-02-01

    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 host 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.

  14. 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

    2016-10-01

    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.

  15. 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

    2016-10-01

    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. PMID:27510097

  16. Automatable Measurement of Gas Exchange Rate in Streams: Oxygen-Carbon Method

    NASA Astrophysics Data System (ADS)

    Pennington, R.; Haggerty, R.; Argerich, A.; Wondzell, S. M.

    2015-12-01

    Gas exchange rates between streams and the atmosphere are critically important to measurement of in-stream ecologic processes, as well as fate and transport of hazardous pollutants such as mercury and PCBs. Methods to estimate gas exchange rates include empirical relations to hydraulics, and direct injection of a tracer gas such as propane or SF6. Empirical relations are inconsistent and inaccurate, particularly for lower order, high-roughness streams. Gas injections are labor-intensive, and measured gas exchange rates are difficult to extrapolate in time since they change with discharge and stream geometry. We propose a novel method for calculation of gas exchange rates utilizing O2, pCO2, pH, and temperature data. Measurements, which can be automated using data loggers and probes, are made on the upstream and downstream end of the study reach. Gas exchange rates are then calculated from a solution to the transport equations for oxygen and dissolved inorganic carbon. Field tests in steep, low order, high roughness streams of the HJ Andrews Experimental Forest indicate the method to be viable along stream reaches with high downstream gas concentration gradients and high rates of gas transfer velocity. Automated and continuous collection of oxygen and carbonate chemistry data is increasingly common, thus the method may be used to estimate gas exchange rates through time, and is well suited for interactivity with databases.

  17. A pilot study of a new spectrophotometry device to measure tissue oxygen saturation.

    PubMed

    Abel, Gemma; Allen, John; Drinnan, Michael

    2014-09-01

    Tissue oxygen saturation (SO2) measurements have the potential for far wider use than at present but are limited by device availability and portability for many potential applications. A device based on a small, low-cost general-purpose spectrophotometer (the Harrison device) might facilitate wider use. The aim of this study was to compare the Harrison device with a commercial instrument, the LEA O2C.Measurements were carried out on the forearm and finger of 20 healthy volunteers, using a blood pressure cuff on the upper arm to induce different levels of oxygenation. Repeatability of both devices was assessed, and the Bland-Altman method was used to assess agreement between them.The devices showed agreement in overall tracking of changes in SO2. Test-retest agreement for the Harrison device was worse than for O2C, with SD repeatability of 10.6% (forearm) or 18.6% (finger). There was no overall bias between devices, but mean (SD) difference of 1.2 (11.8%) (forearm) or 4.4 (11.5%) (finger) were outside of a clinically acceptable range.Disagreements were attributed to the stability of the Harrison probe and the natural SO2 variations across the skin surface increasing the random error. Therefore, though not equivalent to the LEA O2C, a probe redesign and averaged measurements may help establish the Harrison device as a low cost alternative.

  18. 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.

  19. [A Measurement Method for the Hepatic Functional Reserves Anti Oxygen Fluctuations].

    PubMed

    Liu, Guang-da; Chen, Yong; Wang, Jing-jing

    2015-06-01

    Hepatic functional reserves parameters are the key indictors to assess if the hepatic metabolic function is normal, they are also the important basis to a successful hepatectomy. Currently clinical hepatic functional reserves parameters are achieved through Indocyanine Green (ICG) concentration measurement in the method of pulse dye spectrophotometry, with the assumption that blood oxygen saturation is 100%, this hypothetical bias leads to an error in the calculated value of the hepatic functional reserves parameters. In order to solve this problem, hepatic functional reserves parameters measurement that resist fluctuation from blood is presented. The method is based on the modified Lambert Beer's law and realize the correction of ICG concentration measurement in the method of pulse dye spectrophotometry. While the ICG is injected into the patient's body by the cubital veins, using the data acquisition unit that developed by project team to collect 805 nm, 940 nm wavelengths of transmission signals and 730 nm, 805 nm and 890 nm wavelengths of reflected signals in the fingertip skin synchronously, and then upload 5 sets of data to the computer. Draw the ICG concentration curve according the collected data and blood oxygen saturation before injecting ICG to the human body and then calculate the characteristic parameters, according to the characteristic parameters calculate the hepatic reserves functional parameters. Taking the measurement of effective hepatic blood flow as an example, the relative error was obviously decreased when comparing this method and the pulse dye concentration method with the electromagnetic flowmeter (EMF) measurement which is the most accurate method to measure effective hepatic blood flow (EHBF) respectively. The results demonstrate that this method can improve the accuracy of hepatic reserves parameters, and it can also provide a more accurate detection method of hepatic functional reserves parameters for clinical application.

  20. Continuous measurements of greenhouse gases and atmospheric oxygen at the Namib Desert Atmospheric Observatory

    NASA Astrophysics Data System (ADS)

    Morgan, E. J.; Lavrič, J. V.; Seifert, T.; Chicoine, T.; Day, A.; Gomez, J.; Logan, R.; Sack, J.; Shuuya, T.; Uushona, E. G.; Vincent, K.; Schultz, U.; Brunke, E.-G.; Labuschagne, C.; Thompson, R. L.; Schmidt, S.; Manning, A. C.; Heimann, M.

    2015-06-01

    A new coastal background site has been established for observations of greenhouse gases (GHGs) in the central Namib Desert at Gobabeb, Namibia. The location of the site was chosen to provide observations for a data-poor region in the global sampling network for GHGs. Semi-automated continuous measurements of carbon dioxide, methane, nitrous oxide, carbon monoxide, atmospheric oxygen, and basic meteorology are made at a height of 21 m a.g.l., 50 km from the coast at the northern border of the Namib Sand Sea. Atmospheric oxygen is measured with a differential fuel cell analyzer (DFCA). Carbon dioxide and methane are measured with an early-model cavity ring-down spectrometer (CRDS); nitrous oxide and carbon monoxide are measured with an off-axis integrated cavity output spectrometer (OA-ICOS). Instrument-specific water corrections are employed for both the CRDS and OA-ICOS instruments in lieu of drying. The performance and measurement uncertainties are discussed in detail. As the station is located in a remote desert environment, there are some particular challenges, namely fine dust, high diurnal temperature variability, and minimal infrastructure. The gas handling system and calibration scheme were tailored to best fit the conditions of the site. The CRDS and DFCA provide data of acceptable quality when base requirements for operation are met, specifically adequate temperature control in the laboratory and regular supply of electricity. In the case of the OA-ICOS instrument, performance is significantly improved through the implementation of a drift correction through frequent measurements of a reference cylinder.

  1. 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.

  2. The technology and biology of single-cell RNA sequencing.

    PubMed

    Kolodziejczyk, Aleksandra A; Kim, Jong Kyoung; Svensson, Valentine; Marioni, John C; Teichmann, Sarah A

    2015-05-21

    The differences between individual cells can have profound functional consequences, in both unicellular and multicellular organisms. Recently developed single-cell mRNA-sequencing methods enable unbiased, high-throughput, and high-resolution transcriptomic analysis of individual cells. This provides an additional dimension to transcriptomic information relative to traditional methods that profile bulk populations of cells. Already, single-cell RNA-sequencing methods have revealed new biology in terms of the composition of tissues, the dynamics of transcription, and the regulatory relationships between genes. Rapid technological developments at the level of cell capture, phenotyping, molecular biology, and bioinformatics promise an exciting future with numerous biological and medical applications. PMID:26000846

  3. Molecular circuits for associative learning in single-celled organisms.

    PubMed

    Fernando, Chrisantha T; Liekens, Anthony M L; Bingle, Lewis E H; Beck, Christian; Lenser, Thorsten; Stekel, Dov J; Rowe, Jonathan E

    2009-05-01

    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.

  4. Functionalized nanopipettes: toward label-free, single cell biosensors

    PubMed Central

    Actis, Paolo; Mak, Andy C.

    2010-01-01

    Nanopipette technology has been proven to be a label-free biosensor capable of identifying DNA and proteins. The nanopipette can include specific recognition elements for analyte discrimination based on size, shape, and charge density. The fully electrical read-out and the ease and low-cost fabrication are unique features that give this technology an enormous potential. Unlike other biosensing platforms, nanopipettes can be precisely manipulated with submicron accuracy and used to study single cell dynamics. This review is focused on creative applications of nanopipette technology for biosensing. We highlight the potential of this technology with a particular attention to integration of this biosensor with single cell manipulation platforms. PMID:20730113

  5. 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

  6. 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

  7. Single cell pattern formation and transient cytoskeletal arrays

    PubMed Central

    Bement, William M.; von Dassow, George

    2015-01-01

    A major goal of developmental biology is to explain the emergence of pattern in cell layers, tissues and organs. Developmental biologists now accept that reaction diffusion-based mechanisms are broadly employed in developing organisms to direct pattern formation. Here we briefly consider these mechanisms and then apply some of the concepts derived from them to several processes that occur in single cells: wound repair, yeast budding, and cytokinesis. Two conclusions emerge from this analysis: first, there is considerable overlap at the level of general mechanisms between developmental and single cell pattern formation; second, dynamic structures based on the actin cytoskeleton may be far more ordered