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Sample records for measuring single-cell oxygen

  1. 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

  2. Measuring and Modeling Apoptosis in Single Cells

    PubMed Central

    Spencer, Sabrina L.; Sorger, Peter K.

    2011-01-01

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

  3. Using measures of single-cell physiology and physiological state to understand organismic aging.

    PubMed

    Mendenhall, Alexander; Driscoll, Monica; Brent, Roger

    2016-02-01

    Genetically identical organisms in homogeneous environments have different lifespans and healthspans. These differences are often attributed to stochastic events, such as mutations and 'epimutations', changes in DNA methylation and chromatin that change gene function and expression. But work in the last 10 years has revealed differences in lifespan- and health-related phenotypes that are not caused by lasting changes in DNA or identified by modifications to DNA or chromatin. This work has demonstrated persistent differences in single-cell and whole-organism physiological states operationally defined by values of reporter gene signals in living cells. While some single-cell states, for example, responses to oxygen deprivation, were defined previously, others, such as a generally heightened ability to make proteins, were, revealed by direct experiment only recently, and are not well understood. Here, we review technical progress that promises to greatly increase the number of these measurable single-cell physiological variables and measureable states. We discuss concepts that facilitate use of single-cell measurements to provide insight into physiological states and state transitions. We assert that researchers will use this information to relate cell level physiological readouts to whole-organism outcomes, to stratify aging populations into groups based on different physiologies, to define biomarkers predictive of outcomes, and to shed light on the molecular processes that bring about different individual physiologies. For these reasons, quantitative study of single-cell physiological variables and state transitions should provide a valuable complement to genetic and molecular explanations of how organisms age.

  4. Microfluidic micropipette aspiration for measuring the deformability of single cells.

    PubMed

    Guo, Quan; Park, Sunyoung; Ma, Hongshen

    2012-08-07

    We present a microfluidic technique for measuring the deformability of single cells using the pressure required to deform such cells through micrometre-scale tapered constrictions. Our technique is equivalent to whole-cell micropipette aspiration, but involves considerably simpler operation, less specialized equipment, and less technical skill. Single cells are infused into a microfluidic channel, and then deformed through a series of funnel-shaped constrictions. The constriction openings are sized to create a temporary seal with each cell as it passes through the constriction, replicating the interaction with the orifice of a micropipette. Precisely controlled deformation pressures are generated using an external source and then attenuated 100 : 1 using an on-chip microfluidic circuit. Our apparatus is capable of generating precisely controlled pressures as small as 0.3 Pa in a closed microchannel network, which is impervious to evaporative losses that normally limit the precision of such equipment. Intrinsic cell deformability, expressed as cortical tension, is determined from the threshold deformation pressure using the liquid-drop model. We measured the deformability of several types of nucleated cells and determined the optimal range of constriction openings. The cortical tension of passive human neutrophils was measured to be 37.0 ± 4.8 pN μm(-1), which is consistent with previous micropipette aspiration studies. The cortical tensions of human lymphocytes, RT4 human bladder cancer cells, and L1210 mouse lymphoma cells were measured to be 74.7 ± 9.8, 185.4 ± 25.3, and 235.4 ± 31.0 pN μm(-1) respectively. The precision and usability of our technique demonstrates its potential as a biomechanical assay for wide-spread use in biological and clinical laboratories.

  5. 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.

  6. 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.

  7. 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.

  8. 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

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

    PubMed Central

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

    2011-01-01

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

  10. 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.

  11. 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.

  12. Single Cell Mass Measurement Using Drag ForceInside Lab-on-Chip Microfluidics System.

    PubMed

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

    2015-12-22

    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 (2-7 μm 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.

  13. 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.

  14. Measurement of DNA damage in individual cells using the Single Cell Gel Electrophoresis (Comet) assay.

    PubMed

    Hartley, Janet M; Spanswick, Victoria J; Hartley, John A

    2011-01-01

    The Single Cell Gel Electrophoresis (Comet) assay is a simple, versatile and sensitive method for measuring DNA damage in individual cells, allowing the determination of heterogeneity of response within a cell population. The basic alkaline technique described is for the determination of DNA strand break damage and its repair at a single cell level. Specific modifications to the method use a lower pH ('neutral' assay), or allow the measurement of DNA interstrand cross-links. It can be further adapted to, for example, study specific DNA repair mechanisms, be combined with fluorescent in situ hybridisation, or incorporate lesion specific enzymes.

  15. Single cell stiffness measurement at various humidity conditions by nanomanipulation of a nano-needle.

    PubMed

    Shen, Yajing; Nakajima, Masahiro; Yang, Zhan; Tajima, Hirotaka; Najdovski, Zoran; Homma, Michio; Fukuda, Toshio

    2013-04-12

    This paper presents a method for single cell stiffness measurement based on a nano-needle and nanomanipulation. The nano-needle with a buffering beam was fabricated from an atomic force microscope cantilever by the focused ion beam etching technique. Wild type yeast cells (W303) were prepared and placed on the sample stage inside an environmental scanning electron microscope (ESEM) chamber. The nanomanipulator actuated the nano-needle to press against a single yeast cell. As a result, the deformation of the cell and nano-needle was observed by the ESEM system in real-time. Finally, the stiffness of the single cell was determined based on this deformation information. To reveal the relationship between the cell stiffness and the environmental humidity conditions, the cell stiffness was measured at three different humidity conditions, i.e. 40, 70 and 100%, respectively. The results show that the stiffness of a single cell is reduced with increasing humidity.

  16. Single cell stiffness measurement at various humidity conditions by nanomanipulation of a nano-needle

    NASA Astrophysics Data System (ADS)

    Shen, Yajing; Nakajima, Masahiro; Yang, Zhan; Tajima, Hirotaka; Najdovski, Zoran; Homma, Michio; Fukuda, Toshio

    2013-04-01

    This paper presents a method for single cell stiffness measurement based on a nano-needle and nanomanipulation. The nano-needle with a buffering beam was fabricated from an atomic force microscope cantilever by the focused ion beam etching technique. Wild type yeast cells (W303) were prepared and placed on the sample stage inside an environmental scanning electron microscope (ESEM) chamber. The nanomanipulator actuated the nano-needle to press against a single yeast cell. As a result, the deformation of the cell and nano-needle was observed by the ESEM system in real-time. Finally, the stiffness of the single cell was determined based on this deformation information. To reveal the relationship between the cell stiffness and the environmental humidity conditions, the cell stiffness was measured at three different humidity conditions, i.e. 40, 70 and 100%, respectively. The results show that the stiffness of a single cell is reduced with increasing humidity.

  17. 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

  18. 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.

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

    PubMed

    Guo, Xiaoliang; Zhu, Rong; Zong, Xianli

    2015-10-07

    This paper presents a novel microarray chip integrating cell positioning with in situ, real-time and long-time impedance measurement on a single cell. The microchip integrates a plurality of quadrupole-electrode units (termed positioning electrodes) patterned into an array with pairs of planar electrodes (termed measuring electrodes) located at the centers of each quadrupole-electrode unit. The positioning electrodes are utilized to trap and position living cells onto the measuring electrodes based on negative dielectrophoresis (nDEP), while the measuring electrodes are used to measure impedances of the trapped single cells. Each measuring electrode has a small footprint area of 7 × 7 μm(2) to ensure inhabiting only one single cell on it. However, the electrode with a small surface area has a low double-layer capacitance when it is immersed in a liquid solution, thus generating a large double-layer impedance, which reduces the sensitivity for impedance measurement on the single cell. To enlarge the effective surface areas of the measuring electrodes, a novel surface-modification process is proposed to controllably construct gold nanostructures on the surfaces of the measuring electrodes while the positioning electrodes are unstained. The double layer capacitances of the modified electrodes are increased by about one order after surface-modification. The developed microchip is used to monitor the adhering behavior of a single HeLa cell by measuring its impedance spectra in real time. The measured impedance is analyzed and used to extract cellular electrical parameters, which demonstrated that the cell compresses the electrical double layer in the process of adherence and adheres onto the measuring electrodes after 4-5 hours.

  20. Measurement of Single-Cell Deformability Using Impedance Analysis on Microfluidic Chip

    NASA Astrophysics Data System (ADS)

    Kim, Dongil; Choi, Eunpyo; Choi, Sung Sik; Lee, Sangho; Park, Jungyul; Yun, Kwang-Seok

    2010-12-01

    In this paper, we propose a microfluidic chip that measures the deformability of single cells by an impedance measurement method. The proposed chip is designed to differentiate the deformability of various cells by measuring the length of their stretched membrane indirectly according to the variation of the impedance after applying aspiration pressure to the cell membrane. The length of the stretched cell membrane is proportional to the applied pressure. Lengths of 18 and 21 µm were observed at the same suction pressure for human breast normal cells (MCF-10A) and caner cells (MCF-7), respectively. Electrical measurement was performed using an impedance analyzer at various frequencies. Results revealed that the impedance measurement method can be used to analyze the biomechanical characteristics of single cells, which indicates the state of malignancy of cells.

  1. 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

  2. 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.

  3. 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.

  4. Fluorescence emission spectral shift measurements of membrane potential in single cells.

    PubMed

    Kao, W Y; Davis, C E; Kim, Y I; Beach, J M

    2001-08-01

    Previous measurements of transmembrane potential using the electrochromic probe di-8-ANEPPS have used the excitation spectral shift response by alternating excitation between two wavelengths centered at voltage-sensitive portions of the excitation spectrum and recording at a single wavelength near the peak of the emission spectrum. Recently, the emission spectral shift associated with the change in transmembrane potential has been used for continuous membrane potential monitoring. To characterize this form of the electrochromic response from di-8-ANEPPS, we have obtained fluorescence signals from single cells in response to step changes in transmembrane potentials set with a patch electrode, using single wavelength excitation near the peak of the dye absorption spectrum. Fluorescence changes at two wavelengths near voltage-sensitive portions of the emission spectrum and shifts in the complete emission spectrum were determined for emission from plasma membrane and internal membrane. We found that the fluorescence ratio from either dual-wavelength recordings, or from opposite sides of the emission spectrum, varied linearly with the amplitude of the transmembrane potential step between -80 and +60 mV. Voltage dependence of difference spectra exhibit a crossover point near the peak of the emission spectra with approximately equal gain and loss of fluorescence intensity on each side of the spectrum and equal response amplitude for depolarization and hyperpolarization. These results are consistent with an electrochromic mechanism of action and demonstrate how the emission spectral shift response can be used to measure the transmembrane potential in single cells.

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

    PubMed Central

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

    2015-01-01

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

  6. 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.

  7. 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.

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

    PubMed

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

    2014-06-19

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

  9. 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

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

    PubMed

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

    2014-01-01

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

  11. Measurement of intracellular Ca2+ concentration in single cells using ratiometric calcium dyes.

    PubMed

    Srikanth, Sonal; Gwack, Yousang

    2013-01-01

    Measurement of intracellular Ca(2+) concentration ([Ca(2+)](i)) is useful to study the upstream and downstream events of Ca(2+) signaling. Ca(2+)-binding proteins including EF-hand-containing proteins are important downstream effector molecules after an increase of [Ca(2+)](i). Conversely, these proteins can also act as key modulators for regulation of [Ca(2+)](i) by sensing the Ca(2+) levels in the intracellular organelles and cytoplasm. Here we describe a single-cell Ca(2+) imaging technique that was used to measure the intracellular Ca(2+) levels to examine the function of Ca(2+)-binding proteins, STIM1 and Calcium release-activated Calcium channel regulator 2A (CRACR2A), using ratiometric Ca(2+) dye Fura-2 in adherent and non-adherent cells.

  12. SC3-seq: a method for highly parallel and quantitative measurement of single-cell gene expression.

    PubMed

    Nakamura, Tomonori; Yabuta, Yukihiro; Okamoto, Ikuhiro; Aramaki, Shinya; Yokobayashi, Shihori; Kurimoto, Kazuki; Sekiguchi, Kiyotoshi; Nakagawa, Masato; Yamamoto, Takuya; Saitou, Mitinori

    2015-05-19

    Single-cell mRNA sequencing (RNA-seq) methods have undergone rapid development in recent years, and transcriptome analysis of relevant cell populations at single-cell resolution has become a key research area of biomedical sciences. We here present single-cell mRNA 3-prime end sequencing (SC3-seq), a practical methodology based on PCR amplification followed by 3-prime-end enrichment for highly quantitative, parallel and cost-effective measurement of gene expression in single cells. The SC3-seq allows excellent quantitative measurement of mRNAs ranging from the 10,000-cell to 1-cell level, and accordingly, allows an accurate estimate of the transcript levels by a regression of the read counts of spike-in RNAs with defined copy numbers. The SC3-seq has clear advantages over other typical single-cell RNA-seq methodologies for the quantitative measurement of transcript levels and at a sequence depth required for the saturation of transcript detection. The SC3-seq distinguishes four distinct cell types in the peri-implantation mouse blastocysts. Furthermore, the SC3-seq reveals the heterogeneity in human-induced pluripotent stem cells (hiPSCs) cultured under on-feeder as well as feeder-free conditions, demonstrating a more homogeneous property of the feeder-free hiPSCs. We propose that SC3-seq might be used as a powerful strategy for single-cell transcriptome analysis in a broad range of investigations in biomedical sciences.

  13. 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.

  14. 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.

  15. 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

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

    PubMed

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

    2013-09-01

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

  17. MEMS squeezer for the measurement of single cell rupture force, stiffness change, and hysteresis

    NASA Astrophysics Data System (ADS)

    Barazani, B.; Warnat, S.; Fine, A.; Hubbard, T.

    2017-02-01

    A MEMS squeezer able to compress single living cells underwater until rupture was designed and tested. The relatively large motion range of the device in aqueous media (~2.5 µm) allows provoking cell disruption while measuring cell mechanical properties before and after membrane rupture. An AC driven electrothermal micro actuator with mechanical amplification pressed single cells against a reference back spring. Deformations of the cell and the reference spring were measured with nanoscale resolution using optical Fourier transform techniques. The motion of the reference spring divided by the cell deformation provides the cell stiffness relative to the reference spring constant. An abrupt change in the cell stiffness and the appearance of cracks indicated the cell wall rupture force was reached. A total of 22 baker’s yeast cells (Saccharomyces cerevisiae) were squeezed with the micro device. The average force necessary to rupture the cell membrane was 0.47  ±  0.1 µN. Before rupture the cells had an average stiffness of 9.3  ±  3.1 N m-1 the post-rupture stiffness dropped to 0.94  ±  0.57 N m-1. Cell hysteresis was also measured: cells squeezed and released before reaching the rupture force showed residual deformations below 100 nm, while cells squeezed past the rupture force and then released showed residual deformations between 490 and 990 nm.

  18. Finite Element Analysis of Single Cell Stiffness Measurements Using PZT-Integrated Buckling Nanoneedles

    PubMed Central

    Rad, Maryam Alsadat; Tijjani, Auwal Shehu; Ahmad, Mohd Ridzuan; Auwal, Shehu Muhammad

    2016-01-01

    This paper proposes a new technique for real-time single cell stiffness measurement using lead zirconate titanate (PZT)-integrated buckling nanoneedles. The PZT and the buckling part of the nanoneedle have been modelled and validated using the ABAQUS software. The two parts are integrated together to function as a single unit. After calibration, the stiffness, Young’s modulus, Poisson’s ratio and sensitivity of the PZT-integrated buckling nanoneedle have been determined to be 0.7100 N·m−1, 123.4700 GPa, 0.3000 and 0.0693 V·m·N−1, respectively. Three Saccharomyces cerevisiae cells have been modelled and validated based on compression tests. The average global stiffness and Young’s modulus of the cells are determined to be 10.8867 ± 0.0094 N·m−1 and 110.7033 ± 0.0081 MPa, respectively. The nanoneedle and the cell have been assembled to measure the local stiffness of the single Saccharomyces cerevisiae cells The local stiffness, Young’s modulus and PZT output voltage of the three different size Saccharomyces cerevisiae have been determined at different environmental conditions. We investigated that, at low temperature the stiffness value is low to adapt to the change in the environmental condition. As a result, Saccharomyces cerevisiae becomes vulnerable to viral and bacterial attacks. Therefore, the proposed technique will serve as a quick and accurate process to diagnose diseases at early stage in a cell for effective treatment. PMID:28025571

  19. 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.

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

    PubMed Central

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

    2014-01-01

    Protein concentrations are often regulated by dynamic changes in translation rates. Nevertheless, it has been challenging to directly monitor changes in translation in living cells. We have developed a reporter system to measure real-time changes of translation rates in human or mouse individual cells by conjugating translation regulatory motifs to sequences encoding a nuclear targeted fluorescent protein and a controllable destabilization domain. Application of the method showed that individual cells undergo marked fluctuations in the translation rate of mRNAs whose 5′ terminal oligopyrimidine (5′ TOP) motif regulates the synthesis of ribosomal proteins. Furthermore, we show that small reductions in amino acid levels signal through different mTOR-dependent pathways to control TOP mRNA translation, whereas larger reductions in amino acid levels control translation through eIF2A. Our study demonstrates that dynamic measurements of single-cell activities of translation regulatory motifs can be used to identify and investigate fundamental principles of translation. PMID:24213167

  1. Finite Element Analysis of Single Cell Stiffness Measurements Using PZT-Integrated Buckling Nanoneedles.

    PubMed

    Rad, Maryam Alsadat; Tijjani, Auwal Shehu; Ahmad, Mohd Ridzuan; Auwal, Shehu Muhammad

    2016-12-23

    This paper proposes a new technique for real-time single cell stiffness measurement using lead zirconate titanate (PZT)-integrated buckling nanoneedles. The PZT and the buckling part of the nanoneedle have been modelled and validated using the ABAQUS software. The two parts are integrated together to function as a single unit. After calibration, the stiffness, Young's modulus, Poisson's ratio and sensitivity of the PZT-integrated buckling nanoneedle have been determined to be 0.7100 N·m(-1), 123.4700 GPa, 0.3000 and 0.0693 V·m·N(-1), respectively. Three Saccharomyces cerevisiae cells have been modelled and validated based on compression tests. The average global stiffness and Young's modulus of the cells are determined to be 10.8867 ± 0.0094 N·m(-1) and 110.7033 ± 0.0081 MPa, respectively. The nanoneedle and the cell have been assembled to measure the local stiffness of the single Saccharomyces cerevisiae cells The local stiffness, Young's modulus and PZT output voltage of the three different size Saccharomyces cerevisiae have been determined at different environmental conditions. We investigated that, at low temperature the stiffness value is low to adapt to the change in the environmental condition. As a result, Saccharomyces cerevisiae becomes vulnerable to viral and bacterial attacks. Therefore, the proposed technique will serve as a quick and accurate process to diagnose diseases at early stage in a cell for effective treatment.

  2. Decoding the Regulatory Network for Blood Development from Single-Cell Gene Expression Measurements

    PubMed Central

    Haghverdi, Laleh; Lilly, Andrew J.; Tanaka, Yosuke; Wilkinson, Adam C.; Buettner, Florian; Macaulay, Iain C.; Jawaid, Wajid; Diamanti, Evangelia; Nishikawa, Shin-Ichi; Piterman, Nir; Kouskoff, Valerie; Theis, Fabian J.; Fisher, Jasmin; Göttgens, Berthold

    2015-01-01

    Here we report the use of diffusion maps and network synthesis from state transition graphs to better understand developmental pathways from single cell gene expression profiling. We map the progression of mesoderm towards blood in the mouse by single-cell expression analysis of 3,934 cells, capturing cells with blood-forming potential at four sequential developmental stages. By adapting the diffusion plot methodology for dimensionality reduction to single-cell data, we reconstruct the developmental journey to blood at single-cell resolution. Using transitions between individual cellular states as input, we develop a single-cell network synthesis toolkit to generate a computationally executable transcriptional regulatory network model that recapitulates blood development. Model predictions were validated by showing that Sox7 inhibits primitive erythropoiesis, and that Sox and Hox factors control early expression of Erg. We therefore demonstrate that single-cell analysis of a developing organ coupled with computational approaches can reveal the transcriptional programs that control organogenesis. PMID:25664528

  3. 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

  4. 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

  5. Sugar concentrations along and across the Ricinus communis L. hypocotyl measured by single cell sampling analysis.

    PubMed

    Verscht, Jutta; Tomos, Deri; Komor, Ewald

    2006-11-01

    Single cell sap sampling and analysis were used to measure the longitudinal and radial distribution of sucrose, glucose and fructose in the apical cell division zone and in the basal, elongated zone of the Ricinus hypocotyl. Sucrose and hexose increased in concentration from the apex to the base of the seedling axis. In the cell division zone low hexose and sucrose concentrations prevailed in cortex and pith, with a slightly higher hexose concentration in pith cells. The sucrose concentrations in sieve tubes and in phloem were much higher than in the cortex and pith cells. In the basal zone of the hypocotyl high levels of sucrose in phloem, cortex and pith were found, therefore radial, diffusional sucrose flow away from the phloem was considered unlikely. It is proposed that radial flow of growth-water to the hypocotyl periphery together with the down-regulation of a sucrose transporter at the phloem leads to a preferential sucrose flow to the expanding cortex. The pith cells, which do not experience flow of growth-water, are probably insufficiently supplied with sucrose from the phloem resulting eventually in cell death as the plant grows. Shortage of sucrose supply, experimentally achieved by removal of the endosperm, led to sucrose hydrolysis in the pith. The sucrose levels in the other tissues decreased less. It appears that the hydrolysis to hexose was initiated to maintain the osmotic value in the pith cell sap. It is speculated that high hexose levels in the cells are indicative of insufficient sucrose supply via the phloem and that the pith cells are confronted with that situation during early seedling development.

  6. 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.

  7. 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.

  8. 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.

  9. 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

  10. 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.}

  11. 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.

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

    PubMed

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

    2006-01-01

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

  13. 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.

  14. Single-cell measurements of IgE-mediated FcεRI signaling using an integrated microfluidic platform.

    PubMed

    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.

  15. 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.

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

    DOE PAGES

    Liu, Yanli; Barua, Dipak; Liu, Peng; ...

    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

  17. Direct measurement of cell detachment force on single cells using a new electromechanical method.

    PubMed

    Francis, G W; Fisher, L R; Gamble, R A; Gingell, D

    1987-05-01

    We describe a new device in which an accurately measured force is applied to individual adherent cells while the topography of the adhesion zone is simultaneously monitored. The force is applied via a flexible glass micropipette, attached by suction to the cell under study, and is calculated directly from the measured pipette deflection. Regions of close contact in the adhesion zone are observed using interference reflection microscopy. We have used the device to measure the force required to detach human red blood cells from hydrophobic and hydrophilic glass surfaces, and to detach Dictyostelium discoideum amoebae from a hydrophobic glass surface. The measured forces per unit length of contact perimeter are within an order of magnitude of the tensions required for membrane rupture.

  18. Measuring molecular motions inside single cells with improved analysis of single-particle trajectories

    NASA Astrophysics Data System (ADS)

    Rowland, David J.; Biteen, Julie S.

    2017-04-01

    Single-molecule super-resolution imaging and tracking can measure molecular motions inside living cells on the scale of the molecules themselves. Diffusion in biological systems commonly exhibits multiple modes of motion, which can be effectively quantified by fitting the cumulative probability distribution of the squared step sizes in a two-step fitting process. Here we combine this two-step fit into a single least-squares minimization; this new method vastly reduces the total number of fitting parameters and increases the precision with which diffusion may be measured. We demonstrate this Global Fit approach on a simulated two-component system as well as on a mixture of diffusing 80 nm and 200 nm gold spheres to show improvements in fitting robustness and localization precision compared to the traditional Local Fit algorithm.

  19. High-throughput measurement of single-cell growth rates using serial microfluidic mass sensor arrays

    PubMed Central

    Cermak, Nathan; Olcum, Selim; Delgado, Francisco Feijó; Wasserman, Steven C.; Payer, Kristofor R.; Murakami, Mark; Knudsen, Scott M.; Kimmerling, Robert J.; Stevens, Mark M.; Kikuchi, Yuki; Sandikci, Arzu; Ogawa, Masaaki; Agache, Vincent; Baléras, François; Weinstock, David M.; Manalis, Scott R.

    2016-01-01

    Methods to rapidly assess cell growth would be useful for many applications, including drug susceptibility testing, but current technologies have limited sensitivity or throughput. Here we present an approach to precisely and rapidly measure growth rates of many individual cells simultaneously. We flow cells in suspension through a microfluidic channel with 10–12 resonant mass sensors distributed along its length, weighing each cell repeatedly over the 4–20 min it spends in the channel. Because multiple cells traverse the channel at the same time, we obtain growth rates for >60 cells/h with a resolution of 0.2 pg/h for mammalian cells and 0.02 pg/h for bacteria. We measure the growth of single lymphocytic cells, mouse and human T cells, primary human leukemia cells, yeast, Escherichia coli and Enterococcus faecalis. Our system reveals subpopulations of cells with divergent growth kinetics and enables assessment of cellular responses to antibiotics and antimicrobial peptides within minutes. PMID:27598230

  20. FORMING SELF-ASSEMBLED CELL ARRAYS AND MEASURING THE OXYGEN CONSUMPTION RATE OF A SINGLE LIVE CELL.

    PubMed

    Etzkorn, James R; McQuaide, Sarah C; Anderson, Judy B; Meldrum, Deirdre R; Parviz, Babak A

    2009-06-01

    We report a method for forming arrays of live single cells on a chip using polymer micro-traps made of SU8. We have studied the toxicity of the microfabricated structures and the associated environment for two cell lines. We also report a method for measuring the oxygen consumption rate of a single cell using optical interrogation of molecular oxygen sensors placed in micromachined micro-wells by temporarily sealing the cells in the micro-traps. The new techniques presented here add to the collection of tools available for performing "single-cell" biology. A single-cell self-assembly yield of 61% was achieved with oxygen draw down rates of 0.83, 0.82, and 0.71 fmol/minute on three isolated live A549 cells.

  1. Measurement of oxidatively-induced clustered DNA lesions using a novel adaptation of single cell gel electrophoresis (comet assay).

    PubMed

    Georgakilas, Alexandros G; Holt, Stewart M; Hair, Jessica M; Loftin, Charles W

    2010-12-01

    The two basic groups of complex DNA damage are double-strand breaks (DSBs) and non-DSB oxidatively-induced clustered DNA lesions (OCDLs). The single-cell gel electrophoresis (SCGE) or comet assay has been widely used for the detection of low levels of various types of DNA lesions including single-strand breaks (SSBs), DSBs, and oxidized bases per individual cell. There are limited data on the use of the comet assay for the detection of non-DSB clustered DNA lesions using different repair enzymes as enzymatic probes. This unit discusses a novel adaptation of the comet assay used to measure these unique types of lesions. Until now OCDL yields have been measured using primarily pulsed-field agarose gel electrophoresis. The advantages offered by the current approach are: (1) measurement of OCDL levels per individual cell; (2) use of a small number of cells (∼10,000) and relatively low doses of ionizing radiation (1 to 2 Gy) or low levels of oxidative stress, which are not compatible with standard agarose gel electrophoresis; and finally, (3) the assay is fast and allows direct comparison with pulsed-field gel electrophoresis results.

  2. A new Pt-Rh carbon nitride electrocatalyst for the oxygen reduction reaction in polymer electrolyte membrane fuel cells: Synthesis, characterization and single-cell performance

    NASA Astrophysics Data System (ADS)

    Di Noto, Vito; Negro, Enrico

    In this paper the preparation of a new bimetal electrocatalyst for the oxygen reduction reaction (ORR), which is one of the most important bottlenecks in the operation of polymer electrolyte membrane fuel cells (PEMFCs), is described. This material was synthesized through a pyrolysis process of a zeolitic inorganic-organic polymer electrolyte (Z-IOPE-like) precursor, followed by suitable washing and activation procedures of the product. The electrocatalyst, whose active sites consist of platinum and rhodium, was: (a) extensively characterized from the chemical, structural, morphological and electrochemical points of view and (b) used to prepare a membrane-electrode assembly (MEA) which was tested under operative conditions in a single-cell configuration. It was observed that, with respect to a reference material based on supported platinum, rhodium did not compromise the performance of the electrocatalyst in the ORR. This behaviour was interpreted in the framework of a general model concerning the enhancement of ORR performance in bimetal systems supported on carbon nitrides. Finally, the material shows a slightly better tolerance toward a few common contaminants for the ORR such as methanol and chloride anions, typical of direct methanol fuel cells (DMFCs) and vehicular applications, respectively.

  3. 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

  4. 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.

  5. 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

    2016-01-28

    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.

  6. 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)

  7. Measuring tissue oxygen saturation using NIR spectroscopy

    NASA Astrophysics Data System (ADS)

    Sircan-Kucuksayan, Aslinur; Uyuklu, Mehmet; Canpolat, Murat

    2014-05-01

    Tissue oxygen saturation (StO2) is known quite useful parameter for medical applications. A spectroscopic method has been developed to diagnose pathologic tissues due to lack of normal blood circulation by measuring tissue oxygen saturation. In the study, human blood samples with different level of oxygen saturations have been prepared and spectra were taken using an optical fiber probe to investigate correlation between the oxygen saturations and the spectra. The experimental set up for the spectroscopic measurements was consists of a miniature NIR light spectrometer, an optical fiber probe, a halogen-tungsten light source and a laptop. A linear correlation between the oxygen saturation of the blood samples and the ratio of the light of wavelengths 660 nm to 790 nm has been found from the spectra. Then, oxygen saturations of the blood samples were estimated from the spectroscopic measurements within an error of 2.9%. Furthermore, it has been shown that the linear dependence between the ratio and the oxygen saturation of the blood samples was valid for the blood samples with different hematocrits. Tissue oxygen saturation has been estimated from the spectroscopic measurements were taken from the fingers of healthy volunteers using the correlation between the spectra and blood oxygen saturation. The tissue StO2 measured was 80% as expected. The technique developed to measure tissue oxygen saturation has potential to diagnose premalignant tissues, follow up prognosis of cancerous tissues, and evaluation of ischemia reperfusion tissues.

  8. 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).

  9. Single-Cell Metabolomics.

    PubMed

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

    2017-01-01

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

  10. 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.

  11. Oxygen fugacities directly measured in magmatic gases.

    PubMed

    Sato, M; Wright, T L

    1966-09-02

    An electrochemical device was used to measure the fugacity of oxygen (fo(o2)) 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 fo(o2) normally varies linearly with the reciprocal of the absolute temperature, and that chemical changes occurring in the cooling tholeiitic basalt are reflected in the fo(o2) values measured in the holes.

  12. 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.

  13. 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.

  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.

  15. 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

  16. Label-free isolation of a prostate cancer cell among blood cells and the single-cell measurement of drug accumulation using an integrated microfluidic chip

    PubMed Central

    Khamenehfar, A.; Beischlag, T. V.; Russell, P. J.; Ling, M. T. P.; Nelson, C.; Li, P. C. H.

    2015-01-01

    Circulating tumor cells (CTCs) are found in the blood of patients with cancer. Although these cells are rare, they can provide useful information for chemotherapy. However, isolation of these rare cells from blood is technically challenging because they are small in numbers. An integrated microfluidic chip, dubbed CTC chip, was designed and fabricated for conducting tumor cell isolation. As CTCs usually show multidrug resistance (MDR), the effect of MDR inhibitors on chemotherapeutic drug accumulation in the isolated single tumor cell is measured. As a model of CTC isolation, human prostate cancer cells were mixed with mouse blood cells and the label-free isolation of the tumor cells was conducted based on cell size difference. The major advantages of the CTC chip are the ability for fast cell isolation, followed by multiple rounds of single-cell measurements, suggesting a potential assay for detecting the drug responses based on the liquid biopsy of cancer patients. PMID:26594265

  17. Single-cell nanosurgery.

    PubMed

    Zeigler, Maxwell B; Chiu, Daniel T

    2013-01-01

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

  18. 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.

  19. 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

  20. 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.

  1. Measuring oxygen levels in Caco-2 cultures

    PubMed Central

    Zeitouni, Nathalie E; Fandrey, Joachim; Naim, Hassan Y; von Köckritz-Blickwede, Maren

    2015-01-01

    Purpose Measuring oxygen levels in three different systems of Caco-2 cell culture. Methods Caco-2 cells were cultured in three different systems, using conventional polystyrene 24-well plates, special 24-well gas permeable plates, or on membrane inserts in conventional plates. Optical sensor spots were used to measure dissolved O2 levels in these cultured cells over the course of 6 days under normoxia (143 mmHg) and for 6 hours under hypoxia (7 mmHg). Western blot analysis was used to determine the protein levels of hypoxia-inducible factor 1α (HIF-1α) in the different cultures. Results All culture systems displayed lower O2 levels over time than expected when cultured under normoxia conditions. On average, O2 levels reached as low as 25 mmHg in 24-well plates but remained at 97 and 117 mmHg in gas permeable plates and membrane inserts, respectively. Under hypoxia, 1 mL cell cultures equilibrated to 7 mmHg O2 within the first 60 minutes and dropped to 0.39 and 0.61 mmHg O2 in 24-well and gas permeable plates, respectively, after the 6-hour incubation period. Cultures in membrane inserts did not equilibrate to 7 mmHg by the end of the 6-hour incubation period, where the lowest O2 measurements reached 23.12 mmHg. Western blots of HIF-1α protein level in the whole cell lysates of the different Caco-2 cultures revealed distinct stabilization of HIF-1α after hypoxic incubation for 1, 2, and 4 hours in 24-well plates as well as gas permeable plates. For membrane inserts, notable HIF-1α was seen after 4 hours of hypoxic incubation. Conclusion Cellular oxygen depletion was achieved in different hypoxic Caco-2 culture systems. However, different oxygen levels comparing different culture systems indicate that O2 level should be carefully considered in oxygen-dependent experiments. PMID:27774482

  2. Single cell wound repair

    PubMed Central

    Abreu-Blanco, Maria Teresa; Verboon, Jeffrey M

    2011-01-01

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

  3. Flow Cytometric Single-Cell Analysis for Quantitative in Vivo Detection of Protein-Protein Interactions via Relative Reporter Protein Expression Measurement.

    PubMed

    Wu, Lina; Wang, Xu; Zhang, Jianqiang; Luan, Tian; Bouveret, Emmanuelle; Yan, Xiaomei

    2017-03-07

    Cell-based two-hybrid assays have been key players in identifying pairwise interactions, yet quantitative measurement of protein-protein interactions in vivo remains challenging. Here, we show that by using relative reporter protein expression (RRPE), defined as the level of reporter expression normalized to that of the interacting protein, quantitative analysis of protein interactions in a bacterial adenylate cyclase two-hybrid (BACTH) system can be achieved. A multicolor flow cytometer was used to measure simultaneously the expression levels of one of the two putative interacting proteins and the β-galactosidase (β-gal) reporter protein upon dual immunofluorescence staining. Single-cell analysis revealed that there exists bistability in the BACTH system and the RRPE is an intrinsic characteristic associated with the binding strength between the two interacting proteins. The RRPE-BACTH method provides an efficient tool to confirm interacting pairs of proteins, investigate determinant residues in protein-protein interaction, and compare interaction strength of different pairs.

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

    PubMed Central

    van Ginneken, A C; Giles, W

    1991-01-01

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

  6. 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

  7. 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

  8. Epidermal growth factor receptor targeting alters gene expression and restores the adhesion function of cancerous cells as measured by single cell force spectroscopy.

    PubMed

    Azadi, Shohreh; Tafazzoli-Shadpour, Mohammad; Omidvar, Ramin; Moradi, Lida; Habibi-Anbouhi, Mahdi

    2016-12-01

    Loss of cell-cell adhesion function is a common characteristic of many human epithelial carcinomas that is frequently due to loss of E-cadherin expression. In cancer progression, loss of E-cadherin is associated with invasion and metastasis potential, hence restoration of its function may contribute to the metastasis inhibition. This study examined effect of Epidermal Growth Factor Receptor (EGFR/Her1) blockade on the E-cadherin expression, cellular adherence, and cell elasticity in two human epithelial cancer cell lines, MCF7 and A431. EGFR blocking agents as antibodies or small molecules target EGFR directly. Furthermore, due to intracellular signaling pathways they influence cell behavior and activities. The idea here is to investigate the effect of reduced activity of this signaling pathway using anti-EGFR Antibody (Cetuximab) and tyrosine kinase inhibitor (Lapatinib) on cell-cell adhesion and cell mechanical properties. Real-Time PCR analysis demonstrated that treatment of cells with considered drugs increased the expression of E-cadherin gene among samples. The atomic force microscopy-based single cell force spectroscopy technique was used to measure adhesive force of cancerous cells. Results indicated that inhibition of EGFR activity elevated cell-cell adhesion force, accompanied by stiffening of the cell bodies. In summary, Cetuximab and Lapatinib have been found to mediate cell-cell adhesion by restoration of E-cadherin expression and function. Our data suggest possible therapeutic potential for inhibition of metastasis via the blockade of EGFR signaling.

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

    PubMed Central

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

    2016-01-01

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

  10. 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...

  11. 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.

  12. Measurement of arterial and capillary blood oxygen tension

    PubMed Central

    Johnstone, J. H.

    1966-01-01

    An oxygen electrode system, supplied as an attachment to the Radiometer Astrup micro equipment for blood pH determination (AME I), has been investigated. Determination of blood oxygen tension using this electrode system has been compared with tension measurements using an established Bishop type oxygen electrode and satisfactory agreement was found. The storage of blood for routine estimation of oxygen tension has been investigated. Capillary blood oxygen tension has been measured and compared with that of simultaneously taken arterial blood samples. PMID:5929338

  13. 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.

  14. Dynamics of single-cell gene expression

    PubMed Central

    Longo, Diane; Hasty, Jeff

    2006-01-01

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

  15. Measuring Oxygen Consumption Rate in Caenorhabditis elegans

    PubMed Central

    Palikaras, Konstantinos; Tavernarakis, Nektarios

    2017-01-01

    The rate of oxygen consumption is a vital marker indicating cellular function during lifetime under normal or metabolically challenged conditions. It is used broadly to study mitochondrial function (Artal-Sanz and Tavernarakis, 2009; Palikaras et al., 2015; Ryu et al., 2016) or investigate factors mediating the switch from oxidative phosphorylation to aerobic glycolysis (Chen et al., 2015; Vander Heiden et al., 2009). In this protocol, we describe a method for the determination of oxygen consumption rates in the nematode Caenorhabditis elegans. PMID:28239622

  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. 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.

  18. 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

  19. 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. PMID:27773958

  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. 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

  2. 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.

  3. 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

  4. 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.

  5. 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).

  6. Method measuring oxygen tension and transport within subcutaneous devices

    PubMed Central

    Weidling, John; Sameni, Sara; Lakey, Jonathan R. T.; Botvinick, Elliot

    2014-01-01

    Abstract. Cellular therapies hold promise to replace the implantation of whole organs in the treatment of disease. For most cell types, in vivo viability depends on oxygen delivery to avoid the toxic effects of hypoxia. A promising approach is the in situ vascularization of implantable devices which can mediate hypoxia and improve both the lifetime and utility of implanted cells and tissues. Although mathematical models and bulk measurements of oxygenation in surrounding tissue have been used to estimate oxygenation within devices, such estimates are insufficient in determining if supplied oxygen is sufficient for the entire thickness of the implanted cells and tissues. We have developed a technique in which oxygen-sensitive microparticles (OSMs) are incorporated into the volume of subcutaneously implantable devices. Oxygen partial pressure within these devices can be measured directly in vivo by an optical probe placed on the skin surface. As validation, OSMs have been incorporated into alginate beads, commonly used as immunoisolation devices to encapsulate pancreatic islet cells. Alginate beads were implanted into the subcutaneous space of Sprague–Dawley rats. Oxygen transport through beads was characterized from dynamic OSM signals in response to changes in inhaled oxygen. Changes in oxygen dynamics over days demonstrate the utility of our technology. PMID:25162910

  7. 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.

  8. Herschel Measurements of Molecular Oxygen in Orion

    NASA Astrophysics Data System (ADS)

    Goldsmith, Paul F.; Liseau, René; Bell, Tom A.; Black, John H.; Chen, Jo-Hsin; Hollenbach, David; Kaufman, Michael J.; Li, Di; Lis, Dariusz C.; Melnick, Gary; Neufeld, David; Pagani, Laurent; Snell, Ronald; Benz, Arnold O.; Bergin, Edwin; Bruderer, Simon; Caselli, Paola; Caux, Emmanuel; Encrenaz, Pierre; Falgarone, Edith; Gerin, Maryvonne; Goicoechea, Javier R.; Hjalmarson, Åke; Larsson, Bengt; Le Bourlot, Jacques; Le Petit, Franck; De Luca, Massimo; Nagy, Zsofia; Roueff, Evelyne; Sandqvist, Aage; van der Tak, Floris; van Dishoeck, Ewine F.; Vastel, Charlotte; Viti, Serena; Yıldız, Umut

    2011-08-01

    We report observations of three rotational transitions of molecular oxygen (O2) in emission from the H2 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-1 to 12 km s-1 and widths of 3 km s-1. The beam-averaged column density is N(O2) = 6.5 × 1016 cm-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 O2 relative to H2 is (0.3-7.3) × 10-6. The unusual velocity suggests an association with a ~5'' diameter source, denoted Peak A, the Western Clump, or MF4. The mass of this source is ~10 M sun and the dust temperature is >=150 K. Our preferred explanation of the enhanced O2 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 O2. For this small source, the line ratios require a temperature >=180 K. The inferred O2 column density sime5 × 1018 cm-2 can be produced in Peak A, having N(H2) ~= 4 × 1024 cm-2. An alternative mechanism is a low-velocity (10-15 km s-1) C-shock, which can produce N(O2) up to 1017 cm-2. Herschel is an ESA space observatory with science instruments provided by European-led Principal Investigator consortia and with important participation from NASA.

  9. Measuring blood oxygenation of pulsatile arteries using photoacoustic microscopy

    NASA Astrophysics Data System (ADS)

    Li, Qian; Yu, Tianhao; Li, Lin; Chai, Xinyu; Zhou, Chuanqing

    2016-10-01

    Heart pumps blood through the blood vessels to provide body with oxygen and nutrients. As the result, the blood flow, volume and oxygenation in arteries has a pulsatile nature. Measuring these pulsatile parameters enables more precise monitoring of oxygen metabolic rate and is thus valuable for researches and clinical applications. Photoacoustic microscopy (PAM) is a proven label-free method for in vivo measuring blood oxygenation at single blood vessel level. However, studies using PAM to observe the pulsatile nature of blood oxygenation in arteries were not reported. In this paper, we use optical-resolution PAM (OR-PAM) technology to study the blood oxygenation dynamics of pulsatile arteries. First, the ability of our OR-PAM system to accurately reflect the change of optical absorption in imaged objects is demonstrated in a phantom study. Then the system is used to image exposed cortical blood vessels of cat. The pulsatile nature of blood volume and oxygenation in arteries is clearly reflected in photoacoustic (PA) signals, whereas it's not observable in veins. By using a multi-wavelength laser, the dynamics of the blood oxygenation of pulsatile arteries in cardiac cycles can be measured, based on the spectroscopic method.

  10. Measurement of dissolved oxygen during red wines tank aging with chips and micro-oxygenation.

    PubMed

    Nevares, I; del Alamo, M

    2008-07-21

    Nowadays, micro-oxygenation is a very important technique used in aging wines in order to improve their characteristics. The techniques of wine tank aging imply the use of small doses of oxygen and the addition of wood pieces of oak to the wine. Considering the low dissolved oxygen (DO) levels used by micro-oxygenation technique it is necessary to choose the appropriate measurement principle to apply the precise oxygen dosage in wine at any time, in order to assure its correct assimilation. This knowledge will allow the oenologist to control and run the wine aging correctly. This work is a thorough revision of DO measurement main technologies applied to oenology. It describes the strengths and weaknesses of each of them, and draws a comparison of their workings in wine measurement. Both, the traditional systems by electrochemical probes, and the newest photoluminescence-based probes have been used. These probes adapted to red wines ageing study are then compared. This paper also details the first results of the dissolved oxygen content evolution in red wines during a traditional and alternative tank aging. Samples have been treated by three different ageing systems: oak barrels, stainless-steel tanks with small oak wood pieces (chips) and with bigger oak pieces (staves) with low micro-oxygenation levels. French and American oak barrels manufactured by the same cooperage have been used.

  11. A review on recent upper atmosphere atomic oxygen measurements

    NASA Astrophysics Data System (ADS)

    Kaufmann, Martin; Ern, Manfred; Riese, Martin; Zhu, Yajun

    2016-07-01

    Atomic oxygen is a key player in the upper mesosphere lower and thermosphere chemistry, energy balance, and dynamics. In recent years, a few new global datasets of this species have been presented. They are based on airglow measurements from low earth satellites. Surprisingly, the atomic oxygen abundance differs by 30-50% for similar atmospheric conditions. This paper gives an overview on the various atomic oxygen datasets available so far and presents most recent results obtained from measurements of the SCIAMACHY instrument on Envisat. Differences between the datasets are discussed.

  12. 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.

  13. 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.

  14. Single Cell Electrical Characterization Techniques

    PubMed Central

    Mansor, Muhammad Asraf; Ahmad, Mohd Ridzuan

    2015-01-01

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

  15. Introduction: why analyze single cells?

    PubMed

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

    2012-01-01

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

  16. Single Cell Isolation and Analysis

    PubMed Central

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

    2016-01-01

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

  17. Direct measurement of oxygen stoichiometry in perovskite films

    NASA Astrophysics Data System (ADS)

    Scola, J.; Benamar, A.; Berini, B.; Jomard, F.; Dumont, Y.

    2017-02-01

    We present a direct method to measure the oxygen stoichiometry in an oxide film with an accuracy of about 2%. It is based on a combination of 18O annealing and high mass resolution secondary ion mass spectroscopy. Calibration has been done on a LaNiO3 film whose electrical properties dependence on oxygen stoichiometry are well documented. The method is illustrated with a series of LaNiO3 films grown on SrTiO3 substrates prepared with different oxygen stoichiometries. The large influence of the surface state on oxygen exchange is evidenced in films grown on different substrate orientations or coated with a thin layer of LaAlO3. Oxygen surface exchange and bulk diffusion is then discussed for both LaNiO3 and SrVO3 films.

  18. Analysis of mitochondria isolated from single cells.

    PubMed

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

    2007-01-01

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

  19. [Study of plasma temperature measurements for oxygen discharge].

    PubMed

    Li, Liu-Cheng; Wang, Zeng-Qiang; Li, Gu-Fu; Duo, Li-Ping

    2011-10-01

    A radio-frequency discharge setup was constructed by two shell-shaped copper electrodes and a 30 cm long pyrex glass tube (i. d. = 1.65 cm) to examine the gas temperature of oxygen plasma in electric discharge oxygen iodine laser. The discharge was supplied by a 500 watt, 13.56 MHz radio-frequency power. The gas pressure in the discharge cavity was 1 330 Pa. The temperature of oxygen discharge plasma was measured by using the P branch of O2 (b, v = 0) rotational emission spectrum. Two methods were used to deduce the oxygen gas temperature. They are Boltzman plotting method and computer simulating spectrum method, respectively. Gauss fitting method was used to distinguish spectrum peaks for lower resolution spectrum. The spectrum peak area was used to characterize the optical emission intensity. The gas temperature of oxygen discharge plasma was obtained by Boltzmann plotting method. Alternatively, the optical emission spectrum was simulated by computer modeling with spectrometer slit function which was obtained by He-Ne laser. Consequently, the gas temperature of oxygen plasma was obtained by comparing the computer simulating spectrum and the experimentally observed spectrum according to the least square fitting rule. The measurement results with the two methods agree well. It was concluded that the simple optical technique can be used conveniently in the temperature diagnostics of oxygen radio-frequency discharge plasma.

  20. 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.

  1. Diffuse reflectance spectroscopy for the measurement of tissue oxygen saturation.

    PubMed

    Sircan-Kucuksayan, A; Uyuklu, M; Canpolat, M

    2015-12-01

    Tissue oxygen saturation (StO2) is a useful parameter for medical applications. A spectroscopic method has been developed to detect pathologic tissues, due to a lack of normal blood circulation, by measuring StO2. In this study, human blood samples with different levels of oxygen saturation have been prepared and spectra were acquired using an optical fiber probe to investigate the correlation between the oxygen saturation levels and the spectra. A linear correlation between the oxygen saturation and ratio of the intensities (760 nm to 790 nm) of the spectra acquired from blood samples has been found. In a validation study, oxygen saturations of the blood samples were estimated from the spectroscopic measurements with an error of 2.9%. It has also been shown that the linear dependence between the ratio and the oxygen saturation of the blood samples was valid for the blood samples with different hematocrits. Spectra were acquired from the forearms of 30 healthy volunteers to estimate StO2 prior to, at the beginning of, after 2 min, and at the release of total vascular occlusion. The average StO2 of a forearm before and after the two minutes occlusion was significantly different. The results suggested that optical reflectance spectroscopy is a sensitive method to estimate the StO2 levels of human tissue. The technique developed to measure StO2 has potential to detect ischemia in real time.

  2. The measurement of hemoglobin oxygen saturation using multiwavelength photoacoustic microscopy

    NASA Astrophysics Data System (ADS)

    Deng, Zilin; Yang, Xiaoquan; Yu, Lejun; Gong, Hui

    2009-10-01

    Hemoglobin oxygen saturation (SO2) is one of the most critical functional parameters to the metabolism. In this paper, we mainly introduced some initial results of measuring blood oxygen using multi-wavelength photoacoustic microscopy (PAM). In phantom study, we demonstrate the photoacoustic signal amplitude increases linearly with the concentration of red or blue ink. Then the calculated concentration of red ink in double-ink mixtures with PAM has a 5% difference with the result measured with spectrophotometric analysis. In ex vivo experiment, the measured result exhibt 15% difference between the PAM and spectrophotometric analysis. Experiment results suggest that PAM could be used to determine the SO2 quantitatively.

  3. The measurement of hemoglobin oxygen saturation using multiwavelength photoacoustic microscopy

    NASA Astrophysics Data System (ADS)

    Deng, Zilin; Yang, Xiaoquan; Yu, Lejun; Gong, Hui

    2010-02-01

    Hemoglobin oxygen saturation (SO2) is one of the most critical functional parameters to the metabolism. In this paper, we mainly introduced some initial results of measuring blood oxygen using multi-wavelength photoacoustic microscopy (PAM). In phantom study, we demonstrate the photoacoustic signal amplitude increases linearly with the concentration of red or blue ink. Then the calculated concentration of red ink in double-ink mixtures with PAM has a 5% difference with the result measured with spectrophotometric analysis. In ex vivo experiment, the measured result exhibt 15% difference between the PAM and spectrophotometric analysis. Experiment results suggest that PAM could be used to determine the SO2 quantitatively.

  4. Cerebral Blood Oxygenation Measurement Based on Oxygen-dependent Quenching of Phosphorescence

    PubMed Central

    Sakadžić, Sava; Roussakis, Emmanuel; Yaseen, Mohammad A.; Mandeville, Emiri T.; Srinivasan, Vivek J.; Arai, Ken; Ruvinskaya, Svetlana; Wu, Weicheng; Devor, Anna; Lo, Eng H.; Vinogradov, Sergei A.; Boas, David A.

    2011-01-01

    Monitoring of the spatiotemporal characteristics of cerebral blood and tissue oxygenation is crucial for better understanding of the neuro-metabolic-vascular relationship. Development of new pO2 measurement modalities with simultaneous monitoring of pO2 in larger fields of view with higher spatial and/or temporal resolution will enable greater insight into the functioning of the normal brain and will also have significant impact on diagnosis and treatment of neurovascular diseases such as stroke, Alzheimer's disease, and head injury. Optical imaging modalities have shown a great potential to provide high spatiotemporal resolution and quantitative imaging of pO2 based on hemoglobin absorption in visible and near infrared range of optical spectrum. However, multispectral measurement of cerebral blood oxygenation relies on photon migration through the highly scattering brain tissue. Estimation and modeling of tissue optical parameters, which may undergo dynamic changes during the experiment, is typically required for accurate estimation of blood oxygenation. On the other hand, estimation of the partial pressure of oxygen (pO2) based on oxygen-dependent quenching of phosphorescence should not be significantly affected by the changes in the optical parameters of the tissue and provides an absolute measure of pO2. Experimental systems that utilize oxygen-sensitive dyes have been demonstrated in in vivo studies of the perfused tissue as well as for monitoring the oxygen content in tissue cultures, showing that phosphorescence quenching is a potent technology capable of accurate oxygen imaging in the physiological pO2 range. Here we demonstrate with two different imaging modalities how to perform measurement of pO2 in cortical vasculature based on phosphorescence lifetime imaging. In first demonstration we present wide field of view imaging of pO2 at the cortical surface of a rat. This imaging modality has relatively simple experimental setup based on a CCD camera and a

  5. Progress toward single cell metabolomics

    PubMed Central

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

    2012-01-01

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

  6. Instrument for the measurement of retinal vessel oxygen saturation

    NASA Astrophysics Data System (ADS)

    Drewes, Jonathan J.; Smith, Matthew H.; Denninghoff, Kurt R.; Hillman, Lloyd W.

    1999-06-01

    Retinal vessel oxygen saturation has been suggested as a parameter for monitoring a wide range of conditions including occult blood los and a variety of ophthalmic diseases. We have developed an Eye Oximeter (EOX), that noninvasively measures the oxygen saturation of the blood in individual large retinal vessels using scanning lasers. 1D vessel extinction profiles are obtained at four wavelengths (629, 678, 821 and 899 nm), and the vessel transmittances computed. The oxygen saturation of blood within the vessel is then calculated from the transmittance data. We have performed an in vitro experiment on human blood which demonstrates the calibration of the EOX measurements and validates our oximetry equations. Retinal vessel oxygen saturation was measured in a human subject and found to be 65%O2Sat and 101 - 102%O2Sat in the veins and arteries on the optic disk. Irregularities in the background measured away from the optic disk resulted in a large variance in the calculated saturation when compared to measurements made on the disk.

  7. Disposable screen-printed biosensor for transcutaneous oxygen measurement

    NASA Astrophysics Data System (ADS)

    Lam, Yu-Zhi Liza; Atkinson, John

    2002-12-01

    A disposable transcutaneous oxygen sensor has been designed and fabricated in-house employing screen-printing methods so as to achieve cost-effective and reliable production. The oxygen sensing module of the device is based on the amperometric Clarke cell principle. A three-electrode configuration consisting of miniature gold working and counter-electrodes and a silver/silver chloride reference electrode is screen-printed onto alumina substrate resulting in approximately 15 µm thickness layers. A platinum heater element is integrated into the design to make transcutaneous measurements, typically at 44°C. The performances of different screen-printed membrane materials have been evaluated in both hydrated and dry test conditions using cyclic voltammographs and static tests where the devices are subjected to different oxygen levels. All measurements are made by a fully automated computer-controlled gas rig.

  8. Measuring the Yield of Singlet Oxygen in a Chemical Oxygen Iodine Laser (Postprint)

    DTIC Science & Technology

    2006-08-01

    release; distribution is unlimited 13. SUPPLEMENTARY NOTES *Los Gatos Research, 67 East Evelyn Ave., Suite 3, Mountain View, CA 94041-1518...Kirtland AFB, NM 87117-5776 bLos Gatos Research, 67 East Evelyn Avenue, Suite 3, Mountain View, CA 94041-1518 ABSTRACT A critical parameter...oxygen to be measured. Ongoing work will enable researchers at AFRL and Los Gatos Research to more accurately measure the yield as additional

  9. Single cell studies of the cell cycle and some models

    PubMed Central

    Mitchison, JM

    2005-01-01

    Analysis of growth and division often involves measurements made on cell populations, which tend to average data. The value of single cell analysis needs to be appreciated, and models based on findings from single cells should be taken into greater consideration in our understanding of the way in which cell size and division are co-ordinated. Examples are given of some single cell analyses in mammalian cells, yeast and other microorganisms. There is also a short discussion on how far the results are in accord with simple models. PMID:15703075

  10. 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.

  11. Biological Oxygen Productivity Over The Last Glacial Termination From Triple Oxygen Isotope Measurements

    NASA Astrophysics Data System (ADS)

    Blunier, T.; Bender, M. L.; Hendricks, M. B.

    The atmospheric oxygen isotope signature of O2 is linked to the oxygen signature of seawater through photosynthesis and respiration. Fractionation during these pro- cesses is mass dependent affecting 17O about half as much as 18O. A mass indepen- dent fractionation process takes place during isotope exchange between O2 and CO2 in the stratosphere (Thiemens, 1999; Luz et al., 1999). The magnitude of the mass- independent anomaly in the triple isotope composition of O2 depends on relative rates of biological O2 cycling and photochemical reactions in the stratosphere. Variations of this anomaly thus allows us to estimate changes of mass dependent O2 production by photosynthesis versus mass independent O2-CO2 exchange in the stratosphere. We reconstruct total oxygen productivity for the past from 17O and 18O measure- ments of O2 trapped in ice cores. With a box model we estimate that the total biogenic productivity was only 76-83 % of today for the glacial and was probably still lower than today during the glacial-interglacial transition and the early Holocene. In principle we can calculate the oxygen flux from the ocean biosphere if we know the oxygen flux from the land biosphere. Calculated ocean production is very sensitive to the estimate of land biosphere production. The latter term remains uncertain, however, and we can presently only constrain glacial ocean production to 88 to 140 % of the present value.

  12. 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...

  13. Monitoring microvascular free flaps with tissue oxygen measurement and PET.

    PubMed

    Schrey, Aleksi R; Kinnunen, Ilpo A J; Grénman, Reidar A; Minn, Heikki R I; Aitasalo, Kalle M J

    2008-07-01

    Tissue oxygen measurement and positron emission tomography (PET) were evaluated as methods for predicting ischemia in microvascular free flaps of the head and neck. Ten patients with head and neck squamous cell cancer underwent resection of the tumour followed by microvascular reconstruction with a free flap. Tissue oxygenation of the flap (P(ti)O(2)) was continuously monitored for three postoperative (POP) days and the blood flow of the flap was assessed using oxygen-15 labelled water and PET. In three free flaps a perfusion problem was suspected due to a remarkable drop in P(ti)O(2)-values, due to two anastomosis problems and due to POP turgor. No flap losses occurred. During the blood flow measurements with PET [mean 8.5 mL 100 g(-1) min(-1 )(SD 2.5)], the mean P(ti)O(2) of the flaps [46.8 mmHg (SD 17.0)] appeared to correlate with each other in each patient (p<0.05, n=10). Tissue oxygenation measurement is a feasible monitoring system of free flaps. The perfusion-study with PET correlates with P(ti)O(2)-measurement.

  14. 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.

  15. Atomic force microscopy for the examination of single cell rheology.

    PubMed

    Okajima, Takaharu

    2012-11-01

    Rheological properties of living cells play important roles in regulating their various biological functions. Therefore, measuring cell rheology is crucial for not only elucidating the relationship between the cell mechanics and functions, but also mechanical diagnosis of single cells. Atomic force microscopy (AFM) is becoming a useful technique for single cell diagnosis because it allows us to measure the rheological properties of adherent cells at any region on the surface without any modifications. In this review, we summarize AFM techniques for examining single cell rheology in frequency and time domains. Recent applications of AFM for investigating the statistical analysis of single cell rheology in comparison to other micro-rheological techniques are reviewed, and we discuss what specificity and universality of cell rheology are extracted using AFM.

  16. A disposable in vivo oxygen electrode for the continuous measurement of arterial oxygen tension.

    PubMed

    Gold, M I; Diaz, P M; Feingold, A; Duarte, I; Sohn, Y J; Kallos, T

    1975-08-01

    An evaluation of the accuracy, perdision, and clinical safety of the IBC indwellling catheter electrode for the continuous monitoring of arterial oxygen tension during and after general anesthesia was made in a total of 62 patients. A comparison of the standard bench-type electrode (Radiometer) with the International Biophysics Corporation (IBC) electrode for the measurement of oxygen tension in tonometered blood also was performed. Three hundred and fifty comparisons in 51 patients were made of the indwelling electrode with a standard Radiometer unit and, while there was good correlation, there also was ome scatter. An additional small series of 44 comparisons in 11 patients was performed, the primary difference being that the electrode was maintained intr-arterially for approximately 1 day. In both in vivo studies there was excellent correlation but questionable precidion. Four IBC electrodes and eight Radiometer Pao2 electrodes in an additional study were compared at 11 different tonometered oxygen tensions in blood. The IBC electrodes measured oxyygen tension more accurately than did the Radiometer, and standard deviations were consistently smaller at all of 11 different oxygen tensions for the IBC unit. The authors believe that the poor precision within both in vivo studies might be due to the fact that the IBC probe, which was of unknown accuracy and precision, was compared to a standard device (the Radiometer) which in the in vitro investigation proved to be less accurate and less precise. No complications due to the insertion and maintance of this in vivo electrode were encountered. The authors suggest that the IBC method for measuring Pao2 continuously in vivo be considered as an alternative to intermittent gas analysis of oxygen tension.

  17. 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.

  18. Continuous measurement of scalp tissue oxygen tension and carotid arterial oxygen tension in the fetal lamb.

    PubMed

    Aarnoudse, J G; Oeseburg, B; Kwant, G; Huisjes, H J; Zijlstra, W G

    1980-01-01

    Scalp tissue PO2, carotid arterial PO2 fetal heart rate were continuously measured in the anaesthetized fetal lamb in utero while variations in oxygen supply were brought about. In some experiments the transcutaneously measured fetal scalp PO2 was recorded in addition. Scalp tissue PO2 was measured using specially designed miniature needle-type oxygen electrode, incorporated in an easily applicable spiral scalp electrode as commonly used for fetal heart rate monitoring. The measurements showed that fetal carotid arterial hypoxaemia is always nearly immediately followed by fetal scalp tissue hypoxia, and that the recovery of scalp tissue PO2 after a hypoxic period has a remarkably varying time course. Fetal heart rate usually decreased during hypoxia, but in some instances it did not change or even increased, demonstrating that heart rate is not always a reliable indicator of fetal hypoxia. PO2 values obtained with the transcutaneous method were higher than those with the needle electrode, because of the effect of the heating system of the transcutaneous electrode on tissue blood flow and haemoglobin oxygen affinity. It would seem that during hypoxaemia the decrease in scalp tissue PO2 is poossibly the combined result of the fall in arterial PO2 and a concomitant decrease in blood flow through the skin.

  19. 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.

  20. Analysis of global RNA synthesis at the single cell level following hypoxia.

    PubMed

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

    2014-05-13

    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.

  1. 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.

  2. Microfabricated devices for single cell analysis

    NASA Astrophysics Data System (ADS)

    Gao, Yuanfang

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

  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. Tissue oxygenation and haemodynamics measurement with spatially resolved NIRS

    NASA Astrophysics Data System (ADS)

    Zhang, Y.; Scopesi, F.; Serra, G.; Sun, J. W.; Rolfe, P.

    2010-08-01

    We describe the use of Near Infrared Spectroscopy (NIRS) for the non-invasive investigation of changes in haemodynamics and oxygenation of human peripheral tissues. The goal was to measure spatial variations of tissue NIRS oxygenation variables, namely deoxy-haemoglobin (HHb), oxy-haemoglobin (HbO2), total haemoglobin (HbT), and thereby to evaluate the responses of the peripheral circulation to imposed physiological challenges. We present a skinfat- muscle heterogeneous tissue model with varying fat thickness up to 15mm and a Monte Carlo simulation of photon transport within this model. The mean partial path length and the mean photon visit depth in the muscle layer were derived for different source-detector spacing. We constructed NIRS instrumentation comprising of light-emitting diodes (LED) as light sources at four wavelengths, 735nm, 760nm, 810nm and 850nm and sensitive photodiodes (PD) as the detectors. Source-detector spacing was varied to perform measurements at different depths within forearm tissue. Changes in chromophore concentration in response to venous and arterial occlusion were calculated using the modified Lambert-Beer Law. Studies in fat and thin volunteers indicated greater sensitivity in the thinner subjects for the tissue oxygenation measurement in the muscle layer. These results were consistent with those found using Monte Carlo simulation. Overall, the results of this investigation demonstrate the usefulness of the NIRS instrument for deriving spatial information from biological tissues.

  5. Measured fraction of carboxyhaemoglobin depends on oxygen saturation of haemoglobin.

    PubMed

    Hütler, M; Beneke, R; Littschwager, A; Böning, D

    2001-02-01

    The use of the OSM3 oximeter for measurement of the fraction of carboxyhaemoglobin (FCOHb) in blood allows for estimation of total circulating haemoglobin mass (Hb(tot)) by using the carbon monoxide rebreathing method. To ensure high accuracy of Hb(tot) estimation, potential sources of analytical errors should be identified and adjusted for. Based on observed differences in results of measured FCOHb between simultaneously sampled, arterialized and venous blood samples we investigated the influence of haemoglobin oxygen saturation (sO2) on results of measured FCOHb. Blood from nine healthy non-smokers was tonometered with gas mixtures containing 94% N2 or air and 6% CO2. The resulting oxygenated and deoxygenated specimens were mixed in different proportions to obtain varying sO2 values in the same blood. sO2, fractions of dyshaemoglobins, pO2, pCO2 and pH were measured at each step. FCOHb was significantly (p<0.001) higher in oxygenated (median, range: 0.6%, 0.4-0.9%) compared to deoxygenated (-0.2%, -0.5-0.0%) blood. Regression analysis identified the sO2 as the most important factor explaining 86% of the variance in observed changes in FCOHb. The observed sO2 effect has important implications on calibration procedure of OSM3, accuracy of measured FCOHb, and FCOHb dependent calculations such as estimation of Hb(tot) and related quantities. If the highest accuracy of FCOHb measurement is needed, an sO2 effect on results of measured FCOHb has to be considered and adjusted for.

  6. Oxygen Respiration rates of benthic foraminifera measured under laboratory conditions using oxygen microelectrodes

    NASA Astrophysics Data System (ADS)

    Geslin, Emmanuelle; Risgaard-Petersen, N.; Langlet, D.; Metzger, E.; Jorissen, F.

    2010-05-01

    Oxygen respiration rates of benthic foraminifera are not well documented because of the difficulties to measure them. However, the determination of the respiration rates of benthic foraminifera is important in order: 1) to compare the metabolic rates of different species, of various size, and with different microhabitats in the sediment; 2) to estimate the contribution of benthic foraminifera in the aerobic mineralization of organic matter. Benthic foraminifera from 4 different natural environments were used: three species from the intertidal rocky shore of Yeu island, two species from the muddy Bay of Aiguillon, two species from the Bay of Biscay and eleven species from the Rhône prodelta (France). Living foraminifera were placed in a small tube, in which oxygen gradients were determined using oxygen microelectrodes. Respiration rates were calculated on the basis of the oxygen fluxes measured in the vivinity of the foraminiferal specimens. Foraminiferal biovolumes were estimated on the basis of the overall shape of the various species (for example, Ammonia is assimilated to a half sphere) and the width of the shell walls. The results show a wide range of respiration rates according to the species (around 90 to 5300 pmol. cell-1.day-1) and a clear correlation with the biovolume of the foraminifera. No clear relationship between respiration rates and microhabitat is observed. A comparison with previously published data shows that our estimations are generally lower for the small size species. For example, the respiration rate estimations published recently by Nomaki et al. (Journal of Foraminiferal Research, 37, 281-286, 2007) show a range of 900 to 10 000 pmol. cell-1.day-1. The total contribution of benthic foraminifera in the aerobic mineralization of organic matter is estimated for the studied areas. The first results suggest a minor role of benthic foraminifera in this process, which strongly contrasts with their strong contribution to anaerobic mineralisation

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

    PubMed

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

    2013-09-01

    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)6(3-) and Ru(NH3)6(3+), 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)6(3-) 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.

  8. 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)

  9. 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.

  10. Tissue oxygen tension measurement for monitoring musculocutaneous and cutaneous flaps.

    PubMed

    Hjortdal, V E; Awwad, A M; Gottrup, F; Kirkegaard, L; Gellett, S

    1990-01-01

    In pigs, latissimus dorsi musculocutaneous island flaps and buttock skin island flaps were raised. Subcutaneous (PscO2) and intramuscular oxygen tension (PimO2) were measured using a non-heated needle electrode before, during and after repeated occlusion of the supplying artery or the draining vein. During arterial and venous occlusion, the tissue oxygen tension in the musculocutaneous flap dropped rapidly. A plateau was reached after 15 min. After arterial occlusion the mean value was 20 mmHg (SEM = +/- 5 mmHg, N = 6) in the subcutis and 16 mmHg in the muscle (SEM = +/- 4 mmHg, N = 10). After venous occlusion the mean value was 11 mmHg (SEM = +/- 3 mmHg, N = 6) in the subcutis. In the skin flap the drop of PscO2 was slower, and after 30 min of arterial occlusion the mean value was 29 mmHg (SEM = +/- 9 mmHg, N = 6). This study has shown that tissue oxygen tension measurement can be used as a sensitive indicator of acute impairment of the supplying vessels in island flaps. The method seems to have potential for monitoring free tissue transfers. A comparable decrease in PscO2 was found for arterial and venous impairment.

  11. 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.

  12. 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.

  13. Defining uncertainty and error in planktic foraminiferal oxygen isotope measurements

    NASA Astrophysics Data System (ADS)

    Fraass, A. J.; Lowery, C. M.

    2017-02-01

    Foraminifera are the backbone of paleoceanography. Planktic foraminifera are one of the leading tools for reconstructing water column structure. However, there are unconstrained variables when dealing with uncertainty in the reproducibility of oxygen isotope measurements. This study presents the first results from a simple model of foraminiferal calcification (Foraminiferal Isotope Reproducibility Model; FIRM), designed to estimate uncertainty in oxygen isotope measurements. FIRM uses parameters including location, depth habitat, season, number of individuals included in measurement, diagenesis, misidentification, size variation, and vital effects to produce synthetic isotope data in a manner reflecting natural processes. Reproducibility is then tested using Monte Carlo simulations. Importantly, this is not an attempt to fully model the entire complicated process of foraminiferal calcification; instead, we are trying to include only enough parameters to estimate the uncertainty in foraminiferal δ18O records. Two well-constrained empirical data sets are simulated successfully, demonstrating the validity of our model. The results from a series of experiments with the model show that reproducibility is not only largely controlled by the number of individuals in each measurement but also strongly a function of local oceanography if the number of individuals is held constant. Parameters like diagenesis or misidentification have an impact on both the precision and the accuracy of the data. FIRM is a tool to estimate isotopic uncertainty values and to explore the impact of myriad factors on the fidelity of paleoceanographic records, particularly for the Holocene.

  14. 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.

  15. Method for measurement of volatile oxygenated hydrocarbons in ambient air

    NASA Astrophysics Data System (ADS)

    Leibrock, E.; Slemr, J.

    An automated gas chromatographic method for the quantitative determination of oxygenated (C 2C 5 carbonyls and C 1C 2 alcohols) and some non-oxygenated (C 5C 8) hydrocarbons in ambient air has been developed. The analytical system consists of a gas chromatograph with a cryogenic sampling trap, a precolumn for the separation of water and other interfering compounds, a cryogenic focusing trap and two analytical columns connected in series. Substances are detected either by flame ionization or by a mass spectrometer. Ozone is removed by a potassium iodide scrubber placed upstream the sampling trap. External gas standards generated by a permeation device are used for calibration. The detection limits range between 0.03 and 0.08 ng (depending on the compound), equivalent to 5 to 56 ppt in 1 l of sampled air. The method was tested by an intercomparison with a different gas chromatographic technique for the determination of NMHC. The system has been applied since 1994 for measurements in ambient air. Data obtained during an intensive campaign in summer 1995 at the field station Wank (1778 m a.s.l.) near Garmisch-Partenkirchen, Germany, are reported and compared with NMHC mixing ratios measured simultaneously in the same air masses.

  16. 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

  17. Microfluidics for single-cell genetic analysis.

    PubMed

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

    2014-09-07

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

  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.

  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. Future medical applications of single-cell sequencing in cancer

    PubMed Central

    2011-01-01

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

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

    PubMed

    Navin, Nicholas; Hicks, James

    2011-05-31

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

  2. 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.

  3. 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.

  4. 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

  5. Single cell sequencing: a distinct new field.

    PubMed

    Wang, Jian; Song, Yuanlin

    2017-12-01

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

  6. 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).

  7. Simultaneous Measurement of Dissolved Oxygen Pressure and Oxyhemoglobin Spectra in Solution.

    PubMed

    Litorja, Maritoni; Hwang, Jeeseong C

    2016-01-01

    The measurement of the spatial distribution of oxygen saturation (sO2) in superficial tissues using optical reflectance imaging has been useful in the clinical venue especially in temporally demanding applications such as monitoring tissue oxygenation during surgery. The measurement is based on relative spectrometry of oxy- and deoxyhemoglobin in tissues. We titrated deoxyhemoglobin with oxygen gas and simultaneously measured the dissolved oxygen pressure and the visible absorbance spectra to verify spectral shapes at different saturations. sO2 values derived from the measured pO2 are compared to those derived from the hemoglobin spectra at various stages of oxygenation.

  8. Measuring and interpreting respiratory critical oxygen pressures in roots

    PubMed Central

    Armstrong, William; Webb, Trevor; Darwent, Marcus; Beckett, Peter M.

    2009-01-01

    Background and Aims Respiratory critical oxygen pressures (COPR) determined from O2-depletion rates in media bathing intact or excised roots are unreliable indicators of respiratory O2-dependency in O2-free media and wetlands. A mathematical model was used to help illustrate this, and more relevant polarographic methods for determining COPR in roots of intact plants are discussed. Methods Cortical [O2] near the root apex was monitored indirectly (pea seedlings) from radial oxygen losses (ROL) using sleeving Pt electrodes, or directly (maize) using microelectrodes; [O2] in the root was controlled by manipulating [O2] around the shoots. Mathematical modelling of radial diffusive and respiratory properties of roots used Michaelis–Menten enzyme kinetics. Key Results Respiration declined only when the O2 partial pressure (OPP) in the cortex of root tips fell below 0·5–4·5 kPa, values consistent with depressed respiration near the centre of the stele as confirmed by microelectrode measurements and mathematical modelling. Modelling predictions suggested that the OPP of a significant core at the centre of roots could be below the usual detection limits of O2-microelectrodes but still support some aerobic respiration. Conclusions In O2-free media, as in wetlands, the COPR for roots is likely to be quite low, dependent upon the respiratory demands, dimensions and diffusion characteristics of the stele/stelar meristem and the enzyme kinetics of cytochrome oxidase. Roots of non-wetland plants may not differ greatly in their COPRs from those of wetland species. There is a possibility that trace amounts of O2 may still be present in stelar ‘anaerobic’ cores where fermentation is induced at low cortical OPPs. PMID:18819952

  9. Epigenetics reloaded: the single-cell revolution.

    PubMed

    Bheda, Poonam; Schneider, Robert

    2014-11-01

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

  10. 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.

  11. Single-cell intracellular nano-pH probes.

    PubMed

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

    2015-01-01

    Within a large clonal population, such as cancerous tumor entities, cells are not identical, and the differences between intracellular pH levels of individual cells may be important indicators of heterogeneity that could be relevant in clinical practice, especially in personalized medicine. Therefore, the detection of the intracellular pH at the single-cell level is of great importance to identify and study outlier cells. However, quantitative and real-time measurements of the intracellular pH of individual cells within a cell population is challenging with existing technologies, and there is a need to engineer new methodologies. In this paper, we discuss the use of nanopipette technology to overcome the limitations of intracellular pH measurements at the single-cell level. We have developed a nano-pH probe through physisorption of chitosan onto hydroxylated quartz nanopipettes with extremely small pore sizes (~100 nm). The dynamic pH range of the nano-pH probe was from 2.6 to 10.7 with a sensitivity of 0.09 units. We have performed single-cell intracellular pH measurements using non-cancerous and cancerous cell lines, including human fibroblasts, HeLa, MDA-MB-231 and MCF-7, with the pH nanoprobe. We have further demonstrated the real-time continuous single-cell pH measurement capability of the sensor, showing the cellular pH response to pharmaceutical manipulations. These findings suggest that the chitosan-functionalized nanopore is a powerful nano-tool for pH sensing at the single-cell level with high temporal and spatial resolution.

  12. Multiplex single cell profiling of chromatin accessibility by combinatorial cellular indexing.

    PubMed

    Cusanovich, Darren A; Daza, Riza; Adey, Andrew; Pliner, Hannah A; Christiansen, Lena; Gunderson, Kevin L; Steemers, Frank J; Trapnell, Cole; Shendure, Jay

    2015-05-22

    Technical advances have enabled the collection of genome and transcriptome data sets with single-cell resolution. However, single-cell characterization of the epigenome has remained challenging. Furthermore, because cells must be physically separated before biochemical processing, conventional single-cell preparatory methods scale linearly. We applied combinatorial cellular indexing to measure chromatin accessibility in thousands of single cells per assay, circumventing the need for compartmentalization of individual cells. We report chromatin accessibility profiles from more than 15,000 single cells and use these data to cluster cells on the basis of chromatin accessibility landscapes. We identify modules of coordinately regulated chromatin accessibility at the level of single cells both between and within cell types, with a scalable method that may accelerate progress toward a human cell atlas.

  13. Multiplex Single Cell Profiling of Chromatin Accessibility by Combinatorial Cellular Indexing

    PubMed Central

    Cusanovich, Darren A.; Daza, Riza; Adey, Andrew; Pliner, Hannah; Christiansen, Lena; Gunderson, Kevin L.; Steemers, Frank J.; Trapnell, Cole

    2016-01-01

    Technical advances have enabled the collection of genome and transcriptome datasets with single-cell resolution. However, single-cell characterization of the epigenome has remained challenging. Furthermore, because cells must be physically separated prior to biochemical processing, conventional single-cell preparatory methods scale linearly. We applied combinatorial cellular indexing to measure chromatin accessibility in thousands of single cells per assay, circumventing the need for compartmentalization of individual cells. We report chromatin accessibility profiles from over 15,000 single cells and use these data to cluster cells on the basis of chromatin accessibility landscapes. We identify modules of coordinately regulated chromatin accessibility at the level of single cells both between and within cell types, with a scalable method that may accelerate progress towards a human cell atlas. PMID:25953818

  14. 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.

  15. 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

  16. 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.

  17. 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

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

    PubMed

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

    2015-11-07

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

  19. High Precision Oxygen Measurements as a Tool for CCS Monitoring

    NASA Astrophysics Data System (ADS)

    Trugman, A. T.; Dvonch, C.; Clegg, S. M.; Rahn, T.

    2011-12-01

    CO2 emissions from below ground carbon storage reservoirs can be difficult to discriminate from CO2 produced via natural plant and microbial respiration. However, because respiration produces CO2 and consumes O2 in an approximately 1:1 ratio, it is possible to characterize leakage sources by measurement of simultaneous changes of both O2 and CO2. This approach is complicated by the fact that O2 comprises approximately 21% of the atmosphere, while CO2 is only present in the background atmosphere at ~400 parts per million, making it necessary to accurately measure changes in O2 concentration to six significant figures. Here we describe a portable high precision oxygen measurement system that employs a modified commercial fuel cell analyzer to quantify small changes in O2 concentration. High precision is achieved through precise control of flow and pressure, allowing near part per million precision of O2 and CO2 concentrations. This system has been incorporated into a mobile laboratory and has been deployed to the ZERT controlled release site in Bozeman, Montana and to a natural analog CO2 leak at Soda Springs, Idaho. Samples were collected at ground level, 1 meter, and 3 meters above the CO2 source and are displayed as the ratio of the O2 difference relative to a reference to the CO2 difference in concentration relative to the same reference (ΔO2/ΔCO2). It was observed that at wind speeds ≤ 2 m/s, the ΔO2/ΔCO2 anomaly decreased with height and was still significantly different from background at 3 m. With increasing wind speed, ΔO2/ΔCO2 anomalies decreased to background levels at 1 and 3 m but remained detectable at the ground surface. We will discuss attempts to quantify the CO2 release rate utilizing the measured ΔO2/ΔCO2 elevation profiles and will present complementary eddy covariance data for comparison.

  20. Biological oxygen productivity during the last 60,000 years from triple oxygen isotope measurements

    NASA Astrophysics Data System (ADS)

    Blunier, Thomas; Barnett, Bruce; Bender, Michael L.; Hendricks, Melissa B.

    2002-07-01

    The oxygen isotope signature of atmospheric O2 is linked to the isotopic signature of seawater (H2O) through photosynthesis and respiration. Fractionation during these processes is mass dependent, affecting δ17O about half as much as δ18O. An ``anomalous'' fractionation process, which changes δ17O and δ18O of O2 about equally, takes place during isotope exchange between O2 and CO2 in the stratosphere. The relative rates of biologic O2 production and stratospheric processing determine the relationship between δ17O and δ18O of O2 in the atmosphere. Variations of this relationship thus allow us to estimate changes in the rate of mass-dependent O2 production by photosynthesis versus the rate of O2-CO2 exchange in the stratosphere with about equal fractionations of δ17O and δ18O. In this study we reconstruct total oxygen productivity for the last glacial, the last glacial termination, and the early Holocene from the triple isotope composition of atmospheric oxygen trapped in ice cores. With a box model we estimate that total biogenic productivity was only ~76-83% of today for the glacial and was probably lower than today during the glacial-interglacial transition and the early Holocene. Depending on how reduced the oxygen flux from the land biosphere was during the glacial, the oxygen flux from the glacial ocean biosphere was 88-140% of its present value.

  1. 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.

  2. Technologies for Single-Cell Isolation.

    PubMed

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

    2015-07-24

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

  3. 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

  4. 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

  5. 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...

  6. Dissolved oxygen distribution during micro-oxygenation. Determination of representative measurement points in hydroalcoholic solution and wines.

    PubMed

    Nevares, I; del Alamo, M; Gonzalez-Muñoz, C

    2010-02-15

    Red wine tank aging is monitored by organoleptic analysis, therefore, it is necessary to use an objective parameter representing the process. Among the possible parameters to be checked, it stands out the knowledge of dissolved oxygen because it offers the possibility of anticipating undesirable situations that bring about too much oxidation. Dissolved oxygen measurement, with non-intrusive luminescent technology is becoming an effective alternative. Uncertainty arises when trying to choose the measuring point able to represent the entire tank since previous works have considered the existence of gradients throughout the volume of the treated wine. This paper shows the results obtained from the study of the existence and the quantification of gradients of the dissolved oxygen in a 15% hydroalcoholic solution during the micro-oxygenation process. Different measuring point placements are studied and the solutions to monitor the process by controlling a representative point are set out. A successful monitoring of a red wine tank aging with alternative oak products and adaptative micro-oxygenation has proved that an objective control of the process is, indeed, possible.

  7. Current techniques for single-cell lysis.

    PubMed

    Brown, Robert B; Audet, Julie

    2008-10-06

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

  8. Single-cell sequencing in cancer research.

    PubMed

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

    2016-01-01

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

  9. Origin of Individuality of Two Daughter Cells during the Division Process Examined by the Simultaneous Measurement of Growth and Swimming Property Using an On-Chip Single-Cell Cultivation System

    PubMed Central

    Umehara, Senkei; Inoue, Ippei; Wakamoto, Yuichi; Yasuda, Kenji

    2007-01-01

    We examined the origin of individuality of two daughter cells born from an isolated single Escherichia coli mother cell during its cell division process by monitoring the change in its swimming behavior and tumbling frequency using an on-chip single-cell cultivation system. By keeping the isolated condition of an observed single cell, we compared its growth and swimming property within a generation and over up to seven generations. It revealed that running speed decreased as cell length smoothly increased within each generation, whereas tumbling frequency fluctuated among generations. Also found was an extraordinary tumbling mode characterized by the prolonged duration of pausing in predivisional cells after cell constriction. The observed prolonged pausing may imply the coexistence of two distinct control systems in a predivisional cell, indicating that individuality of daughter cells emerges after a mother cell initiates constriction and before it gets physically separated into two new cell bodies. PMID:17496044

  10. 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.

  11. Single Cell Genomics: Advances and Future Perspectives

    PubMed Central

    Macaulay, Iain C.; Voet, Thierry

    2014-01-01

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

  12. 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.

  13. 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

  14. 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.

  15. 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.

  16. Using near-infrared spectroscopy to measure cerebral metabolic rate of oxygen under multiple levels of arterial oxygenation in piglets.

    PubMed

    Tichauer, Kenneth M; Elliott, Jonathan T; Hadway, Jennifer A; Lee, David S; Lee, Ting-Yim; St Lawrence, Keith

    2010-09-01

    Improving neurological care of neonates has been impeded by the absence of suitable techniques for measuring cerebral hemodynamics and energy metabolism at the bedside. Currently, near-infrared spectroscopy (NIRS) appears to be the technology best suited to fill this gap, and techniques have been proposed to measure both cerebral blood flow (CBF) and cerebral metabolic rate of oxygen (CMRO2). We have developed a fast and reliable bolus-tracking method of determining CMRO2 that combines measurements of CBF and cerebral venous oxygenation [venous oxygen saturation (CSvO2)]. However, this method has never been validated at different levels of arterial oxygenation [arterial oxygen saturation (SaO2)], which can be highly variable in the clinical setting. In this study, NIRS measurements of CBF, CSvO2, and CMRO2 were obtained over a range of SaO2 in newborn piglets (n=12); CSvO2 values measured directly from sagittal sinus blood samples were collected for validation. Two alternative NIRS methods that measure CSvO2 by manipulating venous oxygenation (i.e., head tilt and partial venous occlusion methods) were also employed for comparison. Statistically significant correlations were found between each NIRS technique and sagittal sinus blood oxygenation (P<0.05). Correlation slopes were 1.03 (r=0.91), 0.73 (r=0.73), and 0.73 (r=0.81) for the bolus-tracking, head tilt, and partial venous occlusion methods, respectively. The bolus-tracking technique displayed the best correlation under hyperoxic (SaO2=99.9±0.03%) and normoxic (SaO2=86.9±6.6%) conditions and was comparable to the other techniques under hypoxic conditions (SaO2=40.7±9.9%). The reduced precision of the bolus-tracking method under hypoxia was attributed to errors in CSvO2 measurement that were magnified at low SaO2 levels. In conclusion, the bolus-tracking technique of measuring CSvO2, and therefore CMRO2, is accurate and robust for an SaO2>50% but provides reduced accuracy under more severe hypoxic levels.

  17. 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.}

  18. 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

  19. 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.

  20. 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.

  1. Measurement of oxygen saturation in small retinal vessels with adaptive optics confocal scanning laser ophthalmoscope.

    PubMed

    Li, Hao; Lu, Jing; Shi, Guohua; Zhang, Yudong

    2011-11-01

    We have used an adaptive optics confocal scanning laser ophthalmoscope to assess oxygen saturation in small retinal vessels. Images of the vessels with a diameter smaller than 50 μm are recorded at oxygen sensitive and isosbestic wavelengths (680 and 796 nm, respectively). The vessel optical densities (ODs) are determined by a computer algorithm. Then, OD ratios (ODRs), which are inversely proportional to oxygen saturation, are calculated. The results show that arterial ODRs are significantly smaller than venous ODRs, indicating that oxygen saturation in the artery is higher than that in the vein. To the best of our knowledge, this is the first noninvasive measurement of oxygen saturation in small retinal vessels.

  2. 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.

  3. 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.

  4. 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

  5. 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…

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

    PubMed

    Jordan, Bertrand

    2014-12-01

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

  7. Line Profile Measurements of Atomic Oxygen at 1300 A with a VUV Raman Shifter

    NASA Technical Reports Server (NTRS)

    Sharma, Surendra P.; Exberger, Richard J.; Meyer, Scott A.; Gilmore, John O.

    1994-01-01

    We are currently developing an atomic oxygen diagnostic to study the degree of oxygen dissociation in ground-based facilities. The absorption of the (sub 3)P - (sup 3)S(sup 0) resonance triplet in the vacuum ultraviolet is a direct measure of the ground state number density of atomic oxygen. Although the integrated line strength is well known for these transitions, the line profile is not. We report the results of a series of experiments in which the line profile is measured in shock-heated oxygen. An ArF excimer laser and a hydrogen Raman shifter generate tunable VUV radiation at the resonance wavelength. The test gas is dissociated oxygen, generated in the Electric Arc Shock Tube (EAST) Facility at NASA-Ames Research Center. By measuring the absorption of known concentrations of atomic oxygen, we are able to study the absorption line profile. The results will serve as a calibration to apply this diagnostic in other flowfields.

  8. 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

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

    PubMed

    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

    2016-10-14

    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.

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. Digital microfluidic immunocytochemistry in single cells

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-10-01

    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.

  16. 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

  17. Atomic oxygen-metal surface studies as applied to mass spectrometer measurements of upper planetary atmospheres

    NASA Technical Reports Server (NTRS)

    Sjolander, G. W.

    1976-01-01

    The problem of atomic oxygen loss in mass spectrometer ion sources can be reduced to an understanding of the possible surface interactions between oxygen atoms and the metal surface of the ion source. Results are presented for an experimental study in which an atomic oxygen beam apparatus and a mass spectrometer were used to measure the oxygen atom reflection, recombination, general surface reaction, and occlusion probabilities on six different engineering surfaces as a function of atomic oxygen exposure. The materials studied are gold, Nichrome V, aluminum, titanium, silver, and platinum. The variation in measured reflection probability seems to occur with metals that form oxides, Nichrome V being stable in terms of reflection stability. Recombination is observed an all surfaces except aluminum and platinum. Variation in the complete set of measurements in a single experiment is the result of varying surface conditions.

  18. Measuring vertical oxygen profiles in the hyporheic zone using planar optodes

    NASA Astrophysics Data System (ADS)

    Vieweg, M.; Fleckenstein, J. H.; Schmidt, C.

    2012-04-01

    On of the key parameters, controlling biogeochemical reactions in the hyporheic zone (HZ) is the distribution of oxygen. A reliable measurement of the vertical oxygen distribution is an important tool to understand the dynamic fluctuations of the aerobic zone within the HZ. With repeated measurements of continuous profiles, mixing of surface water and groundwater as well as the consumption of oxygen can be evaluated. We present a novel approach for the in situ measurements of vertical oxygen distribution in the riverbed using a planar optode. The luminescence based optode measurement enables a non invasive measurement without consumption of oxygen, no creation of preferential flow paths and only minimal disturbance of the flow field. Possible atmospheric contamination by pumping pore water into a vessel can be avoided and the readings are independent of flow velocity. A self manufactured planar optode is wrapped around an acrylic tube and installed in the riverbed. The measurement is performed by vertically moving a profiler-piston inside the acrylic tube. The piston holds a robust polymer optical fibre which emits a modulated light signal through the acrylic glass to the optode-foil and transmits the induced luminescence signal back to a commercially available trace oxygen meter. Temperature compensation is accomplished using a depth-oriented temperature probe nearby and processing the raw data within a Matlab script. Robust and unbiased oxygen profiles are obtained by averaging multiple consecutive measurements. To ensure a constant velocity of the profiler for replicating the exact measuring depths, an electric motor device is used. First results at our test site show a variable oxygen profile down to 40 cm depth which is strongly influenced by stream level and upwelling groundwater conditions. The measured oxygen profiles will serve as input parameter for a 3D solute transport and chemical reaction subsurface model of the HZ.

  19. 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.

  20. Single cell elemental analysis using nuclear microscopy

    NASA Astrophysics Data System (ADS)

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

    1999-04-01

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

  1. Membrane Transport of Singlet Oxygen Monitored by Dipole Potential Measurements

    PubMed Central

    Sokolov, Valerij S.; Pohl, Peter

    2009-01-01

    Abstract The efficiency of photodynamic reactions depends on 1), the penetration depth of the photosensitizer into the membrane and 2), the sidedness of the target. Molecules which are susceptible to singlet oxygen (1O2) experience less damage when separated from the photosensitizer by the membrane. Since 1O2 lifetime in the membrane environment is orders of magnitude longer than the time required for nonexcited oxygen (O2) to cross the membrane, this observation suggests that differences between the permeabilities or membrane partition of 1O2 and O2 exist. We investigated this hypothesis by releasing 1O2 at one side of a planar membrane while monitoring the kinetics of target damage at the opposite side of the same membrane. Damage to the target, represented by dipole-modifying molecules (phloretin or phlorizin), was indicated by changes in the interleaflet dipole potential difference Δϕb. A simple analytical model allowed estimation of the 1O2 interleaflet concentration difference from the rate at which Δϕb changed. It confirmed that the lower limit of 1O2 permeability is ∼2 cm/s; i.e., it roughly matches O2 permeability as predicted by Overton's rule. Consequently, the membrane cannot act as a barrier to 1O2 diffusion. Differences in the reaction rates at the cytoplasmic and extracellular membrane leaflets may be attributed only to 1O2 quenchers inside the membrane. PMID:18931253

  2. 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)

  3. 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.

  4. 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.

  5. 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.

  6. 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.

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

    PubMed Central

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

    2016-01-01

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

  8. 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.

  9. 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.

  10. 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

  11. A lenslet-based device for measuring oxygen saturation in the retina

    NASA Astrophysics Data System (ADS)

    Ramella-Roman, Jessica C.; Kandimalla, H.; Dinga, R.; Nabili, A.; Mathews, Scott A.; Nguyen, Q. D.

    2007-02-01

    Diabetic retinopathy (DR) is a complication of diabetes affecting up to 80% of all diabetic patients. DR can lead to blindness and reduced quality of life. Some authors have hypothesized that changes in the flow dynamics associated with DR as well as changes in retinal oxygenation can lead to macular edema. Measurements of oxygen saturation in the retina could help understand the real mechanisms behind this condition. We present a novel spectroscopic imaging device to measure oxygen saturation in the retina. Our system uses a lenslet array to spatially and spectrocopically divide a fundus image. A three wavelengths algorithm is used to calculate oxygen saturation in small vessels. Only wavelengths in the 500 - 580 nm range are considered in order to minimize the wavelength dependence of the scattering from erythrocytes. Preliminary testing on healthy subjects showed values of oxygen saturation comparable to the one reported in the literature.

  12. 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.

  13. New Oxygen Isotope Measurements of Four Stardust Impact Crater Residues Show IDP-Like Compositions

    NASA Astrophysics Data System (ADS)

    Snead, C. J.; McKeegan, K. D.

    2015-07-01

    We have measured the oxygen isotope compositions of four Stardust impact crater residues. These analyses reveal compositions that are similar to those found in interplanetary dust particles, antarctic micrometeorites and CI chondrite components.

  14. Measuring respiration of cultured cell with oxygen electrode as a metabolic indicator for drug screening.

    PubMed

    Amano, Y; Okumura, C; Yoshida, M; Katayama, H; Unten, S; Arai, J; Tagawa, T; Hoshina, S; Hashimoto, H; Ishikawa, H

    1999-03-01

    New trend in methods for assessing pharmacological action to bacteria and cell is to measure their metabolic activities induced, while the conventional methods used population growth. We focused on respiration volume as an indicator of cell metabolism, and developed inexpensive disposable oxygen electrode sensor and multi-channel dissolved oxygen meters (DOX-10 and DOX-96KB). Using these instruments, cytotoxicity was measured for 48 hrs and the method showed superior features to conventional methods in its handiness of one step assay, and excellent adaptability to automated systems. Total usability of this oxygen electrode method is being evaluated in bacterial drug susceptibility test, anticancer drug susceptibility test, and alternatives to animal experiment.

  15. Visualization and analysis of single-cell RNA-seq data by kernel-based similarity learning.

    PubMed

    Wang, Bo; Zhu, Junjie; Pierson, Emma; Ramazzotti, Daniele; Batzoglou, Serafim

    2017-04-01

    We present single-cell interpretation via multikernel learning (SIMLR), an analytic framework and software which learns a similarity measure from single-cell RNA-seq data in order to perform dimension reduction, clustering and visualization. On seven published data sets, we benchmark SIMLR against state-of-the-art methods. We show that SIMLR is scalable and greatly enhances clustering performance while improving the visualization and interpretability of single-cell sequencing data.

  16. 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

  17. Quantitative assessments of glycolysis from single cells.

    PubMed

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

    2015-06-01

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

  18. Quantitative assessments of glycolysis from single cells

    PubMed Central

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

    2015-01-01

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

  19. Single cell systems biology by super-resolution imaging and combinatorial labeling

    PubMed Central

    Lubeck, Eric; Cai, Long

    2012-01-01

    Fluorescence microscopy is a powerful quantitative tool for exploring regulatory networks in single cells. However, the number of molecular species that can be measured simultaneously is limited by the spectral separability of fluorophores. Here we demonstrate a simple but general strategy to drastically increase the capacity for multiplex detection of molecules in single cells by using optical super-resolution microscopy (SRM) and combinatorial labeling. As a proof of principle, we labeled mRNAs with unique combinations of fluorophores using Fluorescence in situ Hybridization (FISH), and resolved the sequences and combinations of fluorophores with SRM. We measured the mRNA levels of 32 genes simultaneously in single S. cerevisiae cells. These experiments demonstrate that combinatorial labeling and super-resolution imaging of single cells provides a natural approach to bring systems biology into single cells. PMID:22660740

  20. 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

  1. 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

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

    PubMed

    Pott, Sebastian; Lieb, Jason D

    2015-08-21

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

  3. Optimal wavelengths for optoacoustic measurements of blood oxygen saturation in biological tissues

    PubMed Central

    Perekatova, Valeriya; Subochev, Pavel; Kleshnin, Mikhail; Turchin, Ilya

    2016-01-01

    The non-invasive measurement of blood oxygen saturation in blood vessels is a promising clinical application of optoacoustic imaging. Nevertheless, precise optoacoustic measurements of blood oxygen saturation are limited because of the complexities of calculating the spatial distribution of the optical fluence. In the paper error in the determination of blood oxygen saturation, associated with the use of approximate methods of optical fluence evaluation within the blood vessel, was investigated for optoacoustic measurements at two wavelengths. The method takes into account both acoustic pressure noise and the error in determined values of the optical scattering and absorption coefficients used for the calculation of the fluence. It is shown that, in conditions of an unknown (or partially known) spatial distribution of fluence at depths of 2 to 8 mm, minimal error in the determination of blood oxygen saturation is achieved at wavelengths of 658 ± 40 nm and 1069 ± 40 nm. PMID:27867709

  4. Microvascular oxygen tension and flow measurements in rodent cerebral cortex during baseline conditions and functional activation

    PubMed Central

    Yaseen, Mohammad A; Srinivasan, Vivek J; Sakadžić, Sava; Radhakrishnan, Harsha; Gorczynska, Iwona; Wu, Weicheng; Fujimoto, James G; Boas, David A

    2011-01-01

    Measuring cerebral oxygen delivery and metabolism microscopically is important for interpreting macroscopic functional magnetic resonance imaging (fMRI) data and identifying pathological changes associated with stroke, Alzheimer's disease, and brain injury. Here, we present simultaneous, microscopic measurements of cerebral blood flow (CBF) and oxygen partial pressure (pO2) in cortical microvessels of anesthetized rats under baseline conditions and during somatosensory stimulation. Using a custom-built imaging system, we measured CBF with Fourier-domain optical coherence tomography (OCT), and vascular pO2 with confocal phosphorescence lifetime microscopy. Cerebral blood flow and pO2 measurements displayed heterogeneity over distances irresolvable with fMRI and positron emission tomography. Baseline measurements indicate O2 extraction from pial arterioles and homogeneity of ascending venule pO2 despite large variation in microvessel flows. Oxygen extraction is linearly related to flow in ascending venules, suggesting that flow in ascending venules closely matches oxygen demand of the drained territory. Oxygen partial pressure and relative CBF transients during somatosensory stimulation further indicate arteriolar O2 extraction and suggest that arterioles contribute to the fMRI blood oxygen level dependent response. Understanding O2 supply on a microscopic level will yield better insight into brain function and the underlying mechanisms of various neuropathologies. PMID:21179069

  5. Measuring Oxygen Cost During Level Walking in Individuals with Acquired Brain Injury in the Clinical Setting

    PubMed Central

    Dawes, Helen; Collett, Johnathen; Ramsbottom, Roger; Howells, Ken; Sackley, Cath; Wade, Derick

    2004-01-01

    This study examined the test-retest reliability of oxygen cost (ml·kg-1·min-1) during level walking in individuals with acquired brain injury (ABI). Ten individuals with ABI (5 men, 5 women) (Traumatic brain injury, 1, central pontine myelinolysis, 1, stroke 8) and 21 healthy controls (11 men, 10 women). Measurements of gross and net (walking minus resting) oxygen consumption (ml·kg-1·min-1), and oxygen cost (ml·kg-1·min-1) during level walking at self-selected speeds. Measurements were taken on two occasions within one week. Oxygen cost was significantly lower (p < 0.05) in individuals with ABI on the second test versus the first test. Percentage variability in oxygen cost from test to re-test ranged from 14.7 to 17.3% in the control group and from 17.4 to 20.8% in the brain injury group. Clinical populations may demonstrate a significant decrease in oxygen cost between testing occasions. Individuals require at least one period of familiarisation if oxygen cost is used as an outcome measure during level walking in clinical groups. The amount of familiarisation has yet to be investigated in individuals with ABI. Key Points Individuals with brain injury during level walking May demonstrate a significant decrease in oxygen cost between testing occasions. May require at least one period of familiarisation if oxygen cost is used as an outcome measure The degree of familiarisation required in this clinical group needs further investigation PMID:24482582

  6. Continuous measurement of transcutaneous oxygen tension of neonates under general anesthesia.

    PubMed

    Welle, P; Hayden, W; Miller, T

    1980-06-01

    Neonates present unique challenges to the anesthesiologist because of their susceptibility to oxygen toxicity and because clinical assessment of the degree of an infant's hypoxia is more difficult than in the adult. Equipment is now available for the continuous noninvasive measurement of transcutaneous oxygen tension. We used this equipment to monitor nine different neonates undergoing ten surgical procedures requiring general anesthesia. We found that certain of the infants were above and below what we considered to be a safe range for the transcutaneous oxygen tension for a significant portion of the surgery. Additionally, the manipulations of the surgeon and anesthesiologists were seen to cause sudden and large fluctuations in the transcutaneous oxygen tension. By providing the anesthesiologist with continuous and immediate data on the cardiorespiratory status of the infant, transcutaneous oxygen monitoring makes itself a valuable addition to the equipment used in the intraoperative monitoring of the neonate.

  7. 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.

  8. 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

  9. In situ single cell detection via microfluidic magnetic bead assay

    PubMed Central

    KC, Pawan; Zhang, Ge; Zhe, Jiang

    2017-01-01

    We present a single cell detection device based on magnetic bead assay and micro Coulter counters. This device consists of two successive micro Coulter counters, coupled with a high gradient magnetic field generated by an external magnet. The device can identify single cells in terms of the transit time difference of the cell through the two micro Coulter counters. Target cells are conjugated with magnetic beads via specific antibody and antigen binding. A target cell traveling through the two Coulter counters interacts with the magnetic field, and have a longer transit time at the 1st counter than that at the 2nd counter. In comparison, a non-target cell has no interaction with the magnetic field, and hence has nearly the same transit times through the two counters. Each cell passing through the two counters generates two consecutive voltage pulses one after the other; the pulse widths and magnitudes indicating the cell’s transit times through the counters and the cell’s size respectively. Thus, by measuring the pulse widths (transit times) of each cell through the two counters, each single target cell can be differentiated from non-target cells even if they have similar sizes. We experimentally proved that the target human umbilical vein endothelial cells (HUVECs) and non-target rat adipose-derived stem cells (rASCs) have significant different transit time distribution, from which we can determine the recognition regions for both cell groups quantitatively. We further demonstrated that within a mixed cell population of rASCs and HUVECs, HUVECs can be detected in situ and the measured HUVECs ratios agree well with the pre-set ratios. With the simple device structure and easy sample preparation, this method is expected to enable single cell detection in a continuous flow and can be applied to facilitate general cell detection applications such as stem cell identification and enumeration. PMID:28222140

  10. 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.

  11. [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.

  12. 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

  13. 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.

  14. 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)

  15. 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.

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

    PubMed

    Grün, Dominic; van Oudenaarden, Alexander

    2015-11-05

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

  17. Measurement of Oxygen Consumption Using the Canadian Clearance Diving Apparatus (CCDA)

    DTIC Science & Technology

    1988-08-01

    tem for the measurement of Vo in water. * iii INTRODUCTION In semi- closed circuit underwater breathing apparatus (SCCBA) the oxygen partial pressure...13 9 9 0 0 ii ABSTRACT The Canadian Clearance Diving Apparatus (CCDA) was modified to serve as a 100% oxygen rebreathing ...were open- circuit , compressed-air systems based upon the principles used in the LRBS. In the LRBA and UMAS, the subject inspired gas from a bag

  18. Effects of incremental exercise on cerebral oxygenation measured by near-infrared spectroscopy: a systematic review.

    PubMed

    Rooks, Cherie R; Thom, Nathaniel J; McCully, Kevin K; Dishman, Rod K

    2010-10-01

    We conducted a systematic review and meta-regression analysis to quantify effects of exercise on brain hemodynamics measured by near-infrared spectroscopy (NIRS). The results indicate that acute incremental exercise (categorized relative to aerobic capacity (VO(2)peak) as low - <30% VO(2)peak; moderate - ≥30% VO(2)peak to <60% VO(2)peak; hard - ≥60% VO(2)peak to oxygenated hemoglobin (O(2)Hb) or other measures of oxygen level (O(2)Hbdiff) or saturation (SCO(2)) (0.92±0.67, 1.17), deoxygenated hemoglobin (dHb) (0.87±0.56, 1.19), and blood volume estimated by total hemoglobin (tHb) (1.21±0.84, 1.59). After peaking at hard intensities, cerebral oxygen levels dropped during very hard intensities. People who were aerobically trained attained higher levels of cortical oxygen, dHb, and tHb than untrained people during very hard intensities. Among untrained people, a marked drop in oxygen levels and a small increase in dHb at very hard intensities accompanied declines in tHb, implying reduced blood flow. In 6 studies of 222 patients with heart or lung conditions, oxygenation and dHb were lowered or unchanged during exercise compared to baseline. In conclusion, prefrontal oxygenation measured with NIRS in healthy people showed a quadratic response to incremental exercise, rising between moderate and hard intensities, then falling at very hard intensities. Training status influenced the responses. While methodological improvements in measures of brain oxygen are forthcoming, these results extend the evidence relevant to existing models of central limitations to maximal exercise.

  19. Measurement of mitochondrial oxygen consumption rates in mouse primary neurons and astrocytes.

    PubMed

    Ribeiro, Sofia M; Giménez-Cassina, Alfredo; Danial, Nika N

    2015-01-01

    The introduction of microplate-based assays that measure extracellular fluxes in intact, living cells has revolutionized the field of cellular bioenergetics. Here, we describe a method for real time assessment of mitochondrial oxygen consumption rates in primary mouse cortical neurons and astrocytes. This method requires the Extracellular Flux Analyzer Instrument (XF24, Seahorse Biosciences), which uses fluorescent oxygen sensors in a microplate assay format.

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

    PubMed

    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.

  1. Real time measurement of myocardial oxygen dynamics during cardiac ischemia-reperfusion of rats.

    PubMed

    Lee, Gi-Ja; Kim, Seung Ki; Kang, Sung Wook; Kim, Ok-Kyun; Chae, Su-Jin; Choi, Samjin; Shin, Jae Ho; Park, Hun-Kuk; Chung, Joo-Ho

    2012-11-21

    Because oxygen plays a critical role in the pathophysiology of myocardial injury during subsequent reperfusion, as well as ischemia, the accurate measurement of myocardial oxygen tension is crucial for the assessment of myocardial viability by ischemia-reperfusion (IR) injury. Therefore, we utilized a sol-gel derived electrochemical oxygen microsensor to monitor changes in oxygen tension during myocardial ischemia-reperfusion. We also analyzed differences in oxygen tension recovery in post-ischemic myocardium depending on ischemic time to investigate the correlation between recovery parameters for oxygen tension and the severity of IR injury. An oxygen sensor was built using a xerogel-modified platinum microsensor and a coiled Ag/AgCl reference electrode. Rat hearts were randomly divided into 5 groups: control (0 min ischemia), I-10 (10 min ischemia), I-20 (20 min ischemia), I-30 (30 min ischemia), and I-40 (40 min ischemia) groups (n = 3 per group, respectively). After the induction of ischemia, reperfusion was performed for 60 min. As soon as the ischemia was initiated, oxygen tension rapidly declined to near zero levels. When reperfusion was initiated, the changes in oxygen tension depended on ischemic time. The normalized peak level of oxygen tension during the reperfusion episode was 188 ± 27 in group I-10, 120 ± 24 in group I-20, 12.5 ± 10.6 in group I-30, and 1.24 ± 1.09 in group I-40 (p < 0.001, n = 3, respectively). After 60 min of reperfusion, the normalized restoration level was 129 ± 30 in group I-10, 88 ± 4 in group I-20, 3.40 ± 4.82 in group I-30, and 0.99 ± 0.94 in group I-40 (p < 0.001, n = 3, respectively). The maximum and restoration values of oxygen tension in groups I-30 and I-40 after reperfusion were lower than pre-ischemic values. In particular, oxygen tension in the I-40 group was not recovered at all. These results were also demonstrated by TTC staining. We suggest that these recovery parameters could be utilized as an index of

  2. Standardization for oxygen isotope ratio measurement - still an unsolved problem.

    PubMed

    Kornexl; Werner; Gehre

    1999-07-01

    Numerous organic and inorganic laboratory standards were gathered from nine European and North American laboratories and were analyzed for their delta(18)O values with a new on-line high temperature pyrolysis system that was calibrated using Vienna standard mean ocean water (VSMOW) and standard light Antartic precipitation (SLAP) internationally distributed reference water samples. Especially for organic materials, discrepancies between reported and measured values were high, ranging up to 2 per thousand. The reasons for these discrepancies are discussed and the need for an exact and reliable calibration of existing reference materials, as well as for the establishment of additional organic and inorganic reference materials is stressed. Copyright 1999 John Wiley & Sons, Ltd.

  3. Measurements of electron attachment by oxygen molecule in proportional counter

    NASA Astrophysics Data System (ADS)

    Tosaki, M.; Kawano, T.; Isozumi, Y.

    2013-11-01

    We present pulse height measurements for 5-keV Auger electrons from a radioactive 55Fe source mounted at the inner cathode surface of cylindrical proportional counter, which is operated with CH4 admixed dry air or N2. A clear shift of the pulse height has been observed by varying the amount of the admixtures; the number of electrons, created in the primary ionization by Auger electrons, is decreased by the electron attachment of the admixtures during their drift from the place near the source to the anode wire. The large gas amplification (typically 104) in the secondary ionization of proportional counter makes it possible to investigate a small change in the number of primary electrons. The electron attenuation cross-section of O2 has been evaluated by analyzing the shifts of the pulse height caused by the electron attachment to dry air and N2.

  4. 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.

  5. Determination of oxygen stoichiometry of oxide fuel during high temperature vapour pressure measurement

    NASA Astrophysics Data System (ADS)

    Beneš, O.; Konings, R. J. M.; Colle, J.-Y.

    2015-07-01

    This study presents an original approach of oxygen stoichiometry determination during high temperature (>2000 K) measurements of vapour pressure using the Knudsen effusion mass spectrometry technique. The method has been developed taking into account the vapour pressure measurements of series of (U1-x,Pux)O2-δ samples with x(Pu) = 0.25, 0.5, 0.75 together with pure UO2-δ and PuO2-δ end-members coupled with equilibrium calculations based on thermodynamic assessment of the U-Pu-O system. The presented method consists of two steps; in the first step the oxygen potential of the oxide phase is determined based on the measured partial vapour pressures of UO(g), UO2(g), PuO(g) and PuO2(g) gaseous species and during the second step the thus determined oxygen potential is linked with the matching oxygen stoichiometry of the sample. From the obtained results it has been demonstrated that it is possible to accurately estimate the oxygen stoichiometry of the mixed oxide fuel samples knowing the description of the oxygen potential of the corresponding end-members only.

  6. Robust high-performance nanoliter-volume single-cell multiple displacement amplification on planar substrates

    PubMed Central

    Leung, Kaston; Klaus, Anders; Lin, Bill K.; Laks, Emma; Biele, Justina; Lai, Daniel; Bashashati, Ali; Huang, Yi-Fei; Aniba, Radhouane; Moksa, Michelle; Steif, Adi; Mes-Masson, Anne-Marie; Hirst, Martin; Shah, Sohrab P.; Aparicio, Samuel; Hansen, Carl L.

    2016-01-01

    The genomes of large numbers of single cells must be sequenced to further understanding of the biological significance of genomic heterogeneity in complex systems. Whole genome amplification (WGA) of single cells is generally the first step in such studies, but is prone to nonuniformity that can compromise genomic measurement accuracy. Despite recent advances, robust performance in high-throughput single-cell WGA remains elusive. Here, we introduce droplet multiple displacement amplification (MDA), a method that uses commercially available liquid dispensing to perform high-throughput single-cell MDA in nanoliter volumes. The performance of droplet MDA is characterized using a large dataset of 129 normal diploid cells, and is shown to exceed previously reported single-cell WGA methods in amplification uniformity, genome coverage, and/or robustness. We achieve up to 80% coverage of a single-cell genome at 5× sequencing depth, and demonstrate excellent single-nucleotide variant (SNV) detection using targeted sequencing of droplet MDA product to achieve a median allelic dropout of 15%, and using whole genome sequencing to achieve false and true positive rates of 9.66 × 10−6 and 68.8%, respectively, in a G1-phase cell. We further show that droplet MDA allows for the detection of copy number variants (CNVs) as small as 30 kb in single cells of an ovarian cancer cell line and as small as 9 Mb in two high-grade serous ovarian cancer samples using only 0.02× depth. Droplet MDA provides an accessible and scalable method for performing robust and accurate CNV and SNV measurements on large numbers of single cells. PMID:27412862

  7. 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.

  8. Hand-Held and Integrated Single-Cell Pipettes

    PubMed Central

    2015-01-01

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

  9. 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.

  10. 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.

  11. Nanowire-based single-cell endoscopy

    NASA Astrophysics Data System (ADS)

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

    2012-03-01

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

  12. High time resolution measurements of the thermosphere from Fabry-Perot Interferometer measurements of atomic oxygen

    NASA Astrophysics Data System (ADS)

    Ford, E. A. K.; Aruliah, A. L.; Griffin, E. M.; McWhirter, I.

    2007-06-01

    Recent advances in the performance of CCD detectors have enabled a high time resolution study of the high latitude upper thermosphere with Fabry-Perot Interferometers (FPIs) to be performed. 10-s integration times were used during a campaign in April 2004 on an FPI located in northern Sweden in the auroral oval. The FPI is used to study the thermosphere by measuring the oxygen red line emission at 630.0 nm, which emits at an altitude of approximately 240 km. Previous time resolutions have been 4 min at best, due to the cycle of look directions normally observed. By using 10 s rather than 40 s integration times, and by limiting the number of full cycles in a night, high resolution measurements down to 15 s were achievable. This has allowed the maximum variability of the thermospheric winds and temperatures, and 630.0 nm emission intensities, at approximately 240 km, to be determined as a few minutes. This is a significantly greater variability than the often assumed value of 1 h or more. A Lomb-Scargle analysis of this data has shown evidence of gravity wave activity with waves with short periods. Gravity waves are an important feature of mesosphere-lower thermosphere (MLT) dynamics, observed using many techniques and providing an important mechanism for energy transfer between atmospheric regions. At high latitudes gravity waves may be generated in-situ by localised auroral activity. Short period waves were detected in all four clear nights when this experiment was performed, in 630.0 nm intensities and thermospheric winds and temperatures. Waves with many periodicities were observed, from periods of several hours, down to 14 min. These waves were seen in all parameters over several nights, implying that this variability is a typical property of the thermosphere.

  13. 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.

  14. 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.

  15. 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.

  16. 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

  17. Polarographic Electrode Measures of Cerebral Tissue Oxygenation: Implications for Functional Brain Imaging

    PubMed Central

    Bartlett, Kate; Saka, Mohamad; Jones, Myles

    2008-01-01

    The changes in blood flow, blood volume and oxygenation that accompany focal increases in neural activity are collectively referred to as the hemodynamic response and form the basis of non-invasive neuroimaging techniques such as blood oxygen level dependent (BOLD) functional magnetic resonance imaging. A principle factor influencing blood oxygenation, the cerebral metabolic rate of oxygen consumption is poorly understood and as such, data from imaging techniques are difficult to interpret in terms of the underlying neural activity. In particular how neurometabolic changes vary temporally, spatially and in magnitude remains uncertain. Furthermore knowledge of which aspects of neural activity are closely reflected by metabolic changes is essential for the correct interpretation of cognitive neuroscience studies in terms of information processing. Polarographic electrode measurements of cerebral tissue oxygenation in animal models following presentation of sensory stimuli have started to address these issues. Early studies demonstrated both increases and decreases in tissue oxygenation following neural activation. However a recent series of elegant studies in the cat visual system demonstrated a tight spatial and temporal coupling between evoked peri-synaptic activity and oxygen consumption following presentation of visual stimuli. PMID:27873951

  18. Observations on intrauterine oxygen tension measured by fibre-optic microsensors.

    PubMed

    Ottosen, Lars D M; Hindkaer, Johnny; Husth, Merete; Petersen, Dorrit Elschner; Kirk, John; Ingerslev, Hans Jakob

    2006-09-01

    Understanding the biology of reproductive organs is essential for the development of assisted reproductive techniques. There is at present no direct evidence for either the concentration and dynamics of intrauterine oxygen tension at the endometrial surface, nor its importance for the receptiveness of the endometrium. In this study a new method measured mid-cycle (ranging from day 12-18) endometrial surface oxygen tension in 21 patients referred to intrauterine insemination (IUI). Time series was measured online for a period of 5-10 min. The (mean) individual oxygen tension among patients varied from 4 to 27% air saturation. Overall mean oxygen tension among all patients was 11.8% air saturation. Within the same patient, considerable time-related variations were observed. Some patients exhibited rhythmic oscillations with a frequency in the order of 1 min, whereas others did not show any regular patterns. A good description of endometrial surface oxygen concentration and dynamics was thus obtained, but given the relatively small number of participants, an association with pregnancy following insemination could not be established. Further studies using this new method could elucidate the association between individual intrauterine activity, embryo implantation and endometrial surface oxygen tension.

  19. Near infrared noninvasive quantitative measurement of oxygen content in hepatic tissues

    NASA Astrophysics Data System (ADS)

    Wang, Zhicheng; Tian, Yan; Tian, Jinwen; Liu, Jian; Xie, Zeping

    2006-09-01

    A new noninvasive measurement of oxygen contents in hepatic tissues using near-infrared technique according to physiological characteristics is proposed. The procedure can be divided into three categories. First a quantitative formula is introduced to measure oxygen contents in hepatic tissues based on the relationship between absorption coefficient and typical wavelengths, where 760nm and 850nm infrared wavebands are utilized in this paper. Second, many characteristics such as waveforms of oxygen contents in hepatic tissues, cross correlation of blood-oxygen and power spectrum of oxygen contents, are analyzed detailedly with regard to different symptoms in hepatic tissues. Finally, a conclusion can be drawn that waveforms of oxygen contents, cross correlation and power spectrum are three main features, which can well depict the symptoms of hepatic tissues. The proposed method is applied to examine 143 people, including 40 normal people and 103 patients with different symptoms in hepatic tissues. The false probability is 8.3% and the missing probability is 13.7% under specified criterion. The clinical experiments show that our proposed method is simple but effective and can be used to routine examinations or intensive care units for liverish patients.

  20. In-situ measurement of photosynthetic oxygen production in the water column.

    PubMed

    Häder, D P; Schäfer, J

    1994-09-01

    A novel hardware device is described to determine photosynthetic and respiratory oxygen uptake and release, respectively, in organisms in their natural habitat even under the rough conditions of their marine environment. Both macroalgae and phytoplankton can be utilized and oxygen exchange can be determined in solar radiation. The chamber can be used at or above the water surface or can be lowered into the water column. The data of oxygen concentration, irradiance and temperature are constantly monitored by a laptop computer and stored in disk files. The experimental data measured in some macroalgae as well as in phytoplankton indicate that the irradiance window for positive net photosynthetic oxygen production is fairly limited under natural conditions; at too low irradiances respiration exceeds photosynthesis and at too high irradiances photosynthesis is shut down by photoinhibition, at least in species not adapted to unattenuated solar radiation.

  1. 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.

  2. Flame temperature measurements by radar resonance-enhanced multiphoton ionization of molecular oxygen.

    PubMed

    Wu, Yue; Sawyer, Jordan; Zhang, Zhili; Adams, Steven F

    2012-10-01

    Here we report nonintrusive local rotational temperature measurements of molecular oxygen, based on coherent microwave scattering (radar) from resonance-enhanced multiphoton ionization (REMPI) in room air and hydrogen/air flames. Analyses of the rotational line strengths of the two-photon molecular oxygen C(3)Π(v=2)←X(3)Σ(v'=0) transition have been used to determine the hyperfine rotational state distribution of the ground X(3)Σ(v'=0) state. Rotationally resolved 2+1 REMPI spectra of the molecular oxygen C(3)Π(v=2)←X(3)Σ(v'=0) transition at different temperatures were obtained experimentally by radar REMPI. Rotational temperatures have been determined from the resulting Boltzmann plots. The measurements in general had an accuracy of ~±60 K in the hydrogen/air flames at various equivalence ratios. Discussions about the decreased accuracy for the temperature measurement at elevated temperatures have been presented.

  3. Measuring Flow Rate in Crystalline Bedrock Wells Using the Dissolved Oxygen Alteration Method.

    PubMed

    Vitale, Sarah A; Robbins, Gary A

    2017-03-22

    Determination of vertical flow rates in a fractured bedrock well can aid in planning and implementing hydraulic tests, water quality sampling, and improving interpretations of water quality data. Although flowmeters are highly accurate in flow rate measurement, the high cost and logistics may be limiting. In this study the dissolved oxygen alteration method (DOAM) is expanded upon as a low-cost alternative to determine vertical flow rates in crystalline bedrock wells. The method entails altering the dissolved oxygen content in the wellbore through bubbler aeration, and monitoring the vertical advective movement of the dissolved oxygen over time. Measurements were taken for upward and downward flows, and under ambient and pumping conditions. Vertical flow rates from 0.06 to 2.30 Lpm were measured. To validate the method, flow rates determined with the DOAM were compared to pump discharge rates and found to be in agreement within 2.5%.

  4. 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

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

    PubMed

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

    2015-08-12

    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.

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

    PubMed Central

    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

  7. 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

  8. 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).

  9. Microfluidic single-cell analysis for systems immunology.

    PubMed

    Junkin, Michael; Tay, Savaş

    2014-04-07

    The immune system constantly battles infection and tissue damage, but exaggerated immune responses lead to allergies, autoimmunity and cancer. Discrimination of self from foreign and the fine-tuning of immunity are achieved by information processing pathways, whose regulatory mechanisms are little understood. Cell-to-cell variability and stochastic molecular interactions result in diverse cellular responses to identical signaling inputs, casting doubt on the reliability of traditional population-averaged analyses. Furthermore, dynamic molecular and cellular interactions create emergent properties that change over multiple time scales. Understanding immunity in the face of complexity and noisy dynamics requires time-dependent analysis of single-cells in a proper context. Microfluidic systems create precisely defined microenvironments by controlling fluidic and surface chemistries, feature sizes, geometries and signal input timing, and thus enable quantitative multi-parameter analysis of single cells. Such qualities allow observable dynamic environments approaching in vivo levels of biological complexity. Seamless parallelization of functional units in microfluidic devices allows high-throughput measurements, an essential feature for statistically meaningful analysis of naturally variable biological systems. These abilities recapitulate diverse scenarios such as cell-cell signaling, migration, differentiation, antibody and cytokine production, clonal selection, and cell lysis, thereby enabling accurate and meaningful study of immune behaviors in vitro.

  10. 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.

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

    PubMed

    Wu, Hongjin; Wang, Charles; Wu, Shixiu

    2016-11-16

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

  12. 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

  13. Noninvasive measurement of cerebral hemoglobin oxygen saturation using two near infrared spectroscopy approaches.

    PubMed

    Quaresima, V; Sacco, S; Totaro, R; Ferrari, M

    2000-04-01

    Spatially resolved spectroscopy (SRS) is a new near infrared spectroscopy (NIRS) method that, using the multi-distance approach, measures local cerebral cortex hemoglobin oxygen saturation [J. Matcher, P. Kirkpatrick, K. Nahid, M. Cope, and D. T. Delpy, Proc. SPIE 2389, 486-495 (1995)]. Using a conventional continuous wave NIRS photometer, cerebral venous oxygen saturation (SvO2) can be calculated from oxyhemoglobin and total hemoglobin rise induced by partial occlusion of jugular vein [C. E. Elwell, S. J. Matcher, L. Tyszczuk, J. H. Meek, and D. T. Delpy, Adv. Exp. Med. Biol. 411, 453-460 (1997)]. The aim of this study was to compare direct measurements of forehead tissue oxygenation index (TOI) with the calculated SvO2 during venous occlusion in 16 adult volunteers using a clinical two-channel SRS oximeter (NIRO-300). Measured TOI and calculated SvO2 values of either right or left forehead did not significantly differ. A good agreement between the two NIRS methods was also demonstrated. On 16 other subjects, no significant differences were found between the right and left forehead TOI values measured simultaneously, and between the TOI values measured by channel 1 or 2 on the same side. The results confirm that cerebral cortex hemoglobin oxygen saturation, measured directly by the SRS method, reflects predominantly the saturation of the intracranial venous compartment of circulation.

  14. Muscle oxygenation measurement in humans by noninvasive optical spectroscopy and Locally Weighted Regression.

    PubMed

    Arakaki, Lorilee S L; Schenkman, Kenneth A; Ciesielski, Wayne A; Shaver, Jeremy M

    2013-06-27

    We have developed a method to make real-time, continuous, noninvasive measurements of muscle oxygenation (Mox) from the surface of the skin. A key development was measurement in both the visible and near infrared (NIR) regions. Measurement of both oxygenated and deoxygenated myoglobin and hemoglobin resulted in a more accurate measurement of Mox than could be achieved with measurement of only the deoxygenated components, as in traditional near-infrared spectroscopy (NIRS). Using the second derivative with respect to wavelength reduced the effects of scattering on the spectra and also made oxygenated and deoxygenated forms more distinguishable from each other. Selecting spectral bands where oxygenated and deoxygenated forms absorb filtered out noise and spectral features unrelated to Mox. NIR and visible bands were scaled relative to each other in order to correct for errors introduced by normalization. Multivariate Curve Resolution (MCR) was used to estimate Mox from spectra within each data set collected from healthy subjects. A Locally Weighted Regression (LWR) model was built from calibration set spectra and associated Mox values from 20 subjects using 2562 spectra. LWR and Partial Least Squares (PLS) allow accurate measurement of Mox despite variations in skin pigment or fat layer thickness in different subjects. The method estimated Mox in five healthy subjects with an RMSE of 5.4%.

  15. Gravisensing in single-celled systems

    NASA Astrophysics Data System (ADS)

    Braun, M.; Limbach, C.

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

  16. Emergent collective chemotaxis without single-cell gradient sensing

    PubMed Central

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

    2016-01-01

    Many eukaryotic cells chemotax, sensing and following chemical gradients. However, experiments have shown that even under conditions when single cells cannot chemotax, small clusters may still follow a gradient. This behavior has been 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 (CIL), 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 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. PMID:26991203

  17. 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.

  18. Use of an oxygen-insensitive microscale biosensor for methane to measure methane concentration profiles in a rice paddy.

    PubMed

    Damgaard, L R; Revsbech, N P; Reichardt, W

    1998-03-01

    An oxygen-insensitive microscale biosensor for methane was constructed by furnishing a previously described biosensor with an oxygen guard. The guard consisted of a glass capillary containing heterotrophic bacteria, which consumed oxygen diffusing through the tip membrane, thus preventing it from diffusing into the methane-sensing unit. Oxygen microprofiles were measured through the oxygen guard capillary, demonstrating the principle and limitations of the method. When the tip of the guard capillary was exposed to 100% oxygen at 21 degrees C, heterotrophic oxygen consumption prevented oxygen from diffusing further than 170 mum into the capillary, whereas atmospheric levels of oxygen were consumed within 50 mum. The capacity of the oxygen guard for scavenging oxygen decreased with decreasing temperature, and atmospheric levels of oxygen caused oxygen penetration to 200 mum at 5 degrees C. The sensors could be manufactured with tip diameters as small as 25 mum, and response times were about 1 min at room temperature. Pore water profiles of methane concentrations in a rice paddy soil were measured, and a strong correlation between the depths of oxygen penetration and methane appearance was observed as a function of the light regimen; this finding confirmed the role of microbenthic photosynthesis in limiting methane emissions from surfaces of waterlogged sediments and soils.

  19. Long-term growth data of Escherichia coli at a single-cell level

    PubMed Central

    Tanouchi, Yu; Pai, Anand; Park, Heungwon; Huang, Shuqiang; Buchler, Nicolas E.; You, Lingchong

    2017-01-01

    Long-term, single-cell measurement of bacterial growth is extremely valuable information, particularly in the study of homeostatic aspects such as cell-size and growth rate control. Such measurement has recently become possible due to the development of microfluidic technology. Here we present data from single-cell measurements of Escherichia coli growth over 70 generations obtained for three different growth conditions. The data were recorded every minute, and contain time course data of cell length and fluorescent intensity of constitutively expressed yellow fluorescent protein. PMID:28350394

  20. 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.

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

    PubMed

    Efroni, Idan; Birnbaum, Kenneth D

    2016-04-05

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

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

    PubMed

    Ståhlberg, Anders; Rusnakova, Vendula; Kubista, Mikael

    2013-03-01

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

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

    PubMed

    Liang, Jialong; Cai, Wanshi; Sun, Zhongsheng

    2014-10-20

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

  4. 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.

  5. 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

  6. Measurement of the oxygen isotopic composition of nitrate in seawater and freshwater using the denitrifier method

    USGS Publications Warehouse

    Casciotti, K.L.; Sigman, D.M.; Hastings, M. Galanter; Böhlke, J.K.; Hilkert, A.

    2002-01-01

    We report a novel method for measurement of the oxygen isotopic composition (18O/16O) of nitrate (NO3-) from both seawater and freshwater. The denitrifier method, based on the isotope ratio analysis of nitrous oxide generated from sample nitrate by cultured denitrifying bacteria, has been described elsewhere for its use in nitrogen isotope ratio (15N/14N) analysis of nitrate.1Here, we address the additional issues associated with 18O/16O analysis of nitrate by this approach, which include (1) the oxygen isotopic difference between the nitrate sample and the N2O analyte due to isotopic fractionation associated with the loss of oxygen atoms from nitrate and (2) the exchange of oxygen atoms with water during the conversion of nitrate to N2O. Experiments with 18O-labeled water indicate that water exchange contributes less than 10%, and frequently less than 3%, of the oxygen atoms in the N2O product for Pseudomonas aureofaciens. In addition, both oxygen isotope fractionation and oxygen atom exchange are consistent within a given batch of analyses. The analysis of appropriate isotopic reference materials can thus be used to correct the measured 18O/16O ratios of samples for both effects. This is the first method tested for 18O/16O analysis of nitrate in seawater. Benefits of this method, relative to published freshwater methods, include higher sensitivity (tested down to 10 nmol and 1 μM NO3-), lack of interference by other solutes, and ease of sample preparation.

  7. Optical measurements of the dependence of chemoreception on oxygen pressure in the cat carotid body.

    PubMed

    Rumsey, W L; Iturriaga, R; Spergel, D; Lahiri, S; Wilson, D F

    1991-10-01

    The relationship between oxygen pressure (PO2) in the carotid body and carotid sinus nerve discharge was evaluated in the isolated perfused/superfused cat carotid body using the oxygen-dependent quenching of phosphorescence. Images of phosphorescence intensity arising from Pd-coproporphyrin within the microcirculation of the carotid body provided measurements of intravascular PO2. These measurements were substantiated by determining phosphorescence life-time. The carotid body was perfused in the isolated state via the common carotid artery with N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid-buffered Tyrode solution, pH 7.4, at a constant pressure of 80 mmHg. Superfusion was maintained with similar media equilibrated with 100% argon. PO2 in the exchange vessels was markedly less than that in the perfusate entering the carotid artery, 23 +/- 3 and 45 +/- 3 Torr for normoxic (111 +/- 15 Torr) and hyperoxic (345 +/- 72 Torr) perfusates, respectively. Chemosensory discharge rose slowly in response to a brief interruption of perfusate flow as PO2 steadily declined from either of these capillary PO2 values to approximately 10 Torr. Between approximately 10 and 3 Torr, chemosensory discharge increased strikingly, concomitant with an enhanced rate of oxygen disappearance, from -36 +/- 4 to -69 +/- 13 (92% change) and -28 +/- 3 to -48 +/- 3 (71% change) Torr/s for normoxic and hyperoxic perfusates, respectively. As PO2 fell below approximately 3 Torr, oxygen disappearance slowed and neural activity decayed. Thus the relationships between microvascular PO2 and chemosensory discharge and between oxygen disappearance and neural discharge suggest that oxygen metabolism in the carotid body determines the expression of oxygen chemoreception.

  8. 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.

  9. 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.

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

    DOE PAGES

    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

  11. 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.

  12. 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.

  13. Inferring fitness landscapes and selection on phenotypic states from single-cell genealogical data

    PubMed Central

    Kussell, Edo

    2017-01-01

    Recent advances in single-cell time-lapse microscopy have revealed non-genetic heterogeneity and temporal fluctuations of cellular phenotypes. While different phenotypic traits such as abundance of growth-related proteins in single cells may have differential effects on the reproductive success of cells, rigorous experimental quantification of this process has remained elusive due to the complexity of single cell physiology within the context of a proliferating population. We introduce and apply a practical empirical method to quantify the fitness landscapes of arbitrary phenotypic traits, using genealogical data in the form of population lineage trees which can include phenotypic data of various kinds. Our inference methodology for fitness landscapes determines how reproductivity is correlated to cellular phenotypes, and provides a natural generalization of bulk growth rate measures for single-cell histories. Using this technique, we quantify the strength of selection acting on different cellular phenotypic traits within populations, which allows us to determine whether a change in population growth is caused by individual cells’ response, selection within a population, or by a mixture of these two processes. By applying these methods to single-cell time-lapse data of growing bacterial populations that express a resistance-conferring protein under antibiotic stress, we show how the distributions, fitness landscapes, and selection strength of single-cell phenotypes are affected by the drug. Our work provides a unified and practical framework for quantitative measurements of fitness landscapes and selection strength for any statistical quantities definable on lineages, and thus elucidates the adaptive significance of phenotypic states in time series data. The method is applicable in diverse fields, from single cell biology to stem cell differentiation and viral evolution. PMID:28267748

  14. 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.

  15. 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.

  16. 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.

  17. 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...

  18. Measurements of oxygen tension in native and transplanted rat pancreatic islets.

    PubMed

    Carlsson, P O; Liss, P; Andersson, A; Jansson, L

    1998-07-01

    This study was performed to measure the oxygen tension before and after revascularization of pancreatic islets transplanted beneath the renal capsule and to investigate to what extent this was affected by acute and chronic hyperglycemia. In addition, the oxygen tension in islets within the pancreas was determined. PO2 was measured with a modified Clark electrode (tip 2-6 microm o.d.). Within native pancreatic islets, the mean PO2 was higher (31-37 mmHg) than within the exocrine pancreas (20-23 mmHg). The mean oxygen tension in the transplanted islets the day after implantation was half of that recorded in native islets (14-19 mmHg) and did not differ between normoglycemic and diabetic recipients. At 1 month after transplantation, when revascularization had occurred, the mean PO2 in the islet grafts was 9-15 mmHgf in normoglycemic animals but was lower (6-8 mmHg) in diabetic animals, whereas the blood perfusion of the transplants, as measured with laser-Doppler flowmetry (probe diameter 0.45 mm), was similar in both groups. The mean oxygen tension in the superficial renal cortex surrounding the implanted islets was similar in all groups and remained stable at 13-21 mmHg. Intravenous administration of D-glucose (1 g/kg) did not affect the oxygen tension in any of the investigated tissues. We conclude that the mean PO2 in islets implanted under the renal capsule is markedly lower than in native islets, not only in the immediate posttransplantation period but also 1 month after implantation, i.e., when revascularization has occurred. Furthermore, persistent hyperglycemia in the recipient leads to a further decrease in graft oxygen tension. To what extent this may contribute to islet graft failure is at present unknown.

  19. 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

  20. 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

    2013-09-07

    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.

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

    NASA Astrophysics Data System (ADS)

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

    2014-01-01

    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.

  2. 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.

  3. 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.

  4. 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.

  5. Dynamic staining of bacteria at a single-cell level

    NASA Astrophysics Data System (ADS)

    Nuñez, Vicente; Upadhyayula, Srigokul; Lin, Adam; Chau, Kenny; Vullev, Valentine I.

    2011-05-01

    Bacterial infectious diseases remain one of the major health hazards nation- and worldwide. The expedience of detection and identification of bacterial pathogens determines how early the diagnosis is, and hence, what the treatment and the outcome of the illness would be. As we have previously reported, the dynamics of fluorescence staining provides venues for the development of expedient assays for detection and identification of bacterial species[1]. We measured the kinetics of bacterial staining with cyanine and thioflavin dyes and investigated their photophysical properties. We demonstrated that the pseudo first-order kinetic constants of the fluorescence staining processes have species specificity without contrition dependence. Combining the dynamics of staining with real-time fluorescence microscopy we characterized the fluorescence staining process at the single-cell level with improved sensitivity and contrast.

  6. Whole-genome molecular haplotyping of single cells.

    PubMed

    Fan, H Christina; Wang, Jianbin; Potanina, Anastasia; Quake, Stephen R

    2011-01-01

    Conventional experimental methods of studying the human genome are limited by the inability to independently study the combination of alleles, or haplotype, on each of the homologous copies of the chromosomes. We developed a microfluidic device capable of separating and amplifying homologous copies of each chromosome from a single human metaphase cell. Single-nucleotide polymorphism (SNP) array analysis of amplified DNA enabled us to achieve completely deterministic, whole-genome, personal haplotypes of four individuals, including a HapMap trio with European ancestry (CEU) and an unrelated European individual. The phases of alleles were determined at ∼99.8% accuracy for up to ∼96% of all assayed SNPs. We demonstrate several practical applications, including direct observation of recombination events in a family trio, deterministic phasing of deletions in individuals and direct measurement of the human leukocyte antigen haplotypes of an individual. Our approach has potential applications in personal genomics, single-cell genomics and statistical genetics.

  7. Cerebral and muscle oxygen saturation measurement by a frequency-domain near-infrared spectroscopic technique

    NASA Astrophysics Data System (ADS)

    Ferrari, Marco; De Blasi, Roberto A.; Fantini, Sergio; Franceschini, Maria-Angela; Barbieri, Beniamino B.; Quaresima, Valentina; Gratton, Enrico

    1995-05-01

    Absorption and reduced scattering coefficients at 715 and 825 nm as well as hemoglobin saturation and content of the forehead and the forearm were measured by a 110 MHz frequency-domain multisource instrument. The absolute data obtained by the frequency- domain spectrometer were compared with oxygenation changes measured by a continuous wave instrument during quadriceps ischemia and postural changes. These preliminary results indicate that portable frequency-domain instruments could be very helpful to investigate brain and muscle pathophysiology.

  8. 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.

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

    PubMed

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

    2007-03-07

    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.

  10. Terahertz Limb Sounder for Lower Thermosphere Wind, Temperature, and Atomic Oxygen Density Measurements

    NASA Astrophysics Data System (ADS)

    Yee, J. H.; Boldt, J.; Wu, D. L.; Mehdi, I.; Schlecht, E.

    2015-12-01

    In this paper, we present the concept of a high-sensitivity heterodyne spectrometer operating at Terahertz (THz) frequency for global lower thermospheric neutral wind, temperature and atomic oxygen density measurements from a low earth orbit. The instrument, THz Limb Sounder (TLS) is aimed to provide, for the first time, global neutral wind/temperature/density profile measurements globally during day and night, with focus at altitudes of 100-150 km where most of the ion-neutral energy/momentum couplings take place. It is an ambient-temperature Schottky diode based all solid-state heterodyne spectrometer designed to extend the limb sounding technique employed by Microwave Limb Sounder for density/temperature/wind measurements by measuring the Doppler line shape of atomic oxygen (OI) fine structure emission at 2.06THz. This atomic oxygen line emission is very bright and distributed nearly uniformly globally (at all latitudes including high latitude aurora particle precipitation regions) and temporally (at all local times during both day and night), thus ideal for thermospheric remote sensing. TLS is an ambient-temperature Schottky diode based heterodyne receiver system The TLS instrument concept, measurement methodology, receiver performance, and the expected measurement capability will be presented and discussed in this paper.

  11. 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.

  12. 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

  13. 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.

  14. 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.

  15. Surface Measurements of Oxygen, Carbon Dioxide and Water Vapor Fluxes Using AN Automated Chamber System

    NASA Astrophysics Data System (ADS)

    Fentabil, M. M.; Fazackerley, S.; Nichol, C. F.

    2011-12-01

    The various soil respiration measurements that are available depend on measuring the emitted CO2 flux as organic carbon is respired aerobically. Comparative studies among the four well known methods (the open-flow infra-red gas analyzer method; the closed chamber method; the dynamic closed chamber method; and the alkali absorption method) under field conditions and laboratory experiments show differences. The discrepancies observed in these methods under field conditions could be evaluated by incorporating O2 flux measurement in addition to CO2 flux measurement. However, this is hampered by the absence of suitable equipment for measuring oxygen influx at the soil surface. A system to measure O2 fluxes at the soil surface using chamber methods has been developed. A gas handling subsystem and O2 analyser has been incorporated into an existing non-steady state automated chamber system originally designed for CO2 and H2O flux monitoring. The system consists of four 60 L soil chambers connected to temperature-controlled datalogger housing. During a measurement cycle, the chamber lid closes and the system measures changes of CO2/H2O and depletion of O2in the chamber headspace. Samples for CO2 /H2O circulate in a closed path between the chamber and an IR gas analyser. An air sample for O2 analysis is sub-sampled from this circulating air stream. Air samples for O2 are first dried using a two-stage Nafion drier. Mass flows and pressures are balanced at the pascal level prior to the gas passing into a ppm level fuel-cell oxygen analyser (Sable Systems Oxzilla). The chamber sample airstream is measured against a reference gas identically handled in a parallel air stream. A makeup gas of dry N2 is injected back to the chamber to return equimolar amounts of gas to replace the wet air removed, and hence prevent pressure fluctuation in the headspace of the chamber. The implementation of automated control of gas drying and sampling, pressure balancing, flow regulation and self

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

    PubMed Central

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

    2011-01-01

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

  17. 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.

  18. Single Cell Characterization of Prostate Cancer Circulating Tumor Cells

    DTIC Science & Technology

    2011-08-01

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

  19. 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

  20. Single cell sequencing approaches for complex biological systems.

    PubMed

    Baslan, Timour; Hicks, James

    2014-06-01

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

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

    PubMed

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

    2017-02-17

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

  2. 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.

  3. 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.

  4. 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.

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

    PubMed Central

    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

  6. 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.

  7. 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.

  8. Benchmark oxygen-oxygen pair-distribution function of ambient water from x-ray diffraction measurements with a wide Q-range.

    PubMed

    Skinner, Lawrie B; Huang, Congcong; Schlesinger, Daniel; Pettersson, Lars G M; Nilsson, Anders; Benmore, Chris J

    2013-02-21

    Four recent x-ray diffraction measurements of ambient liquid water are reviewed here. Each of these measurements represents a significant development of the x-ray diffraction technique applied to the study of liquid water. Sources of uncertainty from statistical noise, Q-range, Compton scattering, and self-scattering are discussed. The oxygen-hydrogen contribution to the measured x-ray scattering pattern was subtracted using literature data to yield an experimental determination, with error bars, of the oxygen-oxygen pair-distribution function, g(OO)(r), which essentially describes the distribution of molecular centers. The extended Q-range and low statistical noise of these measurements has significantly reduced truncation effects and related errors in the g(OO)(r) functions obtained. From these measurements and error analysis, the position and height of the nearest neighbor maximum in g(OO)(r) were found to be 2.80(1) Å and 2.57(5) respectively. Numerical data for the coherent differential x-ray scattering cross-section I(X)(Q), the oxygen-oxygen structure factor S(OO)(Q), and the derived g(OO)(r) are provided as benchmarks for calibrating force-fields for water.

  9. 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.

  10. Systematic single-cell analysis of Pichia pastoris reveals secretory capacity limits productivity.

    PubMed

    Love, Kerry Routenberg; Politano, Timothy J; Panagiotou, Vasiliki; Jiang, Bo; Stadheim, Terrance A; Love, J Christopher

    2012-01-01

    Biopharmaceuticals represent the fastest growing sector of the global pharmaceutical industry. Cost-efficient production of these biologic drugs requires a robust host organism for generating high titers of protein during fermentation. Understanding key cellular processes that limit protein production and secretion is, therefore, essential for rational strain engineering. Here, with single-cell resolution, we systematically analysed the productivity of a series of Pichia pastoris strains that produce different proteins both constitutively and inducibly. We characterized each strain by qPCR, RT-qPCR, microengraving, and imaging cytometry. We then developed a simple mathematical model describing the flux of folded protein through the ER. This combination of single-cell measurements and computational modelling shows that protein trafficking through the secretory machinery is often the rate-limiting step in single-cell production, and strategies to enhance the overall capacity of protein secretion within hosts for the production of heterologous proteins may improve productivity.

  11. 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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

    Doron, Guy; Brecht, Michael

    2015-09-19

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

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

    PubMed Central

    Doron, Guy; Brecht, Michael

    2015-01-01

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

  15. The Application of Micropipette Aspiration in Molecular Mechanics of Single Cells.

    PubMed

    Lee, Lap Man; Liu, Allen P

    2014-11-01

    Micropipette aspiration is arguably the most classical technique in mechanical measurements and manipulations of single cells. Despite its simplicity, micropipette aspiration has been applied to a variety of experimental systems that span different length scales to study cell mechanics, nanoscale molecular mechanisms in single cells, bleb growth, and nucleus dynamics, to name a few. Enabled by micro/nanotechnology, several novel microfluidic devices have been developed recently with better accuracy, sensitivity, and throughput. Further technical advancements of microfluidics-based micropipette aspiration would have broad applications in both fundamental cell mechanics studies and for disease diagnostics.

  16. Singlet-oxygen generation at gas-liquid interfaces: A significant artifact in the measurement of singlet-oxygen yields from ozone-biomolecule reactions

    SciTech Connect

    Kanofsky, J.R.; Sima, P.D. )

    1993-09-01

    Several ozone-biomolecule reactions have previously been shown to generate singlet oxygen in high yields. For some of these ozone-biomolecule reactions, we now show that the apparent singlet-oxygen yields determined from measurements of 1270 nm chemiluminescence were artifactually elevated by production of gas-phase singlet oxygen. The gas-phase singlet oxygen results from the reaction of gas-phase ozone with biomolecules near the surface of the solution. Through the use of a flow system that excludes air from the reaction chamber, accurate singlet-oxygen yields can be obtained. The revised singlet-oxygen yields (mol 1O2 per mol O3) for the reactions of ozone with cysteine, reduced glutathione, NADH, NADPH, human albumin, methionine, uric acid and oxidized glutathione are 0.23 +/- 0.02, 0.26 +/- 0.2, 0.48 +/- 0.04, 0.41 +/- 0.01, 0.53 +/- 0.06, 1.11 +/- 0.04, 0.73 +/- 0.05 and 0.75 +/- 0.01, respectively. These revised singlet-oxygen yields are still substantial.

  17. 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

  18. Consistent calculation of aquatic gross production from oxygen triple isotope measurements

    NASA Astrophysics Data System (ADS)

    Kaiser, J.

    2011-04-01

    Oxygen triple isotope measurements can be used to calculate aquatic gross oxygen production rates. Past studies have emphasised the appropriate definition of the 17O excess and often used an approximation to derive production rates from the 17O excess. Here, I show that the calculation can be phrased more consistently and without any approximations using the relative 17O/16O and 18O/16O isotope ratio differences directly. The 17O excess is merely a mathematical construct and the derived production rate is independent of its definition, provided all calculations are performed with a consistent definition. I focus on the mixed layer, but also show how time series of triple oxygen measurements below the mixed layer can be used to derive gross production. In the calculation of mixed layer productivity, I explicitly include isotopic fractionation during gas invasion and evasion, which requires the oxygen supersaturation s to be measured as well. I also suggest how bubble injection could be considered in the same mathematical framework. I distinguish between concentration steady state and isotopic steady state and show that only the latter needs to be assumed in the calculation. It is even possible to derive an estimate of the net production rate in the mixed layer that is independent of the assumption of concentration steady state. I review measurements of the parameters required for the calculation of gross production rates and show how their systematic uncertainties as well as the use of different published calculation methods can cause large variations in the production rates for the same underlying isotope ratios. In particular, the 17O excess of dissolved O2 in equilibrium with atmospheric O2 and the 17O excess of photosynthetic O2 need to be re-measured. Because of these uncertainties, all calculation parameters should always be fully documented and the measured isotope ratio differences as well as the oxygen supersaturation should be permanently archived, so that

  19. Synchronizing stochastic circadian oscillators in single cells of Neurospora crassa

    NASA Astrophysics Data System (ADS)

    Deng, Zhaojie; Arsenault, Sam; Caranica, Cristian; Griffith, James; Zhu, Taotao; Al-Omari, Ahmad; Schüttler, Heinz-Bernd; Arnold, Jonathan; Mao, Leidong

    2016-10-01

    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.

  20. 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.

  1. 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

  2. Long-term measurement of renal cortical and medullary tissue oxygenation and perfusion in unanesthetized sheep.

    PubMed

    Calzavacca, Paolo; Evans, Roger G; Bailey, Michael; Lankadeva, Yugeesh R; Bellomo, Rinaldo; May, Clive N

    2015-05-15

    The role of renal cortical and medullary hypoxia in the development of acute kidney injury is controversial, partly due to a lack of techniques for the long-term measurement of intrarenal oxygenation and perfusion in conscious animals. We have, therefore, developed a methodology to chronically implant combination probes to chronically measure renal cortical and medullary tissue perfusion and oxygen tension (tPO2) in conscious sheep and evaluated their responsiveness and reliability. A transit-time flow probe and a vascular occluder were surgically implanted on the left renal artery. At the same operation, dual fiber-optic probes, comprising a fluorescence optode to measure tPO2 and a laser-Doppler probe to assess tissue perfusion, were inserted into the renal cortex and medulla. In recovered conscious sheep (n = 8) breathing room air, mean 24-h cortical and medullary tPO2 were similar (31.4 ± 0.6 and 29.7 ± 0.7 mmHg, respectively). In the renal cortex and medulla, a 20% reduction in renal blood flow (RBF) decreased perfusion (14.6 ± 8.6 and 41.2 ± 8.5%, respectively) and oxygenation (48.1 ± 8.5 and 72.4 ± 8.5%, respectively), with greater decreases during a 50% reduction in RBF. At autopsy, minimal fibrosis was observed around the probes. In summary, we have developed a technique to chronically implant fiber-optic probes in the renal cortex and medulla for recording tissue perfusion and oxygenation over many days. In normal resting conscious sheep, cortical and medullary tPO2 were similar. The responses to and recovery from renal artery occlusion, together with the consistent measurements over a 24-h period, demonstrate the responsiveness and stability of the probes.

  3. 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.

  4. 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

  5. Noninvasive approaches to measuring respiratory patterns using a PtTFPP based phase-lifetime self-referencing oxygen optrode

    NASA Astrophysics Data System (ADS)

    Porterfield, D. Marshall; Rickus, Jenna L.; Kopelman, Raoul

    2006-10-01

    Optically transduced sensors (optrodes, or optodes) offer significant advantages over polarographic techniques for measuring oxygen. In biology and medicine, how we make measurements is very important, and this is especially true in terms of physiological exchange. Cellular and tissue oxygenation is a function of background concentration and respiratory demand, and in pure physical terms this is best expressed in terms of molecular flux based on Fick's law. Measuring dynamic flux from biological systems requires sensing technology that can measure activity in multiple dimensions. Here we report the development of a self-referencing oxygen optrode (SRO) for reliably making noninvasive measurements of oxygen flux from a variety of biological systems. The self-referencing microsensor technique was adapted to operate optrodic oxygen sensors through the integration of optical sensing instrumentation with software-controlled data acquisition and micro-stepping motion control. This allows the sensor to scan biologically active gradients of oxygen flux directly, as it relates to cellular and tissue respiratory activity. The technique was validated first using artificially generated oxygen gradients, which are theoretically modelled and compare with measured signals. Subsequently, the SRO was applied in basic research applications to non-invasively measure molecular oxygen flux from a variety of animal and plant systems.

  6. Quantitative measurement of neuronal degeneration in organotypic hippocampal cultures after combined oxygen/glucose deprivation.

    PubMed

    Strasser, U; Fischer, G

    1995-04-01

    Organotypic hippocampal cultures were used to study cell degeneration during the recovery period after defined periods (30 and 60 min) of combined oxygen/glucose deprivation mimicking transient ischemic conditions. Staining with the fluorescent dye propidium iodide allowed detection of damaged cells. Fluorescence intensity was measured by an image analysis system and used to quantify cell damage at different time points during the recovery period (up to 22 h). At 30 min of oxygen/glucose deprivation cells in the CA1 area were relatively more sensitive compared to CA3 and dentate gyrus cells, with respect to the time course of degeneration and the percentage of affected cells. Expanding the oxygen/glucose deprivation period from 30 to 60 min drastically increased the percentage of cells dying in all hippocampal areas. Still, however, cells in CA1 degenerated faster compared to those in the CA3 area and dentate gyrus. A histological analysis of toluidine blue as well as MAP2-immunostained sections revealed that almost all neurons degenerated in all hippocampal areas following the 60-min deprivation period, whereas GFAP-stained astrocytes appeared to be unaffected. Therefore, neuronal degeneration could be quantified by taking the fluorescence intensity values 22 h after 60 min of oxygen/glucose deprivation as 100% neuronal damage. The possibility to quantify neuronal damage in organotypic cultures offers a useful tool for detailed studies on mechanisms of neuronal cell death in a cell culture system which is closer to in situ conditions than monolayer cell cultures.

  7. 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.

  8. 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

  9. Massively multiplex single-cell Hi-C.

    PubMed

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

    2017-03-01

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

  10. 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.

  11. 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.

  12. Direct measurement of oxygen consumption rates from attached and unattached cells in a reversibly sealed, diffusionally isolated sample chamber

    PubMed Central

    Strovas, Timothy J.; McQuaide, Sarah C.; Anderson, Judy B.; Nandakumar, Vivek; Kalyuzhnaya, Marina G.; Burgess, Lloyd W.; Holl, Mark R.; Meldrum, Deirdre R.; Lidstrom, Mary E.

    2011-01-01

    Oxygen consumption is a fundamental component of metabolic networks, mitochondrial function, and global carbon cycling. To date there is no method available that allows for replicate measurements on attached and unattached biological samples without compensation for extraneous oxygen leaking into the system. Here we present the Respiratory Detection System, which is compatible with virtually any biological sample. The RDS can be used to measure oxygen uptake in microliter-scale volumes with a reversibly sealed sample chamber, which contains a porphyrin-based oxygen sensor. With the RDS, one can maintain a diffusional seal for up to three hours, allowing for the direct measurement of respiratory function of samples with fast or slow metabolic rates. The ability to easily measure oxygen uptake in small volumes with small populations or dilute samples has implications in cell biology, environmental biology, and clinical diagnostics. PMID:21546993

  13. Flagellates as model system for gravity detection of single cells

    NASA Astrophysics Data System (ADS)

    Lebert, Michael; Richter, Peter; Daiker, Viktor; Schuster, Martin; Tebart, Jenny; Strauch, Sebastian M.; Donat-Peter, H.

    Euglena gracilis is a unicellular, photosynthetic organism which uses light and gravity as en-vironmental hints to reach and stay in horizons of the water column which are optimal for growth and reproduction. The orientation in respect to light (so called positive and nega-tive phototaxis, i.e. movement toward or away of a light source) was well known and fairly good understood. In contrast, knowledge about the movement away from the centre of gravity (negative gravitaxis) was rather scarce. Over a century it was unclear whether orientation in respect to the gravity vector is based on a physical or a physiological mechanism. Recent results clearly favour the latter. Knock-down mutants (RNAi) were characterized which define certain key components of the gravitactic signal transduction chain. These key components include a TRP-like channel, a gravitaxis-specific calmodulin and a protein kinase A. The molecular characterization of these components is currently performed and will be presented. Euglena is not only a model system for the close understanding of gravity detection in single cells, but can also be used as photosynthetic component, i.e. oxygen source and carbon dioxide as well as nitrogenic components sink in Closed Environmental Systems (CES). Due CES are systems of choice in times of scarce flight opportunities. They allow a massive sample sharing and combine possibilities to do microgravity research for biologists but also for engineers, physicists and material scientists. Recent attempts include Aquacells and Omegahab. In the near future miniaturized systems (Chinese ShenZhou) as well as advanced CES will be flown or tested, respectively. Current attempts and plans will be presented.

  14. 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.

  15. 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.

  16. 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.

  17. Oxygen saturation in healthy newborn infants immediately after birth measured by pulse oximetry.

    PubMed

    Toth, B; Becker, A; Seelbach-Göbel, B

    2002-04-01

    Pre- and postductal arterial oxygen saturation (SpO2) rates were measured in 50 healthy vaginally delivered newborn infants to establish reference values of SpO2 rates immediately after birth. We compared the SpO2 values in the pre- and postductal areas and assessed the influence of oxitocin and analgetics applied during delivery. Fifty neonates were examined by the 2nd minute (min) of life using Nellcor N-3000 pulse oximeters on the right hand and foot. Measurements were carried out until a SpO2 of 95% was achieved. Heart rates were registered simultaneously. Two min after birth the mean preductal SpO2 was 73% (44-95%) and 67% (34-93%) in the postductal region. SpO2 rates of > 95% were reached after 12 min (2-55 min) preductally and after 14 min (3-55 min) postductally. Our results demonstrate that it takes 12-14 min for healthy neonates to reach an oxygen saturation of 95% prerespectively postductal, in some cases even 55 min. All neonates were in good clinical condition and didn't require any supplemental oxygen. Additionally, we were able to show that epidural anaesthesia (PDA) during delivery increases the heart rate of the newborn infant.

  18. The single-cell chemostat: an agarose-based, microfluidic device for high-throughput, single-cell studies of bacteria and bacterial communities.

    PubMed

    Moffitt, Jeffrey R; Lee, Jeffrey B; Cluzel, Philippe

    2012-04-21

    Optical microscopy of single bacteria growing on solid agarose support is a powerful method for studying the natural heterogeneity in growth and gene expression. While the material properties of agarose make it an excellent substrate for such studies, the sheer number of exponentially growing cells eventually overwhelms the agarose pad, which fundamentally limits the duration and the throughput of measurements. Here we overcome the limitations of exponential growth by patterning agarose pads on the sub-micron-scale. Linear tracks constrain the growth of bacteria into a high density array of linear micro-colonies. Buffer flow through microfluidic lines washes away excess cells and delivers fresh nutrient buffer. Densely patterned tracks allow us to cultivate and image hundreds of thousands of cells on a single agarose pad over 30-40 generations, which drastically increases single-cell measurement throughput. In addition, we show that patterned agarose can facilitate single-cell measurements within bacterial communities. As a proof-of-principle, we study a community of E. coli auxotrophs that can complement the amino acid deficiencies of one another. We find that the growth rate of colonies of one strain decreases sharply with the distance to colonies of the complementary strain over distances of only a few cell lengths. Because patterned agarose pads maintain cells in a chemostatic environment in which every cell can be imaged, we term our device the single-cell chemostat. High-throughput measurements of single cells growing chemostatically should greatly facilitate the study of a variety of microbial behaviours.

  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. 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.

  1. A LED-based phosphorimeter for measurement of microcirculatory oxygen pressure.

    PubMed

    Guerci, Philippe; Ince, Yasin; Heeman, Paul; Faber, Dirk; Ergin, Bulent; Ince, Can

    2017-02-01

    Quantitative measurements of microcirculatory and tissue oxygenation are of prime importance in experimental research. The noninvasive phosphorescence quenching method has given further insight into the fundamental mechanisms of oxygen transport to healthy tissues and in models of disease. Phosphorimeters are devices dedicated to the study of phosphorescence quenching. The experimental applications of phosphorimeters range from measuring a specific oxygen partial pressure (Po2) in cellular organelles such as mitochondria, finding values of Po2 distributed over an organ or capillaries, to measuring microcirculatory Po2 changes simultaneously in several organ systems. Most of the current phosphorimeters use flash lamps as a light excitation source. However, a major drawback of flash lamps is their inherent plasma glow that persists for tens of microseconds after the primary discharge. This complex distributed excitation pattern generated by the flash lamp can lead to inaccurate Po2 readings unless a deconvolution analysis is performed. Using light-emitting diode (LED), a rectangular shaped light pulse can be generated that provides a more uniformly distributed excitation signal. This study presents the design and calibration process of an LED-based phosphorimeter (LED-P). The in vitro calibration of the LED-P using palladium(II)-meso-tetra(4-carboxyphenyl)-porphyrin (Pd-TCCP) as a phosphorescent dye is presented. The pH and temperature were altered to assess whether the decay times of the Pd-TCCP measured by the LED-P were significantly influenced. An in vivo validation experiment was undertaken to measure renal cortical Po2 in a rat subjected to hypoxic ventilation conditions and ischemia/reperfusion. The benefits of using LEDs as a light excitation source are presented.

  2. Numerical simulation and analysis of accurate blood oxygenation measurement by using optical resolution photoacoustic microscopy

    NASA Astrophysics Data System (ADS)

    Yu, Tianhao; Li, Qian; Li, Lin; Zhou, Chuanqing

    2016-10-01

    Accuracy of photoacoustic signal is the crux on measurement of oxygen saturation in functional photoacoustic imaging, which is influenced by factors such as defocus of laser beam, curve shape of large vessels and nonlinear saturation effect of optical absorption in biological tissues. We apply Monte Carlo model to simulate energy deposition in tissues and obtain photoacoustic signals reaching a simulated focused surface detector to investigate corresponding influence of these factors. We also apply compensation on photoacoustic imaging of in vivo cat cerebral cortex blood vessels, in which signals from different lateral positions of vessels are corrected based on simulation results. And this process on photoacoustic images can improve the smoothness and accuracy of oxygen saturation results.

  3. Optical triple sensor for measuring pH, oxygen and carbon dioxide.

    PubMed

    Weigl, B H; Holobar, A; Trettnak, W; Klimant, I; Kraus, H; O'Leary, P; Wolfbeis, O S

    1994-02-14

    A triple sensor unit consisting of opto-chemical sensors for measurement of pH, oxygen and carbon dioxide in bioreactors is presented. The pH and the CO2 sensor are based on the color change of a pH-sensitive dye immobilized on a polymeric support. The resulting changes in absorption are monitored through optical fibers. The oxygen sensor is based on the quenching of the fluorescence of a metal-organic dye. All three sensors are fully LED compatible. The sensitive membranes consist of plastic films and can be stored and replaced conveniently. The sensors are sterilizable with hydrogen peroxide and ethanol. In addition, the pH sensor is steam sterilizable. Accuracy, resolution and reproducibility fulfill the requirements for use in biotechnological applications. Calibration procedures for each sensor are presented. The working principle and the performance of all three sensors are described, with particular emphasis given to their application in bioreactors.

  4. 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.

  5. [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

  6. Technical note: Consistent calculation of aquatic gross production from oxygen triple isotope measurements

    NASA Astrophysics Data System (ADS)

    Kaiser, J.

    2011-07-01

    Oxygen triple isotope measurements can be used to calculate aquatic gross oxygen production rates. Past studies have emphasised the appropriate definition of the 17O excess and often used an approximation to derive production rates from the 17O excess. Here, I show that the calculation can be phrased more consistently and without any approximations using the relative 17O/16O and 18O/16O isotope ratio differences (delta values) directly. I call this the "dual delta method". The 17O excess is merely a mathematical construct and the derived production rate is independent of its definition, provided all calculations are performed with a consistent definition. I focus on the mixed layer, but also show how time series of triple isotope measurements below the mixed layer can be used to derive gross production. In the calculation of mixed layer productivity, I explicitly include isotopic fractionation during gas invasion and evasion, which requires the oxygen supersaturation s to be measured as well. I also suggest how bubble injection could be considered in the same mathematical framework. I distinguish between concentration steady state and isotopic steady state and show that only the latter needs to be assumed in the calculation. It is even possible to derive an estimate of the net production rate in the mixed layer that is independent of the assumption of concentration steady state. I review measurements of the parameters required for the calculation of gross production rates and show how their systematic uncertainties as well as the use of different published calculation methods can cause large variations in the production rates for the same underlying isotope ratios. In particular, the 17O excess of dissolved O2 in equilibrium with atmospheric O2 and the 17O excess of photosynthetic O2 need to be re-measured. Because of these uncertainties, all calculation parameters should always be fully documented and the measured relative isotope ratio differences as well as the

  7. Genome-wide base-resolution mapping of DNA methylation in single cells using single-cell bisulfite sequencing (scBS-seq).

    PubMed

    Clark, Stephen J; Smallwood, Sébastien A; Lee, Heather J; Krueger, Felix; Reik, Wolf; Kelsey, Gavin

    2017-03-01

    DNA methylation (DNAme) is an important epigenetic mark in diverse species. Our current understanding of DNAme is based on measurements from bulk cell samples, which obscures intercellular differences and prevents analyses of rare cell types. Thus, the ability to measure DNAme in single cells has the potential to make important contributions to the understanding of several key biological processes, such as embryonic development, disease progression and aging. We have recently reported a method for generating genome-wide DNAme maps from single cells, using single-cell bisulfite sequencing (scBS-seq), allowing the quantitative measurement of DNAme at up to 50% of CpG dinucleotides throughout the mouse genome. Here we present a detailed protocol for scBS-seq that includes our most recent developments to optimize recovery of CpGs, mapping efficiency and success rate; reduce hands-on time; and increase sample throughput with the option of using an automated liquid handler. We provide step-by-step instructions for each stage of the method, comprising cell lysis and bisulfite (BS) conversion, preamplification and adaptor tagging, library amplification, sequencing and, lastly, alignment and methylation calling. An individual with relevant molecular biology expertise can complete library preparation within 3 d. Subsequent computational steps require 1-3 d for someone with bioinformatics expertise.

  8. 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.

  9. Droplet microfluidics--a tool for single-cell analysis.

    PubMed

    Joensson, Haakan N; Andersson Svahn, Helene

    2012-12-03

    Droplet microfluidics allows the isolation of single cells and reagents in monodisperse picoliter liquid capsules and manipulations at a throughput of thousands of droplets per second. These qualities allow many of the challenges in single-cell analysis to be overcome. Monodispersity enables quantitative control of solute concentrations, while encapsulation in droplets provides an isolated compartment for the single cell and its immediate environment. The high throughput allows the processing and analysis of the tens of thousands to millions of cells that must be analyzed to accurately describe a heterogeneous cell population so as to find rare cell types or access sufficient biological space to find hits in a directed evolution experiment. The low volumes of the droplets make very large screens economically viable. This Review gives an overview of the current state of single-cell analysis involving droplet microfluidics and offers examples where droplet microfluidics can further biological understanding.

  10. Virtual Microfluidics for digital quantification and single-cell sequencing

    PubMed Central

    Xu, Liyi; Brito, Ilana L.; Alm, Eric J.; Blainey, Paul C.

    2016-01-01

    Interest in highly parallelized analysis of single molecules and single cells is growing rapidly. Here we develop hydrogel-based virtual microfluidics as a simple alternative to complex engineered microfluidic systems for the compartmentalization of nucleic acid amplification reactions. We applied digital multiple displacement amplification (dMDA) to purified DNA templates, cultured bacterial cells, and human microbiome samples in the virtual microfluidics system and demonstrated recovery and whole-genome sequencing of single-cell MDA products. Our results from control samples showed excellent coverage uniformity and markedly reduced chimerism compared with single-cell data obtained from conventional liquid MDA reactions. We also demonstrate the applicability of the hydrogel method for genomic studies of naturally occurring microbes in human microbiome samples. The virtual microfluidics approach is a simple and robust method that will enable many laboratories to perform single-cell genomic analyses. PMID:27479330

  11. Measuring percent oxygen saturation of hemoglobin, percent carboxyhemoglobin and methemoglobin, and concentrations of total hemoglobin and oxygen in blood of man, dog, and baboon.

    PubMed

    Dennis, R C; Valeri, C R

    1980-08-01

    We used an automated four-wavelength spectrometer to measure the concentration of total hemoglobin, percent oxyhemoglobin, carboxyhemoglobin, and methemoglobin, and concentration of oxygen bound to hemoglobin in the blood of humans, dogs, and baboons under clinical and various experimental conditions. Measurements of total hemoglobin and methemoglobin with this simple method were comparable to those with standard spectrometric procedures. Carboxyhemoglobin measurements were comparable to those made with gas chromatography, and measurements of oxygen content were comparable to those made with the galvanic cell method. The new instrument is as accurate as the comparison methods used to evaluate it in all parameters, is reliable, and measurements take only 63 s per sample. In addition, it requires minimal operator training, infrequent need for calibration, and no sample preparation.

  12. 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.

  13. 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.

  14. Comparison between direct and predicted maximal oxygen uptake measurement during cycling.

    PubMed

    Santtila, Matti; Häkkinen, Keijo; Pihlainen, Kai; Kyröläinen, Heikki

    2013-02-01

    Predicted maximal oxygen uptake (VO2max) measurements are based on the assumption of linear relationship between heart rate or power output and oxygen consumption during various intensities. To develop more reliable predicted test for soldiers, the purpose of the present study was to compare the results of direct measurements of VO2max to respective predicted values in cycling (military fitness test). The predicted mean (+/- SD) peak oxygen uptake (VO2peak) value was 45.2 +/- 7.7 mL kg(-1) min(-1) during first week, whereas the respective direct value was 44.8 +/- 8.5 mL kg(-1) min(-1). During the ninth week, the predicted and measured mean (+/-SD) VO2max values were 47.4 +/- 6.7 mL kg(-1) min(-1) and 48.7 +/- 7.3 mL kg(-1) min(-1), respectively. The absolute differences between the methods were -0.42 mL kg(-1) min(-1) (p = 0.46) and 1.28 mL kg(-1) min(-1) (p < 0.05), which correspond to relative values of 0.9% and 2.7%, respectively. A Bland-Altman plot of measured VO2max and predicted VO2max showed no significant trend between the mean and the difference of the 2 methods either before (r = 0.14, p = 0.24) or after the basic military training period (r = 0.11, p = 0.36). Intraclass correlation coefficient varied between r = 0.82 to 0.94. In conclusion, the predicted protocol is fairly accurate (+/-3%) and reliable to predict VO2max values in male soldiers but the use for clinical purposes should be considered individually.

  15. 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

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

    PubMed Central

    Steinmeyer, Joseph D.; Yanik, Mehmet Fatih

    2012-01-01

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

  17. Single Cell Analysis: From Technology to Biology and Medicine.

    PubMed

    Pan, Xinghua

    2014-01-01

    Single-cell analysis heralds a new era that allows "omics" analysis, notably genomics, transcriptomics, epigenomics and proteomics at the single-cell level. It enables the identification of the minor subpopulations that may play a critical role in a biological process of a population of cells, which conventionally are regarded as homogeneous. It provides an ultra-sensitive tool to clarify specific molecular mechanisms and pathways and reveal the nature of cell heterogeneity. It also facilitates the clinical investigation of patients when a very low quantity or a single cell is available for analysis, such as noninvasive prenatal diagnosis and cancer screening, and genetic evaluation for in vitro fertilization. Within a few short years, single-cell analysis, especially whole genomic sequencing and transcriptomic sequencing, is becoming robust and broadly accessible, although not yet a routine practice. Here, with single cell RNA-seq emphasized, an overview of the discipline, progresses, and prospects of single-cell analysis and its applications in biology and medicine are given with a series of logic and theoretical considerations.

  18. Method for Single-Cell Mass and Electrophoretic Mobility Measurement

    DTIC Science & Technology

    2010-02-01

    machining of the many custom components that were needed to conduct the research described in this thesis. This work has been funded by the National Cancer ...Characterization of Bacteria by Buoyant Mass and Electrophoretic Mobility 5.1 Introduction Bacterial biofilms , layers of bacterial cells attached to solid...descriptions of the initial adhesion kinetics of biofilm formation. [19, 20] One approach to this problem is to model the dependence of cellular EPM

  19. [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.

  20. 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

  1. Small signal gain measurement of liquid oxygen under different wavelength laser pump

    NASA Astrophysics Data System (ADS)

    Shi, Zhe; Li, Hui; Zhou, Canhua; Liu, Jinbo; Cai, Xianglong; Hu, Shu; Gai, Baodong; Zhou, Dongjian; Liu, Dong; Guo, Jingwei; Jin, Yuqi

    2015-02-01

    Oxygen molecules existed in pairs under liquid condition, the radiation from vibrational ground state of 1 Δ state to the first vibrational excited state of 3 ∑ state was electronic dipole moment transition allowed, and a photon with wavelength of 1580 nm was emitted. In our experiment, dye laser with wavelength of 581 nm, 634 nm, 764 nm was used to excite liquid oxygen to different excited states, while a tunable OPO was used as the seeder laser, and the small signal gain was measured to be 0.23 cm-1, 0.3 cm-1 and 0.076 cm-1 respectively. The small signal gain (pump by photon of 634 nm) was significantly higher than that of common solid state lasers and chemical lasers. When the fundamental output of a Q-switched Nd:YAG laser was used as the pump source, the corresponding small signal gain was 0.12 cm-1. The profiles of small signal gain form 1579.2 nm to 1580.8 nm were also presented. These results were consistent with theoretical calculation. The high positive gain indicated that the liquid oxygen was a potential medium for high energy laser. A comprehensive parameter optimization was still necessary in order to improve the mall signal gain.

  2. 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.

  3. 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.

  4. 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.

  5. Measurement of blood oxygen saturation using a multichannel fiberoptic oximeter-densitometer.

    PubMed

    Sekelj, P; Retfalvi, S; Lavoie, A

    1977-06-01

    Design principles and performance of a new fiberoptic oximeter-densitometer for measurement of blood oxygen saturation are described. This instrument is capable of performing measurements using either an intravascular catheter, flow-through cuvette, or earpiece. The operations of the flow-through cuvette and earpiece are based on the principles of light transmission, that of the catheter on the principles of hemoreflection. The system incorporates five interference filters permitting the selection of a particular wavelength or combination of wavelengths to perform different modes of operation. Estimates using both catheter and cuvette appeared to be independent of variations over a wide range in hematocrit and flow rate. In the range from 20 to 100% saturation the standard deviation of the differences between the in vitro estimates by the catheter and (or) cuvette and the Van Slyke analyses were 1.62 and 1.73%, respectively. In 53 children with congenital heart disease (22 cyanotic) values calculated from readings by the earpiece were related to values of arterial oxygen saturation as measured by American Optical Company reflection oximeter at the time of cardiac catheterization. In the range from 50 to 100% saturation, the regression line between the two techniques was linear and the standard deviation of the differences was 2.89% (3.12% in cyanotic children alone). The method provides a high degree of compensation for variations in skin pigmentation.

  6. 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.

  7. 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.

  8. Shock-tube measurements of excited oxygen atoms using cavity-enhanced absorption spectroscopy.

    PubMed

    Nations, Marcel; Wang, Shengkai; Goldenstein, Christopher S; Sun, Kai; Davidson, David F; Jeffries, Jay B; Hanson, Ronald K

    2015-10-10

    We report the use of cavity-enhanced absorption spectroscopy (CEAS) using two distributed feedback diode lasers near 777.2 and 844.6 nm for sensitive, time-resolved, in situ measurements of excited-state populations of atomic oxygen in a shock tube. Here, a 1% O2/Ar mixture was shock-heated to 5400-8000 K behind reflected shock waves. The combined use of a low-finesse cavity, fast wavelength scanning of the lasers, and an off-axis alignment enabled measurements with 10 μs time response and low cavity noise. The CEAS absorption gain factors of 104 and 142 for the P35←S520 (777.2 nm) and P0,1,23←S310 (844.6 nm) atomic oxygen transitions, respectively, significantly improved the detection sensitivity over conventional single-pass measurements. This work demonstrates the potential of using CEAS to improve shock-tube studies of nonequilibrium electronic-excitation processes at high temperatures.

  9. Single-cell PCR of genomic DNA enabled by automated single-cell printing for cell isolation.

    PubMed

    Stumpf, F; Schoendube, J; Gross, A; Rath, C; Niekrawietz, S; Koltay, P; Roth, G

    2015-07-15

    Single-cell analysis has developed into a key topic in cell biology with future applications in personalized medicine, tumor identification as well as tumor discovery (Editorial, 2013). Here we employ inkjet-like printing to isolate individual living single human B cells (Raji cell line) and load them directly into standard PCR tubes. Single cells are optically detected in the nozzle of the microfluidic piezoelectric dispenser chip to ensure printing of droplets with single cells only. The printing process has been characterized by using microbeads (10µm diameter) resulting in a single bead delivery in 27 out of 28 cases and relative positional precision of ±350µm at a printing distance of 6mm between nozzle and tube lid. Process-integrated optical imaging enabled to identify the printing failure as void droplet and to exclude it from downstream processing. PCR of truly single-cell DNA was performed without pre-amplification directly from single Raji cells with 33% success rate (N=197) and Cq values of 36.3±2.5. Additionally single cell whole genome amplification (WGA) was employed to pre-amplify the single-cell DNA by a factor of >1000. This facilitated subsequent PCR for the same gene yielding a success rate of 64% (N=33) which will allow more sophisticated downstream analysis like sequencing, electrophoresis or multiplexing.

  10. Single cell electric impedance topography: mapping membrane capacitance.

    PubMed

    Dharia, Sameera; Ayliffe, Harold E; Rabbitt, Richard D

    2009-12-07

    Single-cell electric impedance topography (sceTopo), a technique introduced here, maps the spatial distribution of capacitance (i.e. displacement current) associated with the membranes of isolated, living cells. Cells were positioned in the center of a circular recording chamber surrounded by eight electrodes. Electrodes were evenly distributed on the periphery of the recording chamber. Electric impedance measured between adjacent electrode pairs (10 kHz-5 MHz) was used to construct topographical maps of the spatial distribution of membrane capacitance. Xenopus Oocytes were used as a model cell to develop sceTopo because these cells consist of two visually distinguishable hemispheres, each with distinct membrane composition and structure. Results showed significant differences in the imaginary component of the impedance between the two oocyte hemispheres. In addition, the same circumferential array was used to map the size of the extracellular electrical shunt path around the cell, providing a means to estimate the location and shape of the cell in the recording chamber.

  11. 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

  12. Single cell viability and impact of heating by laser absorption.

    PubMed

    Wetzel, Franziska; Rönicke, Susanne; Müller, Karla; Gyger, Markus; Rose, Daniel; Zink, Mareike; Käs, Josef

    2011-09-01

    Optical traps such as tweezers and stretchers are widely used to probe the mechanical properties of cells. Beyond their large range of applications, the use of infrared laser light in optical traps causes significant heating effects in the cell. This study investigated the effect of laser-induced heating on cell viability. Common viability assays are not very sensitive to damages caused in short periods of time or are not practicable for single cell analysis. We used cell spreading, a vital ability of cells, as a new sensitive viability marker. The optical stretcher, a two beam laser trap, was used to simulate heat shocks that cells typically experience during measurements in optical traps. The results show that about 60% of the cells survived heat shocks without vital damage at temperatures of up to 58 ± 2°C for 0.5 s. By varying the duration of the heat shocks, it was shown that 60% of the cells stayed viable when exposed to 48 ± 2°C for 5 s.

  13. Single cell electric impedance topography: Mapping membrane capacitance

    PubMed Central

    Dharia, Sameera; Ayliffe, Harold E.

    2010-01-01

    Single-cell electric impedance topography (sceTopo), a technique introduced here, maps the spatial distribution of capacitance (i.e. displacement current) associated with the membranes of isolated, living cells. Cells were positioned in the center of a circular recording chamber surrounded by eight electrodes. Electrodes were evenly distributed on the periphery of the recording chamber. Electric impedance measured between adjacent electrode pairs (10 kHz–5 MHz) was used to construct topographical maps of the spatial distribution of membrane capacitance. Xenopus Oocytes were used as a model cell to develop sceTopo because these cells consist of two visually distinguishable hemispheres, each with distinct membrane composition and structure. Results showed significant differences in the imaginary component of the impedance between the two oocyte hemispheres. In addition, the same circumferential array was used to map the size of the extracellular electrical shunt path around the cell, providing a means to estimate the location and shape of the cell in the recording chamber. PMID:19904403

  14. 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.

  15. 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.

    2015-02-01

    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 working tank.

  16. Bioinformatics approaches to single-cell analysis in developmental biology.

    PubMed

    Yalcin, Dicle; Hakguder, Zeynep M; Otu, Hasan H

    2016-03-01

    Individual cells within the same population show various degrees of heterogeneity, which may be better handled with single-cell analysis to address biological and clinical questions. Single-cell analysis is especially important in developmental biology as subtle spatial and temporal differences in cells have significant associations with cell fate decisions during differentiation and with the description of a particular state of a cell exhibiting an aberrant phenotype. Biotechnological advances, especially in the area of microfluidics, have led to a robust, massively parallel and multi-dimensional capturing, sorting, and lysis of single-cells and amplification of related macromolecules, which have enabled the use of imaging and omics techniques on single cells. There have been improvements in computational single-cell image analysis in developmental biology regarding feature extraction, segmentation, image enhancement and machine learning, handling limitations of optical resolution to gain new perspectives from the raw microscopy images. Omics approaches, such as transcriptomics, genomics and epigenomics, targeting gene and small RNA expression, single nucleotide and structural variations and methylation and histone modifications, rely heavily on high-throughput sequencing technologies. Although there are well-established bioinformatics methods for analysis of sequence data, there are limited bioinformatics approaches which address experimental design, sample size considerations, amplification bias, normalization, differential expression, coverage, clustering and classification issues, specifically applied at the single-cell level. In this review, we summarize biological and technological advancements, discuss challenges faced in the aforementioned data acquisition and analysis issues and present future prospects for application of single-cell analyses to developmental biology.

  17. Simultaneous in-situ measurements of neutral temperature and oxygen in the mesosphere during the WADIS sounding rocket project

    NASA Astrophysics Data System (ADS)

    Strelnikov, Boris; Lübken, Franz-Josef; Rapp, Markus; Grygalashvyly, Mykhaylo; Löhle, Stefan; Eberhart, Martin; Fasoulas, Stefanos; Hedin, Jonas; Gumbel, Jörg; Khaplanov, Mikhail; Stegman, Jacek; Friedrich, Martin

    2016-04-01

    The WADIS project (Wave propagation and dissipation in the middle atmosphere: energy budget and distribution of trace constituents) aimed at studying waves, their dissipation, and effects on trace constituents. The project comprised two sounding rocket campaigns conducted at the Andøya Space Center (69 °N, 16 °E). One sounding rocket was launched in summer 2013 and one in winter 2015. In-situ measurements delivered high resolution altitude-profiles of neutral temperature and density, as well as plasma and oxygen densities. Atomic oxygen was measured by two different techniques. Airglow photometers operated by MISU measured emissions from excited molecular oxygen at 1.27 um (daytime summer launch) and 762 nm (night-time winter launch), both of which can be used to infer altitude profiles of atomic oxygen. This is a well-proven technique and has been applied to sounding rocket and satellite measurements in the past. Solid electrolyte sensors (FIPEX) operated by IRS is a new technique for sounding rockets, which yielded atomic oxygen density profiles with a height resolution better than 10 m. The neutral air density and temperature was measured by the CONE instrument also with very heigh altitude resolution and precision. All these instruments were mounted on the same deck of the sounding rocket and, therefore delivered real common volume measurements. In this paper we present simultaneous in-situ temperature and oxygen density measurements and discuss how variability of these quantities may influence temperature derivations from OH airglow observations at mesopause heights.

  18. Local Nanomechanical Motion In Single Cells.

    NASA Astrophysics Data System (ADS)

    Pelling, Andrew; Gimzewski, James

    2004-03-01

    We present new evidence that the nanoscale motion of the cell wall of Saccharomyces cerevisiae exhibits local bionanomechanical motion at characteristic frequencies and which is not caused by random or Brownian processes. This motion is measured with the AFM tip which acts as a nanomechanical sensor, permitting the motion of the cell wall to be recorded as a function of time, applied force, etc. We present persuasive evidence which shows that the local nanomechanical motion is characteristic of metabolic processes taking place inside the cell. This is demonstrated by clear differences between living cells and living cells treated with a metabolic inhibitor. This inhibitor specifically targets cytochrome oxidase inside the mitochondria and inhibits ATP production. The cells observed in this study display characteristic local cell wall motion with amplitudes between 1 and 3 nm and frequencies between 500 and 1700 Hz. The motion is temperature dependant which also suggests the mechanism for the observed motion has biological origins. In addition to a stringent series of control experiments we also discuss local measurements of the cell's mechanical properties and their influence on the observed bionanomechanical motion.

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

    PubMed

    Severin, E; Stellmach, J

    1984-01-01

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

  20. Pulse oximetry in the pulmonary tissue for the non-invasive measurement of mixed venous oxygen saturation.

    PubMed

    Nitzan, Meir; Nitzan, Itamar

    2013-08-01

    The oxygen saturation of the systemic arterial blood is associated with the adequacy of respiration, and can be measured non-invasively by pulse oximetry in the systemic tissue. The oxygen saturation of the blood in the pulmonary artery, the mixed venous blood, reflects the balance between oxygen supply to the systemic tissues and their oxygen demand. The mixed venous oxygen saturation has also clinical significance because it is used in Fick equation for the quantitative measurement of cardiac output. At present the measurement of the mixed venous oxygen saturation is invasive and requires insertion of a Swan-Ganz catheter into the pulmonary artery. We suggest a noninvasive method for the measurement of the mixed venous oxygen saturation in infants, pulmonary pulse oximetry. The method is similar to the systemic pulse oximetry, which is based on the different light absorption curves of oxygenated and deoxygenated hemoglobin and on the analysis of photoplethysmographic curves in two wavelengths. The proposed pulmonary pulse oximeter includes light-sources of two wavelengths in the infrared, which illuminate the pulmonary tissue through the thoracic wall. Part of the light which is scattered back from the pulmonary tissue and passes through the thoracic wall is detected, and for each wavelength a pulmonary photoplethysmographic curve is obtained. The pulmonary photoplethysmographic curves reflect blood volume increase during systole in the pulmonary arteries in the lung tissue, which contain mixed venous blood. The ratio R of the amplitude-to-baseline ratio for the two wavelengths is related to the mixed venous oxygen saturation through equations derived for the systemic pulse oximetry. The method requires the use of extinction coefficients values for oxygenated and deoxygenated hemoglobin, which can be found in the literature.

  1. RECENT ADVANCES IN HIGH TEMPERATURE ELECTROLYSIS AT IDAHO NATIONAL LABORATORY: SINGLE CELL TESTS

    SciTech Connect

    X. Zhang; J. E. O'Brien; R. C. O'Brien

    2012-07-01

    An experimental investigation on the performance and durability of single solid oxide electrolysis cells (SOECs) is under way at the Idaho National Laboratory. In order to understand and mitigate the degradation issues in high temperature electrolysis, single SOECs with different configurations from several manufacturers have been evaluated for initial performance and long-term durability. A new test apparatus has been developed for single cell and small stack tests from different vendors. Single cells from Ceramatec Inc. show improved durability compared to our previous stack tests. Single cells from Materials and Systems Research Inc. (MSRI) demonstrate low degradation both in fuel cell and electrolysis modes. Single cells from Saint Gobain Advanced Materials (St. Gobain) show stable performance in fuel cell mode, but rapid degradation in the electrolysis mode. Electrolyte-electrode delamination is found to have significant impact on degradation in some cases. Enhanced bonding between electrolyte and electrode and modification of the microstructure help to mitigate degradation. Polarization scans and AC impedance measurements are performed during the tests to characterize the cell performance and degradation.

  2. 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.

  3. 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

  4. Gene Expression Correlates with the Number of Herpes Viral Genomes Initiating Infection in Single Cells

    PubMed Central

    Cohen, Efrat M.

    2016-01-01

    Viral gene expression varies significantly among genetically identical cells. The sources of these variations are not well understood and have been suggested to involve both deterministic host differences and stochastic viral host interactions. For herpesviruses, only a limited number of incoming viral genomes initiate expression and replication in each infected cell. To elucidate the effect of this limited number of productively infecting genomes on viral gene expression in single cells, we constructed a set of fluorescence-expressing genetically tagged herpes recombinants. The number of different barcodes originating from a single cell is a good representative of the number of incoming viral genomes replicating (NOIVGR) in that cell. We identified a positive correlation between the NOIVGR and viral gene expression, as measured by the fluorescent protein expressed from the viral genome. This correlation was identified in three distinct cell-types, although the average NOIVGR per cell differed among these cell-types. Among clonal single cells, high housekeeping gene expression levels are not supportive of high viral gene expression, suggesting specific host determinants effecting viral infection. We developed a model to predict NOIVGR from cellular parameters, which supports the notion that viral gene expression is tightly linked to the NOIVGR in single-cells. Our results support the hypothesis that the stochastic nature of viral infection and host cell determinants contribute together to the variability observed among infected cells. PMID:27923068

  5. First respiration estimates of cold-seep vesicomyid bivalves from in situ total oxygen uptake measurements.

    PubMed

    Decker, Carole; Caprais, Jean-Claude; Khripounoff, Alexis; Olu, Karine

    2012-04-01

    Vesicomyid bivalves are one of the most abundant symbiont-bearing species inhabiting deep-sea reducing ecosystems. Nevertheless, except for the hydrothermal vent clam Calyptogena magnifica, their metabolic rates have not been documented, and only assessed with ex situ experiments. In this study, gathering benthic chamber measurements and biomass estimation, we give the first in situ assessment of the respiration rate of these bivalves. The giant pockmark Regab, located at 3160m depth along the Congo-Angola margin, is a cold-seep site characterised by dense assemblages of two species of vesicomyids: Christineconcha regab and Laubiericoncha chuni with high dominance of C. regab. Two sites with dense aggregates of vesicomyids were selected to measure total oxygen uptake (TOU), and methane fluxes using IFREMER's benthic chamber CALMAR deployed by the ROV Quest 4000 (MARUM). Photographs were taken and bivalves were sampled using blade corers to estimate density and biomass. Total oxygen uptake was higher at Site 2 compared to Site 1 (respectively 492 mmol.m(-2).d(-1) and 332 mmol.m(-2).d(-1)). However, given vesicomyid densities and biomass, mean oxygen consumption rates were similar at both sites (1.9 to 2.5 μmol.g total dry mass(-1).h(-1) at the Site 1 and 1.8 to 2.3 μmol.g total dry mass(-1).h(-1) at Site 2). These respiration rates are higher than published ex situ estimates for cold-seep or hydrothermal vent bivalves. Although methane fluxes at the base of sulphide production were clearly higher at Site 2 (14.6 mmol.m(-2).d(-1)) than at Site 1 (0.3 mmol.m(-2).d(-1)), they do not seem to influence the respiration rates of these bivalves associated to sulphide-oxidizing symbionts.

  6. Tissue blood flow and oxygen consumption measured with near-infrared frequency-domain spectroscopy

    NASA Astrophysics Data System (ADS)

    Paunescu, Lelia Adelina

    2001-12-01

    For decades, researchers have contributed with new ways of applying physics' principles to medicine. Moreover, researchers were involved in developing new, non-invasive instrumentation for medical applications. Recently, application of optical techniques in biology and medicine became an important field. Researchers found a non- invasive approach of using visible and near-infrared light as a probe for tissue investigation. Optical methods can contribute to medicine by offering the possibility of rapid, low-resolution, functional images and real-time devices. Near-infrared spectroscopy (NIRS) is a useful technique for the investigation of biological tissues because of the relatively low absorption of water and high absorption of oxy- and deoxy-hemoglobin in the near- infrared region of 750-900 nm. Due to these properties, the near-infrared light can penetrate biological tissues in the range of 0.5-2 cm, offering investigation possibility of deep tissues and differentiate among healthy and diseased tissues. This work represents the initial steps towards understanding and improving of the promising near- infrared frequency-domain technique. This instrument has a very important advantage: it can be used non-invasively to investigate many parts of the human body, including the brain. My research consists primarily of in vivo measurements of optical parameters such as absorption and reduced scattering coefficients and consequently, blood parameters such as oxy, deoxy, and total hemoglobin concentrations, tissue oxygen saturation, blood flow and oxygen consumption of skeletal muscle of healthy and diseased subjects. This research gives a solid background towards a ready- to-use instrument that can continuously, in real-time, measure blood parameters and especially blood oxygenation. This is a very important information in emergency medicine, for persons under intensive care, or undergoing surgery, organ transplant or other interventions.

  7. New principle for measuring arterial blood oxygenation, enabling motion-robust remote monitoring

    PubMed Central

    van Gastel, Mark; Stuijk, Sander; de Haan, Gerard

    2016-01-01

    Finger-oximeters are ubiquitously used for patient monitoring in hospitals worldwide. Recently, remote measurement of arterial blood oxygenation (SpO2) with a camera has been demonstrated. Both contact and remote measurements, however, require the subject to remain static for accurate SpO2 values. This is due to the use of the common ratio-of-ratios measurement principle that measures the relative pulsatility at different wavelengths. Since the amplitudes are small, they are easily corrupted by motion-induced variations. We introduce a new principle that allows accurate remote measurements even during significant subject motion. We demonstrate the main advantage of the principle, i.e. that the optimal signature remains the same even when the SNR of the PPG signal drops significantly due to motion or limited measurement area. The evaluation uses recordings with breath-holding events, which induce hypoxemia in healthy moving subjects. The events lead to clinically relevant SpO2 levels in the range 80–100%. The new principle is shown to greatly outperform current remote ratio-of-ratios based methods. The mean-absolute SpO2-error (MAE) is about 2 percentage-points during head movements, where the benchmark method shows a MAE of 24 percentage-points. Consequently, we claim ours to be the first method to reliably measure SpO2 remotely during significant subject motion. PMID:27924930

  8. Validation of manometric microrespirometers for measuring oxygen consumption in small arthropods

    PubMed Central

    Melvin, Richard G.; Ballard, J. William O.; Williams, Joseph B.

    2008-01-01

    Scientists have used numerous techniques to measure organismal metabolic rate, including assays of oxygen (O2) consumption and carbon dioxide (CO2) production. Relatively few studies have directly compared estimates of metabolic rate on the same groups of animals as determined by different assay methods. This study directly compared measures of the metabolic rate of three lines of Drosophila simulans as determined either from direct measures of CO2 production using infrared gas analysis (IRGA), or from estimates of O2 consumption based on manometeric techniques. Determinations of metabolic rate of the same cohorts of flies using these two methods produced results that often differed widely. Typically metabolic rate as determined by the manometric method was significantly greater than that determined by CO2 output. These differences are difficult to explain by simple biotic or abiotic factor/s. Because of the idiosyncratic nature of these differences it is not possible to use a simple factor to convert from metabolic rate measurements done using manometric techniques to those expected from direct measures of CO2 output or O2 consumption. Although manometric devices are simple to construct and use, measurements of metabolic rate made with this method can vary significantly from measurements made by directly assaying CO2 production or O2 consumption. PMID:18606168

  9. 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

  10. Sensitive Technique Developed Using Atomic Force Microscopy to Measure the Low-Earth-Orbit Atomic Oxygen Erosion of Polymers

    NASA Technical Reports Server (NTRS)

    deGroh, Kim D.; Banks, Bruce A.; Clark, Gregory W.; Hammerstrom, Anne; Youngstrom, Erica; Kaminski, Carolyn; Fine, Elizabeth; Marx, Laura

    2001-01-01

    A recession measurement technique has been developed at the NASA Glenn Research Center to determine the atomic oxygen durability of polymers exposed to the space environment for short durations. Polymers such as polyimide Kapton and Teflon FEP (fluorinated ethylene propylene, DuPont) are commonly used in spacecraft because of their desirable properties, such as flexibility, low density, and in the case of FEP, low solar absorptance and high thermal emittance. Polymers on the exterior of spacecraft in the low- Earth-orbit environment are exposed to energetic atomic oxygen, resulting in erosion and potential structural loss. 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. Because long-term space exposure data are rare and very costly, short-term exposures, such as on the space shuttles, are often relied on for atomic oxygen erosion determination. The most common technique for determining E is through mass-loss measurements. For limited-duration exposure experiments, such as shuttle flight experiments, the atomic oxygen fluence is often so small that mass-loss measurements are not sensitive enough. Therefore, a recession measurement technique has been developed at Glenn to obtain accurate erosion yields of polymers exposed to low atomic oxygen fluences.

  11. Determination of Sediment Oxygen Demand in the Ziya River Watershed, China: Based on Laboratory Core Incubation and Microelectrode Measurements

    PubMed Central

    Rong, Nan; Shan, Baoqing; Wang, Chao

    2016-01-01

    A study coupling sedimentcore incubation and microelectrode measurementwas 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. PMID:26907307

  12. Spatially resolved measurement of singlet delta oxygen by radar resonance-enhanced multiphoton ionization.

    PubMed

    Wu, Yue; Zhang, Zhili; Ombrello, Timothy M

    2013-07-01

    Coherent microwave Rayleigh scattering (Radar) from resonance-enhanced multiphoton ionization (REMPI) was demonstrated to directly and nonintrusively measure singlet delta oxygen, O(2)(a(1)Δ(g)), with high spatial resolution. Two different approaches, photodissociation of ozone and microwave discharge plasma in an argon and oxygen flow, were utilized for O(2)(a(1)Δ(g)) generation. The d(1)Π(g)←a(1)Δ(g) (3-0) and d(1)Π(g)←a(1)Δ(g) (1-0) bands of O(2)(a(1)Δ(g)) were detected by Radar REMPI for two different flow conditions. Quantitative absorption measurements using sensitive off-axis integrated cavity output spectroscopy (ICOS) was used simultaneously to evaluate the accuracy and sensitivity of the Radar REMPI technique. The detection limit of Radar REMPI was found to be comparable to the ICOS technique with a detection threshold of approximately 10(14) molecules/cm(3) but with a spatial resolution that was 8 orders of magnitude smaller than the ICOS technique.

  13. Measurement of the fractional oxygenation of leghemoglobin in intact detached pea nodules by reflectance spectroscopy

    SciTech Connect

    Monroe, J.D.; Owens, T.G. ); LaRue, T.A. )

    1989-10-01

    A method is presented for the rapid measurement of the spectral properties of detached nodules of pea (Pisum sativum L. cv Sparkle) by diffuse reflectance spectroscopy. After correcting the spectra for surface light scattering, the spectrum of leghemoglobin is obtained. From this, the fractional oxygenation of leghemoglobin and the internal O{sub 2} concentration can be calculated. With this method, we determined internal O{sub 2} while measuring nitrogenase activity (C{sub 2}H{sub 2}) in detached pea nodules over a range of external O{sub 2} concentrations. Nitrogenase activity was maximum when leghemoglobin was 25% oxygenated, corresponding to a calculated free O{sub 2} concentration of 45 nanomolar in infected cells. Advantages of this method over previous methods which employed transmitted light are: (a) many nodules can be assayed simultaneously, (b) nitrogenase activity (C{sub 2}H{sub 2}) can be determined at the same time as spectra are recorded, and (c) spectra can be obtained from nodules submerged in buffer containing metabolic effectors.

  14. Reconstructing seasonal climate from high-resolution carbon and oxygen isotope measurements across tree rings

    NASA Astrophysics Data System (ADS)

    Schubert, B.; Jahren, H.

    2014-12-01

    Intra-annual records of carbon (δ13C) and oxygen (δ18O) isotope measurements across tree rings reveal significant changes in δ13C and δ18O value across each growing season. We previously found that across a broad range of climate regimes, the seasonal change in δ13C measured within tree rings reflects changes in seasonal precipitation amount, and demonstrated its utility for quantifying seasonal paleo-precipitation from non-permineralized, fossil wood. Here we produce an equation relating intra-ring changes in δ18O to seasonal changes in temperature and precipitation amount, but the equation yields for unknowns (summer and winter precipitation amounts, and cold and warm month mean temperatures). By combining high-resolution δ13C and δ18O records with independent estimates of mean annual temperature and mean annual precipitation, we show how our general, global relationships could be used to quantify seasonal climate information from fossil sites. We validate our approach using high-resolution δ13C and δ18O data from trees growing at five modern sites (Hawaii, Alaska, Norway, Guyana, and Kenya). The reconstructed estimates of seasonal precipitation and temperature showed excellent agreement with the known climate data for each site (precipitation: R2 = 0.98; temperature: R2 = 0.91). These results confirm that across diverse sites and tree species, seasonal climate information can be accurately quantified using a combination of carbon and oxygen intra-ring isotope profiles.

  15. Assessment of skin flaps using optically based methods for measuring blood flow and oxygenation.

    PubMed

    Payette, Jeri R; Kohlenberg, Elicia; Leonardi, Lorenzo; Pabbies, Arone; Kerr, Paul; Liu, Kan-Zhi; Sowa, Michael G

    2005-02-01

    The objective of this study was to compare two noninvasive techniques, laser Doppler and optical spectroscopy, for monitoring hemodynamic changes in skin flaps. Animal models for assessing these changes in microvascular free flaps and pedicle flaps were investigated. A 2 x 3-cm free flap model based on the epigastric vein-artery pair and a reversed MacFarlane 3 x 10-cm pedicle flap model were used in this study. Animals were divided into four groups, with groups 1 (n = 6) and 2 (n = 4) undergoing epigastric free flap surgery and groups 3 (n = 3) and 4 (n = 10) undergoing pedicle flap surgery. Groups 1 and 4 served as controls for each of the flap models. Groups 2 and 3 served as ischemia-reperfusion models. Optical spectroscopy provides a measure of hemoglobin oxygen saturation and blood volume, and the laser Doppler method measures blood flow. Optical spectroscopy proved to be consistently more reliable in detecting problems with arterial in flow compared with laser Doppler assessments. When spectroscopy was used in an imaging configuration, oxygen saturation images of the entire flap were generated, thus creating a visual picture of global flap health. In both single-point and imaging modes the technique was sensitive to vessel manipulation, with the immediate post operative images providing an accurate prediction of eventual outcome. This series of skin flap studies suggests a potential role for optical spectroscopy and spectroscopic imaging in the clinical assessment of skin flaps.

  16. In vivo measurements of the influence of the skin on cerebral oxygenation changes measured with near-infrared spectrophotometry (NIRS)

    NASA Astrophysics Data System (ADS)

    Klaessens, John H. G. M.; van Os, Sandra H. G.; Hopman, Jeroen C. W.; Liem, K. D.; van de Bor, Margot; Thijssen, Johan M.

    2004-07-01

    Goal: To investigate the influence of skin on the accuracy and precision of regional cerebral oxygenation measurements using CW-NIRS and to reduce the inter individual variability of NIRS measurements by normalization with data from an extra wavelength. Method: Three piglets (7.8-9.3 kg) were anesthetized, paralyzed and mechanically ventilated. Receiving optodes were placed over the left and right hemisphere (C3, C4 EEG placement code) and one emitting optode on Cz position (optode distance=1.8cm). Optical densities (OD) were measured for 3 wavelengths (767, 850, 905 nm) (OXYMON) during stable normoxic, mild and deep hypoxemic conditions (SaO2=100%, 80% and 60%) of one minute in each region. This was repeated 3 times: all optodes with skin (condition 1); one receiving optode directly on the skull (2); emitting and also receiving optode on the skull (3). The absolute cO2Hb, cHHb, ctHb concentrations (μmol/L) were calculated from the OD's and changes with respect to the SaO2=100% condition were estimated. Because ODs varied over a large range, the light intensity was externally attenuated to adapt to the range of the spectrophotometer. The data were then corrected for these attenuation effects and for pathlength changes caused by skin removal using the OD at the independent wavelength (λ=975nm). Results: Removal of the skin resulted in an increase of the absorption values (average 0.25 OD in condition 2 and 0.42 OD in condition 3 with respect to condition 1). The change from normoxic to medium, and to deep hypoxic conditions produced a decrease of cO2Hb (-15, and -29 μmol/L, respectively), an increase in cHHb (+16, and +35 μmol/L) and in ctHb (+1, and +5 μmol/L). Total skin removal yielded an extra change in cO2Hb (-5, -1 μmol/L), cHHb (+8, +9 μmol/L), and ctHb (+3, +8 μmol /L). The coefficient of variability of the absolute concentration changes was considerably decreased by the normalization of densities by the density obtained at 795 nm. Conclusion: Skin

  17. Isolation and Characterization of Single Cells from Zebrafish Embryos

    PubMed Central

    Samsa, Leigh Ann; Fleming, Nicole; Magness, Scott; Qian, Li; Liu, Jiandong

    2017-01-01

    The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been described at the morphological level, little is known about the molecular basis of cellular changes that occur as the organism develops. With recent advancements in microfluidics and multiplexing technologies, it is now possible to characterize gene expression in single cells. This allows for investigation of heterogeneity between individual cells of specific cell populations to identify and classify cell subtypes, characterize intermediate states that occur during cell differentiation, and explore differential cellular responses to stimuli. This study describes a protocol to isolate viable, single cells from zebrafish embryos for high throughput multiplexing assays. This method may be rapidly applied to any zebrafish embryonic cell type with fluorescent markers. An extension of this method may also be used in combination with high throughput sequencing technologies to fully characterize the transcriptome of single cells. As proof of principle, the relative abundance of cardiac differentiation markers was assessed in isolated, single cells derived from nkx2.5 positive cardiac progenitors. By evaluation of gene expression at the single cell level and at a single time point, the data support a model in which cardiac progenitors coexist with differentiating progeny. The method and work flow described here is broadly applicable to the zebrafish research community, requiring only a labeled transgenic fish line and access to microfluidics technologies. PMID:27022828

  18. [Recent progress in single-cell RNA-Seq analysis].

    PubMed

    Lu, Wen; Fuchou, Tang

    2014-11-01

    Cell heterogeneity is a general feature of biological tissues. Standard transcriptome analysis approaches require tens of thousands of cells to provide an average view of gene expression and ignore the information of gene expression heterogeneity. The single-cell RNA-Seq technologies profile gene expression at the single-cell level and serve as powerful tools to identify distinct phenotypic cell types within a heterogeneous population. The single-cell RNA-Seq technologies have been developed rapidly in recent years. The methodological progress includes a variety of cDNA amplification methods, the quantitative analysis of the sensitivity and noise of the technologies, and the development of the low-coverage high-throughput single-cell RNA-Seq and the in situ RNA-Seq technologies. Furthermore, the scope of application is extended from early embryonic development to tissue and organ development, immunology and oncology. In this review, we discuss recent progress in methodology and applications of the single-cell RNA-Seq technologies.

  19. Detection of aneuploidy in single cells using comparative genomic hybridization.

    PubMed

    Voullaire, L; Wilton, L; Slater, H; Williamson, R

    1999-09-01

    The ability of comparative genomic hybridization (CGH) to detect aneuploidy following universal amplification of DNA from a single cell, or a small number of cells, was investigated with a view to preimplantation diagnosis following in vitro fertilization, and prenatal diagnosis using fetal erythroblasts obtained from maternal blood. The DNA obtained from lysed single cells was amplified using degenerate oligonucleotide-primed PCR (DOP-PCR). This product was labelled using nick translation and hybridized together with normal reference genomic DNA. The CGH fluorescent ratio profiles obtained could be used to determine aneuploidy with cut-off thresholds of 0.75 and 1.25. Deviation in the profiles in the heterochromatic regions was reduced by using, as a reference sample, normal genomic DNA that had also undergone DOP-PCR. Single cells known to be trisomic for chromosomes 13, 18 or 21 were analysed using this technique. The resolution of CGH with amplified DNA from a single cell is of the order of 40 Mb, sufficient for the diagnosis of trisomy 21, and possibly segmental aneuploidy of equivalent size. These results, and those of others, demonstrate that diagnosis of chromosomal aneuploidy in single cells is possible using CGH with DOP-PCR amplified DNA.

  20. 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.

  1. A comprehensive strategy for the analysis of acoustic compressibility and optical deformability on single cells.

    PubMed

    Yang, Tie; Bragheri, Francesca; Nava, Giovanni; Chiodi, Ilaria; Mondello, Chiara; Osellame, Roberto; Berg-Sørensen, Kirstine; Cristiani, Ilaria; Minzioni, Paolo

    2016-04-04

    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.

  2. Optical measurement of cerebral hemodynamics and oxygen metabolism in neonates with congenital heart defects

    PubMed Central

    Durduran, Turgut; Zhou, Chao; Buckley, Erin M.; Kim, Meeri N.; Yu, Guoqiang; Choe, Regine; Gaynor, J. William; Spray, Thomas L.; Durning, Suzanne M.; Mason, Stefanie E.; Montenegro, Lisa M.; Nicolson, Susan C.; Zimmerman, Robert A.; Putt, Mary E.; Wang, Jiongjiong; Greenberg, Joel H.; Detre, John A.; Yodh, Arjun G.; Licht, Daniel J.

    2010-01-01

    We employ a hybrid diffuse correlation spectroscopy (DCS) and near-infrared spectroscopy (NIRS) monitor for neonates with congenital heart disease (n=33). The NIRS-DCS device measured changes during hypercapnia of oxyhemoglobin, deoxyhemoglobin, and total hemoglobin concentrations; cerebral blood flow (rCBFDCS); and oxygen metabolism (rCMRO2). Concurrent measurements with arterial spin-labeled magnetic resonance imaging (rCBFASL-MRI, n=12) cross-validate rCBFDCS against rCBFASL-MRI, showing good agreement (R=0.7, p=0.01). The study demonstrates use of NIRS-DCS on a critically ill neonatal population, and the results indicate that the optical technology is a promising clinical method for monitoring this population. PMID:20615033

  3. Integrated device for the measurement of systemic and local oxygen transport during physical exercise.

    PubMed

    Pollonini, Luca; Re, Rebecca; Simpson, Richard J; Dacso, Clifford C

    2012-01-01

    Current methods for monitoring exercise exertion rely upon heart rate monitors, which represent a crude and lagging indicator of conditioning. The rationale for the present study is that both systemic and local metabolic mechanisms are responsible for physical performance, and therefore they should be simultaneously quantified to achieve an objective assessment of human conditioning. We propose a compact, wearable near-infrared spectroscopy (NIRS) device integrated with electrocardiography (ECG) and photoplethysmography (PPG) to simultaneously assess the cardiovascular and local response to exercise. The system was tested on subjects performing a graded maximal exercise by comparing our readings with metabolic variables measured with respiratory gas analysis. We found strong correlations between local deoxyhemoglobin concentration [HHb], heart rate and oxygen uptake, as well as between oxyhemoglobin concentration [HbO(2)] and stroke volume. This study shows that combined NIRS, ECG and PPG measurements yield useful information to understand the interplay between systemic and local muscular responses to exercise.

  4. Optical measurement of cerebral hemodynamics and oxygen metabolism in neonates with congenital heart defects

    NASA Astrophysics Data System (ADS)

    Durduran, Turgut; Zhou, Chao; Buckley, Erin M.; Kim, Meeri N.; Yu, Guoqiang; Choe, Regine; Gaynor, J. William; Spray, Thomas L.; Durning, Suzanne M.; Mason, Stefanie E.; Montenegro, Lisa M.; Nicolson, Susan C.; Zimmerman, Robert A.; Putt, Mary E.; Wang, Jiongjiong; Greenberg, Joel H.; Detre, John A.; Yodh, Arjun G.; Licht, Daniel J.

    2010-05-01

    We employ a hybrid diffuse correlation spectroscopy (DCS) and near-infrared spectroscopy (NIRS) monitor for neonates with congenital heart disease (n=33). The NIRS-DCS device measured changes during hypercapnia of oxyhemoglobin, deoxyhemoglobin, and total hemoglobin concentrations; cerebral blood flow (rCBFDCS); and oxygen metabolism (rCMRO2). Concurrent measurements with arterial spin-labeled magnetic resonance imaging (rCBFASL-MRI, n=12) cross-validate rCBFDCS against rCBFASL-MRI, showing good agreement (R=0.7, p=0.01). The study demonstrates use of NIRS-DCS on a critically ill neonatal population, and the results indicate that the optical technology is a promising clinical method for monitoring this population.

  5. Measured oxygen fugacities of the Angra dos Reis achondrite as a function of temperature

    USGS Publications Warehouse

    Brett, R.; Stephen, Huebner J.; Sato, M.

    1977-01-01

    Measurements of the oxygen fugacity (f{hook}O2) as a function of temperature (T) were made on an interior bulk sample of the cumulate achondrite, Angra dos Reis. Data clustered between the f{hook}O2-T relationship of the iron-wu??stite assemblage and 1.2 log atm units above iron-wu??stite. Interpretation of the data indicates that, throughout most of the cooling history of the meteorite, f{hook}O2 values were defined by equilibria involving iron-bearing species at values close to the f{hook}O2 of the assemblage iron-wu??stite. Measured f{hook}O2 data are compatible with crystallization and cooling at pressures greater than 50 bars. ?? 1977.

  6. Evapotranspiration partitioning through in-situ oxygen isotope measurements in an oasis cropland

    NASA Astrophysics Data System (ADS)

    Wen, Xue-Fa

    2016-04-01

    The oxygen isotope compositions of ecosystem water pools and fluxes are useful tracers in the water cycle. As part of the Heihe Watershed Allied Telemetry Experimental Research (HiWATER) program, high-frequency and near-continuous in situ measurements of 18O composition of atmospheric vapor (δv) and of evapotranspiration (δET) were made with the flux-gradient method using a cavity ring-down spectroscopy water vapor isotope analyzer. At the sub-daily scale, we found, in conjunction with intensive isotopic measurements of other ecosystem water pools, that the differences between 18O composition of transpiration (δT) and of xylem water (δx) were negligible in early afternoon (13:00-15:00 Beijing time) when ET approached the daytime maximum, indicating isotopic steady state. At the daily scale, for the purpose of flux partitioning, δT was approximated by δx at early afternoon hours, and the 18O composition of soil evaporation (δE) was obtained from the Craig-Gordon model with a moisture-dependent soil resistance. The relative contribution of transpiration to evapotranspiration ranged from 0.71 to 0.96 with a mean of 0.87 ± 0.052 for the growing season according to the isotopic labeling, which was good agreement with soil lysimeter measurements showing a mean transpiration fraction of 0.86 ± 0.058. At the growing season scale, the predicted18O composition of runoff water was within the range of precipitation and irrigation water according to the isotopic mass conservation. The 18O mass conservation requires that the decreased δ18O of ET should be balanced by enhanced δ18O of runoff water. (Wen, XF*, Yang, B, Sun, XM, Lee, X. 2015. Evapotranspiration partitioning through in-situ oxygen isotope measurements in an oasis cropland. Agricultural and Forest Meteorology , doi:10.1016/j.agrformet.2015.12.003).

  7. Dynamic Measurements of Greenhouse Gas Respirations Caused by Changing Oxygen Levels

    NASA Astrophysics Data System (ADS)

    Fleck, D.; Saad, N.

    2015-12-01

    The necessity for constant monitoring of greenhouse gases (GHGs) is clearly evident now more than ever. Moreover, interpreting and understanding the processes that dictate the production and consumption of these gases will allow for proper management of GHGs in order to mitigate its detrimental climate effects. Presence of oxygen, or lack of it, is the driving force for determining pathways within biochemical redox reactions. Experiments to find correlations between oxygen and greenhouse gases have helped us understand photosynthesis, denitrification and beyond. Within the past few years measurements of O2 and nitrous oxide have been used over a wide ranging array of disciplines; from studying avenues for redox chemistry to characterizing gas profiles in sputum of cystic fibrosis patients. We present a full analysis solution, based on cavity ring-down spectroscopy, for simultaneous measurements of N2O, CO2, CH4, H2O, NH3, and O2 concentrations in soil flux, in order to better understand dynamics of ecological and biogeochemical processes. The stability and high temporal resolution of the five-species CRDS analyzer, coupled with a continuous high-precision O2 measurement (1-σ <200ppm) produces a complete picture of biogeochemical processes, for which a multitude of additional research experiments can be conceived. Adding another dimension to explore to help determine the rate at which these greenhouse gases are produced or consumed, allows scientists to further address fundamental scientific questions. Data is presented showing precision, drift and limitations of the O2 sensor measurement as well as the validity of spectroscopic corrections with the CRDS analyzer caused by changing O2. Experimental data is also presented to explore correlations of soil respiration rates of N2O, CO2 and CH4 due to differing soil O2 contents at varying timescales from minutes to days.

  8. 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

  9. Measuring oxygen tension modulation, induced by a new pre-radiotherapy therapeutic, in a mammary window chamber mouse model

    NASA Astrophysics Data System (ADS)

    Schafer, Rachel; Gmitro, Arthur F.

    2015-03-01

    Tumor regions under hypoxic or low oxygen conditions respond less effectively to many treatment strategies, including radiation therapy. A novel investigational therapeutic, NVX-108 (NuvOx Pharma), has been developed to increase delivery of oxygen through the use of a nano-emulsion of dodecofluoropentane. By raising pO2 levels prior to delivering radiation, treatment efficacy may be improved. To aid in evaluating the novel drug, oxygen tension was quantitatively measured, spatially and temporally, to record the effect of administrating NVX-108 in an orthotopic mammary window chamber mouse model of breast cancer. The oxygen tension was measured through the use of an oxygen-sensitive coating, comprised of phosphorescent platinum porphyrin dye embedded in a polystyrene matrix. The coating, applied to the surface of the coverslip of the window chamber through spin coating, is placed in contact with the mammary fat pad to record the oxygenation status of the surface tissue layer. Prior to implantation of the window chamber, a tumor is grown in the SCID mouse model by injection of MCF-7 cells into the mammary fat pad. Two-dimensional spatial distributions of the pO2 levels were obtained through conversion of measured maps of phosphorescent lifetime. The resulting information on the spatial and temporal variation of the induced oxygen modulation could provide valuable insight into the optimal timing between administration of NVX-108 and radiation treatment to provide the most effective treatment outcome.

  10. Single cell electroporation using proton beam fabricated biochips

    NASA Astrophysics Data System (ADS)

    Homhuan, S.; Zhang, B.; Sheu, F.-S.; Bettiol, A. A.; Watt, F.

    2010-05-01

    We report the design and fabrication of a novel single cell electroporation biochip fabricated by the Proton Beam Writing technique (PBW), a new technique capable of direct-writing high-aspect-ratio nano and microstructures. The biochip features nickel micro-electrodes with straight-side walls between which individual cells are positioned. By applying electrical impulses across the electrodes, SYTOX® Green nucleic acid stain is incorporated into mouse neuroblastoma (N2a) cells. When the stain binds with DNA inside the cell nucleus, green fluorescence is observed upon excitation from a halogen lamp. Three parameters; electric field strength, pulse duration, and the number of pulses have been considered and optimized for the single cell electroporation. The results show that our biochip gives successfully electroporated cells . This single cell electroporation system represents a promising method for investigating the introduction of a wide variety of fluorophores, nanoparticles, quantum dots, DNAs and proteins into cells.

  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 RNA-seq: advances and future challenges.

    PubMed

    Saliba, Antoine-Emmanuel; Westermann, Alexander J; Gorski, Stanislaw A; Vogel, Jörg

    2014-08-01

    Phenotypically identical cells can dramatically vary with respect to behavior during their lifespan and this variation is reflected in their molecular composition such as the transcriptomic landscape. Single-cell transcriptomics using next-generation transcript sequencing (RNA-seq) is now emerging as a powerful tool to profile cell-to-cell variability on a genomic scale. Its application has already greatly impacted our conceptual understanding of diverse biological processes with broad implications for both basic and clinical research. Different single-cell RNA-seq protocols have been introduced and are reviewed here-each one with its own strengths and current limitations. We further provide an overview of the biological questions single-cell RNA-seq has been used to address, the major findings obtained from such studies, and current challenges and expected future developments in this booming field.

  13. Massively parallel digital transcriptional profiling of single cells.

    PubMed

    Zheng, Grace X Y; Terry, Jessica M; Belgrader, Phillip; Ryvkin, Paul; Bent, Zachary W; Wilson, Ryan; Ziraldo, Solongo B; Wheeler, Tobias D; McDermott, Geoff P; Zhu, Junjie; Gregory, Mark T; Shuga, Joe; Montesclaros, Luz; Underwood, Jason G; Masquelier, Donald A; Nishimura, Stefanie Y; Schnall-Levin, Michael; Wyatt, Paul W; Hindson, Christopher M; Bharadwaj, Rajiv; Wong, Alexander; Ness, Kevin D; Beppu, Lan W; Deeg, H Joachim; McFarland, Christopher; Loeb, Keith R; Valente, William J; Ericson, Nolan G; Stevens, Emily A; Radich, Jerald P; Mikkelsen, Tarjei S; Hindson, Benjamin J; Bielas, Jason H

    2017-01-16

    Characterizing the transcriptome of individual cells is fundamental to understanding complex biological systems. We describe a droplet-based system that enables 3' mRNA counting of tens of thousands of single cells per sample. Cell encapsulation, of up to 8 samples at a time, takes place in ∼6 min, with ∼50% cell capture efficiency. To demonstrate the system's technical performance, we collected transcriptome data from ∼250k single cells across 29 samples. We validated the sensitivity of the system and its ability to detect rare populations using cell lines and synthetic RNAs. We profiled 68k peripheral blood mononuclear cells to demonstrate the system's ability to characterize large immune populations. Finally, we used sequence variation in the transcriptome data to determine host and donor chimerism at single-cell resolution from bone marrow mononuclear cells isolated from transplant patients.

  14. 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.

  15. Dense transcript profiling in single cells by image correlation decoding

    PubMed Central

    Coskun, Ahmet F.; Cai, Long

    2016-01-01

    Recent work in sequential fluorescent in-situ hybridization (FISH) has demonstrated the ability to uniquely encode a large number of molecular species in single cells. However, the multiplexing capacity is practically limited by the density of the barcoded objects in the cell. Here, we present a general method using image correlation to resolve the temporal barcodes in sequential hybridization experiments, allowing high density objects to be decoded. Using this correlation FISH (corrFISH) approach, we profiled the gene expression of ribosomal proteins in single cells in cell cultures and in mouse thymus tissue sections. In tissues, corrFISH revealed cell type specific gene expression of ribosomal proteins. The combination of sequential barcoding FISH and correlation analyses provides a general strategy for multiplexing a large number of RNA molecules and potentially other high copy number molecules in single cells. PMID:27271198

  16. 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

  17. Massively parallel digital transcriptional profiling of single cells

    PubMed Central

    Zheng, Grace X. Y.; Terry, Jessica M.; Belgrader, Phillip; Ryvkin, Paul; Bent, Zachary W.; Wilson, Ryan; Ziraldo, Solongo B.; Wheeler, Tobias D.; McDermott, Geoff P.; Zhu, Junjie; Gregory, Mark T.; Shuga, Joe; Montesclaros, Luz; Underwood, Jason G.; Masquelier, Donald A.; Nishimura, Stefanie Y.; Schnall-Levin, Michael; Wyatt, Paul W.; Hindson, Christopher M.; Bharadwaj, Rajiv; Wong, Alexander; Ness, Kevin D.; Beppu, Lan W.; Deeg, H. Joachim; McFarland, Christopher; Loeb, Keith R.; Valente, William J.; Ericson, Nolan G.; Stevens, Emily A.; Radich, Jerald P.; Mikkelsen, Tarjei S.; Hindson, Benjamin J.; Bielas, Jason H.

    2017-01-01

    Characterizing the transcriptome of individual cells is fundamental to understanding complex biological systems. We describe a droplet-based system that enables 3′ mRNA counting of tens of thousands of single cells per sample. Cell encapsulation, of up to 8 samples at a time, takes place in ∼6 min, with ∼50% cell capture efficiency. To demonstrate the system's technical performance, we collected transcriptome data from ∼250k single cells across 29 samples. We validated the sensitivity of the system and its ability to detect rare populations using cell lines and synthetic RNAs. We profiled 68k peripheral blood mononuclear cells to demonstrate the system's ability to characterize large immune populations. Finally, we used sequence variation in the transcriptome data to determine host and donor chimerism at single-cell resolution from bone marrow mononuclear cells isolated from transplant patients. PMID:28091601

  18. 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.

  19. Wishbone identifies bifurcating developmental trajectories from single-cell data

    PubMed Central

    Setty, Manu; Tadmor, Michelle D; Reich-Zeliger, Shlomit; Angel, Omer; Salame, Tomer Meir; Kathail, Pooja; Choi, Kristy; Bendall, Sean; Friedman, Nir; Pe’er, Dana

    2016-01-01

    Recent single-cell analysis technologies offer an unprecedented opportunity to elucidate developmental pathways. Here we present Wishbone, an algorithm for positioning single cells along bifurcating developmental trajectories with high resolution. Wishbone uses multi-dimensional single-cell data, such as mass cytometry or RNA-seq data, as input and orders cells according to their developmental progression by pinpointing bifurcation points and labeling each cell as pre-bifurcation or as one of two post-bifurcation cell fates. Using 30-channel mass cytometry data, we show that Wishbone accurately recovers the known stages of T cell development in the mouse thymus, including the bifurcation point. We also apply the algorithm to mouse myeloid differentiation and demonstrate its generalization to additional lineages. A comparison of Wishbone to diffusion maps, SCUBA and Monocle shows that it outperforms these methods both in the accuracy of ordering cells and in the correct identification of branch points. PMID:27136076

  20. Exploring viral infection using single-cell sequencing.

    PubMed

    Rato, Sylvie; Golumbeanu, Monica; Telenti, Amalio; Ciuffi, Angela

    2016-11-02

    Single-cell sequencing (SCS) has emerged as a valuable tool to study cellular heterogeneity in diverse fields, including virology. By studying the viral and cellular genome and/or transcriptome, the dynamics of viral infection can be investigated at single cell level. Most studies have explored the impact of cell-to-cell variation on the viral life cycle from the point of view of the virus, by analyzing viral sequences, and from the point of view of the cell, mainly by analyzing the cellular host transcriptome. In this review, we will focus on recent studies that use single-cell sequencing to explore viral diversity and cell variability in response to viral replication.

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

    PubMed

    Yu, Zhilong; Lu, Sijia; Huang, Yanyi

    2014-10-07

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

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

    PubMed

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

    2016-10-07

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

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

    PubMed Central

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

    2011-01-01

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

  4. Optoacoustic measurement of central venous oxygenation for assessment of circulatory shock: clinical study in cardiac surgery patients

    NASA Astrophysics Data System (ADS)

    Petrov, Irene Y.; Prough, Donald S.; Kinsky, Michael; Petrov, Yuriy; Petrov, Andrey; Henkel, S. Nan; Seeton, Roger; Salter, Michael G.; Esenaliev, Rinat O.

    2014-03-01

    Circulatory shock is a dangerous medical condition, in which blood flow cannot provide the necessary amount of oxygen to organs and tissues. Currently, its diagnosis and therapy decisions are based on hemodynamic parameters (heart rate, blood pressure, blood gases) and mental status of a patient, which all have low specificity. Measurement of mixed or central venous blood oxygenation via catheters is more reliable, but highly invasive and associated with complications. Our previous studies in healthy volunteers demonstrated that optoacoustic systems provide non-invasive measurement of blood oxygenation in specific vessels, including central veins. Here we report our first results of a clinical study in coronary artery bypass graft (CABG) surgery patients. We used a medical-grade OPO-based optoacoustic system developed in our laboratory to measure in real time blood oxygenation in the internal jugular vein (IJV) of these patients. A clinical ultrasound imaging system (GE Vivid e) was used for IJV localization. Catheters were placed in the IJV as part of routine care and blood samples taken via the catheters were processed with a CO-oximeter. The optoacoustic oxygenation data were compared to the CO-oximeter readings. Good correlation between the noninvasive and invasive measurements was obtained. The results of these studies suggest that the optoacoustic system can provide accurate, noninvasive measurements of central venous oxygenation that can be used for patients with circulatory shock.

  5. 3D restoration microscopy improves quantification of enzyme-labeled fluorescence-based single-cell phosphatase activity in plankton.

    PubMed

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

    2014-10-01

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

  6. Chlorine measurement in the jet singlet oxygen generator considering the effects of the droplets

    NASA Astrophysics Data System (ADS)

    Goodarzi, Mohamad S.; Saghafifar, Hossein

    2016-09-01

    A new method is presented to measure chlorine concentration more accurately than conventional method in exhaust gases of a jet-type singlet oxygen generator. One problem in this measurement is the existence of micrometer-sized droplets. In this article, an empirical method is reported to eliminate the effects of the droplets. Two wavelengths from a fiber coupled LED are adopted and the measurement is made on both selected wavelengths. Chlorine is measured by the two-wavelength more accurately than the one-wavelength method by eliminating the droplet term in the equations. This method is validated without the basic hydrogen peroxide injection in the reactor. In this case, a pressure meter value in the diagnostic cell is compared with the optically calculated pressure, which is obtained by the one-wavelength and the two-wavelength methods. It is found that chlorine measurement by the two-wavelength method and pressure meter is nearly the same, while the one-wavelength method has a significant error due to the droplets.

  7. 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

  8. 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.

  9. Pooled CRISPR screening with single-cell transcriptome readout.

    PubMed

    Datlinger, Paul; Rendeiro, André F; Schmidl, Christian; Krausgruber, Thomas; Traxler, Peter; Klughammer, Johanna; Schuster, Linda C; Kuchler, Amelie; Alpar, Donat; Bock, Christoph

    2017-03-01

    CRISPR-based genetic screens are accelerating biological discovery, but current methods have inherent limitations. Widely used pooled screens are restricted to simple readouts including cell proliferation and sortable marker proteins. Arrayed screens allow for comprehensive molecular readouts such as transcriptome profiling, but at much lower throughput. Here we combine pooled CRISPR screening with single-cell RNA sequencing into a broadly applicable workflow, directly linking guide RNA expression to transcriptome responses in thousands of individual cells. Our method for CRISPR droplet sequencing (CROP-seq) enables pooled CRISPR screens with single-cell transcriptome resolution, which will facilitate high-throughput functional dissection of complex regulatory mechanisms and heterogeneous cell populations.

  10. 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

  11. A microfluidic device enabling high-efficiency single cell trapping

    PubMed Central

    Jin, D.; Deng, B.; Cai, W.; Tu, L.; Chen, J.; Wu, Q.; Wang, W. H.

    2015-01-01

    Single cell trapping increasingly serves as a key manipulation technique in single cell analysis for many cutting-edge cell studies. Due to their inherent advantages, microfluidic devices have been widely used to enable single cell immobilization. To further improve the single cell trapping efficiency, this paper reports on a passive hydrodynamic microfluidic device based on the “least flow resistance path” principle with geometry optimized in line with corresponding cell types. Different from serpentine structure, the core trapping structure of the micro-device consists of a series of concatenated T and inverse T junction pairs which function as bypassing channels and trapping constrictions. This new device enhances the single cell trapping efficiency from three aspects: (1) there is no need to deploy very long or complicated channels to adjust flow resistance, thus saving space for each trapping unit; (2) the trapping works in a “deterministic” manner, thus saving a great deal of cell samples; and (3) the compact configuration allows shorter flowing path of cells in multiple channels, thus increasing the speed and throughput of cell trapping. The mathematical model of the design was proposed and optimization of associated key geometric parameters was conducted based on computational fluid dynamics (CFD) simulation. As a proof demonstration, two types of PDMS microfluidic devices were fabricated to trap HeLa and HEK-293T cells with relatively significant differences in cell sizes. Experimental results showed 100% cell trapping and 90% single cell trapping over 4 × 100 trap sites for these two cell types, respectively. The space saving is estimated to be 2-fold and the cell trapping speed enhancement to be 3-fold compared to previously reported devices. This device can be used for trapping various types of cells and expanded to trap cells in the order of tens of thousands on 1-cm2 scale area, as a promising tool to pattern large-scale single cells on

  12. Single-cell analysis tools for drug discovery and development.

    PubMed

    Heath, James R; Ribas, Antoni; Mischel, Paul S

    2016-03-01

    The genetic, functional or compositional heterogeneity of healthy and diseased tissues presents major challenges in drug discovery and development. Such heterogeneity hinders the design of accurate disease models and can confound the interpretation of biomarker levels and of patient responses to specific therapies. The complex nature of virtually all tissues has motivated the development of tools for single-cell genomic, transcriptomic and multiplex proteomic analyses. Here, we review these tools and assess their advantages and limitations. Emerging applications of single cell analysis tools in drug discovery and development, particularly in the field of oncology, are discussed.

  13. Oxygen potential measurements in high burnup LWR U0 2 fuel

    NASA Astrophysics Data System (ADS)

    Matzke, Hj.

    1995-05-01

    A miniature solid state galvanic cell was used to measure the oxygen potential Δ overlineG( O2) of reactor irradiated U0 2 fuel at different burnups in the range of 28 to ⩾ 150 GWd d/t M. This very high burnup was achieved in the rim region of a fuel with a cross section average burnup of 75 GWd d/t M. The fuels had different enrichments and therefore different contributions of fission of 235U and 239Pu. The temperature range covered was 900 to 1350 K. None of the fuels showed a significant oxidation. Rather, if allowance is made for the dissolved rare earth fission products and the Pu formed during irradiation, some of the fuels were very slightly substoichiometric and the highest possible degree of oxidation corresponded to U0 2.001. In general, the Δ overlineG( O2) at 750°C was about -400 kJ/mol, corresponding to the Δ overlineG( O2) of the reaction Mo + O 2 → MoO 2. The implication of these results which are in contrast to commonly assumed ideas that U0 2 fuel oxidizes due to burnup, are discussed and the importance of the fission product Mo and of the zircaloy clad as oxygen buffers is outlined.

  14. Direct measurement of local oxygen concentration in the bone marrow of live animals

    NASA Astrophysics Data System (ADS)

    Spencer, Joel A.; Ferraro, Francesca; Roussakis, Emmanuel; Klein, Alyssa; Wu, Juwell; Runnels, Judith M.; Zaher, Walid; Mortensen, Luke J.; Alt, Clemens; Turcotte, Raphaël; Yusuf, Rushdia; Côté, Daniel; Vinogradov, Sergei A.; Scadden, David T.; Lin, Charles P.

    2014-04-01

    Characterization of how the microenvironment, or niche, regulates stem cell activity is central to understanding stem cell biology and to developing strategies for the therapeutic manipulation of stem cells. Low oxygen tension (hypoxia) is commonly thought to be a shared niche characteristic in maintaining quiescence in multiple stem cell types. However, support for the existence of a hypoxic niche has largely come from indirect evidence such as proteomic analysis, expression of hypoxia inducible factor-1α (Hif-1α) and related genes, and staining with surrogate hypoxic markers (for example, pimonidazole). Here we perform direct in vivo measurements of local oxygen tension (pO2) in the bone marrow of live mice. Using two-photon phosphorescence lifetime microscopy, we determined the absolute pO2 of the bone marrow to be quite low (<32 mm Hg) despite very high vascular density. We further uncovered heterogeneities in local pO2, with the lowest pO2 (~9.9 mm Hg, or 1.3%) found in deeper peri-sinusoidal regions. The endosteal region, by contrast, is less hypoxic as it is perfused with small arteries that are often positive for the marker nestin. These pO2 values change markedly after radiation and chemotherapy, pointing to the role of stress in altering the stem cell metabolic microenvironment.

  15. Estimation of the viscosities of silicate liquids at high pressure from measurements of oxygen diffusivity

    NASA Technical Reports Server (NTRS)

    Rubie, D. C.; Ross, C. R., II; Carroll, M. R.; Elphick, S. C.

    1992-01-01

    The dynamics and evolution of a magma ocean depend significantly on the structure and physical properties of silicate liquids at high pressure. Viscosity is particularly important because of its effect on the dynamics of convection and crystal setting. Traditionally, the viscosities of silicate liquids have been measured at high pressure by falling sphere viscometry but, due to some limitations of this technique, few measurements have been made at pressures exceeding 2.5 GPa. An alternative approach, which has previously been proven up to 2 GPa, is to use high pressure measurements of oxygen self-diffusivity (D) to estimate viscosity (eta) from the Eyring relationship eta = kT/D lambda (where k is the Boltzmann constant, T is absolute temperature, and lambda is the diffusive jump distance). We have performed preliminary experiments on Na2Si4O9 liquid in order to explore the feasibility of this method up to pressures in excess of 10 GPa. Diffusion couples were prepared from glass starting materials with one half of the sample enriched in O-18 and the other half containing the natural abundance (0.2 wt percent). Diffusion experiments were performed at 1600 to 1800 C at pressures in the range of 2.5 to 10 GPa for times up to 6 minutes using a 1200 ton multianvil apparatus. Oxygen diffusivities were derived from the resulting O-18 concentration profiles, which were analyzed using an ion microprobe. At 1800 C, oxygen diffusivities increase continuously from 1 x 10(exp -10) m(exp -2) s(exp -1) at 2.5 Gpa to 5 x 10(exp -10) m(exp -2) s(exp -1) at 10 GPa. From the Eyring relationship, the viscosity of Na2SiO9 liquid is predicted to decrease by approx. 0.7 log units over this pressure range at 1800 C. These trends, which can be related to changes in the structure of the liquid at high pressure, are in agreement with results of molecular dynamics calculations. These results suggest that it should be possible to estimate the viscosities of silicate liquids up to at least 15

  16. From Molecules to Cells to Organisms: Understanding Health and Disease with Multidimensional Single-Cell Methods

    NASA Astrophysics Data System (ADS)

    Candia, Julián

    2013-03-01

    The multidimensional nature of many single-cell measurements (e.g. multiple markers measured simultaneously using Fluorescence-Activated Cell Sorting (FACS) technologies) offers unprecedented opportunities to unravel emergent phenomena that are governed by the cooperative action of multiple elements across different scales, from molecules and proteins to cells and organisms. We will discuss an integrated analysis framework to investigate multicolor FACS data from different perspectives: Singular Value Decomposition to achieve an effective dimensional reduction in the data representation, machine learning techniques to separate different patient classes and improve diagnosis, as well as a novel cell-similarity network analysis method to identify cell subpopulations in an unbiased manner. Besides FACS data, this framework is versatile: in this vein, we will demonstrate an application to the multidimensional single-cell shape analysis of healthy and prematurely aged cells.

  17. BAYESIAN HIERARCHICAL MODELING FOR SIGNALING PATHWAY INFERENCE FROM SINGLE CELL INTERVENTIONAL DATA1

    PubMed Central

    Luo, Ruiyan; Zhao, Hongyu

    2011-01-01

    Recent technological advances have made it possible to simultaneously measure multiple protein activities at the single cell level. With such data collected under different stimulatory or inhibitory conditions, it is possible to infer the causal relationships among proteins from single cell interventional data. In this article we propose a Bayesian hierarchical modeling framework to infer the signaling pathway based on the posterior distributions of parameters in the model. Under this framework, we consider network sparsity