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Sample records for measuring single-cell oxygen

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-03-01

    A noninvasive, self-referencing biosensor/probe system has been integrated into the Columbia University Radiological Research Accelerator Facility Microbeam II end station. A single-cell oxygen consumption measurement has been conducted with this type of oxygen probe in 37° C Krebs-Ringer Bicarbonate buffer immediately before and after a single-cell microbeam irradiation. It is the first such measurement made for a microbeam irradiation, and a six fold increment of oxygen flux induced during a 15-s period of time has been observed following radiation exposure. The experimental procedure and the results are discussed. PMID:25335641

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

    PubMed Central

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

    2015-01-01

    A noninvasive, self-referencing biosensor/probe system has been integrated into the Columbia University Radiological Research Accelerator Facility Microbeam II end station. A single-cell oxygen consumption measurement has been conducted with this type of oxygen probe in 37°C Krebs–Ringer Bicarbonate buffer immediately before and after a single-cell microbeam irradiation. It is the first such measurement made for a microbeam irradiation, and a six fold increment of oxygen flux induced during a 15-s period of time has been observed following radiation exposure. The experimental procedure and the results are discussed. PMID:25335641

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

    PubMed Central

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

    2009-01-01

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

  5. 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

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

    NASA Technical Reports Server (NTRS)

    Colvin, James; Schallhorn, Paul; Ramohalli, Kumar

    1991-01-01

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

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

    NASA Astrophysics Data System (ADS)

    Gao, Yang

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

  8. A photoacoustic technique to measure the properties of single cells

    NASA Astrophysics Data System (ADS)

    Strohm, Eric M.; Berndl, Elizabeth S. L.; Kolios, Michael C.

    2013-03-01

    We demonstrate a new technique to non-invasively determine the diameter and sound speed of single cells using a combined ultrasonic and photoacoustic technique. Two cell lines, B16-F1 melanoma cells and MCF7 breast cancer cells were examined using this technique. Using a 200 MHz transducer, the ultrasound backscatter from a single cell in suspension was recorded. Immediately following, the cell was irradiated with a 532 nm laser and the resulting photoacoustic wave recorded by the same transducer. The melanoma cells contain optically absorbing melanin particles, which facilitated photoacoustic wave generation. MCF7 cells have negligible optical absorption at 532 nm; the cells were permeabilized and stained with trypan blue prior to measurements. The measured ultrasound and photoacoustic power spectra were compared to theoretical equations with the cell diameter and sound speed as variables (Anderson scattering model for ultrasound, and a thermoelastic expansion model for photoacoustics). The diameter and sound speed were extracted from the models where the spectral shape matched the measured signals. However the photoacoustic spectrum for the melanoma cell did not match theory, which is likely because melanin particles are located around the cytoplasm, and not within the nucleus. Therefore a photoacoustic finite element model of a cell was developed where the central region was not used to generate a photoacoustic wave. The resulting power spectrum was in better agreement with the measured signal than the thermoelastic expansion model. The MCF7 cell diameter obtained using the spectral matching method was 17.5 μm, similar to the optical measurement of 16 μm, while the melanoma cell diameter obtained was 22 μm, similar to the optical measurement of 21 μm. The sound speed measured from the MCF7 and melanoma cell was 1573 and 1560 m/s, respectively, which is within acceptable values that have been published in literature.

  9. 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. PMID:26616110

  10. High Throughput Micropatterning of Optical Oxygen Sensor for Single Cell Analysis

    PubMed Central

    Zhu, Haixin; Tian, Yanqing; Bhushan, Shivani; Su, Fengyu; Meldrum, Deirdre R.

    2012-01-01

    In this paper, we present our results from process development and characterization of optical oxygen sensors that are patterned by traditional UV lithography. An oxygen sensitive luminescent probe, platinum octaethylporphyrin (PtOEP), was encapsulated in commercially purchased photoresist (AZ5214) to form uniform thin sensor films on fused silica substrates. Plasticizer ethoxylated trimethylolpropane triacrylate (SR454) was added to the dye-photoresist sensor mixtures to improve the oxygen sensitivity. The optimum sensor mixture composition that can be patterned with maximum sensitivity was identified. The microfabrication process conditions, cell adherence and oxygen sensitivity results from patterned structures were characterized in detail. Down to 3 µm features have been fabricated on fused silica substrates using the developed techniques. The result implies the developed methods can provide a feasible way to miniaturize the optical sensor system for single cell analysis with precise control of sensor volume and response PMID:23066352

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

    NASA Astrophysics Data System (ADS)

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

    2015-08-01

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

  12. Simultaneous Multiplexed Measurement of RNA and Proteins in Single Cells

    PubMed Central

    Darmanis, Spyros; Gallant, Caroline Julie; Marinescu, Voichita Dana; Niklasson, Mia; Segerman, Anna; Flamourakis, Georgios; Fredriksson, Simon; Assarsson, Erika; Lundberg, Martin; Nelander, Sven; Westermark, Bengt; Landegren, Ulf

    2015-01-01

    Summary Significant advances have been made in methods to analyze genomes and transcriptomes of single cells, but to fully define cell states, proteins must also be accessed as central actors defining a cell’s phenotype. Methods currently used to analyze endogenous protein expression in single cells are limited in specificity, throughput, or multiplex capability. Here, we present an approach to simultaneously and specifically interrogate large sets of protein and RNA targets in lysates from individual cells, enabling investigations of cell functions and responses. We applied our method to investigate the effects of BMP4, an experimental therapeutic agent, on early-passage glioblastoma cell cultures. We uncovered significant heterogeneity in responses to treatment at levels of RNA and protein, with a subset of cells reacting in a distinct manner to BMP4. Moreover, we found overall poor correlation between protein and RNA at the level of single cells, with proteins more accurately defining responses to treatment. PMID:26748716

  13. 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.

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

    NASA Astrophysics Data System (ADS)

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

    2015-06-01

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

  15. 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. PMID:26133851

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

    SciTech Connect

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

    1987-03-01

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

  17. 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

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

    PubMed

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

    2015-12-01

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

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

    PubMed Central

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

    2015-01-01

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

  20. 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

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

    PubMed

    Guo, Xiaoliang; Zhu, Rong; Zong, Xianli

    2015-10-01

    This paper presents a novel microarray chip integrating cell positioning with in situ, real-time and long-time impedance measurement on a single cell. The microchip integrates a plurality of quadrupole-electrode units (termed positioning electrodes) patterned into an array with pairs of planar electrodes (termed measuring electrodes) located at the centers of each quadrupole-electrode unit. The positioning electrodes are utilized to trap and position living cells onto the measuring electrodes based on negative dielectrophoresis (nDEP), while the measuring electrodes are used to measure impedances of the trapped single cells. Each measuring electrode has a small footprint area of 7 × 7 μm(2) to ensure inhabiting only one single cell on it. However, the electrode with a small surface area has a low double-layer capacitance when it is immersed in a liquid solution, thus generating a large double-layer impedance, which reduces the sensitivity for impedance measurement on the single cell. To enlarge the effective surface areas of the measuring electrodes, a novel surface-modification process is proposed to controllably construct gold nanostructures on the surfaces of the measuring electrodes while the positioning electrodes are unstained. The double layer capacitances of the modified electrodes are increased by about one order after surface-modification. The developed microchip is used to monitor the adhering behavior of a single HeLa cell by measuring its impedance spectra in real time. The measured impedance is analyzed and used to extract cellular electrical parameters, which demonstrated that the cell compresses the electrical double layer in the process of adherence and adheres onto the measuring electrodes after 4-5 hours. PMID:26282920

  2. Nanofork for single cells adhesion measurement via ESEM-nanomanipulator system.

    PubMed

    Ahmad, Mohd Ridzuan; Nakajima, Masahiro; Kojima, Masaru; Kojima, Seiji; Homma, Michio; Fukuda, Toshio

    2012-03-01

    In this paper, single cells adhesion force was measured using a nanofork. The nanofork was used to pick up a single cell on a line array substrate inside an environmental scanning electron microscope (ESEM). The line array substrate was used to provide small gaps between the single cells and the substrate. Therefore, the nanofork could be inserted through these gaps in order to successfully pick up a single cell. Adhesion force was measured during the cell pick-up process from the deflection of the cantilever beam. The nanofork was fabricated using focused ion beam (FIB) etching process while the line array substrate was fabricated using nanoimprinting technology. As to investigate the effect of contact area on the strength of the adhesion force, two sizes of gap distance of line array substrate were used, i.e., 1 μm and 2 μm. Results showed that cells attached on the 1 μm gap line array substrate required more force to be released as compared to the cells attached on the 1 μm gap line array substrate. PMID:22275723

  3. Measurement of nitric oxide in single cells and tissue using a porphyrinic microsensor.

    PubMed

    Malinski, T; Huk, I

    2001-05-01

    This unit describes the preparation and applications of porphyrinic sensors for quantitative measurement of nitric oxide (NO) in single cells and in tissues. The determination of NO is based on the electrochemical oxidation of NO on a carbon fiber electrode covered with a thin layer of a conducting polymeric metalloporphyrin catalyst, overlaid with another thin film of Nafion, a cation exchange material. The electric current generated during NO oxidation on the surface of the polymeric porphyrin is linearly proportional to the concentration of NO, so this current is used as an analytical signal which can be measured in either the amperometric or the voltammetric mode. Both methods provide a quantitative signal. This unit describes the electrochemical setup for measurement of NO in single cells and tissue. Support protocols describe porphyrin synthesis, sensor preparation, and sensor calibration. PMID:18428525

  4. 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.

  5. 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

  6. Quantitative single-cell gene expression measurements of multiple genes in response to hypoxia treatment.

    PubMed

    Zeng, Jia; Wang, Jiangxin; Gao, Weimin; Mohammadreza, Aida; Kelbauskas, Laimonas; Zhang, Weiwen; Johnson, Roger H; Meldrum, Deirdre R

    2011-07-01

    Cell-to-cell heterogeneity in gene transcription plays a central role in a variety of vital cell processes. To quantify gene expression heterogeneity patterns among cells and to determine their biological significance, methods to measure gene expression levels at the single-cell level are highly needed. We report an experimental technique based on the DNA-intercalating fluorescent dye SYBR green for quantitative expression level analysis of up to ten selected genes in single mammalian cells. The method features a two-step procedure consisting of a step to isolate RNA from a single mammalian cell, synthesize cDNA from it, and a qPCR step. We applied the method to cell populations exposed to hypoxia, quantifying expression levels of seven different genes spanning a wide dynamic range of expression in randomly picked single cells. In the experiment, 72 single Barrett's esophageal epithelial (CP-A) cells, 36 grown under normal physiological conditions (controls) and 36 exposed to hypoxia for 30 min, were randomly collected and used for measuring the expression levels of 28S rRNA, PRKAA1, GAPDH, Angptl4, MT3, PTGES, and VEGFA genes. The results demonstrate that the method is sensitive enough to measure alterations in gene expression at the single-cell level, clearly showing heterogeneity within a cell population. We present technical details of the method development and implementation, and experimental results obtained by use of the procedure. We expect the advantages of this technique will facilitate further developments and advances in the field of single-cell gene expression profiling on a nanotechnological scale, and eventually as a tool for future point-of-care medical applications. PMID:21614642

  7. Frequency measurement of the prototype storage ring stainless steel single cell cavity

    SciTech Connect

    Reisinger, E.A.

    1992-07-29

    Frequency measurements were made on the stainless steel single cell cavity after prototype storage ring at the Advanced Photon Source with various port terminations, using two small loops. The cavity contains six larger ports. The top and bottom ports have a diameter of 144 mm, the front and back ports (beam ports) have a diameter of 140 mm, and the two side ports have a diameter of 120 mm. The cavity also have four smaller ports of diameter 34.8 mm, which contain an E-probe, a H-loop, and two field probes.

  8. 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.

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

    NASA Astrophysics Data System (ADS)

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

    2016-04-01

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

  10. 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.

  11. Measuring single cell mass, volume, and density with dual suspended microchannel resonators.

    PubMed

    Bryan, Andrea K; Hecht, Vivian C; Shen, Wenjiang; Payer, Kristofor; Grover, William H; Manalis, Scott R

    2014-02-01

    Cell size, measured as either volume or mass, is a fundamental indicator of cell state. Far more tightly regulated than size is density, the ratio between mass and volume, which can be used to distinguish between cell populations even when volume and mass appear to remain constant. Here we expand upon a previous method for measuring cell density involving a suspended microchannel resonator (SMR). We introduce a new device, the dual SMR, as a high-precision instrument for measuring single-cell mass, volume, and density using two resonators connected by a serpentine fluidic channel. The dual SMR designs considered herein demonstrate the critical role of channel geometry in ensuring proper mixing and damping of pressure fluctuations in microfluidic systems designed for precision measurement. We use the dual SMR to compare the physical properties of two well-known cancer cell lines: human lung cancer cell H1650 and mouse lymphoblastic leukemia cell line L1210. PMID:24296901

  12. Measurement of Protein Tyrosine Phosphatase Activity in Single Cells by Capillary Electrophoresis

    PubMed Central

    Phillips, Ryan M.; Bair, Eric; Lawrence, David S.; Sims, Christopher E.; Allbritton, Nancy L.

    2013-01-01

    A fluorescent peptide substrate was used to measure dephosphorylation by protein tyrosine phosphatases (PTP) in cell lysates, and single cells and to investigate the effect of environmental toxins on PTP activity in these systems. Dephosphorylation of the substrate by PTPN1 and PTPN2 obeyed Michaelis-Menten kinetics, with KM values of 770 ± 250 nM and 290 ± 54 nM, respectively. Dose-response curves and IC50 values were determined for the inhibition of these two enzymes by the environmental toxins Zn2+ and 1,2-naphthoquinone, as well as pervanadate. In A431 cell lysates, the reporter was a poor substrate for peptidases (degradation rate of 100 ± 8.2 fmol min−1 mg−1) but an excellent substrate for phosphatases (dephosphorylation rate of 1.4 ± 0.3 nmol min−1 mg−1). Zn2+, 1,2-naphthoquinone and pervanadate inhibited dephosphorylation of the reporter in cell lysates with IC50 values of 470 nM, 35 μM, and 100 nM, respectively. Dephosphorylation of the reporter following loading into living single cells occurred at rates of at least 2 pmol min−1 mg−1. When single cells were exposed to 1,2-naphthoquinone (50 μM), Zn2+ (100 μM), and pervandate (1 mM), dephosphorylation was inhibited with median values and first and third quartile values of 41 (Q1 = 0%, Q3 = 96%), 50 (Q1 = 46%, Q3 = 74%), and 53% (Q1 = 36%, Q3 = 77%), respectively, demonstrating both the impact of these toxic exposures on cell signaling and the heterogeneity of response between cells. This approach will provide a valuable tool for the study of PTP dynamics, particularly in small, heterogeneous populations such as human biopsy specimens. PMID:23682679

  13. 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.

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

    PubMed

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

    2015-12-15

    The motility of cilia and flagella is driven by thousands of dynein motors that hydrolyze adenosine triphosphate (ATP). Despite decades of genetic, biochemical, structural, and biophysical studies, some aspects of ciliary motility remain elusive, such as the regulation of beating patterns and the energetic efficiency of these nanomachines. In this study, we introduce an experimental method to measure ATP consumption of actively beating axonemes on a single-cell level. We encapsulated individual sea urchin sperm with demembranated flagellum inside water-in-oil emulsion droplets and measured the axoneme's ATP consumption by monitoring fluorescence intensity of a fluorophore-coupled reporter system for ATP turnover in the droplet. Concomitant phase contrast imaging allowed us to extract a linear dependence between the ATP consumption rate and the flagellar beating frequency, with ∼2.3 × 10(5) ATP molecules consumed per beat of a demembranated flagellum. Increasing the viscosity of the aqueous medium led to modified beating waveforms of the axonemes and to higher energy consumption per beat cycle. Our single-cell experimental platform provides both new insights, to our knowledge, into the beating mechanism of flagella and a powerful tool for future studies. PMID:26682814

  15. 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

  16. 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.

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

    PubMed Central

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

    2015-01-01

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

  18. 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.

  19. 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

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

    PubMed Central

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

    2013-01-01

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

  1. Oxygen pressure measurement using singlet oxygen emission

    SciTech Connect

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

    2005-05-15

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

  2. 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

  3. Single Cell Mass Cytometry Adapted to Measurements of the Cell Cycle1

    PubMed Central

    Behbehani, Gregory K.; Bendall, Sean C.; Clutter, Matthew R.; Fantl, Wendy J.; Nolan, Garry P.

    2013-01-01

    Mass cytometry is a recently introduced technology that utilizes transition element isotope-tagged antibodies for protein detection on a single-cell basis. By circumventing the limitations of emission spectral overlap associated with fluorochromes utilized in traditional flow cytometry, mass cytometry currently allows measurement of up to 40 parameters per cell. Recently a comprehensive mass cytometry analysis was described for the hematopoietic differentiation program in human bone marrow from a healthy donor. The present study describes approaches to delineate cell cycle stages utilizing iododeoxyuridine (IdU) to mark cells in S phase, simultaneously with antibodies against cyclin B1, cyclin A, and phosphorylated histone H3 (S28) that characterize the other cell cycle phases. Protocols were developed in which an antibody against phosphorylated retinoblastoma protein (Rb) at serines 807 and 811 was used to separate cells in G0 and G1 phases of the cell cycle. This mass cytometry method yielded cell cycle distributions of both normal and cancer cell populations that were equivalent to those obtained by traditional fluorescence cytometry techniques. We applied this to map the cell cycle phases of cells spanning the hematopoietic hierarchy in healthy human bone marrow as a prelude to later studies with cancers and other disorders of this lineage. PMID:22693166

  4. 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. PMID:25722368

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

    PubMed Central

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

    2015-01-01

    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. PMID:25722368

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

    PubMed Central

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

    2015-01-01

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

  7. Nanowell-Based Immunoassays for Measuring Single-Cell Secretion: Characterization of Transport and Surface Binding

    PubMed Central

    2015-01-01

    Arrays of subnanoliter wells (nanowells) provide a useful system to isolate single cells and analyze their secreted proteins. Two general approaches have emerged: one that uses open arrays and local capture of secreted proteins, and a second (called microengraving) that relies on closed arrays to capture secreted proteins on a solid substrate, which is subsequently removed from the array. However, the design and operating parameters for efficient capture from these two approaches to analyze single-cell secretion have not been extensively considered. Using numerical simulations, we analyzed the operational envelope for both open and closed formats, as a function of the spatial distribution of capture ligands, their affinities for the protein, and the rates of single-cell secretion. Based on these analyses, we present a modified approach to capture secreted proteins in-well for highly active secreting cells. This simple method for in-well detection should facilitate rapid identification of cell lines with high specific productivities. PMID:25347613

  8. Direct Measurement of Aluminum Uptake and Distribution in Single Cells of Chara corallina1

    PubMed Central

    Taylor, Gregory J.; McDonald-Stephens, Julie L.; Hunter, Douglas B.; Bertsch, Paul M.; Elmore, David; Rengel, Zdenko; Reid, Robert J.

    2000-01-01

    Quantitative information on the uptake and distribution of Al at the cellular level is required to understand mechanisms of Al toxicity, but direct measurement of uptake across the plasma membrane has remained elusive. We measured rates of Al transport across membranes in single cells of Chara corallina using the rare 26Al isotope, an emerging technology (accelerator mass spectrometry), and a surgical technique for isolating subcellular compartments. Accumulation of Al in the cell wall dominated total uptake (71–318 μg m−2 min−1), although transport across the plasma membrane was detectable (71–540 ng m−2 min−1) within 30 min of exposure. Transport across the tonoplast was initially negligible, but accelerated to rates approximating uptake across the plasma membrane. The avacuolate protoplasm showed signs of saturation after 60 min, but continued movement across the plasma membrane was supported by sequestration in the vacuole. Saturation of all compartments was observed after 12 to 24 h. Accumulation of Al in the cell wall reflected variation in {Al3+} induced by changes in Al supply or complexing ligands, but was unaffected by pH. In contrast, transport across the plasma membrane peaked at pH 4.3 and increased when {Al3+} was reduced by complexing ligands. Cold temperature (4°C) reduced accumulation in the cell wall and protoplasm, whereas 2,4-dinitrophenol and m-chlorocarbonylcyanidephenyl hydrazone increased membrane transport by 12- to 13-fold. Our data suggest that the cell wall is the major site of Al accumulation. Nonetheless, membrane transport occurs within minutes of exposure and is supported by subsequent sequestration in the vacuole. The rapid delivery of Al to the protoplasm suggests that intracellular lesions may be possible. PMID:10889247

  9. Measurement of free cytosolic calcium in single cells: method and application.

    PubMed

    Raue, F; Zink, A

    1992-05-01

    Intracellular calcium [Ca2+]i acts as an important intracellular messenger system for secretion and synthesis, cell growth and differentiation. In order to demonstrate definitively that a change in [Ca2+]i is responsible for a physiological event, one has to measure [Ca2+]i directly within intact cells and correlate the time course of any [Ca2+]i changes with the biological response. Measurement of [Ca2+]i was done in a single cell preloaded with fluorescent Ca indicator fura2 using a fluorescent unit (lonoquant) consisting of an inverted microscope (Zeiss IM 35) equipped with a mercury lamp and a rotating filter wheel containing filters at wavelengths of 340 and 380 nm. Cells were alternately excited and emission signals of fura 2-loaded cells were collected by a photomultiplier and recorded on-line on a computer screen. As a model system, the rat C-cell carcinoma cell line rMTC 6-23 secreting calcitonin was used. An acute elevation of extracellular calcium resulted in an increase in [Ca2+]i within 5 sec and rapid release of preformed calcitonin. This tight linkage between extracellular calcium and [Ca2+]i is mediated via Ca influx through voltage-dependent Ca channels. These channels are modulated by intracellular cAMP, yielding a rhythmic oscillation of [Ca2+]i, as well as by extracellular somatostatin blocking the Ca channel and the increase of [Ca2+]i via a pertussis toxin sensitive Gi protein. The change in [Ca2+]i is associated with changes in calcitonin secretion, confirming the stimulus secretion coupling via voltage-dependent Ca channels in C-cells. PMID:1354776

  10. Single Cell Multiplex Protein Measurements through Rare Earth Element Immunolabeling, Laser Capture Microdissection and Inductively Coupled Mass Spectrometry

    PubMed Central

    Liba, Amir; Wanagat, Jonathan

    2016-01-01

    Complex diseases such as heart disease, stroke, cancer, and aging are the primary causes of death in the US. These diseases cause heterogeneous conditions among cells, conditions that cannot be measured in tissue homogenates and require single cell approaches. Understanding protein levels within tissues is currently assayed using various molecular biology techniques (e.g., Western blots) that rely on milligram to gram quantities of tissue homogenates or immunofluorescent (IF) techniques that are limited by spectral overlap. Tissue homogenate studies lack references to tissue structure and mask signals from individual or rare cellular events. Novel techniques are required to bring protein measurement sensitivity to the single cell level and offer spatiotemporal resolution and scalability. We are developing a novel approach to protein quantification by exploiting the inherently low concentration of rare earth elements (REE) in biological systems. By coupling REE-antibody immunolabeling of cells with laser capture microdissection (LCM) and ICP-QQQ, we are achieving multiplexed protein measurement in histological sections of single cells. This approach will add to evolving single cell techniques and our ability to understand cellular heterogeneity in complex biological systems and diseases.

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

    PubMed Central

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

    2013-01-01

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

  12. Single cell adhesion force measurement for cell viability identification using an AFM cantilever-based micro putter

    NASA Astrophysics Data System (ADS)

    Shen, Yajing; Nakajima, Masahiro; Kojima, Seiji; Homma, Michio; Kojima, Masaru; Fukuda, Toshio

    2011-11-01

    Fast and sensitive cell viability identification is a key point for single cell analysis. To address this issue, this paper reports a novel single cell viability identification method based on the measurement of single cell shear adhesion force using an atomic force microscopy (AFM) cantilever-based micro putter. Viable and nonviable yeast cells are prepared and put onto three kinds of substrate surfaces, i.e. tungsten probe, gold and ITO substrate surfaces. A micro putter is fabricated from the AFM cantilever by focused ion beam etching technique. The spring constant of the micro putter is calibrated using the nanomanipulation approach. The shear adhesion force between the single viable or nonviable cell and each substrate is measured using the micro putter based on the nanorobotic manipulation system inside an environmental scanning electron microscope. The adhesion force is calculated based on the deflection of the micro putter beam. The results show that the adhesion force of the viable cell to the substrate is much larger than that of the nonviable cell. This identification method is label free, fast, sensitive and can give quantitative results at the single cell level.

  13. 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.

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

    PubMed Central

    Tankus, Ariel; Yeshurun, Yehezkel; Fried, Itzhak

    2010-01-01

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

  15. 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. PMID:26963790

  16. 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

  17. 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

  18. 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.}

  19. 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.

  20. 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.

  1. 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.

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

    DOE PAGESBeta

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

    2013-03-27

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

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

    PubMed

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

    2009-03-01

    In the non-heterocyst, marine cyanobacterium Trichodesmium nitrogen fixation is confined to the photoperiod and occurs coevally with oxygenic photosynthesis although nitrogenase is irreversibly inactivated by oxygen. In previous studies it was found that regulation of photosynthesis for nitrogen fixation involves Mehler reaction and various activity states with reversible coupling of photosynthetic components. We now investigated these activity states in more detail. Spectrally resolved fluorescence kinetic measurements of single cells revealed that they were related to alternate uncoupling and coupling of phycobilisomes from and to the photosystems, changing the effective cross-section of PSII. Therefore, we isolated and purified the phycobiliproteins of Trichodesmium via ion exchange chromatography and recorded their UV/VIS absorption, fluorescence excitation and fluorescence emission spectra. After describing these spectra by mathematical equations via the Gauss-Peak-Spectra method, we used them to deconvolute the in vivo fluorescence spectra of Trichodesmium cells. This revealed that the contribution of different parts of the phycobilisome antenna to fluorescence quenching changed during the daily activity cycle, and that individual phycobiliproteins can be reversibly coupled to the photosystems, while the expression levels of these proteins did not change much during the daily activity cycle. Thus we propose that variable phycobilisome coupling plays a key role in the regulation of photosynthesis for nitrogen fixation in Trichodesmium. PMID:19186173

  4. Measuring bacterial adhesion at environmental interfaces with single-cell and single-molecule techniques

    NASA Astrophysics Data System (ADS)

    Camesano, Terri A.; Liu, Yatao; Datta, Meera

    2007-06-01

    A synopsis is provided of techniques currently used to quantify the interactions between bacterial cells and surfaces. Focus is placed on techniques which allow for direct probing of nano, pico, or femto-scale interaction forces between bacteria and surfaces of relevance for environmental science and engineering. We focus on bacterial adhesion measurements and surface characterizations via techniques that measure forces on individual bacterial cells or cellular macromolecules, particularly atomic force microscopy (AFM) and related force spectroscopy. However, we also include overviews of other techniques useful for evaluating cellular forces, such as optical tweezers, evanescent wave scattering-based techniques (i.e. total internal reflection microscopy (TIRM) and total internal reflection aqueous fluorescence (TIRAF) microscopy) and the quartz crystal microbalance (QCM). These latter techniques, while most are not providing direct measurements of forces of adhesion, can be used to explain adhesion and interaction forces in bacterial systems. We review the operating principles, advantages and limitations of each technique, and key bacterial adhesion studies from each area are presented. Qualitative and quantitative methodologies for relating force measurements to bacterial attachment, particularly to bacterial retention in porous media, are discussed.

  5. Single Cell Measurement of Dopamine Release with Simultaneous Voltage-clamp and Amperometry

    PubMed Central

    Saha, Kaustuv; Swant, Jarod; Khoshbouei, Habibeh

    2012-01-01

    After its release into the synaptic cleft, dopamine exerts its biological properties via its pre- and post-synaptic targets1. The dopamine signal is terminated by diffusion2-3, extracellular enzymes4, and membrane transporters5. The dopamine transporter, located in the peri-synaptic cleft of dopamine neurons clears the released amines through an inward dopamine flux (uptake). The dopamine transporter can also work in reverse direction to release amines from inside to outside in a process called outward transport or efflux of dopamine5. More than 20 years ago Sulzer et al. reported the dopamine transporter can operate in two modes of activity: forward (uptake) and reverse (efflux)5. The neurotransmitter released via efflux through the transporter can move a large amount of dopamine to the extracellular space, and has been shown to play a major regulatory role in extracellular dopamine homeostasis6. Here we describe how simultaneous patch clamp and amperometry recording can be used to measure released dopamine via the efflux mechanism with millisecond time resolution when the membrane potential is controlled. For this, whole-cell current and oxidative (amperometric) signals are measured simultaneously using an Axopatch 200B amplifier (Molecular Devices, with a low-pass Bessel filter set at 1,000 Hz for whole-cell current recording). For amperometry recording a carbon fiber electrode is connected to a second amplifier (Axopatch 200B) and is placed adjacent to the plasma membrane and held at +700 mV. The whole-cell and oxidative (amperometric) currents can be recorded and the current-voltage relationship can be generated using a voltage step protocol. Unlike the usual amperometric calibration, which requires conversion to concentration, the current is reported directly without considering the effective volume7. Thus, the resulting data represent a lower limit to dopamine efflux because some transmitter is lost to the bulk solution. PMID:23207721

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

    NASA Astrophysics Data System (ADS)

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

    1999-03-01

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

  7. 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.

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

    PubMed

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

    2016-01-01

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

  9. 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

  10. Quantification of the specific membrane capacitance of single cells using a microfluidic device and impedance spectroscopy measurement.

    PubMed

    Tan, Qingyuan; Ferrier, Graham A; Chen, Brandon K; Wang, Chen; Sun, Yu

    2012-09-01

    The specific membrane capacitance (SMC) is an electrical parameter that correlates with both the electrical activity and morphology of the plasma membrane, which are physiological markers for cellular phenotype and health. We have developed a microfluidic device that enables impedance spectroscopy measurements of the SMC of single biological cells. Impedance spectra induced by single cells aspirated into the device are captured over a moderate frequency range (5 kHz-1 MHz). Maximum impedance sensitivity is achieved using a tapered microfluidic channel, which effectively routes electric fields across the cell membranes. The SMC is extracted by curve-fitting impedance spectra to an equivalent circuit model. From our measurement, acute myeloid leukemia (AML) cells are found to exhibit larger SMC values in hypertonic solutions as compared with those in isotonic solutions. In addition, AML cell phenotypes (AML2 and NB4) exhibiting varying metastatic potential yield distinct SMC values (AML2: 16.9 ± 1.9 mF/m(2) (n = 23); NB4: 22.5 ± 4.7 mF/m(2) (n = 23)). Three-dimensional finite element simulations of the microfluidic device confirm the feasibility of this approach. PMID:23940502

  11. Measuring Oxygen Isotopes with COSIMA

    NASA Astrophysics Data System (ADS)

    Paquette, J. A.; Engrand, C.; Stenzel, O.; Hilchenbach, M.

    2014-12-01

    Oxygen isotopes in a variety of solar system solids show non-mass-dependent fractionation, i.e. are fractionated along a slope = 1 line in a three isotope plot, rather than the equilibrium fractionation line whose slope is close to 0.5 (Clayton, 1973). Many models have been put forward to explain this observation, such as galactic chemical evolution (Clayton, 1988), photochemical self-shielding (Thiemens and Jackson, 1987; Clayton, 2002; Yurimoto and Kuramoto, 2004; Lyons and Young, 2005), quantum chemical explanations (Hathorn and Marcus, 1999, 2000; Gao and Marcus, 2002; Marcus, 2004), the processing of solids via nebular lightning (Nuth et al, 2011), and others. Some of the models were invalidated when the Genesis results showed that the oxygen isotopic fractionation of solar wind (and hence of the Sun) was relatively much richer in 16O than such bodies as the Earth or the Moon. Whatever the process that produced non-mass-dependent fractionation in some chondrules and calcium aluminum inclusions, its signature may also be detectable in other solar system solids. If at least some cometary dust was produced in the inner nebula and only later transported outward to be incorporated into comets, then such dust may also show some degree of non-mass-dependent fractionation. The COSIMA instrument on the Rosetta spacecraft (Kissel et al 2009) is a secondary ion mass spectrometer designed to measure the composition of cometary dust. Using calibration data from the COSIMA reference model and flight data if possible, measurement all three isotopes of oxygen will be attempted, and the results compared to other solar system bodies.

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

    PubMed

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

    2016-06-01

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

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

    PubMed

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

    2016-04-01

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

  14. 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)

  15. 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.

  16. Single-cell western blotting

    PubMed Central

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

    2014-01-01

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

  17. A new toolbox for assessing single cells.

    PubMed

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

    2014-01-01

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

  18. Measurements Of Singlet Oxygen In Photodynamic Therapy

    NASA Astrophysics Data System (ADS)

    Profio, A. E.; Shu, Kuang-Hsien

    1989-06-01

    Photochemical reactions are used in photodynamic therapy of cancer and other disease. The cytotoxic agent in photochemotherapy is usually singlet oxygen. Thus measurements of singlet oxygen production or concentration may allow prediction of the biological response. The decrease in fluorescence of L-tryptophan because of reaction with singlet oxygen, the decrease in absorbance of a dye such as RNO subject to secondary oxidation by singlet oxygen, and the decrease in fluorescence of the most common photosensitizer, dihematoporphyrin ether/ester (DHE) because of photobleaching, have been investigated in solutions in vitro. The most promising method for dosimetry and prediction of biological response appears to be the photobleaching of DHE.

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

    PubMed

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

    2003-01-01

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

  20. Oxygen measurements in thin ribbon silicon

    NASA Astrophysics Data System (ADS)

    Hyland, S. L.; Ast, D. G.; Baghdadi, A.

    1987-03-01

    The oxygen content of thin silicon ribbons grown by the dendritic web technique was measured using a modification of the ASTM method based on Fourier transform infrared spectroscopy. Web silicon was found to have a high oxygen content, ranging from 13 to 19 ppma, calculated from the absorption peak associated with interstitial oxygen and using the new ASTM conversion coefficient. The oxygen concentration changed by about 10 percent along the growth direction of the ribbon. In some samples, a shoulder was detected on the absorption peak. A similar shoulder in Czochralski grown material has been variously interpreted in the literature as due to a complex of silicon, oxygen, and vacancies, or to a phase of SiO2 developed along dislocations in the material. In the case of web silicon, it is not clear which is the correct interpretation.

  1. SINGLE CELL GENOME SEQUENCING

    PubMed Central

    Yilmaz, Suzan; Singh, Anup K.

    2011-01-01

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

  2. 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.

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

    PubMed Central

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

    2013-01-01

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

  4. Single-cell measurements of enzyme levels as a predictive tool for cellular fates during organic acid production.

    PubMed

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

    2013-12-01

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

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

    PubMed

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

    2016-07-19

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

  6. 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.

  7. A Method for Detecting Circulating Tumor Cells Based on the Measurement of Single-Cell Metabolism in Droplet-Based Microfluidics.

    PubMed

    Del Ben, Fabio; Turetta, Matteo; Celetti, Giorgia; Piruska, Aigars; Bulfoni, Michela; Cesselli, Daniela; Huck, Wilhelm T S; Scoles, Giacinto

    2016-07-18

    The number of circulating tumor cells (CTCs) in blood is strongly correlated with the progress of metastatic cancer. Current methods to detect CTCs are based on immunostaining or discrimination of physical properties. Herein, a label-free method is presented exploiting the abnormal metabolic behavior of cancer cells. A single-cell analysis technique is used to measure the secretion of acid from individual living tumor cells compartmentalized in microfluidically prepared, monodisperse, picoliter (pL) droplets. As few as 10 tumor cells can be detected in a background of 200 000 white blood cells and proof-of-concept data is shown on the detection of CTCs in the blood of metastatic patients. PMID:27247024

  8. 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.

  9. Single-cell proteins

    SciTech Connect

    Litchfield, J.H.

    1983-02-11

    Both photosynthetic and nonphotosynthetic microorganisms, grown on various carbon and energy sources, are used in fermentation processes for the production of single-cell proteins. Commercial-scale production has been limited to two algal processes, one bacterial process, and several yeast and fungal processes. High capital and operating costs and the need for extensive nutritional and toxicological assessments have limited the development and commercialization of new processes. Any increase in commercial-scale production appears to be limited to those regions of the world where low-cost carbon and energy sources are available and conventional animal feedstuff proteins, such as soybean meal or fish meal, are in short supply. (Refs. 59).

  10. 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

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

    PubMed

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

    2016-01-01

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

  12. 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.

  13. Single cell optical transfection.

    PubMed

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

    2010-06-01

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

  14. 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

  15. 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.

  16. 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

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

    PubMed

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

    2016-01-01

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

  18. 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...

  19. Measuring tissue oxygen tension: a review.

    PubMed

    Sheffield, P J

    1998-01-01

    Because of technological advances in tissue oximetry, clinicians and scientists have a better understanding of the role of oxygen in wound healing. In wound care and hyperbaric medicine applications, an oximeter is principally used with vascular assessment to help determine amputation level and to estimate healing potential. With the current emphasis on cost savings in the managed care setting, transcutaneous oximetry (PtcO2) has gained importance as a tool for predicting potential candidates for hyperbaric oxygen (HBO2) therapy. It is used to identify the presence of hypoxia in wounded tissue, to predict the responders to hyperoxia and in some instances to determine when HBO2 treatment is complete. This literature review describes the principal current methods for measuring tissue O2 and the values obtained in normal and wounded tissue under both normobaric and hyperbaric conditions. The review includes the Jefferson C. Davis Wound Care and Hyperbaric Medicine Center protocol for PtcO2 assessment of potential HBO2 candidates and suggestions for obtaining reproducible PtcO2 data. PMID:9789339

  20. Chemical Analysis of Single Cells

    NASA Astrophysics Data System (ADS)

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

    2008-07-01

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

  1. Quantification noise in single cell experiments

    PubMed Central

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

    2011-01-01

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

  2. 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

  3. 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…

  4. 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

  5. Aquatic Respiration Rate Measurements at Low Oxygen Concentrations

    PubMed Central

    Holtappels, Moritz; Tiano, Laura; Kalvelage, Tim; Lavik, Gaute; Revsbech, Niels Peter; Kuypers, Marcel M. M.

    2014-01-01

    Despite its huge ecological importance, microbial oxygen respiration in pelagic waters is little studied, primarily due to methodological difficulties. Respiration measurements are challenging because of the required high resolution of oxygen concentration measurements. Recent improvements in oxygen sensing techniques bear great potential to overcome these limitations. Here we compare 3 different methods to measure oxygen consumption rates at low oxygen concentrations, utilizing amperometric Clark type sensors (STOX), optical sensors (optodes), and mass spectrometry in combination with 18-18O2 labeling. Oxygen concentrations and consumption rates agreed well between the different methods when applied in the same experimental setting. Oxygen consumption rates between 30 and 400 nmol L−1 h−1 were measured with high precision and relative standard errors of less than 3%. Rate detection limits in the range of 1 nmol L−1 h−1 were suitable for rate determinations in open ocean water and were lowest at the lowest applied O2 concentration. PMID:24586724

  6. 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.

  7. Oxygen saturation resolution influences regularity measurements.

    PubMed

    Garde, Ainara; Karlen, Walter; Dehkordi, Parastoo; Ansermino, J Mark; Dumont, Guy A

    2014-01-01

    The measurement of regularity in the oxygen saturation (SpO(2)) signal has been suggested for use in identifying subjects with sleep disordered breathing (SDB). Previous work has shown that children with SDB have lower SpO(2) regularity than subjects without SDB (NonSDB). Regularity was measured using non-linear methods like approximate entropy (ApEn), sample entropy (SamEn) and Lempel-Ziv (LZ) complexity. Different manufacturer's pulse oximeters provide SpO(2) at various resolutions and the effect of this resolution difference on SpO(2) regularity, has not been studied. To investigate this effect, we used the SpO(2) signal of children with and without SDB, recorded from the Phone Oximeter (0.1% resolution) and the same SpO(2) signal rounded to the nearest integer (artificial 1% resolution). To further validate the effect of rounding, we also used the SpO(2) signal (1% resolution) recorded simultaneously from polysomnography (PSG), as a control signal. We estimated SpO(2) regularity by computing the ApEn, SamEn and LZ complexity, using a 5-min sliding window and showed that different resolutions provided significantly different results. The regularity calculated using 0.1% SpO(2) resolution provided no significant differences between SDB and NonSDB. However, the artificial 1% resolution SpO(2) provided significant differences between SDB and NonSDB, showing a more random SpO(2) pattern (lower SpO(2) regularity) in SDB children, as suggested in the past. Similar results were obtained with the SpO(2) recorded from PSG (1% resolution), which further validated that this SpO(2) regularity change was due to the rounding effect. Therefore, the SpO(2) resolution has a great influence in regularity measurements like ApEn, SamEn and LZ complexity that should be considered when studying the SpO(2) pattern in children with SDB. PMID:25570437

  8. 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.

  9. 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

  10. Non-invasive measurement of blood oxygen levels.

    PubMed

    Beyerl, D

    1982-05-01

    Comparison of transcutaneous (TC) monitoring of blood oxygen levels to arterial blood gas analyses was made on patients at rest with room air, during exercise, and at rest with oxygen. Three different transcutaneous monitors were evaluated: Novametrix TC O2 Mette, Biochem Sensomat, and Radiometer TC M1. The Hewlett-Packard ear oximeter for measuring oxygen saturation was also compared to oxygen saturation values calculated on the Severinghaus slide rule. Using at least one measured PO2 as a baseline, either TC monitoring or ear oximetry were valuable tools in monitoring pulmonary function. PMID:7102718

  11. Mass Cytometry: Single Cells, Many Features.

    PubMed

    Spitzer, Matthew H; Nolan, Garry P

    2016-05-01

    Technology development in biological research often aims to either increase the number of cellular features that can be surveyed simultaneously or enhance the resolution at which such observations are possible. For decades, flow cytometry has balanced these goals to fill a critical need by enabling the measurement of multiple features in single cells, commonly to examine complex or hierarchical cellular systems. Recently, a format for flow cytometry has been developed that leverages the precision of mass spectrometry. This fusion of the two technologies, termed mass cytometry, provides measurement of over 40 simultaneous cellular parameters at single-cell resolution, significantly augmenting the ability of cytometry to evaluate complex cellular systems and processes. In this Primer, we review the current state of mass cytometry, providing an overview of the instrumentation, its present capabilities, and methods of data analysis, as well as thoughts on future developments and applications. PMID:27153492

  12. 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

  13. 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.

  14. 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.

  15. Automated micropipette aspiration of single cells.

    PubMed

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

    2013-06-01

    This paper presents a system for mechanically characterizing single cells using automated micropipette aspiration. Using vision-based control and position control, the system controls a micromanipulator, a motorized translation stage, and a custom-built pressure system to position a micropipette (4 μm opening) to approach a cell, form a seal, and aspirate the cell into the micropipette for quantifying the cell's elastic and viscoelastic parameters as well as viscosity. Image processing algorithms were developed to provide controllers with real-time visual feedback and to accurately measure cell deformation behavior on line. Experiments on both solid-like and liquid-like cells demonstrated that the system is capable of efficiently performing single-cell micropipette aspiration and has low operator skill requirements. PMID:23508635

  16. 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).

  17. 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

  18. Single cell-resolution western blotting.

    PubMed

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

    2016-08-01

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

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

    PubMed

    Antoniu, Sabina; Mihaltan, Florin

    2014-06-01

    Dyspnea is a common symptom in many advanced malignant and non-malignant diseases and often is refractory to the usual therapies. In such circumstances palliative care approaches are necessary and among them palliative care oxygen therapy can be applied although currently its effectiveness is rather uncertain. Palliative oxygen therapy can be given on either continuous basis or on demand. Often the continuous palliative oxygen therapy is seen as long-term oxygen therapy although their aims are rather different. Palliative oxygen therapy was evaluated in populations with mixed underlying diseases, with outcome measures not only the most appropriate for the setting and therefore these limitations might have influenced the overall perceived therapeutic benefit. Therefore an evaluation of this method in subsets defined based on the etiology and pathogenic mechanisms and with appropriate outcome measures would help to better define the criteria for its indication and would increase its acceptability. PMID:24741999

  20. 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.

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

    EPA Science Inventory

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

  2. Oxygen partial pressure measurement in the HVOF gun tail flame

    SciTech Connect

    Korpiola, K.; Hirvonen, J.P.; Jalkanen, H.; Laas, L.; Rossi, F.

    1995-12-31

    An important aspect of the HVOF thermal spray process is the turbulent mixing of the spray jet with the surrounding air. The air mixing into the jet causes undesirable oxidation of the sprayed coating. In this paper a low cost and accurate method to determine the degree of air mixing is presented. This method was used to measure for the first time the partial pressure of oxygen in the thermal spray flame. The measuring method is based on electrochemical determination of oxygen potential in the tail flame using a solid electrolyte cell. The oxygen partial pressure in the HVOF-gun tail flame was measured with the fuel-to-oxygen ratio, the fuel flow rate and the stand-off distance as variables. The oxygen content of the tail flame was measured and found to vary between 4 to 17% depending on fuel to oxygen ratios and stand-off distances. Such high oxygen contents are several magnitudes too high if serious oxidation in the coating is to be avoided.

  3. Measuring Intracellular Ca2+ Changes in Human Sperm using Four Techniques: Conventional Fluorometry, Stopped Flow Fluorometry, Flow Cytometry and Single Cell Imaging

    PubMed Central

    Mata-Martínez, Esperanza; José, Omar; Torres-Rodríguez, Paulina; Solís-López, Alejandra; Sánchez-Tusie, Ana A.; Sánchez-Guevara, Yoloxochitl; Treviño, Marcela B.; Treviño, Claudia L.

    2013-01-01

    Spermatozoa are male reproductive cells especially designed to reach, recognize and fuse with the egg. To perform these tasks, sperm cells must be prepared to face a constantly changing environment and to overcome several physical barriers. Being in essence transcriptionally and translationally silent, these motile cells rely profoundly on diverse signaling mechanisms to orient themselves and swim in a directed fashion, and to contend with challenging environmental conditions during their journey to find the egg. In particular, Ca2+-mediated signaling is pivotal for several sperm functions: activation of motility, capacitation (a complex process that prepares sperm for the acrosome reaction) and the acrosome reaction (an exocytotic event that allows sperm-egg fusion). The use of fluorescent dyes to track intracellular fluctuations of this ion is of remarkable importance due to their ease of application, sensitivity, and versatility of detection. Using one single dye-loading protocol we utilize four different fluorometric techniques to monitor sperm Ca2+ dynamics. Each technique provides distinct information that enables spatial and/or temporal resolution, generating data both at single cell and cell population levels. PMID:23728309

  4. 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.

  5. 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.

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

    NASA Astrophysics Data System (ADS)

    Chudyk, Wayne; Tonaszuck, David; Pohlig, Kenneth

    1993-04-01

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

  7. 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.

  8. HERSCHEL MEASUREMENTS OF MOLECULAR OXYGEN IN ORION

    SciTech Connect

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

    2011-08-20

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

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

    NASA Astrophysics Data System (ADS)

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

    2016-04-01

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

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

    PubMed

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

    2014-01-01

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

  11. Langley 8-foot high-temperature tunnel oxygen measurement system

    NASA Technical Reports Server (NTRS)

    Sprinkle, Danny R.; Chen, Tony D.; Chaturvedi, Sushil K.

    1991-01-01

    In order to ensure that there is a proper amount of oxygen necessary for sustaining test engine operation for hypersonic propulsion systems testing at the NASA Langley 8-foot high-temperature tunnel, a quickly responding real-time measurement system of test section oxygen concentration has been designed and tested at Langley. It is built around a zirconium oxide-based sensor which develops a voltage proportional to the oxygen partial pressure of the test gas. The voltage signal is used to control the amount of oxygen being injected into the combustor air. The physical operation of the oxygen sensor is described, as well as the sampling system used to extract the test gas from the tunnel test section. Results of laboratory tests conducted to verify sensor accuracy and response time performance are discussed, as well as the final configuration of the system to be installed in the tunnel.

  12. [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. PMID:22250527

  13. 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.

  14. Noninvasive measurement of cerebral oxygen saturation and cerebral phronetal function

    NASA Astrophysics Data System (ADS)

    Li, Shengli; Zhang, Aiyu; Xu, Min; Jin, Taiyi

    1998-08-01

    Using the Near-Infrared Spectroscopy (NIRS), the noninvasive measurement of cerebral oxygen concentration can be achieved in vivo based on the Lambert-Beer Law. In this paper, we discuss the possibility of studying higher brain functions through combining cerebral oxygen saturation and cerebral function measurement. Event-related experiments are introduced to measure the cerebral phronetal function. Time domain curves show sight differences among these experiment results. However, with the aid of DFT, experiment data of all five human volunteers show the frequency near 20 Hz or 40 Hz is evoked depending on the difficulty of the mental tasks. The results demonstrate the feasibility of cerebral functions study by means of cerebral oxygen saturation measurement analyzed in the frequency domain.

  15. 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.

  16. 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.

  17. Gloxy: an oxygen-sensitive coal for accurate measurement of low oxygen tensions in biological systems.

    PubMed

    James, P E; Grinberg, O Y; Goda, F; Panz, T; O'Hara, J A; Swartz, H M

    1997-07-01

    This paper describes the characteristics of a new oxygen sensitive, paramagnetic material that has some significant advantages for measurements of tissue pO2 by in vivo EPR. This paramagnetic component of Welsh coal, termed "gloxy" was found to have valuable EPR features that allow accurate measurement of low oxygen tensions in vivo; these include large oxygen-dependent changes in linewidth, a high number of paramagnetic spin centers (resulting in high signal amplitude), and stability in tissue allowing repeated pO2 measurements to be made in vivo with high precision. Renal pO2 was measured deep in the medulla region of isolated perfused kidneys and found to be lower than that in the cortex (1.7 +/- 0.05 and 7.1 +/- 0.3 mm Hg, respectively). The quality of the EPR signal obtained from the renal outer medulla and also from tumors in mice was such that the pO2 measurements were obtained with a precision of +/-3% of the measured pO2 (Kidney: 1.7 +/- 0.05 mmHg; Tumor: 1.37 +/- 0.04 mmHg). In vitro tests on the viability of cells and in vivo studies using Gloxy demonstrate the stability and inertness of this oxygen-sensitive material. PMID:9211379

  18. A Single-Cell Assay for Time Lapse Studies of Exosome Secretion and Cell Behaviors.

    PubMed

    Chiu, Yu-Jui; Cai, Wei; Shih, Yu-Ru V; Lian, Ian; Lo, Yu-Hwa

    2016-07-01

    To understand the inhomogeneity of cells in biological systems, there is a growing demand on the capability of characterizing the properties of individual single cells. Since single-cell studies require continuous monitoring of the cell behaviors, an effective single-cell assay that can support time lapsed studies in a high throughput manner is desired. Most currently available single-cell technologies cannot provide proper environments to sustain cell growth and, proliferation of single cells and convenient, noninvasive tests of single-cell behaviors from molecular markers. Here, a highly versatile single-cell assay is presented that can accommodate different cellular types, enable easy and efficient single-cell loading and culturing, and be suitable for the study of effects of in vitro environmental factors in combination with drug screening. One salient feature of the assay is the noninvasive collection and surveying of single-cell secretions at different time points, producing unprecedented insight of single-cell behaviors based on the biomarker signals from individual cells under given perturbations. Above all, the acquired information is quantitative, for example, measured by the number of exosomes each single-cell secretes for a given time period. Therefore, our single-cell assay provides a convenient, low-cost, and enabling tool for quantitative, time lapsed studies of single-cell properties. PMID:27254278

  19. Temperature measurements from oxygen isotope ratios of fish otoliths.

    PubMed

    Devereux, I

    1967-03-31

    Measurements have shown that the temperature of a fish's habitat can be deduced from the Oxygen isotope ratio of its otoliths (ear bones). Isotope ratios Obtained from fossil otoliths indicate a water temperature which agrees wiht that found by isotope measurements on associated benthonic foraminifera. PMID:6020293

  20. Magnetic levitation of single cells.

    PubMed

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

    2015-07-14

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

  1. Magnetic levitation of single cells

    PubMed Central

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

    2015-01-01

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

  2. A comparison of measurements of the oxygen nightglow and atomic oxygen in the lower thermosphere

    NASA Technical Reports Server (NTRS)

    Siskind, David E.; Sharp, William E.

    1991-01-01

    The relationship between the oxygen nightglow and the atomic oxygen density in the lower thermosphere was investigated. This was done using data from two sounding rocket experiments conducted over White Sands Missile Range (32-deg N, 106-deg W). The first flight was launched on November 2, 1978 while the second was launched on December 7, 1981. Both flights contained resonance lamps to measure the atomic oxygen density. The peak density in both cases was near 1.9 x 10 to the 11th/cu cm. In addition, the 1978 flight contained a photometer to measure the 5577 A green line, while the 1981 flight contained photometers to measure the green line, the UV nightglow, and the 7620 A (0,0) atmospheric band. Empirical models of these airglow features were used to compare with the O density measurements. In the case of the atmospheric band, excellent agreement is seen concerning the shape of the atomic oxygen profile, while some discrepancies were seen with the Herzberg band and the green line. In all cases, the absolute value of our peak O density appeared to be about 2.5 times lower, for a given airglow intensity, than previous measurements.

  3. 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

  4. Microscopic oxygen imaging based on fluorescein bleaching efficiency measurements.

    PubMed

    Beutler, Martin; Heisterkamp, Ines M; Piltz, Bastian; Stief, Peter; De Beer, Dirk

    2014-05-01

    Photobleaching of the fluorophore fluorescein in an aqueous solution is dependent on the oxygen concentration. Therefore, the time-dependent bleaching behavior can be used to measure of dissolved oxygen concentrations. The method can be combined with epi-fluorescence microscopy. The molecular states of the fluorophore can be expressed by a three-state energy model. This leads to a set of differential equations which describe the photobleaching behavior of fluorescein. The numerical solution of these equations shows that in a conventional wide-field fluorescence microscope, the fluorescence of fluorescein will fade out faster at low than at high oxygen concentration. Further simulation showed that a simple ratio function of different time-points during a fluorescence decay recorded during photobleaching could be used to describe oxygen concentrations in an aqueous solution. By careful choice of dye concentration and excitation light intensity the sensitivity in the oxygen concentration range of interest can be optimized. In the simulations, the estimation of oxygen concentration by the ratio function was very little affected by the pH value in the range of pH 6.5-8.5. Filming the fluorescence decay by a charge-coupled-device (ccd) camera mounted on a fluorescence microscope allowed a pixelwise estimation of the ratio function in a microscopic image. Use of a microsensor and oxygen-consuming bacteria in a sample chamber enabled the calibration of the system for quantification of absolute oxygen concentrations. The method was demonstrated on nitrifying biofilms growing on snail and mussel shells, showing clear effects of metabolic activity on oxygen concentrations. PMID:24610786

  5. Nanosecond fluorescence microscopy of single cells

    NASA Astrophysics Data System (ADS)

    Keating, Susan M.; Wensel, Theodore G.

    1990-05-01

    A microscope based time-correlated single photon counting instrument has been used to measure nanosecond fluorescence decays from single cells. The excitation source for the instrument is a frequency doubled train of picosecond pulses from the cavity dumped output of a synchronously pumped dye laser. The dye laser is pumped by a mode-locked argon ion laser. In the microscope, the sample is excited and the emission collected using epi-illumination optics before being transmitted through an adjustable diaphragm, which can be closed to 10 μm in diameter. A Hamamatsu R928 photomultiplier is used to collect the fluorescence which is then analyzed using a non-linear least squares procedure. The microscope has been used to measure the intensity decays of model probes to determine the instrument performance and sensitivity. In addition, intensity and anisotropy decays collected from fura-2 loaded into single adherent rat basophilic leukemia cells were measured to demonstrate that the nanosecond fluorescence microscope can be used to obtain information about the environment and mobility of fluorescent probes in single cells.

  6. Advances in High-Throughput Single-Cell Microtechnologies

    PubMed Central

    Weaver, Westbrook M.; Tseng, Peter; Kunze, Anja; Masaeli, Mahdohkht; Chung, Aram J.; Dudani, Jaideep S.; Kittur, Harsha; Kulkarni, Rajan P.; Di Carlo, Dino

    2013-01-01

    Micro-scale biological tools that have allowed probing of individual cells - from the genetic, to proteomic, to phenotypic level - have revealed important contributions of single cells to direct normal and diseased body processes. In analyzing single cells, sample heterogeneity between and within specific cell types drives the need for high-throughput and quantitative measurement of cellular parameters. In recent years, high-throughput single-cell analysis platforms have revealed rare genetic subpopulations in growing tumors, begun to uncover the mechanisms of antibiotic resistance in bacteria, and described the cell-to-cell variations in stem cell differentiation and immune cell response to activation by pathogens. This review surveys these recent technologies, presenting their strengths and contributions to the field, and identifies needs still unmet toward the development of high-throughput single-cell analysis tools to benefit life science research and clinical diagnostics. PMID:24484889

  7. Cerebral blood oxygenation measurement based on oxygen-dependent quenching of phosphorescence.

    PubMed

    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

  8. Optical manipulation for single-cell studies.

    PubMed

    Ramser, Kerstin; Hanstorp, Dag

    2010-04-01

    In the last decade optical manipulation has evolved from a field of interest for physicists to a versatile tool widely used within life sciences. This has been made possible in particular due to the development of a large variety of imaging techniques that allow detailed information to be gained from investigations of single cells. The use of multiple optical traps has high potential within single-cell analysis since parallel measurements provide good statistics. Multifunctional optical tweezers are, for instance, used to study cell heterogeneity in an ensemble, and force measurements are used to investigate the mechanical properties of individual cells. Investigations of molecular motors and forces on the single-molecule level have led to discoveries that would have been difficult to make with other techniques. Optical manipulation has prospects within the field of cell signalling and tissue engineering. When combined with microfluidic systems the chemical environment of cells can be precisely controlled. Hence the influence of pH, salt concentration, drugs and temperature can be investigated in real time. Fast advancing technical developments of automated and user-friendly optical manipulation tools and cross-disciplinary collaboration will contribute to the routinely use of optical manipulation techniques within the life sciences. PMID:19718682

  9. OXYGEN CONSUMPTION MEASURED WITH MICROCOMPUTER-ASSISTED WARBURG MANOMETRY

    EPA Science Inventory

    The authors have developed and tested an automated system that measures in vitro oxygen consumption by Warburg manometry in as many as 16 units that are under the simultaneous control of a microcomputer which requires attention at the beginning of the study only. The all-glass Su...

  10. 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...

  11. 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. PMID:18231800

  12. 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.

  13. 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.

  14. Oxygen concentration measurement in liquid lead-bismuth eutectic

    SciTech Connect

    Darling, T. W.; Li, N.

    2001-01-01

    Liquid lead-bismuth (Pb-Bi) eutectic (LBE) may see extensive use as a coolant fluid, and perhaps also as a spallation target, in next generation nuclear energy systems. While it is not as reactive as alkali metal liquids, it does present a long term corrosion problem with some materials, notably stainless steels. Mitigation of the corrosion problem may be achieved by producing and maintaining a protective oxide on exposed surfaces, through control of the concentration of dissolved oxygen in the LBE. We have developed an oxygen sensor based on available zirconia-based solid electrolytes used in the automotive industry, which represents a relatively inexpensive source of reproducible and reliable components. We will present the design considerations and characteristics of our sensor unit, and describe its use in the LBE test loop at Los Alamos for measurement and control of dissolved oxygen concentration.

  15. Measuring oxygen uptake in fishes with bimodal respiration.

    PubMed

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

    2016-01-01

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

  16. Single-Cell Genomics for Virology.

    PubMed

    Ciuffi, Angela; Rato, Sylvie; Telenti, Amalio

    2016-01-01

    Single-cell sequencing technologies, i.e., single cell analysis followed by deep sequencing investigate cellular heterogeneity in many biological settings. It was only in the past year that single-cell sequencing analyses has been applied in the field of virology, providing new ways to explore viral diversity and cell response to viral infection, which are summarized in the present review. PMID:27153082

  17. Single-Cell Genomics for Virology

    PubMed Central

    Ciuffi, Angela; Rato, Sylvie; Telenti, Amalio

    2016-01-01

    Single-cell sequencing technologies, i.e., single cell analysis followed by deep sequencing investigate cellular heterogeneity in many biological settings. It was only in the past year that single-cell sequencing analyses has been applied in the field of virology, providing new ways to explore viral diversity and cell response to viral infection, which are summarized in the present review. PMID:27153082

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

    PubMed

    Gaudet, Suzanne; Miller-Jensen, Kathryn

    2016-06-01

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

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

    PubMed Central

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

    2016-01-01

    The ability to correlate single-cell genetic information with cellular phenotypes is of great importance to biology and medicine, as it holds the potential to gain insight into disease pathways that is unavailable from ensemble measurements. We present a microfluidic approach to parallelized, rapid, quantitative analysis of messenger RNA from single cells via RT-qPCR. The approach leverages an array of single-cell RT-qPCR analysis units formed by a set of parallel microchannels concurrently controlled by elastomeric pneumatic valves, thereby enabling parallelized handling and processing of single cells in a drastically simplified operation procedure using a relatively small number of microvalves. All steps for single-cell RT-qPCR, including cell isolation and immobilization, cell lysis, mRNA purification, reverse transcription and qPCR, are integrated on a single chip, eliminating the need for off-chip manual cell and reagent transfer and qPCR amplification as commonly used in existing approaches. Additionally, the approach incorporates optically transparent microfluidic components to allow monitoring of single-cell trapping without the need for molecular labeling that can potentially alter the targeted gene expression and utilizes a polycarbonate film as a barrier against evaporation to minimize the loss of reagents at elevated temperatures during the analysis. We demonstrate the utility of the approach by the transcriptional profiling for the induction of the cyclin-dependent kinase inhibitor 1a and the glyceraldehyde 3-phosphate dehydrogenase in single cells from the MCF-7 breast cancer cell line. Furthermore, the methyl methanesulfonate is employed to allow measurement of the expression of the genes in individual cells responding to a genotoxic stress. PMID:27194954

  20. Microfluidic single-cell whole-transcriptome sequencing.

    PubMed

    Streets, Aaron M; Zhang, Xiannian; Cao, Chen; Pang, Yuhong; Wu, Xinglong; Xiong, Liang; Yang, Lu; Fu, Yusi; Zhao, Liang; Tang, Fuchou; Huang, Yanyi

    2014-05-13

    Single-cell whole-transcriptome analysis is a powerful tool for quantifying gene expression heterogeneity in populations of cells. Many techniques have, thus, been recently developed to perform transcriptome sequencing (RNA-Seq) on individual cells. To probe subtle biological variation between samples with limiting amounts of RNA, more precise and sensitive methods are still required. We adapted a previously developed strategy for single-cell RNA-Seq that has shown promise for superior sensitivity and implemented the chemistry in a microfluidic platform for single-cell whole-transcriptome analysis. In this approach, single cells are captured and lysed in a microfluidic device, where mRNAs with poly(A) tails are reverse-transcribed into cDNA. Double-stranded cDNA is then collected and sequenced using a next generation sequencing platform. We prepared 94 libraries consisting of single mouse embryonic cells and technical replicates of extracted RNA and thoroughly characterized the performance of this technology. Microfluidic implementation increased mRNA detection sensitivity as well as improved measurement precision compared with tube-based protocols. With 0.2 M reads per cell, we were able to reconstruct a majority of the bulk transcriptome with 10 single cells. We also quantified variation between and within different types of mouse embryonic cells and found that enhanced measurement precision, detection sensitivity, and experimental throughput aided the distinction between biological variability and technical noise. With this work, we validated the advantages of an early approach to single-cell RNA-Seq and showed that the benefits of combining microfluidic technology with high-throughput sequencing will be valuable for large-scale efforts in single-cell transcriptome analysis. PMID:24782542

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

    PubMed

    Inomata, Naoki; Toda, Masaya; Ono, Takahito

    2016-09-21

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

  2. Identification of biomarkers to measure HIV-specific mucosal and systemic CD8(+) T-cell immunity using single cell Fluidigm 48.48 Dynamic arrays.

    PubMed

    Trivedi, Shubhanshi; Neeman, Teresa; Jackson, Ronald J; Ranasinghe, Roshanka; Jack, Cameron; Ranasinghe, Charani

    2015-12-16

    Thirty genes composed of cytokines, chemokines, granzymes, perforin and integrins were evaluated in gut and splenic K(d)Gag197-205-specific single CD8(+) T cells using Fluidigm 48.48 Dynamic arrays, with the aim of identifying biomarkers to predict effective mucosal and systemic vaccine efficacy. The mRNA expression profiles were analyzed in three ways: (i) the "number" of K(d)Gag197-205-specific CD8(+) T cells expressing the biomarker, (ii) "level" of mRNA expression using principal component analysis (PCA) and (iii) poly-functionality in relation to RANTES expression. In total, 21 genes were found to be differentially expressed between the vaccine groups and the immune compartments tested. Overall, the PCA indicated that IL-13Rα2 or IL-4R antagonist adjuvanted vaccines that previously induced high-avidity mucosal/systemic CD8(+) T cells with better protective efficacy, the "level" of mRNA expression, specifically RANTES, MIP-1β, and integrin α4 in gut K(d)Gag197-205-specific single CD8(+) T cells, were significantly elevated compared to unadjuvanted vaccine. Furthermore, significantly elevated granzymes/perforin levels were detected in IL-13(-/-) mice given the unadjuvanted vaccine, indicating that the degree of IL-13 inhibition (total, transient or no inhibition) can considerably alter the level of T-cell activity/poly-functionality. When splenic- and gut-K(d)Gag197-205-specific CD8(+) T cells were compared, PC1 vs. PC2 scores revealed that not only RANTES, MIP-1β, and integrin α4 mRNA, but also perforin, granzymes A/B, and integrins β1 and β2 mRNA were elevated in spleen. Collectively, data suggest that RANTES, MIP-1β, perforin, and integrins α4, β1 and β7 mRNA in single HIV-specific CD8(+) T cells could be used as a measure of effective mucosal and systemic vaccine efficacy. PMID:26519547

  3. Single cell protein as an occupational hazard.

    PubMed Central

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

    1983-01-01

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

  4. Electrodeformation for single cell mechanical characterization

    NASA Astrophysics Data System (ADS)

    Chen, Jian; Abdelgawad, Mohamed; Yu, Liming; Shakiba, Nika; Chien, Wei-Yin; Lu, Zhe; Geddie, William R.; Jewett, Michael A. S.; Sun, Yu

    2011-05-01

    This paper presents the use of electrodeformation as a method for single cell mechanical characterization in which mechanical properties of SiHa and ME180 cells (two cervical cancer cell lines) were quantified. Cells were directly placed between two microelectrodes with a rectangular ac electric field applied, and cell deformation was recorded under certain experimental conditions. Numerical simulations were performed to model cell electrodeformation based on the Maxwell stress tensor formulation. In these simulations, effects of cell electrical property variations on their electrodeformed behavior were investigated. By comparing the measured morphological changes with those obtained from numerical simulations, we were able to quantify Young's modulus of SiHa cells (601 ± 183 Pa) and ME180 cells (1463 ± 649 Pa). These values were consistent with Young's modulus values (SiHa: 400 ± 290 Pa and ME180: 1070 ± 580 Pa) obtained from conventional micropipette aspiration.

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

    NASA Technical Reports Server (NTRS)

    Kozul, Jean M.; Hewins, Roger H.; Ulmer, Gene C.

    1988-01-01

    The intrinsic oxygen fugacities (IOFs) of two type B Ca-rich and Al-rich inclusions (CAI) from the Allende meteorite were measured using the solid-electrolyte double-cell IOF technique of Ulmer et al. (1976). The measurements were compared to calculated and experimentally extrapolated fO2 of type B phases. It was found that the IOFs of the type B are 6-8 orders of magnitude more oxidized (H2/H2O = 1-10) than the canonical solar nebular gas (H2/H2O = 100-2000). It is suggested that some local fO2 enhancing mechanism, such as dust or gas concentrations, or the release of oxygen-rich vapors during CAI volatilization in the type B inclusions was in operation at temperatures higher than 700 C.

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

    PubMed

    Kinnunen, Matti; Karmenyan, Artashes

    2015-05-01

    We present and discuss several modern optical methods based on elastic light scattering (ELS), along with their technical features and applications in biomedicine and life sciences. In particular, we review some ELS experiments at the single-cell level and explore new directions of applications. Due to recent developments in experimental systems (as shown in the literature), ELS lends itself to useful applications in the life sciences. Of the developed methods, we cover elastic scattering spectroscopy, optical tweezer-assisted measurement, goniometers, Fourier transform light scattering (FTLS), and microscopic methods. FTLS significantly extends the potential analysis of single cells by allowing monitoring of dynamical changes at the single-cell level. The main aim of our review is to demonstrate developments in the experimental investigation of ELS in single cells including issues related to theoretical “representations” and modeling of biological systems (cells, cellular systems, tissues, and so on). Goniometric measurements of ELS from optically trapped single cells are shown and the importance of the experimental verification of theoretical models of ELS in the context of biomedical applications is discussed. PMID:25760756

  7. Integrated Electrowetting Nanoinjector for Single Cell Transfection

    PubMed Central

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

    2016-01-01

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

  8. 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. Leaf water oxygen isotope measurement by direct equilibration.

    PubMed

    Song, Xin; Barbour, Margaret M

    2016-08-01

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

  10. Single cell correlation fractal dimension of chromatin

    PubMed Central

    Récamier, Vincent; Izeddin, Ignacio; Bosanac, Lana; Dahan, Maxime; Proux, Florence; Darzacq, Xavier

    2014-01-01

    Chromatin is a major nuclear component, and it is an active matter of debate to understand its different levels of spatial organization, as well as its implication in gene regulation. Measurements of nuclear chromatin compaction were recently used to understand how DNA is folded inside the nucleus and to detect cellular dysfunctions such as cancer. Super-resolution imaging opens new possibilities to measure chromatin organization in situ. Here, we performed a direct measure of chromatin compaction at the single cell level. We used histone H2B, one of the 4 core histone proteins forming the nucleosome, as a chromatin density marker. Using photoactivation localization microscopy (PALM) and adaptive optics, we measured the three-dimensional distribution of H2B with nanometric resolution. We computed the distribution of distances between every two points of the chromatin structure, namely the Ripley K(r) distribution. We found that the K(r) distribution of H2B followed a power law, leading to a precise measurement of the correlation fractal dimension of chromatin of 2.7. Moreover, using photoactivable GFP fused to H2B, we observed dynamic evolution of chromatin sub-regions compaction. As a result, the correlation fractal dimension of chromatin reported here can be interpreted as a dynamically maintained non-equilibrium state. PMID:24637833

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

    PubMed

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

    2001-05-01

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

  12. Single-cell genome sequencing: current state of the science.

    PubMed

    Gawad, Charles; Koh, Winston; Quake, Stephen R

    2016-03-01

    The field of single-cell genomics is advancing rapidly and is generating many new insights into complex biological systems, ranging from the diversity of microbial ecosystems to the genomics of human cancer. In this Review, we provide an overview of the current state of the field of single-cell genome sequencing. First, we focus on the technical challenges of making measurements that start from a single molecule of DNA, and then explore how some of these recent methodological advancements have enabled the discovery of unexpected new biology. Areas highlighted include the application of single-cell genomics to interrogate microbial dark matter and to evaluate the pathogenic roles of genetic mosaicism in multicellular organisms, with a focus on cancer. We then attempt to predict advances we expect to see in the next few years. PMID:26806412

  13. 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.

  14. Comparison of two sediment oxygen demand measurement techniques

    SciTech Connect

    Truax, D.D.; Sindala, A.; Sartain, H.

    1995-09-01

    Measuring the sediment oxygen demand rate in deep water can be difficult. Yet this parameter can be a significant component in ecological models of deep water systems. The results of a study to characterize the oxygen demand in the benthos of more than 210 km (130 mi) of the Tennessee-Tombigbee Waterway are presented here. Sampling was performed with a modified Shelby tube apparatus, which allowed the collection of samples at depths of more than 7 m (25 ft) without the use of divers. Results of in-situ analyses performed by the U.S. Environmental Protection Agency (EPA) are correlated with data from a sampling and laboratory methodology developed for this project. The laboratory setup used a sealed, cylindrical chamber through which water was continuously recirculated. The results of both procedures were statistically equivalent throughout the majority of the waterway. Only in areas where the channel bottom consisted of extremely porous media did the sampling and laboratory methodology yield questionable results. The results obtained indicate that sediment oxygen demand of the section of the waterway studied ranged between 0.5 g O{sub 2}/m{sup 2}-day and 1.5 g O{sub 2}/m{sup 2}-day.

  15. 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

  16. Near-infrared measurements of brain oxygenation in stroke.

    PubMed

    Moreau, François; Yang, Runze; Nambiar, Vivek; Demchuk, Andrew M; Dunn, Jeff F

    2016-07-01

    We investigated the feasibility of using frequency-domain near-infrared spectroscopy (fdNIRS) to study brain oxygenation in the first few hours of stroke onset. The OxiplexTS(®) fdNIRS system was used in this study. Using a standard probing protocol based on surface landmarks, we measured brain tHb and [Formula: see text] in healthy volunteers, cadavers, and acute stroke patients within 9 h of stroke onset and 3 days later. We obtained measurements from 11 controls, 5 cadavers, and 5 acute stroke patients. [Formula: see text] values were significantly lower in cadavers compared to the controls and stroke patients. Each stroke patient had at least one area with reduced [Formula: see text] on the stroke side compared to the contralateral side. The evolution of tHb and [Formula: see text] at 3 days differed depending on whether a large infarct occurred. This study shows the proof of principle that quantified measurements of brain oxygenation using NIRS could be used in the hectic environment of acute stroke management. It also highlights the current technical limitations and future challenges in the development of this unique bedside monitoring tool for stroke. PMID:26958577

  17. 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.

  18. 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.

  19. Efficient Synergistic Single-Cell Genome Assembly.

    PubMed

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

    2016-01-01

    As the vast majority of all microbes are unculturable, single-cell sequencing has become a significant method to gain insight into microbial physiology. Single-cell sequencing methods, currently powered by multiple displacement genome amplification (MDA), have passed important milestones such as finishing and closing the genome of a prokaryote. However, the quality and reliability of genome assemblies from single cells are still unsatisfactory due to uneven coverage depth and the absence of scattered chunks of the genome in the final collection of reads caused by MDA bias. In this work, our new algorithm Hybrid De novo Assembler (HyDA) demonstrates the power of coassembly of multiple single-cell genomic data sets through significant improvement of the assembly quality in terms of predicted functional elements and length statistics. Coassemblies contain significantly more base pairs and protein coding genes, cover more subsystems, and consist of longer contigs compared to individual assemblies by the same algorithm as well as state-of-the-art single-cell assemblers SPAdes and IDBA-UD. Hybrid De novo Assembler (HyDA) is also able to avoid chimeric assemblies by detecting and separating shared and exclusive pieces of sequence for input data sets. By replacing one deep single-cell sequencing experiment with a few single-cell sequencing experiments of lower depth, the coassembly method can hedge against the risk of failure and loss of the sample, without significantly increasing sequencing cost. Application of the single-cell coassembler HyDA to the study of three uncultured members of an alkane-degrading methanogenic community validated the usefulness of the coassembly concept. HyDA is open source and publicly available at http://chitsazlab.org/software.html, and the raw reads are available at http://chitsazlab.org/research.html. PMID:27243002

  20. Efficient Synergistic Single-Cell Genome Assembly

    PubMed Central

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

    2016-01-01

    As the vast majority of all microbes are unculturable, single-cell sequencing has become a significant method to gain insight into microbial physiology. Single-cell sequencing methods, currently powered by multiple displacement genome amplification (MDA), have passed important milestones such as finishing and closing the genome of a prokaryote. However, the quality and reliability of genome assemblies from single cells are still unsatisfactory due to uneven coverage depth and the absence of scattered chunks of the genome in the final collection of reads caused by MDA bias. In this work, our new algorithm Hybrid De novo Assembler (HyDA) demonstrates the power of coassembly of multiple single-cell genomic data sets through significant improvement of the assembly quality in terms of predicted functional elements and length statistics. Coassemblies contain significantly more base pairs and protein coding genes, cover more subsystems, and consist of longer contigs compared to individual assemblies by the same algorithm as well as state-of-the-art single-cell assemblers SPAdes and IDBA-UD. Hybrid De novo Assembler (HyDA) is also able to avoid chimeric assemblies by detecting and separating shared and exclusive pieces of sequence for input data sets. By replacing one deep single-cell sequencing experiment with a few single-cell sequencing experiments of lower depth, the coassembly method can hedge against the risk of failure and loss of the sample, without significantly increasing sequencing cost. Application of the single-cell coassembler HyDA to the study of three uncultured members of an alkane-degrading methanogenic community validated the usefulness of the coassembly concept. HyDA is open source and publicly available at http://chitsazlab.org/software.html, and the raw reads are available at http://chitsazlab.org/research.html. PMID:27243002

  1. Oxygen solubilities of media used in electrochemical respiration measurements.

    PubMed

    Rasmussen, Hans N; Rasmussen, Ulla F

    2003-08-01

    Solubility data are presented as equations from which the oxygen concentration of arbitrary media may be calculated with an accuracy of about 1%. These equations, covering the range 5-40 degrees C, are based on measurements with a modification of the physical method of St. Helen and Fatt (I. Fatt, Polarographic Oxygen Sensors, CRC Press, Cleveland (1976)). Solutions of the following compounds were measured: EDTA (ethylenediaminetetraacetic acid), EGTA (ethyleneglycol-bis(2-aminoethylether)tetraacetic acid), glucose, Hepes (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid), lactobionic acid (galactosylgluconic acid), magnesium chloride, mannitol, Mes (2-(N-morpholino)ethanesulfonic acid), potassium chloride, potassium phosphate, sodium chloride, sucrose, taurine, and Tris (tris(hydroxymethyl)aminomethane). The present data are compared with solubility data obtained with the method involving oxidation of calibrated amounts of reduced nicotinamide adenine dinucleotide (e.g., R.W. Estabrook, Methods Enzymol. 10 (1967) 41-47) and with the kinetic method of Reynafarje et al. (Anal. Biochem. 145 (1985) 406-418). It is concluded that the former method produces reliable results provided that some simple precautions are taken. The kinetic method, however, appears to have produced calibration errors up to about 5%. PMID:12842113

  2. Single cell transcriptional analysis reveals novel innate immune cell types.

    PubMed

    Kippner, Linda E; Kim, Jinhee; Gibson, Greg; Kemp, Melissa L

    2014-01-01

    Single-cell analysis has the potential to provide us with a host of new knowledge about biological systems, but it comes with the challenge of correctly interpreting the biological information. While emerging techniques have made it possible to measure inter-cellular variability at the transcriptome level, no consensus yet exists on the most appropriate method of data analysis of such single cell data. Methods for analysis of transcriptional data at the population level are well established but are not well suited to single cell analysis due to their dependence on population averages. In order to address this question, we have systematically tested combinations of methods for primary data analysis on single cell transcription data generated from two types of primary immune cells, neutrophils and T lymphocytes. Cells were obtained from healthy individuals, and single cell transcript expression data was obtained by a combination of single cell sorting and nanoscale quantitative real time PCR (qRT-PCR) for markers of cell type, intracellular signaling, and immune functionality. Gene expression analysis was focused on hierarchical clustering to determine the existence of cellular subgroups within the populations. Nine combinations of criteria for data exclusion and normalization were tested and evaluated. Bimodality in gene expression indicated the presence of cellular subgroups which were also revealed by data clustering. We observed evidence for two clearly defined cellular subtypes in the neutrophil populations and at least two in the T lymphocyte populations. When normalizing the data by different methods, we observed varying outcomes with corresponding interpretations of the biological characteristics of the cell populations. Normalization of the data by linear standardization taking into account technical effects such as plate effects, resulted in interpretations that most closely matched biological expectations. Single cell transcription profiling provides

  3. Photosensitized generation of singlet oxygen in porous silicon studied by simultaneous measurements of luminescence of nanocrystals and oxygen molecules

    SciTech Connect

    Gongalsky, M. B.; Kharin, A. Yu.; Zagorodskikh, S. A.; Osminkina, L. A.; Timoshenko, V. Yu.

    2011-07-01

    Photosensitization of singlet oxygen generation in porous silicon (PSi) was investigated by simultaneous measurements of the photoluminescence (PL) of silicon nanocrystals (nc-Si) and the infrared emission of the {sup 1}{Delta}-state of oxygen molecules at 1270 nm (0.98 eV) at room temperature. Photodegradation of the nc-Si PL properties was found to correlate with the efficiency of singlet oxygen generation. The quantum efficiency of singlet oxygen generation in PSi was estimated to be about 1%, while the lifetime of singlet oxygen was about fifteen ms. The kinetics of nc-Si PL intensity under cw excitation undergoes a power law dependence with the exponent dependent on the photon energy of luminescence. The experimental results are explained with a model of photodegradation controlled by the diffusion of singlet oxygen molecules in a disordered structure of porous silicon.

  4. 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.

  5. Accurate measurement of oxygen consumption in children undergoing cardiac catheterization.

    PubMed

    Li, Jia

    2013-01-01

    Oxygen consumption (VO(2) ) is an important part of hemodynamics using the direct Fick principle in children undergoing cardiac catheterization. Accurate measurement of VO(2) is vital. Obviously, any error in the measurement of VO(2) will translate directly into an equivalent percentage under- or overestimation of blood flows and vascular resistances. It remains common practice to estimate VO(2) values from published predictive equations. Among these, the LaFarge equation is the most commonly used equation and gives the closest estimation with the least bias and limits of agreement. However, considerable errors are introduced by the LaFarge equation, particularly in children younger than 3 years of age. Respiratory mass spectrometry remains the "state-of-the-art" method, allowing highly sensitive, rapid and simultaneous measurement of multiple gas fractions. The AMIS 2000 quadrupole respiratory mass spectrometer system has been adapted to measure VO(2) in children under mechanical ventilation with pediatric ventilators during cardiac catheterization. The small sampling rate, fast response time and long tubes make the equipment a unique and powerful tool for bedside continuous measurement of VO(2) in cardiac catheterization for both clinical and research purposes. PMID:22488802

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

    NASA Astrophysics Data System (ADS)

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

    2010-05-01

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

  7. 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

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

    PubMed

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

    2016-01-01

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

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

    PubMed

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

    2015-11-01

    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. PMID:26405849

  10. Computational analysis of signaling patterns in single cells

    PubMed Central

    Davis, Denise M.; Purvis, Jeremy E.

    2014-01-01

    Signaling proteins are flexible in both form and function. They can bind to multiple molecular partners and integrate diverse types of cellular information. When imaged by time-lapse microscopy, many signaling proteins show complex patterns of activity or localization that vary from cell to cell. This heterogeneity is so prevalent that it has spurred the development of new computational strategies to analyze single-cell signaling patterns. A collective observation from these analyses is that cells appear less heterogeneous when their responses are normalized to, or synchronized with, other single-cell measurements. In many cases, these transformed signaling patterns show distinct dynamical trends that correspond with predictable phenotypic outcomes. When signaling mechanisms are unclear, computational models can suggest putative molecular interactions that are experimentally testable. Thus, computational analysis of single-cell signaling has not only provided new ways to quantify the responses of individual cells, but has helped resolve longstanding questions surrounding many well-studied human signaling proteins including NF-κB, p53, ERK1/2, and CDK2. A number of specific challenges lie ahead for single-cell analysis such as quantifying the contribution of non-cell autonomous signaling as well as the characterization of protein signaling dynamics in vivo. PMID:25263011

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

    PubMed

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

    2016-06-01

    To assess the validity of postexercise measurements in estimating peak oxygen uptake (V̇O2peak) in swimming, we compared oxygen uptake (V̇O2) measurements during supramaximal exercise with various commonly adopted methods, including a recently developed heart rate - V̇O2 modelling procedure. Thirty-one elite swimmers performed a 200-m maximal swim where V̇O2 was measured breath-by-breath using a portable gas analyzer connected to a respiratory snorkel, 1 min before, during, and 3 min postexercise. V̇O2peak(-20-0) was the average of the last 20 s of effort. The following postexercise measures were compared: (i) first 20-s average (V̇O2peak(0-20)); (ii) linear backward extrapolation (BE) of the first 20 s (BE(20)), 30 s, and 3 × 20-, 4 × 20-, and 3 or 4 × 20-s averages; (iii) semilogarithmic BE at 20 s (LOG(20)) and at the other same time intervals as in linear BE; and (iv) predicted V̇O2peak using mathematical modelling (pV̇O2(0-20)]. Repeated-measures ANOVA and post-hoc Bonferroni tests compared V̇O2peak (criterion) and each estimated value. Pearson's coefficient of determination (r(2)) was used to assess correlation. Exercise V̇O2peak(-20-0) (mean ± SD 3531 ± 738 mL·min(-1)) was not different (p > 0.30) from pV̇O2(0-20) (3571 ± 735 mL·min(-1)), BE(20) (3617 ± 708 mL·min(-1)), or LOG(20) (3627 ± 746 mL·min(-1)). pV̇O2(0-20) was very strongly correlated with exercise V̇O2peak (r(2) = 0.962; p < 0.001), and showed a low standard error of the estimate (146 mL·min(-1), 4.1%) and the lowest mean difference (40 mL·min(-1); 1.1%). We confirm that the new modelling procedure based on postexercise V̇O2 and heart rate measurements is a valid and accurate procedure for estimating V̇O2peak in swimmers and avoids the estimation bias produced by other methods. PMID:27226382

  12. Ambient air measurements of monoterpenes, oxygenated terpenes, and sesquiterpenes

    NASA Astrophysics Data System (ADS)

    Bouvier-Brown, N. C.; Goldstein, A. H.

    2007-12-01

    Chemical ozone loss within the forest canopy and the presence of biogenic VOC (BVOC) oxidation products in and above the canopy indirectly suggest the presence of very reactive BVOCs at Blodgett Forest. As a part of the 2007 BEARPEX campaign at this coniferous forest in the Sierra Nevada Mountains of California (1300 m elevation, 38.90° N, 120.63° W,), we quantified ambient concentrations of terpenes using a modified in-situ gas chromatograph with a mass spectrometer and a flame ionization detector (GC-MS-FID). The range of terpenes observed in ambient air matched enclosure based measurements of branch level emissions. To our knowledge, these observations represent the first quantification of the oxygenated monoterpene methyl chavicol and various sesquiterpenes in ambient air. Details of the instrument modifications, diurnal profiles of the terpenes, and comparison to branch level emissions will be presented.

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

    NASA Astrophysics Data System (ADS)

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

    2012-03-01

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

  14. A modified procedure for measuring oxygen-18 content of nitrate

    NASA Astrophysics Data System (ADS)

    Ahmed, M. A.; Aly, A. I. M.; Abdel Monem, N.; Hanafy, M.; Gomaa, H. E.

    2012-11-01

    SummaryMass spectrometric analysis of O-isotopic composition of nitrate has many potential applications in studies of environmental processes. Through this work, rapid, reliable, precise, broadly applicable, catalyst-free, low-priced and less labor intensive procedure for measuring δ18O of nitrate using Isotope Ratio Mass Spectrometer has been developed and implemented. The conditions necessary to effect complete nitrate recovery and complete removal of other oxygen containing anions and dissolved organic carbon (DOC) without scarifying the isotopic signature of nitrate were investigated. The developed procedure consists of two main parts: (1) wet chemistry train for extraction and purification of nitrate from the liquid matrix; (2) off-line pyrolysis of extracted nitrate salt with activated graphite at 550 °C for 30 min. The conditions necessary to effect complete nitrate recovery and complete removal of other oxygen containing compounds were investigated. Dramatic reduction in processing times needed for analysis of δ18O of nitrate at natural abundance level was achieved. Preservation experiments revealed that chloroform (99.8%) is an effective preservative. Isotopic contents of some selected nitrate salts were measured using the modified procedure and some other well established methods at two laboratories in Egypt and Germany. Performance assessment of the whole developed analytical train was made using internationally distributed nitrate isotopes reference materials and real world sample of initial zero-nitrate content. The uncertainty budget was evaluated using the graphical nested hierarchal approach. The obtained results proved the suitability for handling samples of complicated matrices. Reduction of consumables cost by about 80% was achieved.

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

    PubMed Central

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

    2016-01-01

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

  16. 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

  17. Technologies for Single-Cell Isolation.

    PubMed

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

    2015-01-01

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

  18. 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. PMID:25458215

  19. Airborne chemistry single cell level

    NASA Astrophysics Data System (ADS)

    Nilsson, Staffan; Viberg, Peter; Spegel, Peter; Santesson, Sabina; Cedergren, Eila; Degerman, Eva; Johansson, Tomas; Nilsson, Johan

    2002-11-01

    A miniaturized analysis system for the studying of living cells and biochemical reactions in microdrops was developed. Cell studies were performed using single adipocytes in 250-nL drops. Continuous flow-through droplet dispensers, developed in-house, were used for additions to the levitated droplet. Addition of b-adrenergic agonists stimulates the lipolysis in the adipocytes, leading to free fatty acid release and a consequent pH decrease of the surrounding buffer, a change that can be easily followed using a pH-dependent fluorophore continuously monitored by fluorescence imaging detection. An analytical method using capillary electrophoresis and nanospray mass spectrometry for measurement of the cAMP level in activated single adipocytes are now being developed for future use in combination with the levitation technique. The levitation approach was also employed for the screening of nucleation conditions for macromolecules. Here, the acoustic levitator offers a simplified way to determine the main features of the phase diagram (i.e., precipitation diagram). Using the droplet dispensers, different types and amounts of precipitation agents are injected into the levitated drop, allowing a systematic search for nucleation conditions that is not possible using standard crystallization methods. Once the precipitation diagram has been obtained, optimization using standard methods is employed to grow the crystals.

  20. 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). PMID:25863368

  1. Oxygen tension measurement using an automatic blood gas analyser1

    PubMed Central

    Becket, J; Chakrabarti, M K; Gillies, I D S; Orchard, C; Hall, G M; Bourdillon, P J

    1981-01-01

    Two different methods of assessing the reliability of the oxygen electrode of one model of an automatic blood gas analyser (BGA) have been studied. In the first, a single automatic BGA was assessed by using outdated bank blood which was pumped around a small extracorporeal circuit into which known gas mixtures were passed. Oxygen tension was varied between 2 and 16 kPa. In the second, fresh heparinized blood was tonometered with calibrated gases and submitted to the automatic BGA used in the first part of the study and also to three other identical machines. Each of the machines was between 3 and 4 years old. Eighteen different units of blood were used in the first part of the study. The correlation coefficient between the automatic BGA and the Po2 in the extracorporeal circuit varied between 0.29 and 0.99. 31% of the total of 209 measurements made by the automatic BGA were more than 1.2 kPa from the reference value, 25% of them being between 1.2 and 4.0 kPa from the reference value. In the second part of the study, the correlation coefficient between this automatic BGA and the tonometered blood was 0.96. The correlation coefficients for the 3 other identical BGAs were 0.84, 0.97 and 0.88, indicating that the BGA used in the first part of the study was no worse than any of the others. It is suggested that although clinicians are likely to ignore readings of an automatic BGA that are more than 4.0 kPa from the true value and are likely to repeat the investigation, readings between 1.2 and 4.0 kPa from the true value may adversely affect patient management. PMID:7288796

  2. 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

  3. Oxygen tension measurement using an automatic blood gas analyser.

    PubMed

    Becket, J; Orchard, C; Chakrabarti, M K; Hall, G M; Gillies, I D; Bourdillon, P J

    1981-08-01

    Two different methods of assessing the reliability of the oxygen electrode of one model of an automatic blood gas analyser (BGA) have been studied. In the first, a single automatic BGA was assessed by using outdated bank blood which was pumped around a small extracorporeal circuit into which known gas mixtures were passed. Oxygen tension was varied between 2 and 16 kPa. In the second, fresh heparinized blood was tonometered with calibrated gases and submitted to the automatic BGA used in the first part of the study and also to three other identical machines. Each of the machines was between 3 and 4 years old.Eighteen different units of blood were used in the first part of the study. The correlation coefficient between the automatic BGA and the Po(2) in the extracorporeal circuit varied between 0.29 and 0.99. 31% of the total of 209 measurements made by the automatic BGA were more than 1.2 kPa from the reference value, 25% of them being between 1.2 and 4.0 kPa from the reference value. In the second part of the study, the correlation coefficient between this automatic BGA and the tonometered blood was 0.96. The correlation coefficients for the 3 other identical BGAs were 0.84, 0.97 and 0.88, indicating that the BGA used in the first part of the study was no worse than any of the others.It is suggested that although clinicians are likely to ignore readings of an automatic BGA that are more than 4.0 kPa from the true value and are likely to repeat the investigation, readings between 1.2 and 4.0 kPa from the true value may adversely affect patient management. PMID:7288796

  4. 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.

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

    NASA Astrophysics Data System (ADS)

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

    2012-03-01

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

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

    SciTech Connect

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

    1996-03-01

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

  7. 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

  8. NADH-dependent biosensor in Saccharomyces cerevisiae: principle and validation at the single cell level

    PubMed Central

    2014-01-01

    A reporter system was constructed to measure perturbations in the NADH/NAD+ co-factor balance in yeast, by using the green fluorescent protein gene under the control of the GPD2 promoter that is induced under conditions of excess of NADH. High fluorescence levels were obtained in a glycerol 3-phosphate dehydrogenase double deletion strain (gpd1Δgpd2Δ), which is deficient in the ability to regenerate NAD+ via glycerol formation. The responsiveness of the reporter system to externally induced perturbations in NADH oxidation was also evaluated in the gpd1Δgpd2Δ strain background by addition of acetoin, as well as by introduction of a set of heterologous xylose reductases (XRs) having different selectivities for NADH. Addition of acetoin during cell proliferation under oxygen-limited conditions resulted in a more than 2-fold decrease in mean fluorescence intensity as compared to the control experiment. Strains carrying XRs with different selectivities for NADH could be distinguished at the single cell level, so that the XR with the highest selectivity for NADH displayed the lowest fluorescence. In conclusion, the designed system successfully allowed for monitoring perturbations in the cellular redox metabolism caused by environmental changes, or by heterologous gene expression. The reporter system displayed high resolution in distinguishing cytosolic NADH oxidation capacity and hence has potential to be used for high-throughput screening based on the fluorescence of single cells. PMID:25401080

  9. Microwave-induced thermogenetic activation of single cells

    SciTech Connect

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

    2015-04-20

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

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

    PubMed Central

    Trapnell, Cole

    2015-01-01

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

  11. Single cell multiplexed assay for proteolytic activity using droplet microfluidics.

    PubMed

    Ng, Ee Xien; Miller, Miles A; Jing, Tengyang; Chen, Chia-Hung

    2016-07-15

    Cellular enzymes interact in a post-translationally regulated fashion to govern individual cell behaviors, yet current platform technologies are limited in their ability to measure multiple enzyme activities simultaneously in single cells. Here, we developed multi-color Förster resonance energy transfer (FRET)-based enzymatic substrates and use them in a microfluidics platform to simultaneously measure multiple specific protease activities from water-in-oil droplets that contain single cells. By integrating the microfluidic platform with a computational analytical method, Proteolytic Activity Matrix Analysis (PrAMA), we are able to infer six different protease activity signals from individual cells in a high throughput manner (~100 cells/experimental run). We characterized protease activity profiles at single cell resolution for several cancer cell lines including breast cancer cell line MDA-MB-231, lung cancer cell line PC-9, and leukemia cell line K-562 using both live-cell and in-situ cell lysis assay formats, with special focus on metalloproteinases important in metastasis. The ability to measure multiple proteases secreted from or expressed in individual cells allows us to characterize cell heterogeneity and has potential applications including systems biology, pharmacology, cancer diagnosis and stem cell biology. PMID:26995287

  12. 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.

  13. 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

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

    NASA Astrophysics Data System (ADS)

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

    2014-11-01

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

  15. 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.

  16. Single-cell nanotoxicity assays of superparamagnetic iron oxide nanoparticles.

    PubMed

    Eustaquio, Trisha; Leary, James F

    2012-01-01

    Properly evaluating the nanotoxicity of nanoparticles involves much more than bulk-cell assays of cell death by necrosis. Cells exposed to nanoparticles may undergo repairable oxidative stress and DNA damage or be induced into apoptosis. Exposure to nanoparticles may cause the cells to alter their proliferation or differentiation or their cell-cell signaling with neighboring cells in a tissue. Nanoparticles are usually more toxic to some cell subpopulations than others, and toxicity often varies with cell cycle. All of these facts dictate that any nanotoxicity assay must be at the single-cell level and must try whenever feasible and reasonable to include many of these other factors. Focusing on one type of quantitative measure of nanotoxicity, we describe flow and scanning image cytometry approaches to measuring nanotoxicity at the single-cell level by using a commonly used assay for distinguishing between necrotic and apoptotic causes of cell death by one type of nanoparticle. Flow cytometry is fast and quantitative, provided that the cells can be prepared into a single-cell suspension for analysis. But when cells cannot be put into suspension without altering nanotoxicity results, or if morphology, attachment, and stain location are important, a scanning image cytometry approach must be used. Both methods are described with application to a particular type of nanoparticle, a superparamagnetic iron oxide nanoparticle (SPION), as an example of how these assays may be applied to the more general problem of determining the effects of nanomaterial exposure to living cells. PMID:22975957

  17. 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...

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

    PubMed

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

    2004-01-01

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

  19. Characterization of a single cell of Chlorella in a microfluidic channel using amperometric electrode arrays.

    PubMed

    Song, Young Seok; Bai, Seoung Jai

    2014-11-01

    Electrochemical characteristics of O2 and/or mediators secreted by a single cell of Chlorella fusea were analyzed by using amperometric measurements on microelectrodes embedded in a microfluidic device. A single cell was trapped in a microfluidic channel, which simplifies the mass transfer phenomenon, i.e., one-dimensional distribution of solutes in the channel. Such amperometric measurements allowed us to obtain more refined data in a localized space and to understand photosynthetic behavior of algae at the single cell level. In addition, the concentration of a photosynthetic mediator, p-benzoquinone, was numerically calculated by using the finite element method. PMID:24966046

  20. 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

  1. Digital microfluidic immunocytochemistry in single cells.

    PubMed

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

    2015-01-01

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

  2. Tree inference for single-cell data.

    PubMed

    Jahn, Katharina; Kuipers, Jack; Beerenwinkel, Niko

    2016-01-01

    Understanding the mutational heterogeneity within tumors is a keystone for the development of efficient cancer therapies. Here, we present SCITE, a stochastic search algorithm to identify the evolutionary history of a tumor from noisy and incomplete mutation profiles of single cells. SCITE comprises a flexible Markov chain Monte Carlo sampling scheme that allows the user to compute the maximum-likelihood mutation history, to sample from the posterior probability distribution, and to estimate the error rates of the underlying sequencing experiments. Evaluation on real cancer data and on simulation studies shows the scalability of SCITE to present-day single-cell sequencing data and improved reconstruction accuracy compared to existing approaches. PMID:27149953

  3. Quantification of Circadian Rhythms in Single Cells

    PubMed Central

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

    2009-01-01

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

  4. 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.

  5. Single-cell printing based on impedance detection

    PubMed Central

    Schoendube, J.; Wright, D.; Zengerle, R.; Koltay, P.

    2015-01-01

    Label-free isolation of single cells is essential for the growing field of single-cell analysis. Here, we present a device which prints single living cells encapsulated in free-flying picoliter droplets. It combines inkjet printing and impedance flow cytometry. Droplet volume can be controlled in the range of 500 pl–800 pl by piezo actuator displacement. Two sets of parallel facing electrodes in a 50 μm × 55 μm channel are applied to measure the presence and velocity of a single cell in real-time. Polystyrene beads with <5% variation in diameter generated signal variations of 12%–17% coefficients of variation. Single bead efficiency (i.e., printing events with single beads vs. total number of printing events) was 73% ± 11% at a throughput of approximately 9 events/min. Viability of printed HeLa cells and human primary fibroblasts was demonstrated by culturing cells for at least eight days. PMID:25759750

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

    PubMed Central

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

    2015-01-01

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

  7. Single-cell analysis of circadian dynamics in tissue explants

    PubMed Central

    Lande-Diner, Laura; Stewart-Ornstein, Jacob; Weitz, Charles J.; Lahav, Galit

    2015-01-01

    Tracking molecular dynamics in single cells in vivo is instrumental to understanding how cells act and interact in tissues. Current tissue imaging approaches focus on short-term observation and typically nonendogenous or implanted samples. Here we develop an experimental and computational setup that allows for single-cell tracking of a transcriptional reporter over a period of >1 wk in the context of an intact tissue. We focus on the peripheral circadian clock as a model system and measure the circadian signaling of hundreds of cells from two tissues. The circadian clock is an autonomous oscillator whose behavior is well described in isolated cells, but in situ analysis of circadian signaling in single cells of peripheral tissues is as-yet uncharacterized. Our approach allowed us to investigate the oscillatory properties of individual clocks, determine how these properties are maintained among different cells, and assess how they compare to the population rhythm. These experiments, using a wide-field microscope, a previously generated reporter mouse, and custom software to track cells over days, suggest how many signaling pathways might be quantitatively characterized in explant models. PMID:26269583

  8. 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. PMID:25819987

  9. Mie scatter corrections in single cell infrared microspectroscopy.

    PubMed

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

    2016-06-23

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

  10. Single cell microfluidics for systems oncology

    NASA Astrophysics Data System (ADS)

    Fan, Rong

    2012-02-01

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

  11. Computing tumor trees from single cells.

    PubMed

    Davis, Alexander; Navin, Nicholas E

    2016-01-01

    Computational methods have been developed to reconstruct evolutionary lineages from tumors using single-cell genomic data. The resulting tumor trees have important applications in cancer research and clinical oncology.Please see related Research articles: http://genomebiology.biomedcentral.com/articles/10.1186/s13059-016-0929-9 and http://genomebiology.biomedcentral.com/articles/10.1186/s13059-016-0936-x . PMID:27230879

  12. 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.

  13. 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).

  14. Dynamic oxygenation measurements using a phosphorescent coating within a mammary window chamber mouse model

    PubMed Central

    Schafer, Rachel; Gmitro, Arthur F.

    2015-01-01

    Phosphorescent lifetime imaging was employed to measure the spatial and temporal distribution of oxygen partial pressure in tissue under the coverslip of a mammary window chamber breast cancer mouse model. A thin platinum-porphyrin coating, whose phosphorescent lifetime varies monotonically with oxygen partial pressure, was applied to the coverslip surface. Dynamic temporal responses to induced modulations in oxygenation levels were measured using this approach. PMID:25780753

  15. 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.

  16. In situ fiber-optic oxygen consumption measurements from a working mouse heart.

    PubMed

    Zhao, Y; Richman, A; Storey, C; Radford, N B; Pantano, P

    1999-09-01

    Luminescence-based imaging-fiber oxygen sensors (IFOSs) were utilized for the in situ measurement of oxygen consumption from intact perfused mouse hearts. IFOSs were fabricated using a technically expedient, photoinitiated polymerization reaction whereby an oxygen-sensitive polymer matrix was immobilized in a precise location on an imaging fiber's distal face. The oxygen-sensing layer used in this work comprised a transition metal complex, Ru(Ph2phen)3(2+), entrapped in a gaspermeable photopolymerizable siloxane membrane (PS802). The transduction mechanism was based upon the oxygen collisional quenching of the ruthenium complex luminescence; detection was performed utilizing an epi-fluorescence microscope/charge coupled device imaging system. IFOS measurements from working mouse hearts were validated through concurrent, blind, ex situ blood gas analyzer (BGA) measurements. The BGA and IFOS methodologies were utilized successfully to measure oxygen concentrations in aortic and pulmonary artery perfusates from the working mouse heart before and after isoproterenol administration. Coupled with coronary-flow measurements, these data were used to calculate myocardial oxygen consumption. Regression analysis of measurements of myocardial oxygen consumption showed that there was a strong correlation between the values generated by the BGA sampling and those obtained via in situ IFOS methods. To our knowledge, this research represents the first report of in situ fiber-optic sensor monitoring of oxygen content from the intact, beating mouse heart. PMID:10489534

  17. 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.

  18. 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.

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

    NASA Technical Reports Server (NTRS)

    Williams, R. J.; Mullins, O.

    1981-01-01

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

  20. GRAPHITE ELECTRODE FOR THE MEASUREMENT OF REDOX POTENTIAL AND OXYGEN DIFFUSION RATE IN SOIL

    EPA Science Inventory

    The objective of the project was to evaluate control measurements that might be made at land treatment sites to determine the effectiveness of operation in the management of hazardous wastes. Initial studies were on measurement of oxygen concentration and oxygen diffusion rate (O...

  1. 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…

  2. 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.

  3. Clonal genotype and population structure inference from single-cell tumor sequencing.

    PubMed

    Roth, Andrew; McPherson, Andrew; Laks, Emma; Biele, Justina; Yap, Damian; Wan, Adrian; Smith, Maia A; Nielsen, Cydney B; McAlpine, Jessica N; Aparicio, Samuel; Bouchard-Côté, Alexandre; Shah, Sohrab P

    2016-07-01

    Single-cell DNA sequencing has great potential to reveal the clonal genotypes and population structure of human cancers. However, single-cell data suffer from missing values and biased allelic counts as well as false genotype measurements owing to the sequencing of multiple cells. We describe the Single Cell Genotyper (https://bitbucket.org/aroth85/scg), an open-source software based on a statistical model coupled with a mean-field variational inference method, which can be used to address these problems and robustly infer clonal genotypes. PMID:27183439

  4. Fast serial analysis of active cholesterol at the plasma membrane in single cells.

    PubMed

    Tian, Chunxiu; Zhou, Junyu; Wu, Zeng-Qiang; Fang, Danjun; Jiang, Dechen

    2014-01-01

    Previously, our group has utilized the luminol electrochemiluminescence to analyze the active cholesterol at the plasma membrane in single cells by the exposure of one cell to a photomultiplier tube (PMT) through a pinhole. In this paper, fast analysis of active cholesterol at the plasma membrane in single cells was achieved by a multimicroelectrode array without the pinhole. Single cells were directly located on the microelectrodes using cell-sized microwell traps. A cycle of voltage was applied on the microelectrodes sequentially to induce a peak of luminescence from each microelectrode for the serial measurement of active membrane cholesterol. A minimal time of 1.60 s was determined for the analysis of one cell. The simulation and the experimental data exhibited a semisteady-state distribution of hydrogen peroxide on the microelectrode after the reaction of cholesterol oxidase with the membrane cholesterol, which supported the relative accuracy of the serial analysis. An eight-microelectrode array was demonstrated to analyze eight single cells in 22 s serially, including the channel switching time. The results from 64 single cells either activated by low ion strength buffer or the inhibition of intracellular acyl-coA/cholesterol acyltransferase (ACAT) revealed that most of the cells analyzed had the similar active membrane cholesterol, while few cells had more active cholesterol resulting in the cellular heterogeneity. The fast single-cell analysis platform developed will be potentially useful for the analysis of more molecules in single cells using proper oxidases. PMID:24328095

  5. A convenient, optimized pipeline for isolation, fluorescence microscopy and molecular analysis of live single cells

    PubMed Central

    2014-01-01

    Background Heterogeneity within cell populations is relevant to the onset and progression of disease, as well as development and maintenance of homeostasis. Analysis and understanding of the roles of heterogeneity in biological systems require methods and technologies that are capable of single cell resolution. Single cell gene expression analysis by RT-qPCR is an established technique for identifying transcriptomic heterogeneity in cellular populations, but it generally requires specialized equipment or tedious manipulations for cell isolation. Results We describe the optimization of a simple, inexpensive and rapid pipeline which includes isolation and culture of live single cells as well as fluorescence microscopy and gene expression analysis of the same single cells by RT-qPCR. We characterize the efficiency of single cell isolation and demonstrate our method by identifying single GFP-expressing cells from a mixed population of GFP-positive and negative cells by correlating fluorescence microscopy and RT-qPCR. Conclusions Single cell gene expression analysis by RT-qPCR is a convenient means for investigating cellular heterogeneity, but is most useful when correlating observations with additional measurements. We demonstrate a convenient and simple pipeline for multiplexing single cell RT-qPCR with fluorescence microscopy which is adaptable to other molecular analyses. PMID:24834016

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

    PubMed Central

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

    2013-01-01

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

  7. 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

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

    NASA Astrophysics Data System (ADS)

    Meyer, Scott Andrew

    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 times 10 ^{17} and 9 times 10^{17} cm ^{-3}. The broadening coefficient for resonance interactions is deduced from this data, and this value is in accord with available theoretical models.

  9. 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.

  10. 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.