WorldWideScience

Sample records for perform single-cell studies

  1. The Effects of Nonuniform Illumination on the Electrical Performance of a Single Conventional Photovoltaic Cell

    Directory of Open Access Journals (Sweden)

    Damasen Ikwaba Paul

    2015-01-01

    Full Text Available Photovoltaic (PV concentrators are a promising approach for lowering PV electricity costs in the near future. However, most of the concentrators that are currently used for PV applications yield nonuniform flux profiles on the surface of a PV module which in turn reduces its electrical performance if the cells are serially connected. One way of overcoming this effect is the use of PV modules with isolated cells so that each cell generates current that is proportional to the energy flux absorbed. However, there are some cases where nonuniform illumination also exists in a single cell in an isolated cells PV module. This paper systematically studied the effect of nonuniform illumination on various cell performance parameters of a single monocrystalline standard PV cell at low and medium energy concentration ratios. Furthermore, the effect of orientation, size, and geometrical shapes of nonuniform illumination was also investigated. It was found that the effect of nonuniform illumination on various PV cell performance parameters of a single standard PV cell becomes noticeable at medium energy flux concentration whilst the location, size, and geometrical shape of nonuniform illumination have no effect on the performance parameters of the cell.

  2. Understanding InP Nanowire Array Solar Cell Performance by Nanoprobe-Enabled Single Nanowire Measurements.

    Science.gov (United States)

    Otnes, Gaute; Barrigón, Enrique; Sundvall, Christian; Svensson, K Erik; Heurlin, Magnus; Siefer, Gerald; Samuelson, Lars; Åberg, Ingvar; Borgström, Magnus T

    2018-05-09

    III-V solar cells in the nanowire geometry might hold significant synthesis-cost and device-design advantages as compared to thin films and have shown impressive performance improvements in recent years. To continue this development there is a need for characterization techniques giving quick and reliable feedback for growth development. Further, characterization techniques which can improve understanding of the link between nanowire growth conditions, subsequent processing, and solar cell performance are desired. Here, we present the use of a nanoprobe system inside a scanning electron microscope to efficiently contact single nanowires and characterize them in terms of key parameters for solar cell performance. Specifically, we study single as-grown InP nanowires and use electron beam induced current characterization to understand the charge carrier collection properties, and dark current-voltage characteristics to understand the diode recombination characteristics. By correlating the single nanowire measurements to performance of fully processed nanowire array solar cells, we identify how the performance limiting parameters are related to growth and/or processing conditions. We use this understanding to achieve a more than 7-fold improvement in efficiency of our InP nanowire solar cells, grown from a different seed particle pattern than previously reported from our group. The best cell shows a certified efficiency of 15.0%; the highest reported value for a bottom-up synthesized InP nanowire solar cell. We believe the presented approach have significant potential to speed-up the development of nanowire solar cells, as well as other nanowire-based electronic/optoelectronic devices.

  3. Solid Polymer Fuel Cells. Electrode and membrane performance studies

    Energy Technology Data Exchange (ETDEWEB)

    Moeller-Holst, S.

    1996-12-31

    This doctoral thesis studies aspects of fuel cell preparation and performance. The emphasis is placed on preparation and analysis of low platinum-loading solid polymer fuel cell (SPEC) electrodes. A test station was built and used to test cells within a wide range of real operating conditions, 40-150{sup o}C and 1-10 bar. Preparation and assembling equipment for single SPFCs was designed and built, and a new technique of spraying the catalyst layer directly onto the membrane was successfully demonstrated. Low Pt-loading electrodes (0.1 mg Pt/cm{sup 2}) prepared by the new technique exhibited high degree of catalyst utilization. The performance of single cells holding these electrodes is comparable to state-of-the-art SPFCs. Potential losses in single cell performance are ascribed to irreversibilities by analysing the efficiency of the Solid Oxide Fuel Cell by means of the second law of thermodynamics. The water management in membranes is discussed for a model system and the results are relevant to fuel cell preparation and performance. The new spray deposition technique should be commercially interesting as it involves few steps as well as techniques that are adequate for larger scale production. 115 refs., 43 figs., 18 tabs.

  4. Solid Polymer Fuel Cells. Electrode and membrane performance studies

    Energy Technology Data Exchange (ETDEWEB)

    Moeller-Holst, S

    1997-12-31

    This doctoral thesis studies aspects of fuel cell preparation and performance. The emphasis is placed on preparation and analysis of low platinum-loading solid polymer fuel cell (SPEC) electrodes. A test station was built and used to test cells within a wide range of real operating conditions, 40-150{sup o}C and 1-10 bar. Preparation and assembling equipment for single SPFCs was designed and built, and a new technique of spraying the catalyst layer directly onto the membrane was successfully demonstrated. Low Pt-loading electrodes (0.1 mg Pt/cm{sup 2}) prepared by the new technique exhibited high degree of catalyst utilization. The performance of single cells holding these electrodes is comparable to state-of-the-art SPFCs. Potential losses in single cell performance are ascribed to irreversibilities by analysing the efficiency of the Solid Oxide Fuel Cell by means of the second law of thermodynamics. The water management in membranes is discussed for a model system and the results are relevant to fuel cell preparation and performance. The new spray deposition technique should be commercially interesting as it involves few steps as well as techniques that are adequate for larger scale production. 115 refs., 43 figs., 18 tabs.

  5. Effect of interlayer on structure and performance of anode-supported SOFC single cells

    International Nuclear Information System (INIS)

    Eom, Tae Wook; Yang, Hae Kwang; Kim, Kyung Hwan; Yoon, Hyon Hee; Kim, Jong Sung; Park, Sang Joon

    2008-01-01

    To lower the operating temperatures in solid oxide fuel cell (SOFC) operations, anode-supported SOFC single cells with a single dip-coated interlayer were fabricated and the effect of the interlayer on the electrolyte structure and the electrical performance was investigated. For the preparation of SOFC single cells, yttria-stabilized zirconia (YSZ) electrolyte, NiO-YSZ anode, and 50% YSZ-50% strontium-doped lanthanum manganite (LSM) cathode were used. In order to characterize the cells, scanning electron microscopy (SEM) and atomic force microscopy (AFM) were utilized and the gas (air) permeability measurements were conducted for gas tightness estimation. When the interlayer was inserted onto NiO-YSZ anode, the surface roughness of anode was diminished by about 40% and dense crack-free electrolytes were obtained. The electrical performance was enhanced remarkably and the maximum power density was 0.57 W/cm 2 at 800 deg. C and 0.44 W/cm 2 at 700 deg. C. On the other hand, the effect of interlayer on the gas tightness was negligible. The characterization study revealed that the enhancement in the electrical performance was mainly attributed to the increase of ion transmission area of anode/electrolyte interface and the increase of ionic conductivity of dense crack-free electrolyte layer

  6. Using Single-Protein Tracking to Study Cell Migration.

    Science.gov (United States)

    Orré, Thomas; Mehidi, Amine; Massou, Sophie; Rossier, Olivier; Giannone, Grégory

    2018-01-01

    To get a complete understanding of cell migration, it is critical to study its orchestration at the molecular level. Since the recent developments in single-molecule imaging, it is now possible to study molecular phenomena at the single-molecule level inside living cells. In this chapter, we describe how such approaches have been and can be used to decipher molecular mechanisms involved in cell migration.

  7. Single-cell analysis of targeted transcriptome predicts drug sensitivity of single cells within human myeloma tumors.

    Science.gov (United States)

    Mitra, A K; Mukherjee, U K; Harding, T; Jang, J S; Stessman, H; Li, Y; Abyzov, A; Jen, J; Kumar, S; Rajkumar, V; Van Ness, B

    2016-05-01

    Multiple myeloma (MM) is characterized by significant genetic diversity at subclonal levels that have a defining role in the heterogeneity of tumor progression, clinical aggressiveness and drug sensitivity. Although genome profiling studies have demonstrated heterogeneity in subclonal architecture that may ultimately lead to relapse, a gene expression-based prediction program that can identify, distinguish and quantify drug response in sub-populations within a bulk population of myeloma cells is lacking. In this study, we performed targeted transcriptome analysis on 528 pre-treatment single cells from 11 myeloma cell lines and 418 single cells from 8 drug-naïve MM patients, followed by intensive bioinformatics and statistical analysis for prediction of proteasome inhibitor sensitivity in individual cells. Using our previously reported drug response gene expression profile signature at the single-cell level, we developed an R Statistical analysis package available at https://github.com/bvnlabSCATTome, SCATTome (single-cell analysis of targeted transcriptome), that restructures the data obtained from Fluidigm single-cell quantitative real-time-PCR analysis run, filters missing data, performs scaling of filtered data, builds classification models and predicts drug response of individual cells based on targeted transcriptome using an assortment of machine learning methods. Application of SCATT should contribute to clinically relevant analysis of intratumor heterogeneity, and better inform drug choices based on subclonal cellular responses.

  8. Single-cell technologies to study the immune system.

    Science.gov (United States)

    Proserpio, Valentina; Mahata, Bidesh

    2016-02-01

    The immune system is composed of a variety of cells that act in a coordinated fashion to protect the organism against a multitude of different pathogens. The great variability of existing pathogens corresponds to a similar high heterogeneity of the immune cells. The study of individual immune cells, the fundamental unit of immunity, has recently transformed from a qualitative microscopic imaging to a nearly complete quantitative transcriptomic analysis. This shift has been driven by the rapid development of multiple single-cell technologies. These new advances are expected to boost the detection of less frequent cell types and transient or intermediate cell states. They will highlight the individuality of each single cell and greatly expand the resolution of current available classifications and differentiation trajectories. In this review we discuss the recent advancement and application of single-cell technologies, their limitations and future applications to study the immune system. © 2015 The Authors. Immunology Published by John Wiley & Sons Ltd.

  9. Performance study of solar cell arrays based on a Trough Concentrating Photovoltaic/Thermal system

    International Nuclear Information System (INIS)

    Li, Ming; Ji, Xu; Li, Guoliang; Wei, Shengxian; Li, YingFeng; Shi, Feng

    2011-01-01

    Highlights: → The performances of solar cell arrays based on a Trough Concentrating Photovoltaic/Thermal (TCPV/T) system have been studied. → The optimum concentration ratios for the single crystalline silicon cell, the Super cells and the GaAs cells were studied by experiments. → The influences between the solar cell's performance and the series resistances, the working temperature, solar irradiation intensity were explored. - Abstract: The performances of solar cell arrays based on a Trough Concentrating Photovoltaic/Thermal (TCPV/T) system have been studied via both experiment and theoretical calculation. The I-V characteristics of the solar cell arrays and the output performances of the TCPV/T system demonstrated that among the investigated four types of solar cell arrays, the triple junction GaAs cells possessed good performance characteristics and the polysilicon cells exhibited poor performance characteristics under concentrating conditions. The optimum concentration ratios for the single crystalline silicon cell, the Super cells and the GaAs cells were also studied by experiments. The optimum concentration ratios for the single crystalline silicon cells and Super cells were 4.23 and 8.46 respectively, and the triple junction GaAs cells could work well at higher concentration ratio. Besides, some theoretical calculations and experiments were performed to explore the influences of the series resistances and the working temperature. When the series resistances R s changed from 0 Ω to 1 Ω, the maximum power P m of the single crystalline silicon, the polycrystalline silicon, the Super cell and the GaAs cell arrays decreased by 67.78%, 74.93%, 77.30% and 58.07% respectively. When the cell temperature increased by 1 K, the short circuit current of the four types of solar cell arrays decreased by 0.11818 A, 0.05364 A, 0.01387 A and 0.00215 A respectively. The research results demonstrated that the output performance of the solar cell arrays with lower

  10. Single Cell Assay for Analyzing Single Cell Exosome and Endocrine Secretion and Cancer Markers

    Science.gov (United States)

    Chiu, Yu-Jui

    To understand the inhomogeneity of cells in biological systems, there is a growing demand for the capability to characterize the properties of individual single cells. Since single cell studies require continuous monitoring of the cell behaviors instead of a snapshot test at a single time point, 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 cannot provide, for appropriate cell types, proliferation of single cells and convenient, non-invasive tests of single cell behaviors from molecular markers. In this dissertation, I present a highly versatile single-cell assay 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. The salient features of the assay are the non-invasive collection and surveying of single cell secretions at different time points and massively parallel translocation of single cells by user defined criteria, producing very high compatibility to the downstream process such as single cell qPCR and sequencing. Above all, the acquired information is quantitative -- for example, one of the studies is 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.

  11. Performance features of 22-cell, 19Ah single pressure vessel nickel hydrogen battery

    Energy Technology Data Exchange (ETDEWEB)

    Rao, G.M.; Vaidyanathan, H.

    1996-02-01

    Two 22-cells 19Ah Nickel-Hydrogen (Ni-H2) Single Pressure Vessel (SPV) Qual batteries, one each from EPI/Joplin and EPI/Butler, were designed and procured. The two batteries differ in the cell encapsulation technology, stack preload, and activation procedure. Both the Butler and Joplin batteries met the specified requirements when subjected to qualification testing and completed 2100 and 1300 LEO cycles respectively, with nominal performance. This paper discusses advantages, design features, testing procedures, and results of the two single pressure vessel Ni-H2 batteries.

  12. Performance features of 22-cell, 19Ah single pressure vessel nickel hydrogen battery

    Science.gov (United States)

    Rao, Gopalakrishna M.; Vaidyanathan, Hari

    1996-01-01

    Two 22-cells 19Ah Nickel-Hydrogen (Ni-H2) Single Pressure Vessel (SPV) Qual batteries, one each from EPI/Joplin and EPI/Butler, were designed and procured. The two batteries differ in the cell encapsulation technology, stack preload, and activation procedure. Both the Butler and Joplin batteries met the specified requirements when subjected to qualification testing and completed 2100 and 1300 LEO cycles respectively, with nominal performance. This paper discusses advantages, design features, testing procedures, and results of the two single pressure vessel Ni-H2 batteries.

  13. Technologies for Single-Cell Isolation

    Science.gov (United States)

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

    2015-01-01

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

  14. Technologies for Single-Cell Isolation

    Directory of Open Access Journals (Sweden)

    Andre Gross

    2015-07-01

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

  15. Technologies for Single-Cell Isolation.

    Science.gov (United States)

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

    2015-07-24

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

  16. Cell biochemistry studied by single-molecule imaging.

    Science.gov (United States)

    Mashanov, G I; Nenasheva, T A; Peckham, M; Molloy, J E

    2006-11-01

    Over the last decade, there have been remarkable developments in live-cell imaging. We can now readily observe individual protein molecules within living cells and this should contribute to a systems level understanding of biological pathways. Direct observation of single fluorophores enables several types of molecular information to be gathered. Temporal and spatial trajectories enable diffusion constants and binding kinetics to be deduced, while analyses of fluorescence lifetime, intensity, polarization or spectra give chemical and conformational information about molecules in their cellular context. By recording the spatial trajectories of pairs of interacting molecules, formation of larger molecular complexes can be studied. In the future, multicolour and multiparameter imaging of single molecules in live cells will be a powerful analytical tool for systems biology. Here, we discuss measurements of single-molecule mobility and residency at the plasma membrane of live cells. Analysis of diffusional paths at the plasma membrane gives information about its physical properties and measurement of temporal trajectories enables rates of binding and dissociation to be derived. Meanwhile, close scrutiny of individual fluorophore trajectories enables ideas about molecular dimerization and oligomerization related to function to be tested directly.

  17. Performance of planar single cell lanthanum gallate based solid oxide fuel cells

    Energy Technology Data Exchange (ETDEWEB)

    Maffei, N.; Kuriakose, A.K. [Materials Technology Labs., CANMET, Natural Resources Canada, Ottawa, ON (Canada)

    1998-09-01

    A novel synthesis of high purity, single phase strontium-magnesium doped lanthanum gallate through a nitrate route is described. The prepared powder is formed into planar monolithic elements by uniaxial pressing followed by isostatic pressing and sintering. XRD analysis of the sintered elements reveal no detectable secondary phases. The performance of the electrolyte in solid oxide fuel cells (SOFC) with three different anode/cathode combinations tested at 700 C with respect to the J-V and power density is reported. The data show that the characteristics of this SOFC are strongly dependent on the particular anode/cathode system chosen. (orig.)

  18. Performance of planar single cell lanthanum gallate based solid oxide fuel cells

    Science.gov (United States)

    Maffei, N.; Kuriakose, A. K.

    A novel synthesis of high purity, single phase strontium-magnesium doped lanthanum gallate through a nitrate route is described. The prepared powder is formed into planar monolithic elements by uniaxial pressing followed by isostatic pressing and sintering. XRD analysis of the sintered elements reveal no detectable secondary phases. The performance of the electrolyte in solid oxide fuel cells (SOFC) with three different anode/cathode combinations tested at 700°C with respect to the J- V and power density is reported. The data show that the characteristics of this SOFC are strongly dependent on the particular anode/cathode system chosen.

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

    OpenAIRE

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

    2013-01-01

    Blood and tissue are composed of many functionally distinct cell subsets. In immunological studies, these can be measured accurately only using single-cell assays. The characterization of these small cell subsets is crucial to decipher system-level biological changes. For this reason, an increasing number of studies rely on assays that provide single-cell measurements of multiple genes and proteins from bulk cell samples. A common problem in the analysis of such data is to identify biomarkers...

  20. Plant Systems Biology at the Single-Cell Level.

    Science.gov (United States)

    Libault, Marc; Pingault, Lise; Zogli, Prince; Schiefelbein, John

    2017-11-01

    Our understanding of plant biology is increasingly being built upon studies using 'omics and system biology approaches performed at the level of the entire plant, organ, or tissue. Although these approaches open new avenues to better understand plant biology, they suffer from the cellular complexity of the analyzed sample. Recent methodological advances now allow plant scientists to overcome this limitation and enable biological analyses of single-cells or single-cell-types. Coupled with the development of bioinformatics and functional genomics resources, these studies provide opportunities for high-resolution systems analyses of plant phenomena. In this review, we describe the recent advances, current challenges, and future directions in exploring the biology of single-cells and single-cell-types to enhance our understanding of plant biology as a system. Copyright © 2017 Elsevier Ltd. All rights reserved.

  1. Study of double porous silicon surfaces for enhancement of silicon solar cell performance

    Science.gov (United States)

    Razali, N. S. M.; Rahim, A. F. A.; Radzali, R.; Mahmood, A.

    2017-09-01

    In this work, design and simulation of double porous silicon surfaces for enhancement of silicon solar cell is carried out. Both single and double porous structures are constructed by using TCAD ATHENA and TCAD DEVEDIT tools of the SILVACO software respectively. After the structures were created, I-V characteristics and spectral response of the solar cell were extracted using ATLAS device simulator. Finally, the performance of the simulated double porous solar cell is compared with the performance of both single porous and bulk-Si solar cell. The results showed that double porous silicon solar cell exhibited 1.8% efficiency compared to 1.3% and 1.2% for single porous silicon and bulk-Si solar cell.

  2. Experimental Study and Comparison of Various Designs of Gas Flow Fields to PEM Fuel Cells and Cell Stack Performance

    International Nuclear Information System (INIS)

    Liu, Hong; Li, Peiwen; Juarez-Robles, Daniel; Wang, Kai; Hernandez-Guerrero, Abel

    2014-01-01

    In this study, a significant number of experimental tests to proton exchange membrane (PEM) fuel cells were conducted to investigate the effect of gas flow fields on fuel cell performance. Graphite plates with various flow field or flow channel designs, from literature survey and also novel designs by the authors, were used for the PEM fuel cell assembly. The fabricated fuel cells have an effective membrane area of 23.5 cm 2 . The results showed that the serpentine flow channel design is still favorable, giving the best single fuel cell performance amongst all the studied flow channel designs. A novel symmetric serpentine flow field was proposed for a relatively large sized fuel cell application. Four fuel cell stacks each including four cells were assembled using different designs of serpentine flow channels. The output power performances of fuel cell stacks were compared and the novel symmetric serpentine flow field design is recommended for its very good performance.

  3. Single-Cell Whole-Genome Amplification and Sequencing: Methodology and Applications.

    Science.gov (United States)

    Huang, Lei; Ma, Fei; Chapman, Alec; Lu, Sijia; Xie, Xiaoliang Sunney

    2015-01-01

    We present a survey of single-cell whole-genome amplification (WGA) methods, including degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR), multiple displacement amplification (MDA), and multiple annealing and looping-based amplification cycles (MALBAC). The key parameters to characterize the performance of these methods are defined, including genome coverage, uniformity, reproducibility, unmappable rates, chimera rates, allele dropout rates, false positive rates for calling single-nucleotide variations, and ability to call copy-number variations. Using these parameters, we compare five commercial WGA kits by performing deep sequencing of multiple single cells. We also discuss several major applications of single-cell genomics, including studies of whole-genome de novo mutation rates, the early evolution of cancer genomes, circulating tumor cells (CTCs), meiotic recombination of germ cells, preimplantation genetic diagnosis (PGD), and preimplantation genomic screening (PGS) for in vitro-fertilized embryos.

  4. High-performance imaging of stem cells using single-photon emissions

    Science.gov (United States)

    Wagenaar, Douglas J.; Moats, Rex A.; Hartsough, Neal E.; Meier, Dirk; Hugg, James W.; Yang, Tang; Gazit, Dan; Pelled, Gadi; Patt, Bradley E.

    2011-10-01

    Radiolabeled cells have been imaged for decades in the field of autoradiography. Recent advances in detector and microelectronics technologies have enabled the new field of "digital autoradiography" which remains limited to ex vivo specimens of thin tissue slices. The 3D field-of-view (FOV) of single cell imaging can be extended to millimeters if the low energy (10-30 keV) photon emissions of radionuclides are used for single-photon nuclear imaging. This new microscope uses a coded aperture foil made of highly attenuating elements such as gold or platinum to form the image as a kind of "lens". The detectors used for single-photon emission microscopy are typically silicon detectors with a pixel pitch less than 60 μm. The goal of this work is to image radiolabeled mesenchymal stem cells in vivo in an animal model of tendon repair processes. Single-photon nuclear imaging is an attractive modality for translational medicine since the labeled cells can be imaged simultaneously with the reparative processes by using the dual-isotope imaging technique. The details our microscope's two-layer gold aperture and the operation of the energy-dispersive, pixellated silicon detector are presented along with the first demonstration of energy discrimination with a 57Co source. Cell labeling techniques have been augmented by genetic engineering with the sodium-iodide symporter, a type of reporter gene imaging method that enables in vivo uptake of free 99mTc or an iodine isotope at a time point days or weeks after the insertion of the genetically modified stem cells into the animal model. This microscopy work in animal research may expand to the imaging of reporter-enabled stem cells simultaneously with the expected biological repair process in human clinical trials of stem cell therapies.

  5. Single-Cell RNA Sequencing of Glioblastoma Cells.

    Science.gov (United States)

    Sen, Rajeev; Dolgalev, Igor; Bayin, N Sumru; Heguy, Adriana; Tsirigos, Aris; Placantonakis, Dimitris G

    2018-01-01

    Single-cell RNA sequencing (sc-RNASeq) is a recently developed technique used to evaluate the transcriptome of individual cells. As opposed to conventional RNASeq in which entire populations are sequenced in bulk, sc-RNASeq can be beneficial when trying to better understand gene expression patterns in markedly heterogeneous populations of cells or when trying to identify transcriptional signatures of rare cells that may be underrepresented when using conventional bulk RNASeq. In this method, we describe the generation and analysis of cDNA libraries from single patient-derived glioblastoma cells using the C1 Fluidigm system. The protocol details the use of the C1 integrated fluidics circuit (IFC) for capturing, imaging and lysing cells; performing reverse transcription; and generating cDNA libraries that are ready for sequencing and analysis.

  6. Single gold nanoparticle plasmonic spectroscopy for study of chemical-dependent efflux function of single ABC transporters of single live Bacillus subtilis cells.

    Science.gov (United States)

    Browning, Lauren M; Lee, Kerry J; Cherukuri, Pavan K; Huang, Tao; Songkiatisak, Preeyaporn; Warren, Seth; Xu, Xiao-Hong Nancy

    2018-03-26

    ATP-binding cassette (ABC) membrane transporters serve as self-defense transport apparatus in many living organisms and they can selectively extrude a wide variety of substrates, leading to multidrug resistance (MDR). The detailed molecular mechanisms remain elusive. Single nanoparticle plasmonic spectroscopy highly depends upon their sizes, shapes, chemical and surface properties. In our previous studies, we have used the size-dependent plasmonic spectra of single silver nanoparticles (Ag NPs) to study the real-time efflux kinetics of the ABC (BmrA) transporter and MexAB-OprM transporter in single live cells (Gram-positive and Gram-negative bacterium), respectively. In this study, we prepared and used purified, biocompatible and stable (non-aggregated) gold nanoparticles (Au NPs) (12.4 ± 0.9 nm) to study the efflux kinetics of single BmrA membrane transporters of single live Bacillus subtillis cells, aiming to probe chemical dependent efflux functions of BmrA transporters and their potential chemical sensing capability. Similar to those observed using Ag NPs, accumulation of the intracellular Au NPs in single live cells (WT and ΔBmrA) highly depends upon the cellular expression of BmrA and the NP concentration (0.7 and 1.4 nM). The lower accumulation of intracellular Au NPs in WT (normal expression of BmrA) than ΔBmrA (deletion of bmrA) indicates that BmrA extrudes the Au NPs out of the WT cells. The accumulation of Au NPs in the cells increases with NP concentration, suggesting that the Au NPs most likely passively diffuse into the cells, similar to antibiotics. The result demonstrates that such small Au NPs can serve as imaging probes to study the efflux function of the BmrA membrane transporter in single live cells. Furthermore, the dependence of the accumulation rate of intracellular Au NPs in single live cells upon the expression of BmrA and the concentration of the NPs is about twice higher than that of the same sized Ag NPs. This interesting finding

  7. The effect of ethanol concentration on the direct ethanol fuel cell performance and products distribution: A study using a single fuel cell/attenuated total reflectance - Fourier transform infrared spectroscopy

    Science.gov (United States)

    Assumpção, M. H. M. T.; Nandenha, J.; Buzzo, G. S.; Silva, J. C. M.; Spinacé, E. V.; Neto, A. O.; De Souza, R. F. B.

    2014-05-01

    The effect of ethanol concentration on the direct ethanol fuel cell (DEFC) performance and products distribution were studied in situ using a single fuel cell/ATR-FTIR setup. The experiments were performed at 80 °C using commercial Pt3Sn/C as anodic catalyst and the concentrations of ethanol solution were varied from 0.1 to 2.0 mol L-1. An increase in power density was observed with the increase of ethanol concentration to 1.0 mol L-1, while the band intensities analysis in the FTIR spectra revealed an increase of acetic acid/acetaldehyde ratio with the increase of ethanol concentration. Also, from FTIR spectra results, it could be concluded that the acetic acid production follow parallel mechanisms; that is, it does not require the presence of acetaldehyde as an intermediate.

  8. Numerical study of assembly pressure effect on the performance of proton exchange membrane fuel cell

    Energy Technology Data Exchange (ETDEWEB)

    Taymaz, Imdat; Benli, Merthan [Department of Mechanical Engineering, University of Sakarya, 54187 Adapazari (Turkey)

    2010-05-15

    The performance of the fuel cell is affected by many parameters. One of these parameters is assembly pressure that changes the mechanical properties and dimensions of the fuel cell components. Its first duty, however, is to prevent gas or liquid leakage from the cell and it is important for the contact behaviors of fuel cell components. Some leakage and contact problems can occur on the low assembly pressures whereas at high pressures, components of the fuel cell, such as bipolar plates (BPP), gas diffusion layers (GDL), catalyst layers, and membranes, can be damaged. A finite element analysis (FEA) model is developed to predict the deformation effect of assembly pressure on the single channel PEM fuel cell in this study. Deformed fuel cell single channel model is imported to three-dimensional, computational fluid dynamics (CFD) model which is developed for simulating proton exchange membrane (PEM) fuel cells. Using this model, the effect of assembly pressure on fuel cell performance can be calculated. It is found that, when the assembly pressure increases, contact resistance, porosity and thickness of the gas diffusion layer (GDL) decreases. Too much assembly pressure causes GDL to destroy; therefore, the optimal assembly pressure is significant to obtain the highest performance from fuel cell. By using the results of this study, optimum fuel cell design and operating condition parameters can be predicted accordingly. (author)

  9. Influence of Electrode Design and Contacting Layers on Performance of Electrolyte Supported SOFC/SOEC Single Cells

    OpenAIRE

    Mihails Kusnezoff; Nikolai Trofimenko; Martin Müller; Alexander Michaelis

    2016-01-01

    The solid oxide cell is a basis for highly efficient and reversible electrochemical energy conversion. A single cell based on a planar electrolyte substrate as support (ESC) is often utilized for SOFC/SOEC stack manufacturing and fulfills necessary requirements for application in small, medium and large scale fuel cell and electrolysis systems. Thickness of the electrolyte substrate, and its ionic conductivity limits the power density of the ESC. To improve the performance of this cell type i...

  10. Single-cell intracellular nano-pH probes†

    Science.gov (United States)

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

    2016-01-01

    Within a large clonal population, such as cancerous tumor entities, cells are not identical, and the differences between intracellular pH levels of individual cells may be important indicators of heterogeneity that could be relevant in clinical practice, especially in personalized medicine. Therefore, the detection of the intracellular pH at the single-cell level is of great importance to identify and study outlier cells. However, quantitative and real-time measurements of the intracellular pH of individual cells within a cell population is challenging with existing technologies, and there is a need to engineer new methodologies. In this paper, we discuss the use of nanopipette technology to overcome the limitations of intracellular pH measurements at the single-cell level. We have developed a nano-pH probe through physisorption of chitosan onto hydroxylated quartz nanopipettes with extremely small pore sizes (~100 nm). The dynamic pH range of the nano-pH probe was from 2.6 to 10.7 with a sensitivity of 0.09 units. We have performed single-cell intracellular pH measurements using non-cancerous and cancerous cell lines, including human fibroblasts, HeLa, MDA-MB-231 and MCF-7, with the pH nanoprobe. We have further demonstrated the real-time continuous single-cell pH measurement capability of the sensor, showing the cellular pH response to pharmaceutical manipulations. These findings suggest that the chitosan-functionalized nanopore is a powerful nano-tool for pH sensing at the single-cell level with high temporal and spatial resolution. PMID:27708772

  11. Single-cell intracellular nano-pH probes.

    Science.gov (United States)

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

    2015-01-01

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

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

    Science.gov (United States)

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

    2009-06-01

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

  13. Single Cell Oncogenesis

    Science.gov (United States)

    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.

  14. Time-course correlation of biofilm properties and electrochemical performance in single-chamber microbial fuel cells

    KAUST Repository

    Ren, Zhiyong; Ramasamy, Ramaraja P.; Cloud-Owen, Susan Red; Yan, Hengjing; Mench, Matthew M.; Regan, John M.

    2011-01-01

    The relationship between anode microbial characteristics and electrochemical parameters in microbial fuel cells (MFCs) was analyzed by time-course sampling of parallel single-bottle MFCs operated under identical conditions. While voltage stabilized within 4. days, anode biofilms continued growing during the six-week operation. Viable cell density increased asymptotically, but membrane-compromised cells accumulated steadily from only 9% of total cells on day 3 to 52% at 6. weeks. Electrochemical performance followed the viable cell trend, with a positive correlation for power density and an inverse correlation for anode charge transfer resistance. The biofilm architecture shifted from rod-shaped, dispersed cells to more filamentous structures, with the continuous detection of Geobacter sulfurreducens-like 16S rRNA fragments throughout operation and the emergence of a community member related to a known phenazine-producing Pseudomonas species. A drop in cathode open circuit potential between weeks two and three suggested that uncontrolled biofilm growth on the cathode deleteriously affects system performance. © 2010 Elsevier Ltd.

  15. Clustering Single-Cell Expression Data Using Random Forest Graphs.

    Science.gov (United States)

    Pouyan, Maziyar Baran; Nourani, Mehrdad

    2017-07-01

    Complex tissues such as brain and bone marrow are made up of multiple cell types. As the study of biological tissue structure progresses, the role of cell-type-specific research becomes increasingly important. Novel sequencing technology such as single-cell cytometry provides researchers access to valuable biological data. Applying machine-learning techniques to these high-throughput datasets provides deep insights into the cellular landscape of the tissue where those cells are a part of. In this paper, we propose the use of random-forest-based single-cell profiling, a new machine-learning-based technique, to profile different cell types of intricate tissues using single-cell cytometry data. Our technique utilizes random forests to capture cell marker dependences and model the cellular populations using the cell network concept. This cellular network helps us discover what cell types are in the tissue. Our experimental results on public-domain datasets indicate promising performance and accuracy of our technique in extracting cell populations of complex tissues.

  16. Parallel single-cell analysis microfluidic platform

    NARCIS (Netherlands)

    van den Brink, Floris Teunis Gerardus; Gool, Elmar; Frimat, Jean-Philippe; Bomer, Johan G.; van den Berg, Albert; le Gac, Severine

    2011-01-01

    We report a PDMS microfluidic platform for parallel single-cell analysis (PaSCAl) as a powerful tool to decipher the heterogeneity found in cell populations. Cells are trapped individually in dedicated pockets, and thereafter, a number of invasive or non-invasive analysis schemes are performed.

  17. High performance platinum single atom electrocatalyst for oxygen reduction reaction

    Science.gov (United States)

    Liu, Jing; Jiao, Menggai; Lu, Lanlu; Barkholtz, Heather M.; Li, Yuping; Wang, Ying; Jiang, Luhua; Wu, Zhijian; Liu, Di-Jia; Zhuang, Lin; Ma, Chao; Zeng, Jie; Zhang, Bingsen; Su, Dangsheng; Song, Ping; Xing, Wei; Xu, Weilin; Wang, Ying; Jiang, Zheng; Sun, Gongquan

    2017-07-01

    For the large-scale sustainable implementation of polymer electrolyte membrane fuel cells in vehicles, high-performance electrocatalysts with low platinum consumption are desirable for use as cathode material during the oxygen reduction reaction in fuel cells. Here we report a carbon black-supported cost-effective, efficient and durable platinum single-atom electrocatalyst with carbon monoxide/methanol tolerance for the cathodic oxygen reduction reaction. The acidic single-cell with such a catalyst as cathode delivers high performance, with power density up to 680 mW cm-2 at 80 °C with a low platinum loading of 0.09 mgPt cm-2, corresponding to a platinum utilization of 0.13 gPt kW-1 in the fuel cell. Good fuel cell durability is also observed. Theoretical calculations reveal that the main effective sites on such platinum single-atom electrocatalysts are single-pyridinic-nitrogen-atom-anchored single-platinum-atom centres, which are tolerant to carbon monoxide/methanol, but highly active for the oxygen reduction reaction.

  18. Using micro-patterned sensors and cell self-assembly for measuring the oxygen consumption rate of single cells

    International Nuclear Information System (INIS)

    Etzkorn, James R; Parviz, Babak A; Wu, Wen-Chung; Tian, Zhiyuan; Kim, Prince; Jang, Sei-Hum; Jen, Alex K-Y; Meldrum, Deirdre R

    2010-01-01

    We present a method for self-assembling arrays of live single cells on a glass chip using a photopatternable polymer to form micro-traps. We have studied the single-cell self-assembly method and optimized the process to obtain a 52% yield of single-trapped cells. We also report a method to measure the oxygen consumption rate of a single cell using micro-patterned sensors. These molecular oxygen sensors were fabricated around each micro-trap allowing optical interrogation of oxygen concentration in the immediate environment of the trapped cell. Micromachined micro-wells were then used to seal the trap, sensor and cell in order to determine the oxygen consumption rate of single cells. These techniques reported here add to the collection of tools for performing 'singe-cell' biology. An oxygen consumption rate of 1.05 ± 0.28 fmol min −1 was found for a data set consisting of 25 single A549 cells.

  19. An agar gel membrane-PDMS hybrid microfluidic device for long term single cell dynamic study.

    Science.gov (United States)

    Wong, Ieong; Atsumi, Shota; Huang, Wei-Chih; Wu, Tung-Yun; Hanai, Taizo; Lam, Miu-Ling; Tang, Ping; Yang, Jian; Liao, James C; Ho, Chih-Ming

    2010-10-21

    Significance of single cell measurements stems from the substantial temporal fluctuations and cell-cell variability possessed by individual cells. A major difficulty in monitoring surface non-adherent cells such as bacteria and yeast is that these cells tend to aggregate into clumps during growth, obstructing the tracking or identification of single-cells over long time periods. Here, we developed a microfluidic platform for long term single-cell tracking and cultivation with continuous media refreshing and dynamic chemical perturbation capability. The design highlights a simple device-assembly process between PDMS microchannel and agar membrane through conformal contact, and can be easily adapted by microbiologists for their routine laboratory use. The device confines cell growth in monolayer between an agar membrane and a glass surface. Efficient nutrient diffusion through the membrane and reliable temperature maintenance provide optimal growth condition for the cells, which exhibited fast exponential growth and constant distribution of cell sizes. More than 24 h of single-cell tracking was demonstrated on a transcription-metabolism integrated synthetic biological model, the gene-metabolic oscillator. Single cell morphology study under alcohol toxicity allowed us to discover and characterize cell filamentation exhibited by different E. coli isobutanol tolerant strains. We believe this novel device will bring new capabilities to quantitative microbiology, providing a versatile platform for single cell dynamic studies.

  20. A quantitative comparison of single-cell whole genome amplification methods.

    Directory of Open Access Journals (Sweden)

    Charles F A de Bourcy

    Full Text Available Single-cell sequencing is emerging as an important tool for studies of genomic heterogeneity. Whole genome amplification (WGA is a key step in single-cell sequencing workflows and a multitude of methods have been introduced. Here, we compare three state-of-the-art methods on both bulk and single-cell samples of E. coli DNA: Multiple Displacement Amplification (MDA, Multiple Annealing and Looping Based Amplification Cycles (MALBAC, and the PicoPLEX single-cell WGA kit (NEB-WGA. We considered the effects of reaction gain on coverage uniformity, error rates and the level of background contamination. We compared the suitability of the different WGA methods for the detection of copy-number variations, for the detection of single-nucleotide polymorphisms and for de-novo genome assembly. No single method performed best across all criteria and significant differences in characteristics were observed; the choice of which amplifier to use will depend strongly on the details of the type of question being asked in any given experiment.

  1. Single-cell technologies in environmental omics

    KAUST Repository

    Kodzius, Rimantas

    2015-10-22

    Environmental studies are primarily done by culturing isolated microorganisms or by amplifying and sequencing conserved genes. Difficulties understanding the complexity of large numbers of various microorganisms in an environment led to the development of techniques to enrich specific microorganisms for upstream analysis, ultimately leading to single-cell isolation and analyses. We discuss the significance of single-cell technologies in omics studies with focus on metagenomics and metatranscriptomics. We propose that by reducing sample heterogeneity using single-cell genomics, metaomic studies can be simplified.

  2. Single-cell technologies in environmental omics

    KAUST Repository

    Kodzius, Rimantas; Gojobori, Takashi

    2015-01-01

    Environmental studies are primarily done by culturing isolated microorganisms or by amplifying and sequencing conserved genes. Difficulties understanding the complexity of large numbers of various microorganisms in an environment led to the development of techniques to enrich specific microorganisms for upstream analysis, ultimately leading to single-cell isolation and analyses. We discuss the significance of single-cell technologies in omics studies with focus on metagenomics and metatranscriptomics. We propose that by reducing sample heterogeneity using single-cell genomics, metaomic studies can be simplified.

  3. Analysis of the performance of fuel cells PWR with a single enrichment and radial distribution of enrichments

    International Nuclear Information System (INIS)

    Vargas, S.; Gonzalez, J. A.; Alonso, G.; Del Valle, E.; Xolocostli M, J. V.

    2008-01-01

    One of the main challenges in the design of fuel assemblies is the efficient use of uranium achieving burnt homogeneous of the fuel rods as well as the burnt maximum possible of the same ones to the unload. In the case of the assemblies type PWR has been decided actually for fuel assemblies with a single radial enrichment. The present work has like effect to show the because of this decision, reason why a comparison of the neutronic performance of two fuel cells takes place with the same enrichment average but one of them with radial distribution of enrichment and the other with a single enrichment equal to the average. The results shown in the present study of the behavior of the neutron flow as well as the power distribution through of assembly sustain the because of a single radial enrichment. (Author)

  4. The nature and nurture of cell heterogeneity: accounting for macrophage gene-environment interactions with single-cell RNA-Seq.

    Science.gov (United States)

    Wills, Quin F; Mellado-Gomez, Esther; Nolan, Rory; Warner, Damien; Sharma, Eshita; Broxholme, John; Wright, Benjamin; Lockstone, Helen; James, William; Lynch, Mark; Gonzales, Michael; West, Jay; Leyrat, Anne; Padilla-Parra, Sergi; Filippi, Sarah; Holmes, Chris; Moore, Michael D; Bowden, Rory

    2017-01-07

    Single-cell RNA-Seq can be a valuable and unbiased tool to dissect cellular heterogeneity, despite the transcriptome's limitations in describing higher functional phenotypes and protein events. Perhaps the most important shortfall with transcriptomic 'snapshots' of cell populations is that they risk being descriptive, only cataloging heterogeneity at one point in time, and without microenvironmental context. Studying the genetic ('nature') and environmental ('nurture') modifiers of heterogeneity, and how cell population dynamics unfold over time in response to these modifiers is key when studying highly plastic cells such as macrophages. We introduce the programmable Polaris™ microfluidic lab-on-chip for single-cell sequencing, which performs live-cell imaging while controlling for the culture microenvironment of each cell. Using gene-edited macrophages we demonstrate how previously unappreciated knockout effects of SAMHD1, such as an altered oxidative stress response, have a large paracrine signaling component. Furthermore, we demonstrate single-cell pathway enrichments for cell cycle arrest and APOBEC3G degradation, both associated with the oxidative stress response and altered proteostasis. Interestingly, SAMHD1 and APOBEC3G are both HIV-1 inhibitors ('restriction factors'), with no known co-regulation. As single-cell methods continue to mature, so will the ability to move beyond simple 'snapshots' of cell populations towards studying the determinants of population dynamics. By combining single-cell culture, live-cell imaging, and single-cell sequencing, we have demonstrated the ability to study cell phenotypes and microenvironmental influences. It's these microenvironmental components - ignored by standard single-cell workflows - that likely determine how macrophages, for example, react to inflammation and form treatment resistant HIV reservoirs.

  5. Influence of Electrode Design and Contacting Layers on Performance of Electrolyte Supported SOFC/SOEC Single Cells

    Directory of Open Access Journals (Sweden)

    Mihails Kusnezoff

    2016-11-01

    Full Text Available The solid oxide cell is a basis for highly efficient and reversible electrochemical energy conversion. A single cell based on a planar electrolyte substrate as support (ESC is often utilized for SOFC/SOEC stack manufacturing and fulfills necessary requirements for application in small, medium and large scale fuel cell and electrolysis systems. Thickness of the electrolyte substrate, and its ionic conductivity limits the power density of the ESC. To improve the performance of this cell type in SOFC/SOEC mode, alternative fuel electrodes, on the basis of Ni/CGO as well as electrolytes with reduced thickness, have been applied. Furthermore, different interlayers on the air side have been tested to avoid the electrode delamination and to reduce the cell degradation in electrolysis mode. Finally, the influence of the contacting layer on cell performance, especially for cells with an ultrathin electrolyte and thin electrode layers, has been investigated. It has been found that Ni/CGO outperform traditional Ni/8YSZ electrodes and the introduction of a ScSZ interlayer substantially reduces the degradation rate of ESC in electrolysis mode. Furthermore, it was demonstrated that, for thin electrodes, the application of contacting layers with good conductivity and adhesion to current collectors improves performance significantly.

  6. Influence of Electrode Design and Contacting Layers on Performance of Electrolyte Supported SOFC/SOEC Single Cells.

    Science.gov (United States)

    Kusnezoff, Mihails; Trofimenko, Nikolai; Müller, Martin; Michaelis, Alexander

    2016-11-08

    The solid oxide cell is a basis for highly efficient and reversible electrochemical energy conversion. A single cell based on a planar electrolyte substrate as support (ESC) is often utilized for SOFC/SOEC stack manufacturing and fulfills necessary requirements for application in small, medium and large scale fuel cell and electrolysis systems. Thickness of the electrolyte substrate, and its ionic conductivity limits the power density of the ESC. To improve the performance of this cell type in SOFC/SOEC mode, alternative fuel electrodes, on the basis of Ni/CGO as well as electrolytes with reduced thickness, have been applied. Furthermore, different interlayers on the air side have been tested to avoid the electrode delamination and to reduce the cell degradation in electrolysis mode. Finally, the influence of the contacting layer on cell performance, especially for cells with an ultrathin electrolyte and thin electrode layers, has been investigated. It has been found that Ni/CGO outperform traditional Ni/8YSZ electrodes and the introduction of a ScSZ interlayer substantially reduces the degradation rate of ESC in electrolysis mode. Furthermore, it was demonstrated that, for thin electrodes, the application of contacting layers with good conductivity and adhesion to current collectors improves performance significantly.

  7. Performance of single-junction and dual-junction InGaP/GaAs solar cells under low concentration ratios

    International Nuclear Information System (INIS)

    Khan, Aurangzeb; Yamaguchi, Masafumi; Takamoto, Tatsuya

    2004-01-01

    A study of the performance of single-junction InGaP/GaAs and dual-junction InGaP/GaAs tandem cells under low concentration ratios (up to 15 suns), before and after 1 MeV electron irradiation is presented. Analysis of the tunnel junction parameters under different concentrated light illuminations reveals that the peak current (J P ) and valley current (J V ) densities should be greater than the short-circuit current density (J sc ) for better performance. The tunnel junction behavior against light intensity improved after irradiation. This led to the suggestion that the peak current density (J P ) and valley current density (J V ) of the tunnel junction were enhanced after irradiation or the peak current was shifted to higher concentration. The recovery of the radiation damage under concentrated light illumination conditions suggests that the performance of the InGaP/GaAs tandem solar cell can be enhanced even under low concentration ratios

  8. Droplet Microfluidics for Compartmentalized Cell Lysis and Extension of DNA from Single-Cells

    Science.gov (United States)

    Zimny, Philip; Juncker, David; Reisner, Walter

    Current single cell DNA analysis methods suffer from (i) bias introduced by the need for molecular amplification and (ii) limited ability to sequence repetitive elements, resulting in (iii) an inability to obtain information regarding long range genomic features. Recent efforts to circumvent these limitations rely on techniques for sensing single molecules of DNA extracted from single-cells. Here we demonstrate a droplet microfluidic approach for encapsulation and biochemical processing of single-cells inside alginate microparticles. In our approach, single-cells are first packaged inside the alginate microparticles followed by cell lysis, DNA purification, and labeling steps performed off-chip inside this microparticle system. The alginate microparticles are then introduced inside a micro/nanofluidic system where the alginate is broken down via a chelating buffer, releasing long DNA molecules which are then extended inside nanofluidic channels for analysis via standard mapping protocols.

  9. X-ray microanalysis of single and cultured cells

    International Nuclear Information System (INIS)

    Wroblewski, J.; Roomans, G.M.

    1984-01-01

    X-ray microanalysis of single or cultured cells is often a useful alternative or complement to the analysis of the corresponding tissue. It also allows the analysis of individual cells in a cell population. Preparation for X-ray microanalysis poses a number of typical problems. Suspensions of single cells can be prepared by either of two pathways: (1) washing - mounting - drying, or (2) centrifugation - freezing or fixation - sectioning. The washing step in the preparation of single or cultured cells presents the most severe problems. Cultured cells are generally grown on a substrate that is compatible with both the analysis and the culture, washed and dried. In some cases, sectioning of cultured cell monolayers has been performed. Special problems in quantitative analysis occur in those cases where the cells are analyzed on a thick substrate, since the substrate contributes to the spectral background

  10. Sorting of cells of the same size, shape, and cell cycle stage for a single cell level assay without staining

    Directory of Open Access Journals (Sweden)

    Yomo Tetsuya

    2006-06-01

    Full Text Available Abstract Background Single-cell level studies are being used increasingly to measure cell properties not directly observable in a cell population. High-performance data acquisition systems for such studies have, by necessity, developed in synchrony. However, improvements in sample purification techniques are also required to reveal new phenomena. Here we assessed a cell sorter as a sample-pretreatment tool for a single-cell level assay. A cell sorter is routinely used for selecting one type of cells from a heterogeneous mixture of cells using specific fluorescence labels. In this case, we wanted to select cells of exactly the same size, shape, and cell-cycle stage from a population, without using a specific fluorescence label. Results We used four light scatter parameters: the peak height and area of the forward scatter (FSheight and FSarea and side scatter (SSheight and SSarea. The rat pheochromocytoma PC12 cell line, a neuronal cell line, was used for all experiments. The living cells concentrated in the high FSarea and middle SSheight/SSarea fractions. Single cells without cell clumps were concentrated in the low SS and middle FS fractions, and in the higher FSheight/FSarea and SSheight/SSarea fractions. The cell populations from these viable, single-cell-rich fractions were divided into twelve subfractions based on their FSarea-SSarea profiles, for more detailed analysis. We found that SSarea was proportional to the cell volume and the FSarea correlated with cell roundness and elongation, as well as with the level of DNA in the cell. To test the method and to characterize the basic properties of the isolated single cells, sorted cells were cultured in separate wells. The cells in all subfractions survived, proliferated and differentiated normally, suggesting that there was no serious damage. The smallest, roundest, and smoothest cells had the highest viability. There was no correlation between proliferation and differentiation. NGF increases

  11. Realignment process of actin stress fibers in single living cells studied by focused femtosecond laser irradiation

    OpenAIRE

    Yasukuni, Ryohei; Spitz, Jean-Alexis; Meallet-Renault, Rachel; Negishi, Takayuki; Tada, Takuji; Hosokawa, Yoichiroh; Asahi, Tsuyoshi; Shukunami, Chisa; Hiraki, Yuji; Masuhara, Hiroshi

    2007-01-01

    Three-dimensional dissection of a single actin stress fiber in a living cell was performed based on multi-photon absorption of a focused femtosecond laser pulse. The realignment process of an actin stress fiber was investigated after its direct cutting by a single-shot femtosecond laser pulse irradiation by high-speed transmission and fluorescence imaging methods. It was confirmed that mechanical force led by the femtosecond laser cutting propagates to entire cell through the cytockelton in a...

  12. New tools to study biophysical properties of single molecules and single cells

    Directory of Open Access Journals (Sweden)

    Márcio S. Rocha

    2007-03-01

    Full Text Available We present a review on two new tools to study biophysical properties of single molecules and single cells. A laser incident through a high numerical aperture microscope objective can trap small dielectric particles near the focus. This arrangement is named optical tweezers. This technique has the advantage to permit manipulation of a single individual object. We use optical tweezers to measure the entropic elasticity of a single DNA molecule and its interaction with the drug Psoralen. Optical tweezers are also used to hold a kidney cell MDCK away from the substrate to allow precise volume measurements of this single cell during an osmotic shock. This procedure allows us to obtain information about membrane water permeability and regulatory volume increase. Defocusing microscopy is a recent technique invented in our laboratory, which allows the observation of transparent objects, by simply defocusing the microscope in a controlled way. Our physical model of a defocused microscope shows that the image contrast observed in this case is proportional to the defocus distance and to the curvature of the transparent object. Defocusing microscopy is very useful to study motility and mechanical properties of cells. We show here the application of defocusing microscopy to measurements of macrophage surface fluctuations and their influence on phagocytosis.Apresentamos uma revisão de duas novas técnicas para estudar propriedades biofísicas de moléculas únicas e células únicas. Um laser incidindo em uma objetiva de microscópio de grande abertura numérica é capaz de aprisionar pequenas partículas dielétricas na região próxima ao foco. Este aparato é chamado de pinça óptica. Esta técnica tem a grande vantagem de permitir a manipulação de um objeto individual. Usamos a pinça óptica para medir a elasticidade entrópica de uma molécula única de DNA em sua interação com o fármaco Psoralen. A pinça óptica também é usada para segurar

  13. Fluidic Logic Used in a Systems Approach to Enable Integrated Single-cell Functional Analysis

    Directory of Open Access Journals (Sweden)

    Naveen Ramalingam

    2016-09-01

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

  14. Improved performance of single-chamber microbial fuel cells through control of membrane deformation

    KAUST Repository

    Zhang, Xiaoyuan

    2010-03-01

    Cation (CEMs) and anion exchange membrane (AEMs) are commonly used in microbial fuel cells (MFCs) to enhance Coulombic efficiencies (CEs) by reducing thefluxof oxygen through the cathode to bacteriaonthe anode. AEMs typically work better than CEMs, but in initial experiments we observed the opposite using a membrane electrode assembly MFC. The reason was identified to be membrane deformation, which resulted in water and gas trapped between the membrane and cathode. To correct this, stainless steel mesh was used to press the membrane flat against the cathode. With the steel mesh, AEM performance increased to 46±4W/m3 in a single cathode MFC, and 98±14W/m3 in a double-cathode MFC. These power densities were higher than those using a CEM of 32±2W/m3 (single cathode) and 63±6W/m3 (double cathode). Higher pH gradients across the membrane and salt precipitation on the cathode were responsible for the reduced performance of the CEM compared to the AEM. CEs reached over 90% for both membranes at >2A/m2. These results demonstrate the importance of avoiding water accumulation in thin films between membranes and electrodes, and explain additional reasons for poorer performance of CEMs compared to AEMs. © 2009 Elsevier B.V.

  15. Improved performance of single-chamber microbial fuel cells through control of membrane deformation.

    Science.gov (United States)

    Zhang, Xiaoyuan; Cheng, Shaoan; Huang, Xia; Logan, Bruce E

    2010-03-15

    Cation (CEMs) and anion exchange membrane (AEMs) are commonly used in microbial fuel cells (MFCs) to enhance Coulombic efficiencies (CEs) by reducing the flux of oxygen through the cathode to bacteria on the anode. AEMs typically work better than CEMs, but in initial experiments we observed the opposite using a membrane electrode assembly MFC. The reason was identified to be membrane deformation, which resulted in water and gas trapped between the membrane and cathode. To correct this, stainless steel mesh was used to press the membrane flat against the cathode. With the steel mesh, AEM performance increased to 46+/-4 W/m(3) in a single cathode MFC, and 98+/-14 W/m(3) in a double-cathode MFC. These power densities were higher than those using a CEM of 32+/-2 W/m(3) (single cathode) and 63+/-6 W/m(3) (double cathode). Higher pH gradients across the membrane and salt precipitation on the cathode were responsible for the reduced performance of the CEM compared to the AEM. CEs reached over 90% for both membranes at >2A/m(2). These results demonstrate the importance of avoiding water accumulation in thin films between membranes and electrodes, and explain additional reasons for poorer performance of CEMs compared to AEMs. (c) 2009 Elsevier B.V. All rights reserved.

  16. High-efficiency single cell encapsulation and size selective capture of cells in picoliter droplets based on hydrodynamic micro-vortices.

    Science.gov (United States)

    Kamalakshakurup, Gopakumar; Lee, Abraham P

    2017-12-05

    Single cell analysis has emerged as a paradigm shift in cell biology to understand the heterogeneity of individual cells in a clone for pathological interrogation. Microfluidic droplet technology is a compelling platform to perform single cell analysis by encapsulating single cells inside picoliter-nanoliter (pL-nL) volume droplets. However, one of the primary challenges for droplet based single cell assays is single cell encapsulation in droplets, currently achieved either randomly, dictated by Poisson statistics, or by hydrodynamic techniques. In this paper, we present an interfacial hydrodynamic technique which initially traps the cells in micro-vortices, and later releases them one-to-one into the droplets, controlled by the width of the outer streamline that separates the vortex from the flow through the streaming passage adjacent to the aqueous-oil interface (d gap ). One-to-one encapsulation is achieved at a d gap equal to the radius of the cell, whereas complete trapping of the cells is realized at a d gap smaller than the radius of the cell. The unique feature of this technique is that it can perform 1. high efficiency single cell encapsulations and 2. size-selective capturing of cells, at low cell loading densities. Here we demonstrate these two capabilities with a 50% single cell encapsulation efficiency and size selective separation of platelets, RBCs and WBCs from a 10× diluted blood sample (WBC capture efficiency at 70%). The results suggest a passive, hydrodynamic micro-vortex based technique capable of performing high-efficiency single cell encapsulation for cell based assays.

  17. Confocal microscopy and electrophysiological study of single patient corneal endothelium cell cultures

    Science.gov (United States)

    Tatini, Francesca; Rossi, Francesca; Coppi, Elisabetta; Magni, Giada; Fusco, Irene; Menabuoni, Luca; Pedata, Felicita; Pugliese, Anna Maria; Pini, Roberto

    2016-04-01

    The characterization of the ion channels in corneal endothelial cells and the elucidation of their involvement in corneal pathologies would lead to the identification of new molecular target for pharmacological treatments and to the clarification of corneal physiology. The corneal endothelium is an amitotic cell monolayer with a major role in preserving corneal transparency and in regulating the water and solute flux across the posterior surface of the cornea. Although endothelial cells are non-excitable, they express a range of ion channels, such as voltage-dependent Na+ channels and K+ channels, L-type Ca2 channels and many others. Interestingly, purinergic receptors have been linked to a variety of conditions within the eye but their presence in the endothelium and their role in its pathophysiology is still uncertain. In this study, we were able to extract endothelial cells from single human corneas, thus obtaining primary cultures that represent the peculiarity of each donor. Corneas were from tissues not suitable for transplant in patients. We characterized the endothelial cells by confocal microscopy, both within the intact cornea and in the primary endothelial cells cultures. We also studied the functional role of the purinergic system (adenosine, ATP and their receptors) by means of electrophysiological recordings. The experiments were performed by patch clamp recordings and confocal time-lapse microscopy and our results indicate that the application of purinergic compounds modulates the amplitude of outward currents in the isolated endothelial cells. These findings may lead to the proposal of new therapies for endothelium-related corneal diseases.

  18. Single cell analysis of normal and leukemic hematopoiesis.

    Science.gov (United States)

    Povinelli, Benjamin J; Rodriguez-Meira, Alba; Mead, Adam J

    2018-02-01

    The hematopoietic system is well established as a paradigm for the study of cellular hierarchies, their disruption in disease and therapeutic use in regenerative medicine. Traditional approaches to study hematopoiesis involve purification of cell populations based on a small number of surface markers. However, such population-based analysis obscures underlying heterogeneity contained within any phenotypically defined cell population. This heterogeneity can only be resolved through single cell analysis. Recent advances in single cell techniques allow analysis of the genome, transcriptome, epigenome and proteome in single cells at an unprecedented scale. The application of these new single cell methods to investigate the hematopoietic system has led to paradigm shifts in our understanding of cellular heterogeneity in hematopoiesis and how this is disrupted in disease. In this review, we summarize how single cell techniques have been applied to the analysis of hematopoietic stem/progenitor cells in normal and malignant hematopoiesis, with a particular focus on recent advances in single-cell genomics, including how these might be utilized for clinical application. Copyright © 2017. Published by Elsevier Ltd.

  19. The application of single cell gel electrophoresis or comet assay to human monitoring studies

    Directory of Open Access Journals (Sweden)

    Valverde Mahara

    1999-01-01

    Full Text Available Objective. In the search of new human genotoxic biomarkers, the single cell gel electrophoresis assay has been proposed as a sensible alternative. Material and methods. This technique detects principally single strand breaks as well as alkali-labile and repair-retarded sites. Results. Herein we present our experience using the single cell gel electrophoresis assay in human population studies, both occupationally and environmentally exposed. Conclusions. We discuss the assay feasibility as a genotoxic biomarker.

  20. Single-cell-derived mesenchymal stem cells overexpressing Csx/Nkx2.5 and GATA4 undergo the stochastic cardiomyogenic fate and behave like transient amplifying cells

    International Nuclear Information System (INIS)

    Yamada, Yoji; Sakurada, Kazuhiro; Takeda, Yukiji; Gojo, Satoshi; Umezawa, Akihiro

    2007-01-01

    Bone marrow-derived stromal cells can give rise to cardiomyocytes as well as adipocytes, osteocytes, and chondrocytes in vitro. The existence of mesenchymal stem cells has been proposed, but it remains unclear if a single-cell-derived stem cell stochastically commits toward a cardiac lineage. By single-cell marking, we performed a follow-up study of individual cells during the differentiation of 9-15c mesenchymal stromal cells derived from bone marrow cells. Three types of cells, i.e., cardiac myoblasts, cardiac progenitors and multipotent stem cells were differentiated from a single cell, implying that cardiomyocytes are generated stochastically from a single-cell-derived stem cell. We also demonstrated that overexpression of Csx/Nkx2.5 and GATA4, precardiac mesodermal transcription factors, enhanced cardiomyogenic differentiation of 9-15c cells, and the frequency of cardiomyogenic differentiation was increased by co-culturing with fetal cardiomyocytes. Single-cell-derived mesenchymal stem cells overexpressing Csx/Nkx2.5 and GATA4 behaved like cardiac transient amplifying cells, and still retained their plasticity in vivo

  1. Single-cell sequencing in stem cell biology.

    Science.gov (United States)

    Wen, Lu; Tang, Fuchou

    2016-04-15

    Cell-to-cell variation and heterogeneity are fundamental and intrinsic characteristics of stem cell populations, but these differences are masked when bulk cells are used for omic analysis. Single-cell sequencing technologies serve as powerful tools to dissect cellular heterogeneity comprehensively and to identify distinct phenotypic cell types, even within a 'homogeneous' stem cell population. These technologies, including single-cell genome, epigenome, and transcriptome sequencing technologies, have been developing rapidly in recent years. The application of these methods to different types of stem cells, including pluripotent stem cells and tissue-specific stem cells, has led to exciting new findings in the stem cell field. In this review, we discuss the recent progress as well as future perspectives in the methodologies and applications of single-cell omic sequencing technologies.

  2. Break-in and Performance Issues on a single cell PBI-based PEM Fuel Cell

    DEFF Research Database (Denmark)

    Kær, Søren Knudsen; Jespersen, Jesper Lebæk

    of the fuel cell, even though break-in of a fuel cell implemented in a commercial application would most likely not be feasible. In the present work a commercially available PBI-based high temperature MEA is subject to a break-in procedure, as specified by the manufacturer. The cell was operated at 160 °C...... during the break-in procedure at a current density of 0.2 A/cm2. The performance of the cell was measured over the 100 hour break-in period and a polarization curve was recorded after completion of break-in. The performance change was minimal during the break-in cycle. However, in the first hour of op......-eration a significant performance decrease of 30 mV was observed. Hereafter a performance in-crease started and the overall performance change during the break-in procedure was a voltage in-crease of 35 mV corresponding to a rate of 240 μV/hr. The performance increase was however fast-est in the first 50 hours...

  3. Identification of innate lymphoid cells in single-cell RNA-Seq data.

    Science.gov (United States)

    Suffiotti, Madeleine; Carmona, Santiago J; Jandus, Camilla; Gfeller, David

    2017-07-01

    Innate lymphoid cells (ILCs) consist of natural killer (NK) cells and non-cytotoxic ILCs that are broadly classified into ILC1, ILC2, and ILC3 subtypes. These cells recently emerged as important early effectors of innate immunity for their roles in tissue homeostasis and inflammation. Over the last few years, ILCs have been extensively studied in mouse and human at the functional and molecular level, including gene expression profiling. However, sorting ILCs with flow cytometry for gene expression analysis is a delicate and time-consuming process. Here we propose and validate a novel framework for studying ILCs at the transcriptomic level using single-cell RNA-Seq data. Our approach combines unsupervised clustering and a new cell type classifier trained on mouse ILC gene expression data. We show that this approach can accurately identify different ILCs, especially ILC2 cells, in human lymphocyte single-cell RNA-Seq data. Our new model relies only on genes conserved across vertebrates, thereby making it in principle applicable in any vertebrate species. Considering the rapid increase in throughput of single-cell RNA-Seq technology, our work provides a computational framework for studying ILC2 cells in single-cell transcriptomic data and may help exploring their conservation in distant vertebrate species.

  4. Single Nanowire Probe for Single Cell Endoscopy and Sensing

    Science.gov (United States)

    Yan, Ruoxue

    adaptable to average bio-lab environment. These probes are mechanically robust and flexible and can withstand repeated bending and deformation without significant deterioration in optical performance, which offers an ideal instrumental platform for out subsequent effort of using these nanoprobes in chemical sensing as well as single cell endoscopy and spot delivery. Parameters affecting the coupling efficiency and output power of the nanoprobe were studied and chemical etched of single mode fiber with small cone angle was established to be optimized for highly effective optical nanoprobes. The versatility of the nanoprobe design was first tested by transforming the nanowire probe into a pH sensor with near-field photopolymerization of a copolymer containing pH sensitive dye on the tip of the nanowire. The pH-sensitive nanoprobe was able to report the pH difference in micro-droplets containing buffer solution with the excitation of light waveguided on the nanoprobe with internal calibration, fast response time and good photostability and reversibility. Such nanoprobe sensors are ideal for high definition spatial and temporal sensing of concentration profile, especially for the kinetic processes in single cell studies for which chemical probes of minute sizes and fast response are desired. The nanoprobe was then applied into spot cargo delivery and in-situ single cell endoscopy. It was demonstrated that nanowire-based optical probe can deliver payloads into the cell with a high spatiotemporal precision, guide and confine visible light into intracellular compartments selectively and detect optical signals from the subcellular regions with high spatial resolution. The nanoprobe was proven to be biocompatible and non-invasive. The effective optical coupling between the fiber optics and the nanowire enables highly localized excitation and detection, limiting the probe volume to the close proximity of the nanowire. None the less, this versatile technique does not rely on any

  5. Plasticity of marrow mesenchymal stem cells from human first-trimester fetus: from single-cell clone to neuronal differentiation.

    Science.gov (United States)

    Zhang, Yihua; Shen, Wenzheng; Sun, Bingjie; Lv, Changrong; Dou, Zhongying

    2011-02-01

    Recent results have shown that bone marrow mesenchymal stem cells (BMSCs) from human first-trimester abortus (hfBMSCs) are closer to embryonic stem cells and perform greater telomerase activity and faster propagation than mid- and late-prophase fetal and adult BMSCs. However, no research has been done on the plasticity of hfBMSCs into neuronal cells using single-cell cloned strains without cell contamination. In this study, we isolated five single cells from hfBMSCs and obtained five single-cell cloned strains, and investigated their biological property and neuronal differentiation potential. We found that four of the five strains showed similar expression profile of surface antigen markers to hfBMSCs, and most of them differentiated into neuron-like cells expressing Nestin, Pax6, Sox1, β-III Tubulin, NF-L, and NSE under induction. One strain showed different expression profile of surface antigen markers from the four strains and hfBMSCs, and did not differentiate toward neuronal cells. We demonstrated for the first time that some of single-cell cloned strains from hfBMSCs can differentiate into nerve tissue-like cell clusters under induction in vitro, and that the plasticity of each single-cell cloned strain into neuronal cells is different.

  6. Single-cell regulome data analysis by SCRAT.

    Science.gov (United States)

    Ji, Zhicheng; Zhou, Weiqiang; Ji, Hongkai

    2017-09-15

    Emerging single-cell technologies (e.g. single-cell ATAC-seq, DNase-seq or ChIP-seq) have made it possible to assay regulome of individual cells. Single-cell regulome data are highly sparse and discrete. Analyzing such data is challenging. User-friendly software tools are still lacking. We present SCRAT, a Single-Cell Regulome Analysis Toolbox with a graphical user interface, for studying cell heterogeneity using single-cell regulome data. SCRAT can be used to conveniently summarize regulatory activities according to different features (e.g. gene sets, transcription factor binding motif sites, etc.). Using these features, users can identify cell subpopulations in a heterogeneous biological sample, infer cell identities of each subpopulation, and discover distinguishing features such as gene sets and transcription factors that show different activities among subpopulations. SCRAT is freely available at https://zhiji.shinyapps.io/scrat as an online web service and at https://github.com/zji90/SCRAT as an R package. hji@jhu.edu. Supplementary data are available at Bioinformatics online. © The Author(s) 2017. Published by Oxford University Press.

  7. Single-cell axotomy of cultured hippocampal neurons integrated in neuronal circuits.

    Science.gov (United States)

    Gomis-Rüth, Susana; Stiess, Michael; Wierenga, Corette J; Meyn, Liane; Bradke, Frank

    2014-05-01

    An understanding of the molecular mechanisms of axon regeneration after injury is key for the development of potential therapies. Single-cell axotomy of dissociated neurons enables the study of the intrinsic regenerative capacities of injured axons. This protocol describes how to perform single-cell axotomy on dissociated hippocampal neurons containing synapses. Furthermore, to axotomize hippocampal neurons integrated in neuronal circuits, we describe how to set up coculture with a few fluorescently labeled neurons. This approach allows axotomy of single cells in a complex neuronal network and the observation of morphological and molecular changes during axon regeneration. Thus, single-cell axotomy of mature neurons is a valuable tool for gaining insights into cell intrinsic axon regeneration and the plasticity of neuronal polarity of mature neurons. Dissociation of the hippocampus and plating of hippocampal neurons takes ∼2 h. Neurons are then left to grow for 2 weeks, during which time they integrate into neuronal circuits. Subsequent axotomy takes 10 min per neuron and further imaging takes 10 min per neuron.

  8. High-dimensional single-cell cancer biology.

    Science.gov (United States)

    Irish, Jonathan M; Doxie, Deon B

    2014-01-01

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

  9. Probing bacterial adhesion at the single-cell level

    DEFF Research Database (Denmark)

    Zeng, Guanghong; Müller, Torsten; Meyer, Rikke Louise

    be considered. We have developed a simple and versatile method to make single-cell bacterial probes for measuring single cell adhesion by force spectroscopy using atomic force microscopy (AFM). A single-cell probe was readily made by picking up a bacterial cell from a glass surface by approaching a tipless AFM...... cantilever coated with the commercial cell adhesive CellTakTM. We applied the method to study adhesion of living cells to abiotic surfaces at the single-cell level. Immobilisation of single bacterial cells to the cantilever was stable for several hours, and viability was confirmed by Live/Dead staining...... on the adhesion force, we explored the bond formation and adhesive strength of four different bacterial strains towards three abiotic substrates with variable hydrophobicity and surface roughness. The adhesion force and final rupture length were dependent on bacterial strains, surfaces properties, and time...

  10. Microfluidics for single cell analysis

    DEFF Research Database (Denmark)

    Jensen, Marie Pødenphant

    Isolation and manipulation of single cells have gained an increasing interest from researchers because of the heterogeneity of cells from the same cell culture. Single cell analysis can ensure a better understanding of differences between individual cells and potentially solve a variety of clinical...... problems. In this thesis lab on a chip systems for rare single cell analysis are investigated. The focus was to develop a commercial, disposable device for circulating tumour cell (CTC) analysis. Such a device must be able to separate rare cells from blood samples and subsequently capture the specific...... cells, and simultaneously be fabricated and operated at low costs and be user-friendly. These challenges were addressed through development of two microfluidic devices, one for rare cell isolation based on pinched flow fractionation (PFF) and one for single cell capture based on hydrodynamic trapping...

  11. Multi-region and single-cell sequencing reveal variable genomic heterogeneity in rectal cancer.

    Science.gov (United States)

    Liu, Mingshan; Liu, Yang; Di, Jiabo; Su, Zhe; Yang, Hong; Jiang, Beihai; Wang, Zaozao; Zhuang, Meng; Bai, Fan; Su, Xiangqian

    2017-11-23

    Colorectal cancer is a heterogeneous group of malignancies with complex molecular subtypes. While colon cancer has been widely investigated, studies on rectal cancer are very limited. Here, we performed multi-region whole-exome sequencing and single-cell whole-genome sequencing to examine the genomic intratumor heterogeneity (ITH) of rectal tumors. We sequenced nine tumor regions and 88 single cells from two rectal cancer patients with tumors of the same molecular classification and characterized their mutation profiles and somatic copy number alterations (SCNAs) at the multi-region and the single-cell levels. A variable extent of genomic heterogeneity was observed between the two patients, and the degree of ITH increased when analyzed on the single-cell level. We found that major SCNAs were early events in cancer development and inherited steadily. Single-cell sequencing revealed mutations and SCNAs which were hidden in bulk sequencing. In summary, we studied the ITH of rectal cancer at regional and single-cell resolution and demonstrated that variable heterogeneity existed in two patients. The mutational scenarios and SCNA profiles of two patients with treatment naïve from the same molecular subtype are quite different. Our results suggest each tumor possesses its own architecture, which may result in different diagnosis, prognosis, and drug responses. Remarkable ITH exists in the two patients we have studied, providing a preliminary impression of ITH in rectal cancer.

  12. Single-Cell Mass Spectrometry Reveals Changes in Lipid and Metabolite Expression in RAW 264.7 Cells upon Lipopolysaccharide Stimulation

    Science.gov (United States)

    Yang, Bo; Patterson, Nathan Heath; Tsui, Tina; Caprioli, Richard M.; Norris, Jeremy L.

    2018-05-01

    It has been widely recognized that individual cells that exist within a large population of cells, even if they are genetically identical, can have divergent molecular makeups resulting from a variety of factors, including local environmental factors and stochastic processes within each cell. Presently, numerous approaches have been described that permit the resolution of these single-cell expression differences for RNA and protein; however, relatively few techniques exist for the study of lipids and metabolites in this manner. This study presents a methodology for the analysis of metabolite and lipid expression at the level of a single cell through the use of imaging mass spectrometry on a high-performance Fourier transform ion cyclotron resonance mass spectrometer. This report provides a detailed description of the overall experimental approach, including sample preparation as well as the data acquisition and analysis strategy for single cells. Applying this approach to the study of cultured RAW264.7 cells, we demonstrate that this method can be used to study the variation in molecular expression with cell populations and is sensitive to alterations in that expression that occurs upon lipopolysaccharide stimulation. [Figure not available: see fulltext.

  13. Single-Cell Mass Spectrometry Reveals Changes in Lipid and Metabolite Expression in RAW 264.7 Cells upon Lipopolysaccharide Stimulation

    Science.gov (United States)

    Yang, Bo; Patterson, Nathan Heath; Tsui, Tina; Caprioli, Richard M.; Norris, Jeremy L.

    2018-03-01

    It has been widely recognized that individual cells that exist within a large population of cells, even if they are genetically identical, can have divergent molecular makeups resulting from a variety of factors, including local environmental factors and stochastic processes within each cell. Presently, numerous approaches have been described that permit the resolution of these single-cell expression differences for RNA and protein; however, relatively few techniques exist for the study of lipids and metabolites in this manner. This study presents a methodology for the analysis of metabolite and lipid expression at the level of a single cell through the use of imaging mass spectrometry on a high-performance Fourier transform ion cyclotron resonance mass spectrometer. This report provides a detailed description of the overall experimental approach, including sample preparation as well as the data acquisition and analysis strategy for single cells. Applying this approach to the study of cultured RAW264.7 cells, we demonstrate that this method can be used to study the variation in molecular expression with cell populations and is sensitive to alterations in that expression that occurs upon lipopolysaccharide stimulation. [Figure not available: see fulltext.

  14. Measuring single-cell density.

    Science.gov (United States)

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

    2011-07-05

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

  15. Single Cell Isolation and Analysis

    Directory of Open Access Journals (Sweden)

    Ping Hu

    2016-10-01

    Full Text Available Increasing evidence shows that the heterogeneity of individual cells within a genetically identical population can be critical to their peculiar function and fate. Conventional cell based assays mainly analysis the average responses from a population cells, while the difference within individual cells may often be masked. The cell size, RNA transcripts and protein expression level are quite different within individual cells and these variations are key point to answer the problems in cancer, neurobiology, stem cell biology, immunology and developmental biology. To better understand the cell-to-cell variations, the single cell analysis can provide much more detailed information which may be helpful for therapeutic decisions in an increasingly personalized medicine. In this review, we will focus on the recent development in single cell analysis, including methods used in single cell isolation, analysis and some application examples. The review provides the historical background to single cell analysis, discusses limitations, and current and future possibilities in this exciting field of research.

  16. Beta-Poisson model for single-cell RNA-seq data analyses.

    Science.gov (United States)

    Vu, Trung Nghia; Wills, Quin F; Kalari, Krishna R; Niu, Nifang; Wang, Liewei; Rantalainen, Mattias; Pawitan, Yudi

    2016-07-15

    Single-cell RNA-sequencing technology allows detection of gene expression at the single-cell level. One typical feature of the data is a bimodality in the cellular distribution even for highly expressed genes, primarily caused by a proportion of non-expressing cells. The standard and the over-dispersed gamma-Poisson models that are commonly used in bulk-cell RNA-sequencing are not able to capture this property. We introduce a beta-Poisson mixture model that can capture the bimodality of the single-cell gene expression distribution. We further integrate the model into the generalized linear model framework in order to perform differential expression analyses. The whole analytical procedure is called BPSC. The results from several real single-cell RNA-seq datasets indicate that ∼90% of the transcripts are well characterized by the beta-Poisson model; the model-fit from BPSC is better than the fit of the standard gamma-Poisson model in > 80% of the transcripts. Moreover, in differential expression analyses of simulated and real datasets, BPSC performs well against edgeR, a conventional method widely used in bulk-cell RNA-sequencing data, and against scde and MAST, two recent methods specifically designed for single-cell RNA-seq data. An R package BPSC for model fitting and differential expression analyses of single-cell RNA-seq data is available under GPL-3 license at https://github.com/nghiavtr/BPSC CONTACT: yudi.pawitan@ki.se or mattias.rantalainen@ki.se Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  17. Performance of a methane-fueled single-cell SOFC stack at various levels of fuel utilization

    International Nuclear Information System (INIS)

    Ahmed, K.; Bolden, R.; Ramprakash and Foger, K.

    1998-01-01

    Fuel-gas mixtures representing 10 to 85% utilization of a methane-steam mixture at S/C=2 were fed to a single cell stack with a Ni-based anode at 875 deg C. Cell voltage and power output were recorded at current densities of 50 to 350 mA/cm 2 . The accompanying anode off-gas composition at some of these conditions were measured using on-line gas chromatograph and compared with the compositions predicted by a thermodynamic model based on the assumption of no carbon formation. Electrical losses were measured at a chosen current density at various levels of fuel utilization by the galvanostatic current-interruption technique. Cell voltage stability was monitored for up to 1000 h at two levels of fuel utilization. The stack performance was simulated using a mathematical model of the stack; the simulations were compared with the stack test data. Copyright (1998) Australasian Ceramic Society

  18. Going single but not solo with podocytes: potentials, limitations, and pitfalls of single-cell analysis.

    Science.gov (United States)

    Schiffer, Mario

    2017-11-01

    Single-cell RNA-sequence (RNA-seq) is a widely used tool to study biological questions in single cells. The discussed study identified 92 genes being predominantly expressed in podocytes based on a 5-fold higher expression compared with endothelial and mesangial cells. In addition to technical pitfalls, the question that is discussed in this commentary is whether results of a single-cell RNAseq study are able to deliver expression data that truly characterize a podocyte. Copyright © 2017 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.

  19. cgCorrect: a method to correct for confounding cell-cell variation due to cell growth in single-cell transcriptomics

    Science.gov (United States)

    Blasi, Thomas; Buettner, Florian; Strasser, Michael K.; Marr, Carsten; Theis, Fabian J.

    2017-06-01

    Accessing gene expression at a single-cell level has unraveled often large heterogeneity among seemingly homogeneous cells, which remains obscured when using traditional population-based approaches. The computational analysis of single-cell transcriptomics data, however, still imposes unresolved challenges with respect to normalization, visualization and modeling the data. One such issue is differences in cell size, which introduce additional variability into the data and for which appropriate normalization techniques are needed. Otherwise, these differences in cell size may obscure genuine heterogeneities among cell populations and lead to overdispersed steady-state distributions of mRNA transcript numbers. We present cgCorrect, a statistical framework to correct for differences in cell size that are due to cell growth in single-cell transcriptomics data. We derive the probability for the cell-growth-corrected mRNA transcript number given the measured, cell size-dependent mRNA transcript number, based on the assumption that the average number of transcripts in a cell increases proportionally to the cell’s volume during the cell cycle. cgCorrect can be used for both data normalization and to analyze the steady-state distributions used to infer the gene expression mechanism. We demonstrate its applicability on both simulated data and single-cell quantitative real-time polymerase chain reaction (PCR) data from mouse blood stem and progenitor cells (and to quantitative single-cell RNA-sequencing data obtained from mouse embryonic stem cells). We show that correcting for differences in cell size affects the interpretation of the data obtained by typically performed computational analysis.

  20. Single-cell MALDI-MS as an analytical tool for studying intrapopulation metabolic heterogeneity of unicellular organisms.

    Science.gov (United States)

    Amantonico, Andrea; Urban, Pawel L; Fagerer, Stephan R; Balabin, Roman M; Zenobi, Renato

    2010-09-01

    Heterogeneity is a characteristic feature of all populations of living organisms. Here we make an attempt to validate a single-cell mass spectrometric method for detection of changes in metabolite levels occurring in populations of unicellular organisms. Selected metabolites involved in central metabolism (ADP, ATP, GTP, and UDP-Glucose) could readily be detected in single cells of Closterium acerosum by means of negative-mode matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS). The analytical capabilities of this approach were characterized using standard compounds. The method was then used to study populations of individual cells with different levels of the chosen metabolites. With principal component analysis and support vector machine algorithms, it was possible to achieve a clear separation of individual C. acerosum cells in different metabolic states. This study demonstrates the suitability of mass spectrometric analysis of metabolites in single cells to measure cell-population heterogeneity.

  1. Performance Degradation Tests of Phosphoric Acid Doped PBI Membrane Based High Temperature PEM Fuel Cells

    DEFF Research Database (Denmark)

    Zhou, Fan; Araya, Samuel Simon; Grigoras, Ionela

    2014-01-01

    Degradation tests of two phosphoric acid (PA) doped PBI membrane based HT-PEM fuel cells were reported in this paper to investigate the effects of start/stop and the presence of methanol in the fuel to the performance degradation. Continuous tests with H2 and simulated reformate which was composed...... of H2, water steam and methanol as the fuel were performed on both single cells. 12-h-startup/12-h-shutdown dynamic tests were performed on the first single cell with pure dry H2 as the fuel and on the second single cell with simulated reformate as the fuel. Along with the tests electrochemical...... techniques such as polarization curves and electrochemical impedance spectroscopy (EIS) were employed to study the degradation mechanisms of the fuel cells. Both single cells showed an increase in the performance in the H2 continuous tests, because of a decrease in the ORR kinetic resistance probably due...

  2. High-performance single CdS nanowire (nanobelt) Schottky junction solar cells with Au/graphene Schottky electrodes.

    Science.gov (United States)

    Ye, Yu; Dai, Yu; Dai, Lun; Shi, Zujin; Liu, Nan; Wang, Fei; Fu, Lei; Peng, Ruomin; Wen, Xiaonan; Chen, Zhijian; Liu, Zhongfan; Qin, Guogang

    2010-12-01

    High-performance single CdS nanowire (NW) as well as nanobelt (NB) Schottky junction solar cells were fabricated. Au (5 nm)/graphene combined layers were used as the Schottky contact electrodes to the NWs (NBs). Typical as-fabricated NW solar cell shows excellent photovoltaic behavior with an open circuit voltage of ∼0.15 V, a short circuit current of ∼275.0 pA, and an energy conversion efficiency of up to ∼1.65%. The physical mechanism of the combined Schottky electrode was discussed. We attribute the prominent capability of the devices to the high-performance Schottky combined electrode, which has the merits of low series resistance, high transparency, and good Schottky contact to the CdS NW (NB). Besides, a promising site-controllable patterned graphene transfer method, which has the advantages of economizing graphene material and free from additional etching process, was demonstrated in this work. Our results suggest that semiconductor NWs (NBs) are promising materials for novel solar cells, which have potential application in integrated nano-optoelectronic systems.

  3. Single-unit-cell layer established Bi 2 WO 6 3D hierarchical architectures: Efficient adsorption, photocatalysis and dye-sensitized photoelectrochemical performance

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Hongwei; Cao, Ranran; Yu, Shixin; Xu, Kang; Hao, Weichang; Wang, Yonggang; Dong, Fan; Zhang, Tierui; Zhang, Yihe

    2017-12-01

    Single-layer catalysis sparks huge interests and gains widespread attention owing to its high activity. Simultaneously, three-dimensional (3D) hierarchical structure can afford large surface area and abundant reactive sites, contributing to high efficiency. Herein, we report an absorbing single-unit-cell layer established Bi2WO6 3D hierarchical architecture fabricated by a sodium dodecyl benzene sulfonate (SDBS)-assisted assembled strategy. The DBS- long chains can adsorb on the (Bi2O2)2+ layers and hence impede stacking of the layers, resulting in the single-unit-cell layer. We also uncovered that SDS with a shorter chain is less effective than SDBS. Due to the sufficient exposure of surface O atoms, single-unit-cell layer 3D Bi2WO6 shows strong selectivity for adsorption on multiform organic dyes with different charges. Remarkably, the single-unit-cell layer 3D Bi2WO6 casts profoundly enhanced photodegradation activity and especially a superior photocatalytic H2 evolution rate, which is 14-fold increase in contrast to the bulk Bi2WO6. Systematic photoelectrochemical characterizations disclose that the substantially elevated carrier density and charge separation efficiency take responsibility for the strengthened photocatalytic performance. Additionally, the possibility of single-unit-cell layer 3D Bi2WO6 as dye-sensitized solar cells (DSSC) has also been attempted and it was manifested to be a promising dye-sensitized photoanode for oxygen evolution reaction (ORR). Our work not only furnish an insight into designing single-layer assembled 3D hierarchical architecture, but also offer a multi-functional material for environmental and energy applications.

  4. Single cell time-lapse analysis reveals that podoplanin enhances cell survival and colony formation capacity of squamous cell carcinoma cells.

    Science.gov (United States)

    Miyashita, Tomoyuki; Higuchi, Youichi; Kojima, Motohiro; Ochiai, Atsushi; Ishii, Genichiro

    2017-01-06

    Tumor initiating cells (TICs) are characterized by high clonal expansion capacity. We previously reported that podoplanin is a TIC-specific marker for the human squamous cell carcinoma cell line A431. The aim of this study is to explore the molecular mechanism underlying the high clonal expansion potential of podoplanin-positive A431cells using Fucci imaging. Single podoplanin-positive cells created large colonies at a significantly higher frequency than single podoplanin-negative cells, whereas no difference was observed between the two types of cells with respect to cell cycle status. Conversely, the cell death ratio of progenies derived from podoplanin-positive single cell was significantly lower than that of cells derived from podoplanin-negative cells. Single A431 cells, whose podoplanin expression was suppressed by RNA interference, exhibited increased cell death ratios and decreased frequency of large colony forming. Moreover, the frequency of large colony forming decreased significantly when podoplanin-positive single cells was treated with a ROCK (Rho-associated coiled-coil kinase) inhibitor, whereas no difference was observed in single podoplanin-negative cells. Our current study cleared that high clonal expansion capacity of podoplanin-positive TICs populations was the result of reduced cell death by podoplanin-mediated signaling. Therefore, podoplanin activity may be a therapeutic target in the treatment of squamous cell carcinomas.

  5. Platforms for Single-Cell Collection and Analysis

    Directory of Open Access Journals (Sweden)

    Lukas Valihrach

    2018-03-01

    Full Text Available Single-cell analysis has become an established method to study cell heterogeneity and for rare cell characterization. Despite the high cost and technical constraints, applications are increasing every year in all fields of biology. Following the trend, there is a tremendous development of tools for single-cell analysis, especially in the RNA sequencing field. Every improvement increases sensitivity and throughput. Collecting a large amount of data also stimulates the development of new approaches for bioinformatic analysis and interpretation. However, the essential requirement for any analysis is the collection of single cells of high quality. The single-cell isolation must be fast, effective, and gentle to maintain the native expression profiles. Classical methods for single-cell isolation are micromanipulation, microdissection, and fluorescence-activated cell sorting (FACS. In the last decade several new and highly efficient approaches have been developed, which not just supplement but may fully replace the traditional ones. These new techniques are based on microfluidic chips, droplets, micro-well plates, and automatic collection of cells using capillaries, magnets, an electric field, or a punching probe. In this review we summarize the current methods and developments in this field. We discuss the advantages of the different commercially available platforms and their applicability, and also provide remarks on future developments.

  6. Platforms for Single-Cell Collection and Analysis.

    Science.gov (United States)

    Valihrach, Lukas; Androvic, Peter; Kubista, Mikael

    2018-03-11

    Single-cell analysis has become an established method to study cell heterogeneity and for rare cell characterization. Despite the high cost and technical constraints, applications are increasing every year in all fields of biology. Following the trend, there is a tremendous development of tools for single-cell analysis, especially in the RNA sequencing field. Every improvement increases sensitivity and throughput. Collecting a large amount of data also stimulates the development of new approaches for bioinformatic analysis and interpretation. However, the essential requirement for any analysis is the collection of single cells of high quality. The single-cell isolation must be fast, effective, and gentle to maintain the native expression profiles. Classical methods for single-cell isolation are micromanipulation, microdissection, and fluorescence-activated cell sorting (FACS). In the last decade several new and highly efficient approaches have been developed, which not just supplement but may fully replace the traditional ones. These new techniques are based on microfluidic chips, droplets, micro-well plates, and automatic collection of cells using capillaries, magnets, an electric field, or a punching probe. In this review we summarize the current methods and developments in this field. We discuss the advantages of the different commercially available platforms and their applicability, and also provide remarks on future developments.

  7. Methodology for single-cell genetic analysis of planktonic foraminifera for studies of protist diversity and evolution

    Directory of Open Access Journals (Sweden)

    Agnes Katharina Maria Weiner

    2016-12-01

    Full Text Available Single-cell genetic analysis is an essential method to investigate the biodiversity and evolutionary ecology of marine protists. In protist groups that do not reproduce under laboratory conditions, this approach provides the only means to directly associate molecular sequences with cell morphology. The resulting unambiguous taxonomic identification of the DNA sequences is a prerequisite for barcoding and analyses of environmental metagenomic data. Extensive single-cell genetic studies have been carried out on planktonic foraminifera over the past 20 years to elucidate their phylogeny, cryptic diversity, biogeography and the relationship between genetic and morphological variability. In the course of these investigations, it has become evident that genetic analysis at the individual specimen level is confronted by innumerable challenges ranging from the negligible amount of DNA present in the single cell to the substantial amount of DNA contamination introduced by endosymbionts or food particles. Consequently, a range of methods has been developed and applied throughout the years for the genetic analysis of planktonic foraminifera in order to enhance DNA amplification success rates. Yet, the description of these methods in the literature rarely occurred with equivalent levels of detail and the different approaches have never been compared in terms of their efficiency and reproducibility. Here, aiming at a standardization of methods, we provide a comprehensive review of all methods that have been employed for the single-cell genetic analysis of planktonic foraminifera. We compile data on success rates of DNA amplification and use these to evaluate the effects of key parameters associated with the methods of sample collection, storage and extraction of single-cell DNA. We show that the chosen methods influence the success rates of single-cell genetic studies, but the differences between them are not sufficient to hinder comparisons between studies

  8. Single-cell qPCR on dispersed primary pituitary cells -an optimized protocol

    Directory of Open Access Journals (Sweden)

    Haug Trude M

    2010-11-01

    Full Text Available Abstract Background The incidence of false positives is a potential problem in single-cell PCR experiments. This paper describes an optimized protocol for single-cell qPCR measurements in primary pituitary cell cultures following patch-clamp recordings. Two different cell harvesting methods were assessed using both the GH4 prolactin producing cell line from rat, and primary cell culture from fish pituitaries. Results Harvesting whole cells followed by cell lysis and qPCR performed satisfactory on the GH4 cell line. However, harvesting of whole cells from primary pituitary cultures regularly produced false positives, probably due to RNA leakage from cells ruptured during the dispersion of the pituitary cells. To reduce RNA contamination affecting the results, we optimized the conditions by harvesting only the cytosol through a patch pipette, subsequent to electrophysiological experiments. Two important factors proved crucial for reliable harvesting. First, silanizing the patch pipette glass prevented foreign extracellular RNA from attaching to charged residues on the glass surface. Second, substituting the commonly used perforating antibiotic amphotericin B with β-escin allowed efficient cytosol harvest without loosing the giga seal. Importantly, the two harvesting protocols revealed no difference in RNA isolation efficiency. Conclusion Depending on the cell type and preparation, validation of the harvesting technique is extremely important as contaminations may give false positives. Here we present an optimized protocol allowing secure harvesting of RNA from single cells in primary pituitary cell culture following perforated whole cell patch clamp experiments.

  9. Effects of external pressure on the performance and ageing of single-layer lithium-ion pouch cells

    Science.gov (United States)

    Mussa, Abdilbari Shifa; Klett, Matilda; Lindbergh, Göran; Lindström, Rakel Wreland

    2018-05-01

    The effects of external compression on the performance and ageing of NMC(1/3)/Graphite single-layer Li-ion pouch cells are investigated using a spring-loaded fixture. The influence of pressure (0.66, 0.99, 1.32, and 1.98 MPa) on impedance is characterized in fresh cells that are subsequently cycled at the given pressure levels. The aged cells are analyzed for capacity fade and impedance rise at the cell and electrode level. The effect of pressure distribution that may occur in large-format cells or in a battery pack is simulated using parallel connected cells. The results show that the kinetic and mass transport resistance increases with pressure in a fresh cell. An optimum pressure around 1.3 MPa is shown to be beneficial to reduce cyclable-lithium loss during cycling. The minor active mass losses observed in the electrodes are independent of the ageing pressure, whereas ageing pressure affects the charge transfer resistance of both NMC and graphite electrodes and the ohmic resistance of the cell. Pressure distribution induces current distribution but the enhanced current throughput at lower pressures cell does not accelerate its ageing. Conclusions from this work can explain some of the discrepancies in non-uniform ageing reported in the literature and indicate coupling between electrochemistry and mechanics.

  10. Single-Molecule Light-Sheet Imaging of Suspended T Cells.

    Science.gov (United States)

    Ponjavic, Aleks; McColl, James; Carr, Alexander R; Santos, Ana Mafalda; Kulenkampff, Klara; Lippert, Anna; Davis, Simon J; Klenerman, David; Lee, Steven F

    2018-05-08

    Adaptive immune responses are initiated by triggering of the T cell receptor. Single-molecule imaging based on total internal reflection fluorescence microscopy at coverslip/basal cell interfaces is commonly used to study this process. These experiments have suggested, unexpectedly, that the diffusional behavior and organization of signaling proteins and receptors may be constrained before activation. However, it is unclear to what extent the molecular behavior and cell state is affected by the imaging conditions, i.e., by the presence of a supporting surface. In this study, we implemented single-molecule light-sheet microscopy, which enables single receptors to be directly visualized at any plane in a cell to study protein dynamics and organization in live, resting T cells. The light sheet enabled the acquisition of high-quality single-molecule fluorescence images that were comparable to those of total internal reflection fluorescence microscopy. By comparing the apical and basal surfaces of surface-contacting T cells using single-molecule light-sheet microscopy, we found that most coated-glass surfaces and supported lipid bilayers profoundly affected the diffusion of membrane proteins (T cell receptor and CD45) and that all the surfaces induced calcium influx to various degrees. Our results suggest that, when studying resting T cells, surfaces are best avoided, which we achieve here by suspending cells in agarose. Copyright © 2018. Published by Elsevier Inc.

  11. Mapping Cellular Hierarchy by Single-Cell Analysis of the Cell Surface Repertoire

    OpenAIRE

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

    2013-01-01

    Stem cell differentiation pathways are most often studied at the population level, whereas critical decisions are executed at the level of single cells. We have established a highly multiplexed, quantitative PCR assay to profile in an unbiased manner a panel of all commonly used cell surface markers (280 genes) from individual cells. With this method we analyzed over 1500 single cells throughout the mouse hematopoietic system, and illustrate its utility for revealing important biological insi...

  12. Parameter Screening in Microfluidics Based Hydrodynamic Single-Cell Trapping

    Directory of Open Access Journals (Sweden)

    B. Deng

    2014-01-01

    Full Text Available Microfluidic cell-based arraying technology is widely used in the field of single-cell analysis. However, among developed devices, there is a compromise between cellular loading efficiencies and trapped cell densities, which deserves further analysis and optimization. To address this issue, the cell trapping efficiency of a microfluidic device with two parallel micro channels interconnected with cellular trapping sites was studied in this paper. By regulating channel inlet and outlet status, the microfluidic trapping structure can mimic key functioning units of previously reported devices. Numerical simulations were used to model this cellular trapping structure, quantifying the effects of channel on/off status and trapping structure geometries on the cellular trapping efficiency. Furthermore, the microfluidic device was fabricated based on conventional microfabrication and the cellular trapping efficiency was quantified in experiments. Experimental results showed that, besides geometry parameters, cellular travelling velocities and sizes also affected the single-cell trapping efficiency. By fine tuning parameters, more than 95% of trapping sites were taken by individual cells. This study may lay foundation in further studies of single-cell positioning in microfluidics and push forward the study of single-cell analysis.

  13. Short Peptides Enhance Single Cell Adhesion and Viability onMicroarrays

    Energy Technology Data Exchange (ETDEWEB)

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

    2007-01-19

    Single cell patterning holds important implications forbiology, biochemistry, biotechnology, medicine, and bioinformatics. Thechallenge for single cell patterning is to produce small islands hostingonly single cells and retaining their viability for a prolonged period oftime. This study demonstrated a surface engineering approach that uses acovalently bound short peptide as a mediator to pattern cells withimproved single cell adhesion and prolonged cellular viabilityon goldpatterned SiO2 substrates. The underlying hypothesis is that celladhesion is regulated bythe type, availability, and stability ofeffective cell adhesion peptides, and thus covalently bound shortpeptides would promote cell spreading and, thus, single cell adhesion andviability. The effectiveness of this approach and the underlyingmechanism for the increased probability of single cell adhesion andprolonged cell viability by short peptides were studied by comparingcellular behavior of human umbilical cord vein endothelial cells on threemodelsurfaces whose gold electrodes were immobilized with fibronectin,physically adsorbed Arg-Glu-Asp-Val-Tyr, and covalently boundLys-Arg-Glu-Asp-Val-Tyr, respectively. The surface chemistry and bindingproperties were characterized by reflectance Fourier transform infraredspectroscopy. Both short peptides were superior to fibronectin inproducing adhesion of only single cells, whereas the covalently boundpeptide also reduced apoptosis and necrosisof adhered cells. Controllingcell spreading by peptide binding domains to regulate apoptosis andviability represents a fundamental mechanism in cell-materialsinteraction and provides an effective strategy in engineering arrays ofsingle cells.

  14. Technical aspects and recommendations for single-cell qPCR.

    Science.gov (United States)

    Ståhlberg, Anders; Kubista, Mikael

    2018-02-01

    Single cells are basic physiological and biological units that can function individually as well as in groups in tissues and organs. It is central to identify, characterize and profile single cells at molecular level to be able to distinguish different kinds, to understand their functions and determine how they interact with each other. During the last decade several technologies for single-cell profiling have been developed and used in various applications, revealing many novel findings. Quantitative PCR (qPCR) is one of the most developed methods for single-cell profiling that can be used to interrogate several analytes, including DNA, RNA and protein. Single-cell qPCR has the potential to become routine methodology but the technique is still challenging, as it involves several experimental steps and few molecules are handled. Here, we discuss technical aspects and provide recommendation for single-cell qPCR analysis. The workflow includes experimental design, sample preparation, single-cell collection, direct lysis, reverse transcription, preamplification, qPCR and data analysis. Detailed reporting and sharing of experimental details and data will promote further development and make validation studies possible. Efforts aiming to standardize single-cell qPCR open up means to move single-cell analysis from specialized research settings to standard research laboratories. Copyright © 2017 Elsevier Ltd. All rights reserved.

  15. High Throughput Single-cell and Multiple-cell Micro-encapsulation

    OpenAIRE

    Lagus, Todd P.; Edd, Jon F.

    2012-01-01

    Microfluidic encapsulation methods have been previously utilized to capture cells in picoliter-scale aqueous, monodisperse drops, providing confinement from a bulk fluid environment with applications in high throughput screening, cytometry, and mass spectrometry. We describe a method to not only encapsulate single cells, but to repeatedly capture a set number of cells (here we demonstrate one- and two-cell encapsulation) to study both isolation and the interactions between cells in groups of ...

  16. Single cell Hi-C reveals cell-to-cell variability in chromosome structure

    Science.gov (United States)

    Schoenfelder, Stefan; Yaffe, Eitan; Dean, Wendy; Laue, Ernest D.; Tanay, Amos; Fraser, Peter

    2013-01-01

    Large-scale chromosome structure and spatial nuclear arrangement have been linked to control of gene expression and DNA replication and repair. Genomic techniques based on chromosome conformation capture assess contacts for millions of loci simultaneously, but do so by averaging chromosome conformations from millions of nuclei. Here we introduce single cell Hi-C, combined with genome-wide statistical analysis and structural modeling of single copy X chromosomes, to show that individual chromosomes maintain domain organisation at the megabase scale, but show variable cell-to-cell chromosome territory structures at larger scales. Despite this structural stochasticity, localisation of active gene domains to boundaries of territories is a hallmark of chromosomal conformation. Single cell Hi-C data bridge current gaps between genomics and microscopy studies of chromosomes, demonstrating how modular organisation underlies dynamic chromosome structure, and how this structure is probabilistically linked with genome activity patterns. PMID:24067610

  17. Single-Cell RNA-Seq Analysis of Infiltrating Neoplastic Cells at the Migrating Front of Human Glioblastoma

    Directory of Open Access Journals (Sweden)

    Spyros Darmanis

    2017-10-01

    Full Text Available Summary: Glioblastoma (GBM is the most common primary brain cancer in adults and is notoriously difficult to treat because of its diffuse nature. We performed single-cell RNA sequencing (RNA-seq on 3,589 cells in a cohort of four patients. We obtained cells from the tumor core as well as surrounding peripheral tissue. Our analysis revealed cellular variation in the tumor’s genome and transcriptome. We were also able to identify infiltrating neoplastic cells in regions peripheral to the core lesions. Despite the existence of significant heterogeneity among neoplastic cells, we found that infiltrating GBM cells share a consistent gene signature between patients, suggesting a common mechanism of infiltration. Additionally, in investigating the immunological response to the tumors, we found transcriptionally distinct myeloid cell populations residing in the tumor core and the surrounding peritumoral space. Our data provide a detailed dissection of GBM cell types, revealing an abundance of information about tumor formation and migration. : Darmanis et al. perform single-cell transcriptomic analyses of neoplastic and stromal cells within and proximal to primary glioblastomas. The authors describe a population of neoplastic-infiltrating glioblastoma cells as well as a putative role of tumor-infiltrating immune cells in supporting tumor growth. Keywords: single cell, RNA-seq, glioma, glioblastoma, GBM, brain, heterogeneity, infiltrating, diffuse, checkpoint

  18. Single cell detection using a magnetic zigzag nanowire biosensor.

    Science.gov (United States)

    Huang, Hao-Ting; Ger, Tzong-Rong; Lin, Ya-Hui; Wei, Zung-Hang

    2013-08-07

    A magnetic zigzag nanowire device was designed for single cell biosensing. Nanowires with widths of 150, 300, 500, and 800 nm were fabricated on silicon trenches by electron beam lithography, electron beam evaporation, and lift-off processes. Magnetoresistance measurements were performed before and after the attachment of a single magnetic cell to the nanowires to characterize the magnetic signal change due to the influence of the magnetic cell. Magnetoresistance responses were measured in different magnetic field directions, and the results showed that this nanowire device can be used for multi-directional detection. It was observed that the highest switching field variation occurred in a 150 nm wide nanowire when the field was perpendicular to the substrate plane. On the other hand, the highest magnetoresistance ratio variation occurred in a 800 nm wide nanowire also when the field was perpendicular to the substrate plane. Besides, the trench-structured substrate proposed in this study can fix the magnetic cell to the sensor in a fluid environment, and the stray field generated by the corners of the magnetic zigzag nanowires has the function of actively attracting the magnetic cells for detection.

  19. RoboSCell: An automated single cell arraying and analysis instrument

    KAUST Repository

    Sakaki, Kelly; Foulds, Ian G.; Liu, William; Dechev, Nikolai; Burke, Robert Douglas; Park, Edward

    2009-01-01

    Single cell research has the potential to revolutionize experimental methods in biomedical sciences and contribute to clinical practices. Recent studies suggest analysis of single cells reveals novel features of intracellular processes, cell-to-cell

  20. Matrigel Mattress: A Method for the Generation of Single Contracting Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

    Science.gov (United States)

    Feaster, Tromondae K; Cadar, Adrian G; Wang, Lili; Williams, Charles H; Chun, Young Wook; Hempel, Jonathan E; Bloodworth, Nathaniel; Merryman, W David; Lim, Chee Chew; Wu, Joseph C; Knollmann, Björn C; Hong, Charles C

    2015-12-04

    The lack of measurable single-cell contractility of human-induced pluripotent stem cell-derived cardiac myocytes (hiPSC-CMs) currently limits the utility of hiPSC-CMs for evaluating contractile performance for both basic research and drug discovery. To develop a culture method that rapidly generates contracting single hiPSC-CMs and allows quantification of cell shortening with standard equipment used for studying adult CMs. Single hiPSC-CMs were cultured for 5 to 7 days on a 0.4- to 0.8-mm thick mattress of undiluted Matrigel (mattress hiPSC-CMs) and compared with hiPSC-CMs maintained on a control substrate (method enables the rapid generation of robustly contracting hiPSC-CMs and enhances maturation. This new method allows quantification of contractile performance at the single-cell level, which should be valuable to disease modeling, drug discovery, and preclinical cardiotoxicity testing. © 2015 American Heart Association, Inc.

  1. Capturing Three-Dimensional Genome Organization in Individual Cells by Single-Cell Hi-C.

    Science.gov (United States)

    Nagano, Takashi; Wingett, Steven W; Fraser, Peter

    2017-01-01

    Hi-C is a powerful method to investigate genome-wide, higher-order chromatin and chromosome conformations averaged from a population of cells. To expand the potential of Hi-C for single-cell analysis, we developed single-cell Hi-C. Similar to the existing "ensemble" Hi-C method, single-cell Hi-C detects proximity-dependent ligation events between cross-linked and restriction-digested chromatin fragments in cells. A major difference between the single-cell Hi-C and ensemble Hi-C protocol is that the proximity-dependent ligation is carried out in the nucleus. This allows the isolation of individual cells in which nearly the entire Hi-C procedure has been carried out, enabling the production of a Hi-C library and data from individual cells. With this new method, we studied genome conformations and found evidence for conserved topological domain organization from cell to cell, but highly variable interdomain contacts and chromosome folding genome wide. In addition, we found that the single-cell Hi-C protocol provided cleaner results with less technical noise suggesting it could be used to improve the ensemble Hi-C technique.

  2. Cloning of Plasmodium falciparum by single-cell sorting.

    Science.gov (United States)

    Miao, Jun; Li, Xiaolian; Cui, Liwang

    2010-10-01

    Malaria parasite cloning is traditionally carried out mainly by using the limiting dilution method, which is laborious, imprecise, and unable to distinguish multiply-infected RBCs. In this study, we used a parasite engineered to express green fluorescent protein (GFP) to evaluate a single-cell sorting method for rapidly cloning Plasmodium falciparum. By dividing a two-dimensional scattergram from a cell sorter into 17 gates, we determined the parameters for isolating singly-infected erythrocytes and sorted them into individual cultures. Pre-gating of the engineered parasites for GFP allowed the isolation of almost 100% GFP-positive clones. Compared with the limiting dilution method, the number of parasite clones obtained by single-cell sorting was much higher. Molecular analyses showed that parasite isolates obtained by single-cell sorting were highly homogenous. This highly efficient single-cell sorting method should prove very useful for cloning both P. falciparum laboratory populations from genetic manipulation experiments and clinical samples. Copyright 2010 Elsevier Inc. All rights reserved.

  3. Cloning of Plasmodium falciparum by single-cell sorting

    Science.gov (United States)

    Miao, Jun; Li, Xiaolian; Cui, Liwang

    2010-01-01

    Malaria parasite cloning is traditionally carried out mainly by using the limiting dilution method, which is laborious, imprecise, and unable to distinguish multiply-infected RBCs. In this study, we used a parasite engineered to express green fluorescent protein (GFP) to evaluate a single-cell sorting method for rapidly cloning Plasmodium falciparum. By dividing a two dimensional scattergram from a cell sorter into 17 gates, we determined the parameters for isolating singly-infected erythrocytes and sorted them into individual cultures. Pre-gating of the engineered parasites for GFP allowed the isolation of almost 100% GFP-positive clones. Compared with the limiting dilution method, the number of parasite clones obtained by single-cell sorting was much higher. Molecular analyses showed that parasite isolates obtained by single-cell sorting were highly homogenous. This highly efficient single-cell sorting method should prove very useful for cloning both P. falciparum laboratory populations from genetic manipulation experiments and clinical samples. PMID:20435038

  4. Simultaneous genomic identification and profiling of a single cell using semiconductor-based next generation sequencing

    Directory of Open Access Journals (Sweden)

    Manabu Watanabe

    2014-09-01

    Full Text Available Combining single-cell methods and next-generation sequencing should provide a powerful means to understand single-cell biology and obviate the effects of sample heterogeneity. Here we report a single-cell identification method and seamless cancer gene profiling using semiconductor-based massively parallel sequencing. A549 cells (adenocarcinomic human alveolar basal epithelial cell line were used as a model. Single-cell capture was performed using laser capture microdissection (LCM with an Arcturus® XT system, and a captured single cell and a bulk population of A549 cells (≈106 cells were subjected to whole genome amplification (WGA. For cell identification, a multiplex PCR method (AmpliSeq™ SNP HID panel was used to enrich 136 highly discriminatory SNPs with a genotype concordance probability of 1031–35. For cancer gene profiling, we used mutation profiling that was performed in parallel using a hotspot panel for 50 cancer-related genes. Sequencing was performed using a semiconductor-based bench top sequencer. The distribution of sequence reads for both HID and Cancer panel amplicons was consistent across these samples. For the bulk population of cells, the percentages of sequence covered at coverage of more than 100× were 99.04% for the HID panel and 98.83% for the Cancer panel, while for the single cell percentages of sequence covered at coverage of more than 100× were 55.93% for the HID panel and 65.96% for the Cancer panel. Partial amplification failure or randomly distributed non-amplified regions across samples from single cells during the WGA procedures or random allele drop out probably caused these differences. However, comparative analyses showed that this method successfully discriminated a single A549 cancer cell from a bulk population of A549 cells. Thus, our approach provides a powerful means to overcome tumor sample heterogeneity when searching for somatic mutations.

  5. Single-cell analysis of growth and cell division of the anaerobe Desulfovibrio vulgaris Hildenborough

    Directory of Open Access Journals (Sweden)

    Anouchka eFievet

    2015-12-01

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

  6. Single cell low dose studies of bystander cell killing with targeted ultrasoft x-rays

    International Nuclear Information System (INIS)

    Schettino, G.; Prise, K.M.; Folkard, M.; Vojnovic, B.; Michael, B.D.; Wu, L.; Held, K.D.

    2003-01-01

    Full text: Bystander responses have attracted considerable interest in the recent years and several investigations have reported a binary behavior with the effect triggered by very small doses and immediately reaching a plateau. The Ultrasoft X-ray Microprobe in operation at the GCI is a facility designed to precisely assess the biological response of individual cells in vitro irradiated with a sub-micron size X-ray beam . Although recent improvements have upgrade the facility with AlK and TiK X-rays, most of the bystander studies have been performed using CK X-rays of 278 eV. The high sensitivity and the accurate irradiation and revisiting of the individual samples allowed us to investigate specific characteristics of the bystander phenomenon. In particular, evidences of a dose dependency of cell killing by bystander effect have been found at doses below 0.2 Gy where no differences is observed between all cell and single cell irradiation. Recent improvements have also allowed us to individually identify the phase of the cell cycle of all samples exposed. Although the G2-S phase have been found the most sensitive in responding to the bystander signal (a factor of 1.3), cells in the G1 phase also respond significantly while the phase of the irradiated cell doesn't seem to play a critical role. The time scale of the bystander effect has also been investigated by irradiating the same sample(s) twice with a few hours gap between exposures. Results indicate that the bystander signal is transmitted within a few minutes from the irradiation as replacing the medium immediately after irradiation does not influence the response, and it doesn't depend on the number of cells irradiated (up to 5). However, after a resting time of a few hours (3 h), the system seems to reset itself and a second irradiation has been shown to trigger a further bystander effect. Finally, by considering the total amount of energy deposited in to the sample population as critical parameter (instead

  7. Lessons from single-cell transcriptome analysis of oxygen-sensing cells.

    Science.gov (United States)

    Zhou, Ting; Matsunami, Hiroaki

    2018-05-01

    The advent of single-cell RNA-sequencing (RNA-Seq) technology has enabled transcriptome profiling of individual cells. Comprehensive gene expression analysis at the single-cell level has proven to be effective in characterizing the most fundamental aspects of cellular function and identity. This unbiased approach is revolutionary for small and/or heterogeneous tissues like oxygen-sensing cells in identifying key molecules. Here, we review the major methods of current single-cell RNA-Seq technology. We discuss how this technology has advanced the understanding of oxygen-sensing glomus cells in the carotid body and helped uncover novel oxygen-sensing cells and mechanisms in the mice olfactory system. We conclude by providing our perspective on future single-cell RNA-Seq research directed at oxygen-sensing cells.

  8. Single-Cell Genomics: Approaches and Utility in Immunology.

    Science.gov (United States)

    Neu, Karlynn E; Tang, Qingming; Wilson, Patrick C; Khan, Aly A

    2017-02-01

    Single-cell genomics offers powerful tools for studying immune cells, which make it possible to observe rare and intermediate cell states that cannot be resolved at the population level. Advances in computer science and single-cell sequencing technology have created a data-driven revolution in immunology. The challenge for immunologists is to harness computing and turn an avalanche of quantitative data into meaningful discovery of immunological principles, predictive models, and strategies for therapeutics. Here, we review the current literature on computational analysis of single-cell RNA-sequencing data and discuss underlying assumptions, methods, and applications in immunology, and highlight important directions for future research. Copyright © 2016 Elsevier Ltd. All rights reserved.

  9. Study of a Microfluidic Chip Integrating Single Cell Trap and 3D Stable Rotation Manipulation

    Directory of Open Access Journals (Sweden)

    Liang Huang

    2016-08-01

    Full Text Available Single cell manipulation technology has been widely applied in biological fields, such as cell injection/enucleation, cell physiological measurement, and cell imaging. Recently, a biochip platform with a novel configuration of electrodes for cell 3D rotation has been successfully developed by generating rotating electric fields. However, the rotation platform still has two major shortcomings that need to be improved. The primary problem is that there is no on-chip module to facilitate the placement of a single cell into the rotation chamber, which causes very low efficiency in experiment to manually pipette single 10-micron-scale cells into rotation position. Secondly, the cell in the chamber may suffer from unstable rotation, which includes gravity-induced sinking down to the chamber bottom or electric-force-induced on-plane movement. To solve the two problems, in this paper we propose a new microfluidic chip with manipulation capabilities of single cell trap and single cell 3D stable rotation, both on one chip. The new microfluidic chip consists of two parts. The top capture part is based on the least flow resistance principle and is used to capture a single cell and to transport it to the rotation chamber. The bottom rotation part is based on dielectrophoresis (DEP and is used to 3D rotate the single cell in the rotation chamber with enhanced stability. The two parts are aligned and bonded together to form closed channels for microfluidic handling. Using COMSOL simulation and preliminary experiments, we have verified, in principle, the concept of on-chip single cell traps and 3D stable rotation, and identified key parameters for chip structures, microfluidic handling, and electrode configurations. The work has laid a solid foundation for on-going chip fabrication and experiment validation.

  10. Chip based single cell analysis for nanotoxicity assessment.

    Science.gov (United States)

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

    2014-05-07

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

  11. The Caenorhabditis elegans Q neuroblasts: A powerful system to study cell migration at single-cell resolution in vivo.

    Science.gov (United States)

    Rella, Lorenzo; Fernandes Póvoa, Euclides E; Korswagen, Hendrik C

    2016-04-01

    During development, cell migration plays a central role in the formation of tissues and organs. Understanding the molecular mechanisms that drive and control these migrations is a key challenge in developmental biology that will provide important insights into disease processes, including cancer cell metastasis. In this article, we discuss the Caenorhabditis elegans Q neuroblasts and their descendants as a tool to study cell migration at single-cell resolution in vivo. The highly stereotypical migration of these cells provides a powerful system to study the dynamic cytoskeletal processes that drive migration as well as the evolutionarily conserved signaling pathways (including different Wnt signaling cascades) that guide the cells along their specific trajectories. Here, we provide an overview of what is currently known about Q neuroblast migration and highlight the live-cell imaging, genome editing, and quantitative gene expression techniques that have been developed to study this process. © 2016 Wiley Periodicals, Inc.

  12. Biomimetic Nanoarchitectures for the Study of T Cell Activation with Single-Molecule Control

    Science.gov (United States)

    Cai, Haogang

    Physical factors in the environment of a cell affect its function and behavior in a variety of ways. There is increasing evidence that, among these factors, the geometric arrangement of receptor ligands plays an important role in setting the conditions for critical cellular processes. The goal of this thesis is to develop new techniques for probing the role of extracellular ligand geometry, with a focus on T cell activation. In this work, top-down molecular-scale nanofabrication and bottom-up selective self-assembly were combined in order to present functional nanomaterials (primarily biomolecules) on a surface with precise spatial control and single-molecule resolution. Such biomolecule nanoarrays are becoming an increasingly important tool in surface-based in vitro assays for biosensing, molecular and cellular studies. The nanoarrays consist of metallic nanodots patterned on glass coverslips using electron beam and nanoimprint lithography, combined with self-aligned pattern transfer. The nanodots were then used as anchors for the immobilization of biological ligands, and backfilled with a protein-repellent passivation layer of polyethylene glycol. The passivation efficiency was improved to minimize nonspecific adsorption. In order to ensure true single-molecule control, we developed an on-chip protocol to measure the molecular occupancy of nanodot arrays based on fluorescence photobleaching, while accounting for quenching effects by plasmonic absorption. We found that the molecular occupancy can be interpreted as a packing problem, with the solution depending on the nanodot size and the concentration of self-assembly reagents, where the latter can be easily adjusted to control the molecular occupancy according to the dot size. The optimized nanoarrays were used as biomimetic architectures for the study of T cell activation with single-molecule control. T cell activation involves an elaborate arrangement of signaling, adhesion, and costimulatory molecules

  13. Full-length single-cell RNA-seq applied to a viral human cancer: applications to HPV expression and splicing analysis in HeLa S3 cells.

    Science.gov (United States)

    Wu, Liang; Zhang, Xiaolong; Zhao, Zhikun; Wang, Ling; Li, Bo; Li, Guibo; Dean, Michael; Yu, Qichao; Wang, Yanhui; Lin, Xinxin; Rao, Weijian; Mei, Zhanlong; Li, Yang; Jiang, Runze; Yang, Huan; Li, Fuqiang; Xie, Guoyun; Xu, Liqin; Wu, Kui; Zhang, Jie; Chen, Jianghao; Wang, Ting; Kristiansen, Karsten; Zhang, Xiuqing; Li, Yingrui; Yang, Huanming; Wang, Jian; Hou, Yong; Xu, Xun

    2015-01-01

    Viral infection causes multiple forms of human cancer, and HPV infection is the primary factor in cervical carcinomas. Recent single-cell RNA-seq studies highlight the tumor heterogeneity present in most cancers, but virally induced tumors have not been studied. HeLa is a well characterized HPV+ cervical cancer cell line. We developed a new high throughput platform to prepare single-cell RNA on a nanoliter scale based on a customized microwell chip. Using this method, we successfully amplified full-length transcripts of 669 single HeLa S3 cells and 40 of them were randomly selected to perform single-cell RNA sequencing. Based on these data, we obtained a comprehensive understanding of the heterogeneity of HeLa S3 cells in gene expression, alternative splicing and fusions. Furthermore, we identified a high diversity of HPV-18 expression and splicing at the single-cell level. By co-expression analysis we identified 283 E6, E7 co-regulated genes, including CDC25, PCNA, PLK4, BUB1B and IRF1 known to interact with HPV viral proteins. Our results reveal the heterogeneity of a virus-infected cell line. It not only provides a transcriptome characterization of HeLa S3 cells at the single cell level, but is a demonstration of the power of single cell RNA-seq analysis of virally infected cells and cancers.

  14. Functional magnetic resonance microscopy at single-cell resolution in Aplysia californica

    Science.gov (United States)

    Radecki, Guillaume; Nargeot, Romuald; Jelescu, Ileana Ozana; Le Bihan, Denis; Ciobanu, Luisa

    2014-01-01

    In this work, we show the feasibility of performing functional MRI studies with single-cell resolution. At ultrahigh magnetic field, manganese-enhanced magnetic resonance microscopy allows the identification of most motor neurons in the buccal network of Aplysia at low, nontoxic Mn2+ concentrations. We establish that Mn2+ accumulates intracellularly on injection into the living Aplysia and that its concentration increases when the animals are presented with a sensory stimulus. We also show that we can distinguish between neuronal activities elicited by different types of stimuli. This method opens up a new avenue into probing the functional organization and plasticity of neuronal networks involved in goal-directed behaviors with single-cell resolution. PMID:24872449

  15. Single Cell Genomics: Approaches and Utility in Immunology

    Science.gov (United States)

    Neu, Karlynn E; Tang, Qingming; Wilson, Patrick C; Khan, Aly A

    2017-01-01

    Single cell genomics offers powerful tools for studying lymphocytes, which make it possible to observe rare and intermediate cell states that cannot be resolved at the population-level. Advances in computer science and single cell sequencing technology have created a data-driven revolution in immunology. The challenge for immunologists is to harness computing and turn an avalanche of quantitative data into meaningful discovery of immunological principles, predictive models, and strategies for therapeutics. Here, we review the current literature on computational analysis of single cell RNA-seq data and discuss underlying assumptions, methods, and applications in immunology, and highlight important directions for future research. PMID:28094102

  16. Numerical Analysis of Hydrodynamic Flow in Microfluidic Biochip for Single-Cell Trapping Application

    Directory of Open Access Journals (Sweden)

    Amelia Ahmad Khalili

    2015-11-01

    Full Text Available Single-cell analysis has become the interest of a wide range of biological and biomedical engineering research. It could provide precise information on individual cells, leading to important knowledge regarding human diseases. To perform single-cell analysis, it is crucial to isolate the individual cells before further manipulation is carried out. Recently, microfluidic biochips have been widely used for cell trapping and single cell analysis, such as mechanical and electrical detection. This work focuses on developing a finite element simulation model of single-cell trapping system for any types of cells or particles based on the hydrodynamic flow resistance (Rh manipulations in the main channel and trap channel to achieve successful trapping. Analysis is carried out using finite element ABAQUS-FEA™ software. A guideline to design and optimize single-cell trapping model is proposed and the example of a thorough optimization analysis is carried out using a yeast cell model. The results show the finite element model is able to trap a single cell inside the fluidic environment. Fluid’s velocity profile and streamline plots for successful and unsuccessful single yeast cell trapping are presented according to the hydrodynamic concept. The single-cell trapping model can be a significant important guideline in designing a new chip for biomedical applications.

  17. Single-cell RNA-seq analysis unveils a prevalent epithelial/mesenchymal hybrid state during mouse organogenesis.

    Science.gov (United States)

    Dong, Ji; Hu, Yuqiong; Fan, Xiaoying; Wu, Xinglong; Mao, Yunuo; Hu, Boqiang; Guo, Hongshan; Wen, Lu; Tang, Fuchou

    2018-03-14

    Organogenesis is crucial for proper organ formation during mammalian embryonic development. However, the similarities and shared features between different organs and the cellular heterogeneity during this process at single-cell resolution remain elusive. We perform single-cell RNA sequencing analysis of 1916 individual cells from eight organs and tissues of E9.5 to E11.5 mouse embryos, namely, the forebrain, hindbrain, skin, heart, somite, lung, liver, and intestine. Based on the regulatory activities rather than the expression patterns, all cells analyzed can be well classified into four major groups with epithelial, mesodermal, hematopoietic, and neuronal identities. For different organs within the same group, the similarities and differences of their features and developmental paths are revealed and reconstructed. We identify mutual interactions between epithelial and mesenchymal cells and detect epithelial cells with prevalent mesenchymal features during organogenesis, which are similar to the features of intermediate epithelial/mesenchymal cells during tumorigenesis. The comprehensive transcriptome at single-cell resolution profiled in our study paves the way for future mechanistic studies of the gene-regulatory networks governing mammalian organogenesis.

  18. A probabilistic cell model in background corrected image sequences for single cell analysis

    Directory of Open Access Journals (Sweden)

    Fieguth Paul

    2010-10-01

    Full Text Available Abstract Background Methods of manual cell localization and outlining are so onerous that automated tracking methods would seem mandatory for handling huge image sequences, nevertheless manual tracking is, astonishingly, still widely practiced in areas such as cell biology which are outside the influence of most image processing research. The goal of our research is to address this gap by developing automated methods of cell tracking, localization, and segmentation. Since even an optimal frame-to-frame association method cannot compensate and recover from poor detection, it is clear that the quality of cell tracking depends on the quality of cell detection within each frame. Methods Cell detection performs poorly where the background is not uniform and includes temporal illumination variations, spatial non-uniformities, and stationary objects such as well boundaries (which confine the cells under study. To improve cell detection, the signal to noise ratio of the input image can be increased via accurate background estimation. In this paper we investigate background estimation, for the purpose of cell detection. We propose a cell model and a method for background estimation, driven by the proposed cell model, such that well structure can be identified, and explicitly rejected, when estimating the background. Results The resulting background-removed images have fewer artifacts and allow cells to be localized and detected more reliably. The experimental results generated by applying the proposed method to different Hematopoietic Stem Cell (HSC image sequences are quite promising. Conclusion The understanding of cell behavior relies on precise information about the temporal dynamics and spatial distribution of cells. Such information may play a key role in disease research and regenerative medicine, so automated methods for observation and measurement of cells from microscopic images are in high demand. The proposed method in this paper is capable

  19. A Single-Cell Biochemistry Approach Reveals PAR Complex Dynamics during Cell Polarization.

    Science.gov (United States)

    Dickinson, Daniel J; Schwager, Francoise; Pintard, Lionel; Gotta, Monica; Goldstein, Bob

    2017-08-21

    Regulated protein-protein interactions are critical for cell signaling, differentiation, and development. For the study of dynamic regulation of protein interactions in vivo, there is a need for techniques that can yield time-resolved information and probe multiple protein binding partners simultaneously, using small amounts of starting material. Here we describe a single-cell protein interaction assay. Single-cell lysates are generated at defined time points and analyzed using single-molecule pull-down, yielding information about dynamic protein complex regulation in vivo. We established the utility of this approach by studying PAR polarity proteins, which mediate polarization of many animal cell types. We uncovered striking regulation of PAR complex composition and stoichiometry during Caenorhabditis elegans zygote polarization, which takes place in less than 20 min. PAR complex dynamics are linked to the cell cycle by Polo-like kinase 1 and govern the movement of PAR proteins to establish polarity. Our results demonstrate an approach to study dynamic biochemical events in vivo. Copyright © 2017 Elsevier Inc. All rights reserved.

  20. Performance of Pd on activated carbon as hydrogen electrode with respect to hydrogen yield in a single cell proton exchange membrane (PEM) water electrolyser

    Energy Technology Data Exchange (ETDEWEB)

    Naga Mahesh, K.; Sarada Prasad, J.; Venkateswer Rao, M.; Himabindu, V. [Centre for Environment, Institute of Science and Technology, Jawaharlal Nehru Technological University Hyderabad, Kukatpally, Hyderabad 500085 (A.P.) (India); Yerramilli, Anjaneyulu [TLGVRC, JSU Box 18739, Jackson State University, Jackson, MS 32917 - 0939 (United States); Raghunathan Rao, P. [Fuel cell section, Heavy Water Division, Bhabha Atomic Research Centre, Trombay, Mumbai - 400 085 (India)

    2009-08-15

    Palladium (Pd) on activated carbon is used as electrocatalyst coated on Nafion 115 membrane as Hydrogen electrode and RuO{sub 2} is coated on other side of membrane used as oxygen electrode. 5 wt% and 10 wt% Pd on activated carbon is prepared as membrane electrode assembly (MEA) and investigated the performance of the same using inhouse prepared 10 cm{sup 2} single cell. The performance of the single cell assembly and the hydrogen yield are reported during electrolysis operation at temperatures 27 C, 45 C and 65 C at 0.1, 0.2, 0.3, 0.4, 0.5 A/cm{sup 2} current densities with respect to voltages. (author)

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

    Science.gov (United States)

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

    2015-08-11

    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.

  2. Unravel lipid accumulation mechanism in oleaginous yeast through single cell systems biology study

    Energy Technology Data Exchange (ETDEWEB)

    Ding, Shiyou; Xiaoliang, Xie

    2017-12-18

    transcriptional profiles at the single-cell level, as follows: 1) We developed hyperspectral Stimulated Raman Scattering (hsSRS) microscope tailored for fast chemical imaging in complex biological systems. It features high numerical aperture of 1.4 for better spatial resolution and reduced cross-phase modulation. The femtosecond infrared laser was selected for deeper penetration and less photodamage for longer in-situ imaging time. hsSRS was achieved through spectral focusing where two femtosecond laser beams were linearly chirped to create overlapping pulses in time domain. We achieved transformed limited spectral resolution of ~15cm for superior clarity in identifying different types of lipids. A set of sophisticated algorithms based on Multivariate Curve Resolution (MCR) was developed to analyze yeast images and track droplets. 2) Lipid production was carried out on selected oleaginous yeast strains. Lipid accumulation was studied in R. glutinis and Y. lipolytica grown in regular and nitrogen starvation media. hsSRS imaging of lipid droplets showed considerable variation among individual Y. lipolytica cells regarding volume and composition, while the R. glutins lipid droplet showed similar Raman response representing more homogenous lipid production. hsSRS analysis revealed the spatial distribution of two major lipids-TAG and sterol ester (SE)- where TAG formed cores were surrounded by SE shells. Significantly elevated level of SE/TAG was observed at cellular and sub-cellular levels which could be attributed to epigenetic variation in cell development. 3) Yeast growth conditions were studied based on their lipid droplets formation. Optimal condition was found in two-stage grown media where the yeast was grown in regular YEPD and transferred to nitrogen starvation media after maturation. The volume and composition of lipid droplets vary significantly depending on the availability of nitrogen source. Large number of cells have been fast screened and analyzed by custom

  3. Epigenetics reloaded: the single-cell revolution.

    Science.gov (United States)

    Bheda, Poonam; Schneider, Robert

    2014-11-01

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

  4. Advances of Single-Cell Sequencing Technique in Tumors

    Directory of Open Access Journals (Sweden)

    Ji-feng FENG

    2017-03-01

    Full Text Available With the completion of human genome project (HGP and the international HapMap project as well as rapid development of high-throughput biochip technology, whole genomic sequencing-targeted analysis of genomic structures has been primarily finished. Application of single cell for the analysis of the whole genomics is not only economical in material collection, but more importantly, the cell will be more purified, and the laboratory results will be more accurate and reliable. Therefore, exploration and analysis of hereditary information of single tumor cells has become the dream of all researchers in the field of basic research of tumors. At present, single-cell sequencing (SCS on malignancies has been widely used in the studies of pathogeneses of multiple malignancies, such as glioma, renal cancer and hematologic neoplasms, and in the studies of the metastatic mechanism of breast cancer by some researchers. This study mainly reviewed the SCS, the mechanisms and the methods of SCS in isolating tumor cells, and application of SCS technique in tumor-related basic research and clinical treatment.

  5. Single-cell analysis reveals early manifestation of cancerous phenotype in pre-malignant esophageal cells.

    Directory of Open Access Journals (Sweden)

    Jiangxin Wang

    Full Text Available Cellular heterogeneity plays a pivotal role in a variety of functional processes in vivo including carcinogenesis. However, our knowledge about cell-to-cell diversity and how differences in individual cells manifest in alterations at the population level remains very limited mainly due to the lack of appropriate tools enabling studies at the single-cell level. We present a study on changes in cellular heterogeneity in the context of pre-malignant progression in response to hypoxic stress. Utilizing pre-malignant progression of Barrett's esophagus (BE as a disease model system we studied molecular mechanisms underlying the progression from metaplastic to dysplastic (pre-cancerous stage. We used newly developed methods enabling measurements of cell-to-cell differences in copy numbers of mitochondrial DNA, expression levels of a set of mitochondrial and nuclear genes involved in hypoxia response pathways, and mitochondrial membrane potential. In contrast to bulk cell studies reported earlier, our study shows significant differences between metaplastic and dysplastic BE cells in both average values and single-cell parameter distributions of mtDNA copy numbers, mitochondrial function, and mRNA expression levels of studied genes. Based on single-cell data analysis, we propose that mitochondria may be one of the key factors in pre-malignant progression in BE.

  6. DIMM-SC: a Dirichlet mixture model for clustering droplet-based single cell transcriptomic data.

    Science.gov (United States)

    Sun, Zhe; Wang, Ting; Deng, Ke; Wang, Xiao-Feng; Lafyatis, Robert; Ding, Ying; Hu, Ming; Chen, Wei

    2018-01-01

    Single cell transcriptome sequencing (scRNA-Seq) has become a revolutionary tool to study cellular and molecular processes at single cell resolution. Among existing technologies, the recently developed droplet-based platform enables efficient parallel processing of thousands of single cells with direct counting of transcript copies using Unique Molecular Identifier (UMI). Despite the technology advances, statistical methods and computational tools are still lacking for analyzing droplet-based scRNA-Seq data. Particularly, model-based approaches for clustering large-scale single cell transcriptomic data are still under-explored. We developed DIMM-SC, a Dirichlet Mixture Model for clustering droplet-based Single Cell transcriptomic data. This approach explicitly models UMI count data from scRNA-Seq experiments and characterizes variations across different cell clusters via a Dirichlet mixture prior. We performed comprehensive simulations to evaluate DIMM-SC and compared it with existing clustering methods such as K-means, CellTree and Seurat. In addition, we analyzed public scRNA-Seq datasets with known cluster labels and in-house scRNA-Seq datasets from a study of systemic sclerosis with prior biological knowledge to benchmark and validate DIMM-SC. Both simulation studies and real data applications demonstrated that overall, DIMM-SC achieves substantially improved clustering accuracy and much lower clustering variability compared to other existing clustering methods. More importantly, as a model-based approach, DIMM-SC is able to quantify the clustering uncertainty for each single cell, facilitating rigorous statistical inference and biological interpretations, which are typically unavailable from existing clustering methods. DIMM-SC has been implemented in a user-friendly R package with a detailed tutorial available on www.pitt.edu/∼wec47/singlecell.html. wei.chen@chp.edu or hum@ccf.org. Supplementary data are available at Bioinformatics online. © The Author

  7. Landscape of Infiltrating T Cells in Liver Cancer Revealed by Single-Cell Sequencing.

    Science.gov (United States)

    Zheng, Chunhong; Zheng, Liangtao; Yoo, Jae-Kwang; Guo, Huahu; Zhang, Yuanyuan; Guo, Xinyi; Kang, Boxi; Hu, Ruozhen; Huang, Julie Y; Zhang, Qiming; Liu, Zhouzerui; Dong, Minghui; Hu, Xueda; Ouyang, Wenjun; Peng, Jirun; Zhang, Zemin

    2017-06-15

    Systematic interrogation of tumor-infiltrating lymphocytes is key to the development of immunotherapies and the prediction of their clinical responses in cancers. Here, we perform deep single-cell RNA sequencing on 5,063 singlecells isolated from peripheral blood, tumor, and adjacent normal tissues from six hepatocellular carcinoma patients. The transcriptional profiles of these individual cells, coupled with assembled T cell receptor (TCR) sequences, enable us to identify 11 T cell subsets based on their molecular and functional properties and delineate their developmental trajectory. Specific subsets such as exhausted CD8 + T cells and Tregs are preferentially enriched and potentially clonally expanded in hepatocellular carcinoma (HCC), and we identified signature genes for each subset. One of the genes, layilin, is upregulated on activated CD8 + T cells and Tregs and represses the CD8 + T cell functions in vitro. This compendium of transcriptome data provides valuable insights and a rich resource for understanding the immune landscape in cancers. Copyright © 2017 Elsevier Inc. All rights reserved.

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

    Science.gov (United States)

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

    2013-10-03

    Stem cell differentiation pathways are most often studied at the population level, whereas critical decisions are executed at the level of single cells. We have established a highly multiplexed, quantitative PCR assay to profile in an unbiased manner a panel of all commonly used cell surface markers (280 genes) from individual cells. With this method, we analyzed over 1,500 single cells throughout the mouse hematopoietic system and illustrate its utility for revealing important biological insights. The comprehensive single cell data set permits mapping of the mouse hematopoietic stem cell differentiation hierarchy by computational lineage progression analysis. Further profiling of 180 intracellular regulators enabled construction of a genetic network to assign the earliest differentiation event during hematopoietic lineage specification. Analysis of acute myeloid leukemia elicited by MLL-AF9 uncovered a distinct cellular hierarchy containing two independent self-renewing lineages with different clonal activities. The strategy has broad applicability in other cellular systems. Copyright © 2013 Elsevier Inc. All rights reserved.

  9. Study on the micro direct ethanol fuel cell (Micro-DEFC) performance

    Science.gov (United States)

    Saisirirat, Penyarat; Joommanee, Bordindech

    2018-01-01

    The direct ethanol fuel cell (DEFC) is selected for this research. DEFC uses ethanol in the fuel cell instead of the more toxic methanol. Ethanol is more attractive than methanol by many reasons. Ethanol is a hydrogen-rich liquid and it has a higher specific energy (8.0 kWh/kg) compared to that of methanol (6.1 kWh/kg). Ethanol can be obtained in great quantity from biomass through a fermentation process from renewable resources such as sugar cane, wheat, corn, and even straw. The use of ethanol would also overcome both the storage and infrastructure challenge of hydrogen for fuel cell applications. The experimental apparatus on the micro direct ethanol fuel cell for measuring the cell performance has been set for this research. The objective is to study the micro direct ethanol fuel cell performance for applying with the portable electronic devices. The cell performance is specified in the terms of cell voltage, cell current and power of the cell at room operating temperature and 1 atm for the pressure and also includes the ethanol fuel consumption. The effect of operating temperature change on the electrical production performance is also studied. The steady-state time for collecting each data value is about 5-10 minutes. The results show that with the increase of concentrations of ethanol by volume, the reactant concentration at the reaction sites increases so the electrochemical rate also increases but when it reaches the saturated point the performance gradually drops.

  10. Tools for Genomic and Transcriptomic Analysis of Microbes at Single-Cell Level

    Directory of Open Access Journals (Sweden)

    Zixi Chen

    2017-09-01

    Full Text Available Microbiologists traditionally study population rather than individual cells, as it is generally assumed that the status of individual cells will be similar to that observed in the population. However, the recent studies have shown that the individual behavior of each single cell could be quite different from that of the whole population, suggesting the importance of extending traditional microbiology studies to single-cell level. With recent technological advances, such as flow cytometry, next-generation sequencing (NGS, and microspectroscopy, single-cell microbiology has greatly enhanced the understanding of individuality and heterogeneity of microbes in many biological systems. Notably, the application of multiple ‘omics’ in single-cell analysis has shed light on how individual cells perceive, respond, and adapt to the environment, how heterogeneity arises under external stress and finally determines the fate of the whole population, and how microbes survive under natural conditions. As single-cell analysis involves no axenic cultivation of target microorganism, it has also been demonstrated as a valuable tool for dissecting the microbial ‘dark matter.’ In this review, current state-of-the-art tools and methods for genomic and transcriptomic analysis of microbes at single-cell level were critically summarized, including single-cell isolation methods and experimental strategies of single-cell analysis with NGS. In addition, perspectives on the future trends of technology development in the field of single-cell analysis was also presented.

  11. Quantitative high-resolution genomic analysis of single cancer cells.

    Science.gov (United States)

    Hannemann, Juliane; Meyer-Staeckling, Sönke; Kemming, Dirk; Alpers, Iris; Joosse, Simon A; Pospisil, Heike; Kurtz, Stefan; Görndt, Jennifer; Püschel, Klaus; Riethdorf, Sabine; Pantel, Klaus; Brandt, Burkhard

    2011-01-01

    During cancer progression, specific genomic aberrations arise that can determine the scope of the disease and can be used as predictive or prognostic markers. The detection of specific gene amplifications or deletions in single blood-borne or disseminated tumour cells that may give rise to the development of metastases is of great clinical interest but technically challenging. In this study, we present a method for quantitative high-resolution genomic analysis of single cells. Cells were isolated under permanent microscopic control followed by high-fidelity whole genome amplification and subsequent analyses by fine tiling array-CGH and qPCR. The assay was applied to single breast cancer cells to analyze the chromosomal region centred by the therapeutical relevant EGFR gene. This method allows precise quantitative analysis of copy number variations in single cell diagnostics.

  12. Performance evaluation of thermally treated graphite felt electrodes for vanadium redox flow battery and their four-point single cell characterization

    Science.gov (United States)

    Mazúr, P.; Mrlík, J.; Beneš, J.; Pocedič, J.; Vrána, J.; Dundálek, J.; Kosek, J.

    2018-03-01

    In our contribution we study the electrocatalytic effect of oxygen functionalization of thermally treated graphite felt on kinetics of electrode reactions of vanadium redox flow battery. Chemical and morphological changes of the felts are analysed by standard physico-chemical characterization techniques. A complex method four-point method is developed and employed for characterization of the felts in a laboratory single-cell. The method is based on electrochemical impedance spectroscopy and load curves measurements of positive and negative half-cells using platinum wire pseudo-reference electrodes. The distribution of ohmic and faradaic losses within a single-cell is evaluated for both symmetric and asymmetric electrode set-up with respect to the treatment conditions. Positive effect of oxygen functionalization is observed only for negative electrode, whereas kinetics of positive electrode reaction is almost unaffected by the treatment. This is in a contradiction to the results of typically employed cyclovoltammetric characterization which indicate that both electrodes are enhanced by the treatment to a similar extent. The developed four-point characterization method can be further used e.g., for the component screening and in-situ durability studies on single-cell scale redox flow batteries of various chemistries.

  13. Single-cell analysis by ICP-MS/MS as a fast tool for cellular bioavailability studies of arsenite.

    Science.gov (United States)

    Meyer, S; López-Serrano, A; Mitze, H; Jakubowski, N; Schwerdtle, T

    2018-01-24

    Single-cell inductively coupled plasma mass spectrometry (SC-ICP-MS) has become a powerful and fast tool to evaluate the elemental composition at a single-cell level. In this study, the cellular bioavailability of arsenite (incubation of 25 and 50 μM for 0-48 h) has been successfully assessed by SC-ICP-MS/MS for the first time directly after re-suspending the cells in water. This procedure avoids the normally arising cell membrane permeabilization caused by cell fixation methods (e.g. methanol fixation). The reliability and feasibility of this SC-ICP-MS/MS approach with a limit of detection of 0.35 fg per cell was validated by conventional bulk ICP-MS/MS analysis after cell digestion and parallel measurement of sulfur and phosphorus.

  14. High throughput single-cell and multiple-cell micro-encapsulation.

    Science.gov (United States)

    Lagus, Todd P; Edd, Jon F

    2012-06-15

    Microfluidic encapsulation methods have been previously utilized to capture cells in picoliter-scale aqueous, monodisperse drops, providing confinement from a bulk fluid environment with applications in high throughput screening, cytometry, and mass spectrometry. We describe a method to not only encapsulate single cells, but to repeatedly capture a set number of cells (here we demonstrate one- and two-cell encapsulation) to study both isolation and the interactions between cells in groups of controlled sizes. By combining drop generation techniques with cell and particle ordering, we demonstrate controlled encapsulation of cell-sized particles for efficient, continuous encapsulation. Using an aqueous particle suspension and immiscible fluorocarbon oil, we generate aqueous drops in oil with a flow focusing nozzle. The aqueous flow rate is sufficiently high to create ordering of particles which reach the nozzle at integer multiple frequencies of the drop generation frequency, encapsulating a controlled number of cells in each drop. For representative results, 9.9 μm polystyrene particles are used as cell surrogates. This study shows a single-particle encapsulation efficiency P(k=1) of 83.7% and a double-particle encapsulation efficiency P(k=2) of 79.5% as compared to their respective Poisson efficiencies of 39.3% and 33.3%, respectively. The effect of consistent cell and particle concentration is demonstrated to be of major importance for efficient encapsulation, and dripping to jetting transitions are also addressed. Continuous media aqueous cell suspensions share a common fluid environment which allows cells to interact in parallel and also homogenizes the effects of specific cells in measurements from the media. High-throughput encapsulation of cells into picoliter-scale drops confines the samples to protect drops from cross-contamination, enable a measure of cellular diversity within samples, prevent dilution of reagents and expressed biomarkers, and amplify

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

    Science.gov (United States)

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

    2016-09-01

    Single-cell biology is considered a new approach to identify and validate disease-specific biomarkers. However, the concern raised by clinicians is how to apply single-cell measurements for clinical practice, translate the message of single-cell systems biology into clinical phenotype or explain alterations of single-cell gene sequencing and function in patient response to therapies. This study is to address the importance and necessity of single-cell gene sequencing in the identification and development of disease-specific biomarkers, the definition and significance of single-cell biology and single-cell systems biology in the understanding of single-cell full picture, the development and establishment of whole-cell models in the validation of targeted biological function and the figure and meaning of single-molecule imaging in single cell to trace intra-single-cell molecule expression, signal, interaction and location. We headline the important role of single-cell biology in the discovery and development of disease-specific biomarkers with a special emphasis on understanding single-cell biological functions, e.g. mechanical phenotypes, single-cell biology, heterogeneity and organization of genome function. We have reason to believe that such multi-dimensional, multi-layer, multi-crossing and stereoscopic single-cell biology definitely benefits the discovery and development of disease-specific biomarkers. © 2016 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

  16. Experimental study on the effect of cathode flow humidity and temperature on the performance of PEM fuel cell

    Energy Technology Data Exchange (ETDEWEB)

    El-Emam, R.S.; Awad, M.M.; Hamed, A.M.; Tolba, M. [Mansoura Univ., Mansoura (Egypt). Dept. of Mechanical Engineering

    2009-07-01

    The fuel cell is an electrochemical energy conversion device that produces electricity directly from chemical energy, and the by-products are only water and heat. The fuel cell could provide a solution to a whole range of environmental challenges, such as global warming and harmful levels of local pollutants. One of the most promising alternative power generation methods is the proton exchange membrane fuel cell (PEMFC) because of its low operating temperature, relative tolerance for impurities, and high power-density. This paper presented an experimental study on the performance characteristics of a single unit of a PEMFC with an active area of 25 square centimetres using two different cell configurations. The test system was designed to control the temperature and the relative humidity of the cathode feeding gas. Oxygen and air were used as oxidizers, while dry hydrogen was the cell fuel. Two different cell configurations were assembled and integrated into the test stand. The paper described the experimental work and presented the results of the study. It was concluded that low oxygen relative humidity with the dry hydrogen caused membrane drying and ultimately resulted in a degradation of fuel cell power output and cell performance. 17 refs., 17 figs.

  17. Identifying EGFR-Expressed Cells and Detecting EGFR Multi-Mutations at Single-Cell Level by Microfluidic Chip

    Science.gov (United States)

    Li, Ren; Zhou, Mingxing; Li, Jine; Wang, Zihua; Zhang, Weikai; Yue, Chunyan; Ma, Yan; Peng, Hailin; Wei, Zewen; Hu, Zhiyuan

    2018-03-01

    EGFR mutations companion diagnostics have been proved to be crucial for the efficacy of tyrosine kinase inhibitor targeted cancer therapies. To uncover multiple mutations occurred in minority of EGFR-mutated cells, which may be covered by the noises from majority of un-mutated cells, is currently becoming an urgent clinical requirement. Here we present the validation of a microfluidic-chip-based method for detecting EGFR multi-mutations at single-cell level. By trapping and immunofluorescently imaging single cells in specifically designed silicon microwells, the EGFR-expressed cells were easily identified. By in situ lysing single cells, the cell lysates of EGFR-expressed cells were retrieved without cross-contamination. Benefited from excluding the noise from cells without EGFR expression, the simple and cost-effective Sanger's sequencing, but not the expensive deep sequencing of the whole cell population, was used to discover multi-mutations. We verified the new method with precisely discovering three most important EGFR drug-related mutations from a sample in which EGFR-mutated cells only account for a small percentage of whole cell population. The microfluidic chip is capable of discovering not only the existence of specific EGFR multi-mutations, but also other valuable single-cell-level information: on which specific cells the mutations occurred, or whether different mutations coexist on the same cells. This microfluidic chip constitutes a promising method to promote simple and cost-effective Sanger's sequencing to be a routine test before performing targeted cancer therapy.[Figure not available: see fulltext.

  18. New frontiers in single-cell analysis

    OpenAIRE

    Templer, Richard H.; Ces, Oscar

    2008-01-01

    For this special issue of J. R. Soc. Interface we present an overview of the driving forces behind technological advances in the field of single-cell analysis. These range from increasing our understanding of cellular heterogeneity through to the study of rare cells, areas of research that cannot be tackled effectively using current high-throughput population-based averaging techniques.

  19. RoboSCell: An automated single cell arraying and analysis instrument

    KAUST Repository

    Sakaki, Kelly

    2009-09-09

    Single cell research has the potential to revolutionize experimental methods in biomedical sciences and contribute to clinical practices. Recent studies suggest analysis of single cells reveals novel features of intracellular processes, cell-to-cell interactions and cell structure. The methods of single cell analysis require mechanical resolution and accuracy that is not possible using conventional techniques. Robotic instruments and novel microdevices can achieve higher throughput and repeatability; however, the development of such instrumentation is a formidable task. A void exists in the state-of-the-art for automated analysis of single cells. With the increase in interest in single cell analyses in stem cell and cancer research the ability to facilitate higher throughput and repeatable procedures is necessary. In this paper, a high-throughput, single cell microarray-based robotic instrument, called the RoboSCell, is described. The proposed instrument employs a partially transparent single cell microarray (SCM) integrated with a robotic biomanipulator for in vitro analyses of live single cells trapped at the array sites. Cells, labeled with immunomagnetic particles, are captured at the array sites by channeling magnetic fields through encapsulated permalloy channels in the SCM. The RoboSCell is capable of systematically scanning the captured cells temporarily immobilized at the array sites and using optical methods to repeatedly measure extracellular and intracellular characteristics over time. The instrument\\'s capabilities are demonstrated by arraying human T lymphocytes and measuring the uptake dynamics of calcein acetoxymethylester-all in a fully automated fashion. © 2009 Springer Science+Business Media, LLC.

  20. Micro-magnet arrays for specific single bacterial cell positioning

    Energy Technology Data Exchange (ETDEWEB)

    Pivetal, Jérémy, E-mail: jeremy.piv@netcmail.com [Ecole Centrale de Lyon, CNRS UMR 5005, Laboratoire Ampère, F-69134 Écully (France); Royet, David [Ecole Centrale de Lyon, CNRS UMR 5005, Laboratoire Ampère, F-69134 Écully (France); Ciuta, Georgeta [Univ. Grenoble Alpes, Inst NEEL, F-38042 Grenoble (France); CNRS, Inst NEEL, F-38042 Grenoble (France); Frenea-Robin, Marie [Université de Lyon, Université Lyon 1, CNRS UMR 5005, Laboratoire Ampère, F-69622 Villeurbanne (France); Haddour, Naoufel [Ecole Centrale de Lyon, CNRS UMR 5005, Laboratoire Ampère, F-69134 Écully (France); Dempsey, Nora M. [Univ. Grenoble Alpes, Inst NEEL, F-38042 Grenoble (France); CNRS, Inst NEEL, F-38042 Grenoble (France); Dumas-Bouchiat, Frédéric [Univ Limoges, CNRS, SPCTS UMR 7513, 12 Rue Atlantis, F-87068 Limoges (France); Simonet, Pascal [Ecole Centrale de Lyon, CNRS UMR 5005, Laboratoire Ampère, F-69134 Écully (France)

    2015-04-15

    In various contexts such as pathogen detection or analysis of microbial diversity where cellular heterogeneity must be taken into account, there is a growing need for tools and methods that enable microbiologists to analyze bacterial cells individually. One of the main challenges in the development of new platforms for single cell studies is to perform precise cell positioning, but the ability to specifically target cells is also important in many applications. In this work, we report the development of new strategies to selectively trap single bacterial cells upon large arrays, based on the use of micro-magnets. Escherichia coli bacteria were used to demonstrate magnetically driven bacterial cell organization. In order to provide a flexible approach adaptable to several applications in the field of microbiology, cells were magnetically and specifically labeled using two different strategies, namely immunomagnetic labeling and magnetic in situ hybridization. Results show that centimeter-sized arrays of targeted, isolated bacteria can be successfully created upon the surface of a flat magnetically patterned hard magnetic film. Efforts are now being directed towards the integration of a detection tool to provide a complete micro-system device for a variety of microbiological applications. - Highlights: 1.We report a new approach to selectively micropattern bacterial cells individually upon micro-magnet arrays. 2.Permanent micro-magnets of a size approaching that of bacteria could be fabricated using a Thermo-Magnetic Patterning process. 3.Bacterial cells were labeled using two different magnetic labeling strategies providing flexible approach adaptable to several applications in the field of microbiology.

  1. Quantitative high-resolution genomic analysis of single cancer cells.

    Directory of Open Access Journals (Sweden)

    Juliane Hannemann

    Full Text Available During cancer progression, specific genomic aberrations arise that can determine the scope of the disease and can be used as predictive or prognostic markers. The detection of specific gene amplifications or deletions in single blood-borne or disseminated tumour cells that may give rise to the development of metastases is of great clinical interest but technically challenging. In this study, we present a method for quantitative high-resolution genomic analysis of single cells. Cells were isolated under permanent microscopic control followed by high-fidelity whole genome amplification and subsequent analyses by fine tiling array-CGH and qPCR. The assay was applied to single breast cancer cells to analyze the chromosomal region centred by the therapeutical relevant EGFR gene. This method allows precise quantitative analysis of copy number variations in single cell diagnostics.

  2. Microbeam evolution: From single cell irradiation to preclinical studies

    DEFF Research Database (Denmark)

    Ghita, Mihaela; Fernandez-Palomo, Cristian; Fukunaga, Hisanori

    2018-01-01

    Purpose: This review follows the development of microbeam technology from the early days of single cell irradiations, to investigations of specific cellular mechanisms and to the development of new treatment modalities in vivo. A number of microbeam applications are discussed with a focus on prec...... to deliver radiotherapy using plane parallel microbeams, in Microbeam Radiotherapy (MRT)....

  3. Single-cell Hi-C for genome-wide detection of chromatin interactions that occur simultaneously in a single cell.

    Science.gov (United States)

    Nagano, Takashi; Lubling, Yaniv; Yaffe, Eitan; Wingett, Steven W; Dean, Wendy; Tanay, Amos; Fraser, Peter

    2015-12-01

    Hi-C is a powerful method that provides pairwise information on genomic regions in spatial proximity in the nucleus. Hi-C requires millions of cells as input and, as genome organization varies from cell to cell, a limitation of Hi-C is that it only provides a population average of genome conformations. We developed single-cell Hi-C to create snapshots of thousands of chromatin interactions that occur simultaneously in a single cell. To adapt Hi-C to single-cell analysis, we modified the protocol to include in-nucleus ligation. This enables the isolation of single nuclei carrying Hi-C-ligated DNA into separate tubes, followed by reversal of cross-links, capture of biotinylated ligation junctions on streptavidin-coated magnetic beads and PCR amplification of single-cell Hi-C libraries. The entire laboratory protocol can be carried out in 1 week, and although we have demonstrated its use in mouse T helper (TH1) cells, it should be applicable to any cell type or species for which standard Hi-C has been successful. We also developed an analysis pipeline to filter noise and assess the quality of data sets in a few hours. Although the interactome maps produced by single-cell Hi-C are sparse, the data provide useful information to understand cellular variability in nuclear genome organization and chromosome structure. Standard wet and dry laboratory skills in molecular biology and computational analysis are required.

  4. Nanoliter reactors improve multiple displacement amplification of genomes from single cells.

    Directory of Open Access Journals (Sweden)

    Yann Marcy

    2007-09-01

    Full Text Available Since only a small fraction of environmental bacteria are amenable to laboratory culture, there is great interest in genomic sequencing directly from single cells. Sufficient DNA for sequencing can be obtained from one cell by the Multiple Displacement Amplification (MDA method, thereby eliminating the need to develop culture methods. Here we used a microfluidic device to isolate individual Escherichia coli and amplify genomic DNA by MDA in 60-nl reactions. Our results confirm a report that reduced MDA reaction volume lowers nonspecific synthesis that can result from contaminant DNA templates and unfavourable interaction between primers. The quality of the genome amplification was assessed by qPCR and compared favourably to single-cell amplifications performed in standard 50-microl volumes. Amplification bias was greatly reduced in nanoliter volumes, thereby providing a more even representation of all sequences. Single-cell amplicons from both microliter and nanoliter volumes provided high-quality sequence data by high-throughput pyrosequencing, thereby demonstrating a straightforward route to sequencing genomes from single cells.

  5. Single cell transcriptomics of neighboring hyphae of Aspergillus niger

    Science.gov (United States)

    2011-01-01

    Single cell profiling was performed to assess differences in RNA accumulation in neighboring hyphae of the fungus Aspergillus niger. A protocol was developed to isolate and amplify RNA from single hyphae or parts thereof. Microarray analysis resulted in a present call for 4 to 7% of the A. niger genes, of which 12% showed heterogeneous RNA levels. These genes belonged to a wide range of gene categories. PMID:21816052

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

    OpenAIRE

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

    2015-01-01

    Oxygen is transported throughout the body by hemoglobin in red blood cells. While the oxygen affinity of blood is well understood and is routinely assessed in patients by pulse oximetry, variability at the single-cell level has not been previously measured. In contrast, single-cell measurements of red blood cell volume and hemoglobin concentration are taken millions of times per day by clinical hematology analyzers and are important factors in determining the health of the hematologic system....

  7. Effects of coal-derived trace species on performance of molten carbonate fuel cells

    Energy Technology Data Exchange (ETDEWEB)

    1992-05-01

    The Carbonate Fuel Cell is a very promising option for highly efficient generation of electricity from many fuels. If coal-gas is to be used, the interactions of coal-derived impurities on various fuel cell components need to be understood. Thus the effects on Carbonate Fuel Cell performance due to ten different coal-derived contaminants viz., NH{sub 3}, H{sub 2}S, HC{ell}, H{sub 2}Se, AsH{sub 3}, Zn, Pb, Cd, Sn, and Hg, have been studied at Energy Research Corporation. Both experimental and theoretical evaluations were performed, which have led to mechanistic insights and initial estimation of qualitative tolerance levels for each species individually and in combination with other species. The focus of this study was to investigate possible coal-gas contaminant effects on the anode side of the Carbonate Fuel Cell, using both out-of-cell thermogravimetric analysis by isothermal TGA, and fuel cell testing in bench-scale cells. Separate experiments detailing performance decay in these cells with high levels of ammonia contamination (1 vol %) and with trace levels of Cd, Hg, and Sn, have indicated that, on the whole, these elements do not affect carbonate fuel cell performance. However, some performance decay may result when a number of the other six species are present, singly or simultaneously, as contaminants in fuel gas. In all cases, tolerance levels have been estimated for each of the 10 species and preliminary models have been developed for six of them. At this stage the models are limited to isothermal, benchscale (300 cm{sup 2} size) single cells. The information obtained is expected to assist in the development of coal-gas cleanup systems, while the contaminant performance effects data will provide useful basic information for modeling fuel cell endurance in conjunction with integrated gasifier/fuel-cell systems (IGFC).

  8. Seeding of single hemopoietic stem cells and self renewal of committed stem cells

    International Nuclear Information System (INIS)

    Brecher, G.

    1986-01-01

    Single cells and two to five proliferating cells were transfused into mice whose own stem cells had been killed by irradiation. When a small inoculum of 50,000 AB marrow cells was given only 4 of 20 recipients survived, but all 4 had only PGK A enzyme in their peripheral blood cells. The results indicate that the survivors received a single pluripotential stem cell capable of proliferating. Survivors showed no deterioration in their blood picture after many months. It was concluded that there is no clonal succession in the marrow cells. Further studies with transfusions of 100,000 and 10,000,000 marrow cells after lethal irradiation suggest that there is production of committed stem cells with significant self-renewal

  9. Compare analysis for the nanotoxicity effects of different amounts of endocytic iron oxide nanoparticles at single cell level.

    Science.gov (United States)

    Huang, Chen-Yu; Ger, Tzong-Rong; Wei, Zung-Hang; Lai, Mei-Feng

    2014-01-01

    Developing methods that evaluate the cellular uptake of magnetic nanoparticles (MNPs) and nanotoxicity effects at single-cellular level are needed. In this study, magnetophoresis combining fluorescence based cytotoxicity assay was proposed to assess the viability and the single-cellular MNPs uptake simultaneously. Malignant cells (SKHep-1, HepG2, HeLa) were incubated with 10 nm anionic iron oxide nanoparticles. Prussian blue stain was performed to visualize the distribution of magnetic nanoparticles. MTT and fluorescence based assay analyzed the cytotoxicity effects of the bulk cell population and single cell, respectively. DAPI/PI stained was applied to evaluate death mechanism. The number of intracellular MNPs was found to be strongly correlated with the cell death. Significant differences between cellular MNP uptake in living and dead cells were observed. The method could be useful for future study of the nanotoxicity induced by MNPs.

  10. Labeling of mesenchymal stem cells for MRI with single-cell sensitivity

    Directory of Open Access Journals (Sweden)

    Ariza de Schellenberger A

    2016-04-01

    -uptake-dependent biocompatibility studies and cell detection by MRI and future MPI. Additionally, using a 7 T MR imager equipped with a cryocoil resulted in approximately two times higher detection. In conclusion, we established labeling conditions for new high-relaxivity MCP, VSOP, and Resovist® for improved MRI of MSC with single-cell sensitivity. Keywords: magnetic field microdistortions, single-cell imaging, mesenchymal stem cells, VSOP, MCP, Resovist®

  11. Study of molasses / vinasse waste ratio for single cell protein and total microorganisms

    Directory of Open Access Journals (Sweden)

    Marcia Luciana Cazetta

    2006-02-01

    Full Text Available Different molasses/ vinasse ratio were used as substrate to investigate single cell protein and total lipids production by five microorganisms: four yeasts strains: Candida lipolytica, Rhodotorula mucilaginosa, Saccharomyces cerevisiae, a yeast isolated from vinasse lake (denominated LLV98 and a bacterium strain, Corynebacterium glutamicum. The media utilized were: a 50% molasses and 50% vinasse; b 25% molasses and 75% vinasse and c 75% molasses and 25% vinasse. The objective of this work was to study the growth of microorganisms and also evaluate protein and lipids content in the biomass obtained from these by-products. The highest single cell protein production was obtained by S. cerevisiae, 50.35%, followed by R. mucilaginosa, 41.96%. The lowest productions were obtained by C. glutamicum. The higher total lipids productions, more than 26%, were founded in molasses plus vinasse at 50%/50% by S. cerevisiae and C. glutamicum.

  12. Advancing haematopoietic stem and progenitor cell biology through single-cell profiling

    OpenAIRE

    Hamey, Fiona; Nestorowa, Sonia; Wilson, Nicola Kaye; Göttgens, Berthold

    2016-01-01

    Haematopoietic stem and progenitor cells (HSPCs) sit at the top of the haematopoietic hierarchy, and their fate choices need to be carefully controlled to ensure balanced production of all mature blood cell types. As cell fate decisions are made at the level of the individual cells, recent technological advances in measuring gene and protein expression in increasingly large numbers of single cells have been rapidly adopted to study both normal and pathological HSPC function. In this review we...

  13. Reliable single cell array CGH for clinical samples.

    Directory of Open Access Journals (Sweden)

    Zbigniew T Czyż

    Full Text Available BACKGROUND: Disseminated cancer cells (DCCs and circulating tumor cells (CTCs are extremely rare, but comprise the precursors cells of distant metastases or therapy resistant cells. The detailed molecular analysis of these cells may help to identify key events of cancer cell dissemination, metastatic colony formation and systemic therapy escape. METHODOLOGY/PRINCIPAL FINDINGS: Using the Ampli1™ whole genome amplification (WGA technology and high-resolution oligonucleotide aCGH microarrays we optimized conditions for the analysis of structural copy number changes. The protocol presented here enables reliable detection of numerical genomic alterations as small as 0.1 Mb in a single cell. Analysis of single cells from well-characterized cell lines and single normal cells confirmed the stringent quantitative nature of the amplification and hybridization protocol. Importantly, fixation and staining procedures used to detect DCCs showed no significant impact on the outcome of the analysis, proving the clinical usability of our method. In a proof-of-principle study we tracked the chromosomal changes of single DCCs over a full course of high-dose chemotherapy treatment by isolating and analyzing DCCs of an individual breast cancer patient at four different time points. CONCLUSIONS/SIGNIFICANCE: The protocol enables detailed genome analysis of DCCs and thereby assessment of the clonal evolution during the natural course of the disease and under selection pressures. The results from an exemplary patient provide evidence that DCCs surviving selective therapeutic conditions may be recruited from a pool of genomically less advanced cells, which display a stable subset of specific genomic alterations.

  14. Single cell enzyme diagnosis on the chip

    DEFF Research Database (Denmark)

    Jensen, Sissel Juul; Harmsen, Charlotte; Nielsen, Mette Juul

    2013-01-01

    Conventional diagnosis based on ensemble measurements often overlooks the variation among cells. Here, we present a droplet-microfluidics based platform to investigate single cell activities. Adopting a previously developed isothermal rolling circle amplification-based assay, we demonstrate...... detection of enzymatic activities down to the single cell level with small quantities of biological samples, which outcompetes existing techniques. Such a system, capable of resolving single cell activities, will ultimately have clinical applications in diagnosis, prediction of drug response and treatment...

  15. Single-cell intracellular nano-pH probes†

    OpenAIRE

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

    2015-01-01

    Within a large clonal population, such as cancerous tumor entities, cells are not identical, and the differences between intracellular pH levels of individual cells may be important indicators of heterogeneity that could be relevant in clinical practice, especially in personalized medicine. Therefore, the detection of the intracellular pH at the single-cell level is of great importance to identify and study outlier cells. However, quantitative and real-time measurements of the intracellular p...

  16. Impact of NBTI Aging on the Single-Event Upset of SRAM Cells

    CERN Document Server

    Bagatin, M; Gerardin, Simone; Paccagnella, Alessandro; Bagatin, Marta

    2010-01-01

    We analyzed the impact of negative bias temperature instability (NBTI) on the single-event upset rate of SRAM cells through experiments and SPICE simulations. We performed critical charge simulations introducing different degradation patterns in the cells, in three technology nodes, from 180 to 90 nm. The simulations results were checked with alpha-particle and heavy-ion irradiations on a 130-nm technology. Both simulations and experimental results show that NBTI degradation does not significantly affect the single-event upset SRAM cell rate as long as the parametric drift induced by aging is within 10\\%.

  17. Comparison of reverse transcription-quantitative polymerase chain reaction methods and platforms for single cell gene expression analysis.

    Science.gov (United States)

    Fox, Bridget C; Devonshire, Alison S; Baradez, Marc-Olivier; Marshall, Damian; Foy, Carole A

    2012-08-15

    Single cell gene expression analysis can provide insights into development and disease progression by profiling individual cellular responses as opposed to reporting the global average of a population. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is the "gold standard" for the quantification of gene expression levels; however, the technical performance of kits and platforms aimed at single cell analysis has not been fully defined in terms of sensitivity and assay comparability. We compared three kits using purification columns (PicoPure) or direct lysis (CellsDirect and Cells-to-CT) combined with a one- or two-step RT-qPCR approach using dilutions of cells and RNA standards to the single cell level. Single cell-level messenger RNA (mRNA) analysis was possible using all three methods, although the precision, linearity, and effect of lysis buffer and cell background differed depending on the approach used. The impact of using a microfluidic qPCR platform versus a standard instrument was investigated for potential variability introduced by preamplification of template or scaling down of the qPCR to nanoliter volumes using laser-dissected single cell samples. The two approaches were found to be comparable. These studies show that accurate gene expression analysis is achievable at the single cell level and highlight the importance of well-validated experimental procedures for low-level mRNA analysis. Copyright © 2012 Elsevier Inc. All rights reserved.

  18. Photocleavable DNA Barcoding Antibodies for Multiplexed Protein Analysis in Single Cells.

    Science.gov (United States)

    Ullal, Adeeti V; Weissleder, Ralph

    2015-01-01

    We describe a DNA-barcoded antibody sensing technique for single cell protein analysis in which the barcodes are photocleaved and digitally detected without amplification steps (Ullal et al., Sci Transl Med 6:219, 2014). After photocleaving the unique ~70 mer DNA barcodes we use a fluorescent hybridization technology for detection, similar to what is commonly done for nucleic acid readouts. This protocol offers a simple method for multiplexed protein detection using 100+ antibodies and can be performed on clinical samples as well as single cells.

  19. Dielectrophoretic capture and genetic analysis of single neuroblastoma tumor cells

    Directory of Open Access Journals (Sweden)

    Erica L Carpenter

    2014-07-01

    Full Text Available Our understanding of the diversity of cells that escape the primary tumor and seed micrometastases remains rudimentary, and approaches for studying circulating and disseminated tumor cells have been limited by low throughput and sensitivity, reliance on single parameter sorting, and a focus on enumeration rather than phenotypic and genetic characterization. Here we utilize a highly sensitive microfluidic and dielectrophoretic approach for the isolation and genetic analysis of individual tumor cells. We employed fluorescence labeling to isolate 208 single cells from spiking experiments conducted with 11 cell lines, including 8 neuroblastoma cell lines, and achieved a capture sensitivity of 1 tumor cell per 106 white blood cells. Sample fixation or freezing had no detectable effect on cell capture. Point mutations were accurately detected in the whole genome amplification product of captured single tumor cells but not in negative control white blood cells. We applied this approach to capture 144 single tumor cells from 10 bone marrow samples from patients suffering from neuroblastoma. In this pediatric malignancy, high-risk patients often exhibit wide-spread hematogenous metastasis, but access to primary tumor can be difficult or impossible. Here we used flow-based sorting to pre-enrich samples with tumor involvement below 0.02%. For all patients for whom a mutation in the Anaplastic Lymphoma Kinase gene had already been detected in their primary tumor, the same mutation was detected in single cells from their marrow. These findings demonstrate a novel, non-invasive, and adaptable method for the capture and genetic analysis of single tumor cells from cancer patients.

  20. Microfluidic device for continuous single cells analysis via Raman spectroscopy enhanced by integrated plasmonic nanodimers

    KAUST Repository

    Perozziello, Gerardo

    2015-12-11

    In this work a Raman flow cytometer is presented. It consists of a microfluidic device that takes advantages of the basic principles of Raman spectroscopy and flow cytometry. The microfluidic device integrates calibrated microfluidic channels- where the cells can flow one-by-one -, allowing single cell Raman analysis. The microfluidic channel integrates plasmonic nanodimers in a fluidic trapping region. In this way it is possible to perform Enhanced Raman Spectroscopy on single cell. These allow a label-free analysis, providing information about the biochemical content of membrane and cytoplasm of the each cell. Experiments are performed on red blood cells (RBCs), peripheral blood lymphocytes (PBLs) and myelogenous leukemia tumor cells (K562). © 2015 Optical Society of America.

  1. Single-cell mRNA transfection studies: delivery, kinetics and statistics by numbers.

    Science.gov (United States)

    Leonhardt, Carolin; Schwake, Gerlinde; Stögbauer, Tobias R; Rappl, Susanne; Kuhr, Jan-Timm; Ligon, Thomas S; Rädler, Joachim O

    2014-05-01

    In artificial gene delivery, messenger RNA (mRNA) is an attractive alternative to plasmid DNA (pDNA) since it does not require transfer into the cell nucleus. Here we show that, unlike for pDNA transfection, the delivery statistics and dynamics of mRNA-mediated expression are generic and predictable in terms of mathematical modeling. We measured the single-cell expression time-courses and levels of enhanced green fluorescent protein (eGFP) using time-lapse microscopy and flow cytometry (FC). The single-cell analysis provides direct access to the distribution of onset times, life times and expression rates of mRNA and eGFP. We introduce a two-step stochastic delivery model that reproduces the number distribution of successfully delivered and translated mRNA molecules and thereby the dose-response relation. Our results establish a statistical framework for mRNA transfection and as such should advance the development of RNA carriers and small interfering/micro RNA-based drugs. This team of authors established a statistical framework for mRNA transfection by using a two-step stochastic delivery model that reproduces the number distribution of successfully delivered and translated mRNA molecules and thereby their dose-response relation. This study establishes a nice connection between theory and experimental planning and will aid the cellular delivery of mRNA molecules. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

  2. Cell-substrate impedance fluctuations of single amoeboid cells encode cell-shape and adhesion dynamics.

    Science.gov (United States)

    Leonhardt, Helmar; Gerhardt, Matthias; Höppner, Nadine; Krüger, Kirsten; Tarantola, Marco; Beta, Carsten

    2016-01-01

    We show systematic electrical impedance measurements of single motile cells on microelectrodes. Wild-type cells and mutant strains were studied that differ in their cell-substrate adhesion strength. We recorded the projected cell area by time-lapse microscopy and observed irregular oscillations of the cell shape. These oscillations were correlated with long-term variations in the impedance signal. Superposed to these long-term trends, we observed fluctuations in the impedance signal. Their magnitude clearly correlated with the adhesion strength, suggesting that strongly adherent cells display more dynamic cell-substrate interactions.

  3. Cell-substrate impedance fluctuations of single amoeboid cells encode cell-shape and adhesion dynamics

    Science.gov (United States)

    Leonhardt, Helmar; Gerhardt, Matthias; Höppner, Nadine; Krüger, Kirsten; Tarantola, Marco; Beta, Carsten

    2016-01-01

    We show systematic electrical impedance measurements of single motile cells on microelectrodes. Wild-type cells and mutant strains were studied that differ in their cell-substrate adhesion strength. We recorded the projected cell area by time-lapse microscopy and observed irregular oscillations of the cell shape. These oscillations were correlated with long-term variations in the impedance signal. Superposed to these long-term trends, we observed fluctuations in the impedance signal. Their magnitude clearly correlated with the adhesion strength, suggesting that strongly adherent cells display more dynamic cell-substrate interactions.

  4. Application of single-cell technology in cancer research.

    Science.gov (United States)

    Liang, Shao-Bo; Fu, Li-Wu

    2017-07-01

    In this review, we have outlined the application of single-cell technology in cancer research. Single-cell technology has made encouraging progress in recent years and now provides the means to detect rare cancer cells such as circulating tumor cells and cancer stem cells. We reveal how this technology has advanced the analysis of intratumor heterogeneity and tumor epigenetics, and guided individualized treatment strategies. The future prospects now are to bring single-cell technology into the clinical arena. We believe that the clinical application of single-cell technology will be beneficial in cancer diagnostics and treatment, and ultimately improve survival in cancer patients. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  5. Performance comparison of low-temperature direct alcohol fuel cells with different anode catalysts

    Science.gov (United States)

    Zhou, W. J.; Zhou, B.; Li, W. Z.; Zhou, Z. H.; Song, S. Q.; Sun, G. Q.; Xin, Q.; Douvartzides, S.; Goula, M.; Tsiakaras, P.

    Low-temperature polymer electrolyte membrane fuel cells directly fed by methanol and ethanol were investigated employing carbon supported Pt, PtSn and PtRu as anode catalysts, respectively. Employing Pt/C as anode catalyst, both direct methanol fuel cell (DMFC) and direct ethanol fuel cell (DEFC) showed poor performances even in presence of high Pt loading on anode. It was found that the addition of Ru or Sn to the Pt dramatically enhances the electro-oxidation of both methanol and ethanol. It was also found that the single cell adopting PtRu/C as anode shows better DMFC performance, while PtSn/C catalyst shows better DEFC performance. The single fuel cell using PtSn/C as anode catalyst at 90 °C shows similar power densities whenever fueled by methanol or ethanol. The cyclic voltammetry (CV) and single fuel cell tests indicated that PtRu is more suitable for DMFC while PtSn is more suitable for DEFC.

  6. A robust method to analyze copy number alterations of less than 100 kb in single cells using oligonucleotide array CGH.

    Directory of Open Access Journals (Sweden)

    Birte Möhlendick

    Full Text Available Comprehensive genome wide analyses of single cells became increasingly important in cancer research, but remain to be a technically challenging task. Here, we provide a protocol for array comparative genomic hybridization (aCGH of single cells. The protocol is based on an established adapter-linker PCR (WGAM and allowed us to detect copy number alterations as small as 56 kb in single cells. In addition we report on factors influencing the success of single cell aCGH downstream of the amplification method, including the characteristics of the reference DNA, the labeling technique, the amount of input DNA, reamplification, the aCGH resolution, and data analysis. In comparison with two other commercially available non-linear single cell amplification methods, WGAM showed a very good performance in aCGH experiments. Finally, we demonstrate that cancer cells that were processed and identified by the CellSearch® System and that were subsequently isolated from the CellSearch® cartridge as single cells by fluorescence activated cell sorting (FACS could be successfully analyzed using our WGAM-aCGH protocol. We believe that even in the era of next-generation sequencing, our single cell aCGH protocol will be a useful and (cost- effective approach to study copy number alterations in single cells at resolution comparable to those reported currently for single cell digital karyotyping based on next generation sequencing data.

  7. Automated Single Cell Data Decontamination Pipeline

    Energy Technology Data Exchange (ETDEWEB)

    Tennessen, Kristin [Lawrence Berkeley National Lab. (LBNL), Walnut Creek, CA (United States). Dept. of Energy Joint Genome Inst.; Pati, Amrita [Lawrence Berkeley National Lab. (LBNL), Walnut Creek, CA (United States). Dept. of Energy Joint Genome Inst.

    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

  8. Single cell metabolomics

    NARCIS (Netherlands)

    Heinemann, Matthias; Zenobi, Renato

    Recent discoveries suggest that cells of a clonal population often display multiple metabolic phenotypes at the same time. Motivated by the success of mass spectrometry (MS) in the investigation of population-level metabolomics, the analytical community has initiated efforts towards MS-based single

  9. A Microchip for Integrated Single-Cell Gene Expression Profiling and Genotoxicity Detection

    Directory of Open Access Journals (Sweden)

    Hui Dong

    2016-09-01

    Full Text Available Microfluidics-based single-cell study is an emerging approach in personalized treatment or precision medicine studies. Single-cell gene expression holds a potential to provide treatment selections with maximized efficacy to help cancer patients based on a genetic understanding of their disease. This work presents a multi-layer microchip for single-cell multiplexed gene expression profiling and genotoxicity detection. Treated by three drug reagents (i.e., methyl methanesulfonate, docetaxel and colchicine with varied concentrations and time lengths, individual human cancer cells (MDA-MB-231 are lysed on-chip, and the released mRNA templates are captured and reversely transcribed into single strand DNA. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH, cyclin-dependent kinase inhibitor 1A (CDKN1A, and aurora kinase A (AURKA genes from single cells are amplified and real-time quantified through multiplex polymerase chain reaction. The microchip is capable of integrating all steps of single-cell multiplexed gene expression profiling, and providing precision detection of drug induced genotoxic stress. Throughput has been set to be 18, and can be further increased following the same approach. Numerical simulation of on-chip single cell trapping and heat transfer has been employed to evaluate the chip design and operation.

  10. Evaluation of single and stack membraneless enzymatic fuel cells based on ethanol in simulated body fluids.

    Science.gov (United States)

    Galindo-de-la-Rosa, J; Arjona, N; Moreno-Zuria, A; Ortiz-Ortega, E; Guerra-Balcázar, M; Ledesma-García, J; Arriaga, L G

    2017-06-15

    The purpose of this work is to evaluate single and double-cell membraneless microfluidic fuel cells (MMFCs) that operate in the presence of simulated body fluids SBF, human serum and blood enriched with ethanol as fuels. The study was performed using the alcohol dehydrogenase enzyme immobilised by covalent binding through an array composed of carbon Toray paper as support and a layer of poly(methylene blue)/tetrabutylammonium bromide/Nafion and glutaraldehyde (3D bioanode electrode). The single MMFC was tested in a hybrid microfluidic fuel cell using Pt/C as the cathode. A cell voltage of 1.035V and power density of 3.154mWcm -2 were observed, which is the highest performance reported to date. The stability and durability were tested through chronoamperometry and polarisation/performance curves obtained at different days, which demonstrated a slow decrease in the power density on day 10 (14%) and day 20 (26%). Additionally, the cell was tested for ethanol oxidation in simulated body fluid (SBF) with ionic composition similar to human blood plasma. Those tests resulted in 0.93V of cell voltage and a power density close to 1.237mWcm -2 . The double cell MMFC (Stack) was tested using serum and human blood enriched with ethanol. The stack operated with blood in a serial connection showed an excellent cell performance (0.716mWcm -2 ), demonstrating the feasibility of employing human blood as energy source. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. Essentials of single-cell analysis concepts, applications and future prospects

    CERN Document Server

    Santra, Tuhin

    2016-01-01

    This book provides an overview of single-cell isolation, separation, injection, lysis and dynamics analysis as well as a study of their heterogeneity using different miniaturized devices. As an important part of single-cell analysis, different techniques including electroporation, microinjection, optical trapping, optoporation, rapid electrokinetic patterning and optoelectronic tweezers are described in detail. It presents different fluidic systems (e.g. continuous micro/nano-fluidic devices, microfluidic cytometry) and their integration with sensor technology, optical and hydrodynamic stretchers etc., and demonstrates the applications of single-cell analysis in systems biology, proteomics, genomics, epigenomics, cancer transcriptomics, metabolomics, biomedicine and drug delivery systems. It also discusses the future challenges for single-cell analysis, including the advantages and limitations. This book is enjoyable reading material while at the same time providing essential information to scientists in acad...

  12. Single incision laparoscopic pancreas resection for pancreatic metastasis of renal cell carcinoma.

    Science.gov (United States)

    Barbaros, Umut; Sümer, Aziz; Demirel, Tugrul; Karakullukçu, Nazlı; Batman, Burçin; Içscan, Yalın; Sarıçam, Gülay; Serin, Kürçsat; Loh, Wei-Liang; Dinççağ, Ahmet; Mercan, Selçuk

    2010-01-01

    Transumbilical single incision laparoscopic surgery (SILS) offers excellent cosmetic results and may be associated with decreased postoperative pain, reduced need for analgesia, and thus accelerated recovery. Herein, we report the first transumbilical single incision laparoscopic pancreatectomy case in a patient who had renal cell cancer metastasis on her pancreatic corpus and tail. A 59-year-old female who had metastatic lesions on her pancreas underwent laparoscopic subtotal pancreatectomy through a 2-cm umbilical incision. Single incision pancreatectomy was performed with a special port (SILS port) and articulated equipment. The procedure lasted 330 minutes. Estimated blood loss was 100mL. No perioperative complications occurred. The patient was discharged on the seventh postoperative day with a low-volume (20mL/day) pancreatic fistula that ceased spontaneously. Pathology result of the specimen was renal cell cancer metastases. This is the first reported SILS pancreatectomy case, demonstrating that even advanced surgical procedures can be performed using the SILS technique in well-experienced centers. Transumbilical single incision laparoscopic pancreatectomy is feasible and can be performed safely in experienced centers. SILS may improve cosmetic results and allow accelerated recovery for patients even with malignancy requiring advanced laparoscopic interventions.

  13. Toward single cell traction microscopy within 3D collagen matrices

    International Nuclear Information System (INIS)

    Hall, Matthew S.; Long, Rong; Feng, Xinzeng; Huang, YuLing; Hui, Chung-Yuen; Wu, Mingming

    2013-01-01

    Mechanical interaction between the cell and its extracellular matrix (ECM) regulates cellular behaviors, including proliferation, differentiation, adhesion, and migration. Cells require the three-dimensional (3D) architectural support of the ECM to perform physiologically realistic functions. However, current understanding of cell–ECM and cell–cell mechanical interactions is largely derived from 2D cell traction force microscopy, in which cells are cultured on a flat substrate. 3D cell traction microscopy is emerging for mapping traction fields of single animal cells embedded in either synthetic or natively derived fibrous gels. We discuss here the development of 3D cell traction microscopy, its current limitations, and perspectives on the future of this technology. Emphasis is placed on strategies for applying 3D cell traction microscopy to individual tumor cell migration within collagen gels. - Highlights: • Review of the current state of the art in 3D cell traction force microscopy. • Bulk and micro-characterization of remodelable fibrous collagen gels. • Strategies for performing 3D cell traction microscopy within collagen gels

  14. Toward single cell traction microscopy within 3D collagen matrices

    Energy Technology Data Exchange (ETDEWEB)

    Hall, Matthew S. [Department of Biological and Environmental Engineering, Cornell University, Ithaca, NY 14853 (United States); Long, Rong [Department of Mechanical Engineering, University of Alberta, Edmonton, AB, Canada T6G 2G8 (Canada); Feng, Xinzeng [Department of Mechanical and Aerospace Engineering, Cornell University, Ithaca, NY 14853 (United States); Huang, YuLing [Department of Biological and Environmental Engineering, Cornell University, Ithaca, NY 14853 (United States); Hui, Chung-Yuen [Department of Mechanical and Aerospace Engineering, Cornell University, Ithaca, NY 14853 (United States); Wu, Mingming, E-mail: mw272@cornell.edu [Department of Biological and Environmental Engineering, Cornell University, Ithaca, NY 14853 (United States)

    2013-10-01

    Mechanical interaction between the cell and its extracellular matrix (ECM) regulates cellular behaviors, including proliferation, differentiation, adhesion, and migration. Cells require the three-dimensional (3D) architectural support of the ECM to perform physiologically realistic functions. However, current understanding of cell–ECM and cell–cell mechanical interactions is largely derived from 2D cell traction force microscopy, in which cells are cultured on a flat substrate. 3D cell traction microscopy is emerging for mapping traction fields of single animal cells embedded in either synthetic or natively derived fibrous gels. We discuss here the development of 3D cell traction microscopy, its current limitations, and perspectives on the future of this technology. Emphasis is placed on strategies for applying 3D cell traction microscopy to individual tumor cell migration within collagen gels. - Highlights: • Review of the current state of the art in 3D cell traction force microscopy. • Bulk and micro-characterization of remodelable fibrous collagen gels. • Strategies for performing 3D cell traction microscopy within collagen gels.

  15. Single-cell entropy for accurate estimation of differentiation potency from a cell's transcriptome

    Science.gov (United States)

    Teschendorff, Andrew E.; Enver, Tariq

    2017-01-01

    The ability to quantify differentiation potential of single cells is a task of critical importance. Here we demonstrate, using over 7,000 single-cell RNA-Seq profiles, that differentiation potency of a single cell can be approximated by computing the signalling promiscuity, or entropy, of a cell's transcriptome in the context of an interaction network, without the need for feature selection. We show that signalling entropy provides a more accurate and robust potency estimate than other entropy-based measures, driven in part by a subtle positive correlation between the transcriptome and connectome. Signalling entropy identifies known cell subpopulations of varying potency and drug resistant cancer stem-cell phenotypes, including those derived from circulating tumour cells. It further reveals that expression heterogeneity within single-cell populations is regulated. In summary, signalling entropy allows in silico estimation of the differentiation potency and plasticity of single cells and bulk samples, providing a means to identify normal and cancer stem-cell phenotypes. PMID:28569836

  16. Single-Cell Landscape of Transcriptional Heterogeneity and Cell Fate Decisions during Mouse Early Gastrulation

    Directory of Open Access Journals (Sweden)

    Hisham Mohammed

    2017-08-01

    Full Text Available The mouse inner cell mass (ICM segregates into the epiblast and primitive endoderm (PrE lineages coincident with implantation of the embryo. The epiblast subsequently undergoes considerable expansion of cell numbers prior to gastrulation. To investigate underlying regulatory principles, we performed systematic single-cell RNA sequencing (seq of conceptuses from E3.5 to E6.5. The epiblast shows reactivation and subsequent inactivation of the X chromosome, with Zfp57 expression associated with reactivation and inactivation together with other candidate regulators. At E6.5, the transition from epiblast to primitive streak is linked with decreased expression of polycomb subunits, suggesting a key regulatory role. Notably, our analyses suggest elevated transcriptional noise at E3.5 and within the non-committed epiblast at E6.5, coinciding with exit from pluripotency. By contrast, E6.5 primitive streak cells became highly synchronized and exhibit a shortened G1 cell-cycle phase, consistent with accelerated proliferation. Our study systematically charts transcriptional noise and uncovers molecular processes associated with early lineage decisions.

  17. Deconstructing stem cell population heterogeneity: Single-cell analysis and modeling approaches

    Science.gov (United States)

    Wu, Jincheng; Tzanakakis, Emmanuel S.

    2014-01-01

    Isogenic stem cell populations display cell-to-cell variations in a multitude of attributes including gene or protein expression, epigenetic state, morphology, proliferation and proclivity for differentiation. The origins of the observed heterogeneity and its roles in the maintenance of pluripotency and the lineage specification of stem cells remain unclear. Addressing pertinent questions will require the employment of single-cell analysis methods as traditional cell biochemical and biomolecular assays yield mostly population-average data. In addition to time-lapse microscopy and flow cytometry, recent advances in single-cell genomic, transcriptomic and proteomic profiling are reviewed. The application of multiple displacement amplification, next generation sequencing, mass cytometry and spectrometry to stem cell systems is expected to provide a wealth of information affording unprecedented levels of multiparametric characterization of cell ensembles under defined conditions promoting pluripotency or commitment. Establishing connections between single-cell analysis information and the observed phenotypes will also require suitable mathematical models. Stem cell self-renewal and differentiation are orchestrated by the coordinated regulation of subcellular, intercellular and niche-wide processes spanning multiple time scales. Here, we discuss different modeling approaches and challenges arising from their application to stem cell populations. Integrating single-cell analysis with computational methods will fill gaps in our knowledge about the functions of heterogeneity in stem cell physiology. This combination will also aid the rational design of efficient differentiation and reprogramming strategies as well as bioprocesses for the production of clinically valuable stem cell derivatives. PMID:24035899

  18. Quantitative control of mitochondria transfer between live single cells using a microfluidic device

    Directory of Open Access Journals (Sweden)

    Ken-Ichi Wada

    2017-12-01

    Full Text Available Quantitative control of mitochondria transfer between live cells is a promising approach for genetic manipulation of mitochondrial DNA (mtDNA because single mitochondrion transfer to a mtDNA-less (ρ0 cell potentially leads to homoplasmy of mtDNA. In this paper, we describe a method for quantitative control of mitochondria transfer between live single cells. For this purpose, we fabricated novel microfluidic devices having cell paring structures with a 4.1, 5.6 or 10.0 μm-length microtunnel. When cells were fused through a microtunnel using the Sendai virus envelope-based method, a strictured cytoplasmic connection was achieved with a length corresponding to that of the microtunnel. Elongation of the cytoplasmic connection led to a decrease in mitochondria transfer to the fusion partner. Moreover, some cell pairs that fused through a 10.0 μm-length microtunnel showed single mitochondrion transfer. Fused cells were spontaneously disconnected from each other when they were recovered in a normal culture medium. These results suggest that our cell fusion method can perform quantitative control of mitochondria transfer that includes a single mitochondrion transfer.

  19. Single cells for forensic DNA analysis--from evidence material to test tube.

    Science.gov (United States)

    Brück, Simon; Evers, Heidrun; Heidorn, Frank; Müller, Ute; Kilper, Roland; Verhoff, Marcel A

    2011-01-01

    The purpose of this project was to develop a method that, while providing morphological quality control, allows single cells to be obtained from the surfaces of various evidence materials and be made available for DNA analysis in cases where only small amounts of cell material are present or where only mixed traces are found. With the SteREO Lumar.V12 stereomicroscope and UV unit from Zeiss, it was possible to detect and assess single epithelial cells on the surfaces of various objects (e.g., glass, plastic, metal). A digitally operated micromanipulator developed by aura optik was used to lift a single cell from the surface of evidence material and to transfer it to a conventional PCR tube or to an AmpliGrid(®) from Advalytix. The actual lifting of the cells was performed with microglobes that acted as carriers. The microglobes were held with microtweezers and were transferred to the DNA analysis receptacles along with the adhering cells. In a next step, the PCR can be carried out in this receptacle without removing the microglobe. Our method allows a single cell to be isolated directly from evidence material and be made available for forensic DNA analysis. © 2010 American Academy of Forensic Sciences.

  20. Design of a Facility for Studying Shock-Cell Noise on Single and Coaxial Jets

    Directory of Open Access Journals (Sweden)

    Daniel Guariglia

    2018-03-01

    Full Text Available Shock-cell noise occurs in aero-engines when the nozzle exhaust is supersonic and shock-cells are present in the jet. In commercial turbofan engines, at cruise, the secondary flow is often supersonic underexpanded, with the formation of annular shock-cells in the jet and consequent onset of shock-cell noise. This paper aims at describing the design process of the new facility FAST (Free jet AeroacouSTic laboratory at the von Karman Institute, aimed at the investigation of the shock-cell noise phenomenon on a dual stream jet. The rig consists of a coaxial open jet, with supersonic capability for both the primary and secondary flow. A coaxial silencer was designed to suppress the spurious noise coming from the feeding lines. Computational fluid dynamics (CFD simulations of the coaxial jet and acoustic simulations of the silencer have been carried out to support the design choices. Finally, the rig has been validated by performing experimental measurements on a supersonic single stream jet and comparing the results with the literature. Fine-scale PIV (Particle Image Velocimetry coupled with a microphone array in the far field have been used in this scope. Preliminary results of the dual stream jet are also shown.

  1. Single-cell printing to form three-dimensional lines of olfactory ensheathing cells

    International Nuclear Information System (INIS)

    Othon, Christina M; Ringeisen, Bradley R; Wu Xingjia; Anders, Juanita J

    2008-01-01

    Biological laser printing (BioLP(TM)) is a unique tool capable of printing high resolution two- and three-dimensional patterns of living mammalian cells, with greater than 95% viability. These results have been extended to primary cultured olfactory ensheathing cells (OECs), harvested from adult Sprague-Dawley rats. OECs have been found to provide stimulating environments for neurite outgrowth in spinal cord injury models. BioLP is unique in that small load volumes (∼μLs) are required to achieve printing, enabling low numbers of OECs to be harvested, concentrated and printed. BioLP was used to form several 8 mm lines of OECs throughout a multilayer hydrogel scaffold. The line width was as low as 20 μm, with most lines comprising aligned single cells. Fluorescent confocal microscopy was used to determine the functionality of the printed OECs, to monitor interactions between printed OECs, and to determine the extent of cell migration throughout the 3D scaffold. High-resolution printing of low cell count, harvested OECs is an important advancement for in vitro study of cell interactions and functionality. In addition, these cell-printed scaffolds may provide an alternative for spinal cord repair studies, as the single-cell patterns formed here are on relevant size scales for neurite outgrowth

  2. Unleashing the potential of the root hair cell as a single plant cell type model in root systems biology

    Directory of Open Access Journals (Sweden)

    Zhenzhen eQiao

    2013-11-01

    Full Text Available Plant root is an organ composed of multiple cell types with different functions. This multicellular complexity limits our understanding of root biology because –omics studies performed at the level of the entire root reflect the average responses of all cells composing the organ. To overcome this difficulty and allow a more comprehensive understanding of root cell biology, an approach is needed that would focus on one single cell type in the plant root. Because of its biological functions (i.e. uptake of water and various nutrients; primary site of infection by nitrogen-fixing bacteria in legumes, the root hair cell is an attractive single cell model to study root cell response to various stresses and treatments. To fully study their biology, we have recently optimized procedures in obtaining root hair cell samples. We culture the plants using an ultrasound aeroponic system maximizing root hair cell density on the entire root systems and allowing the homogeneous treatment of the root system. We then isolate the root hair cells in liquid nitrogen. Isolated root hair yields could be up to 800 to 1000 mg of plant cells from 60 root systems. Using soybean as a model, the purity of the root hair was assessed by comparing the expression level of genes previously identified as soybean root hair specific between preparations of isolated root hair cells and stripped roots, roots devoid in root hairs. Enlarging our tests to include other plant species, our results support the isolation of large quantities of highly purified root hair cells which is compatible with a systems biology approach.

  3. Effects of coal-derived trace species on performance of molten carbonate fuel cells. Final report

    Energy Technology Data Exchange (ETDEWEB)

    1992-05-01

    The Carbonate Fuel Cell is a very promising option for highly efficient generation of electricity from many fuels. If coal-gas is to be used, the interactions of coal-derived impurities on various fuel cell components need to be understood. Thus the effects on Carbonate Fuel Cell performance due to ten different coal-derived contaminants viz., NH{sub 3}, H{sub 2}S, HC{ell}, H{sub 2}Se, AsH{sub 3}, Zn, Pb, Cd, Sn, and Hg, have been studied at Energy Research Corporation. Both experimental and theoretical evaluations were performed, which have led to mechanistic insights and initial estimation of qualitative tolerance levels for each species individually and in combination with other species. The focus of this study was to investigate possible coal-gas contaminant effects on the anode side of the Carbonate Fuel Cell, using both out-of-cell thermogravimetric analysis by isothermal TGA, and fuel cell testing in bench-scale cells. Separate experiments detailing performance decay in these cells with high levels of ammonia contamination (1 vol %) and with trace levels of Cd, Hg, and Sn, have indicated that, on the whole, these elements do not affect carbonate fuel cell performance. However, some performance decay may result when a number of the other six species are present, singly or simultaneously, as contaminants in fuel gas. In all cases, tolerance levels have been estimated for each of the 10 species and preliminary models have been developed for six of them. At this stage the models are limited to isothermal, benchscale (300 cm{sup 2} size) single cells. The information obtained is expected to assist in the development of coal-gas cleanup systems, while the contaminant performance effects data will provide useful basic information for modeling fuel cell endurance in conjunction with integrated gasifier/fuel-cell systems (IGFC).

  4. Nanoparticle uptake and their co-localization with cell compartments - a confocal Raman microscopy study at single cell level

    International Nuclear Information System (INIS)

    Estrela-Lopis, I; Donath, E; Romero, G; Rojas, E; Moya, S E

    2011-01-01

    Confocal Raman Microscopy, a non-invasive, non-destructive and label-free technique, was employed to study the uptake and localization of nanoparticles (NPs) in the Hepatocarcinoma human cell line HepG2 at the level of single cells. Cells were exposed to carbon nanotubes (CNTs) the surface of which was engineered with polyelectrolytes and lipid layers, aluminium oxide and cerium dioxide nanoparticles. Raman spectra deconvolution was applied to obtain the spatial distributions of NPs together with lipids/proteins in cells. The colocalization of the NPs with different intracellular environments, lipid bodies, protein and DNA, was inferred. Lipid coated CNTs associated preferentially with lipid rich regions, whereas polyelectrolyte coated CNTs were excluded from lipid rich regions. Al 2 O 3 NPs were found in the cytoplasm. CeO 2 NPs were readily taken up and have been observed all over the cell. Raman z-scans proved the intracellular distribution of the respective NPs.

  5. Nanoparticle uptake and their co-localization with cell compartments - a confocal Raman microscopy study at single cell level

    Science.gov (United States)

    Estrela-Lopis, I.; Romero, G.; Rojas, E.; Moya, S. E.; Donath, E.

    2011-07-01

    Confocal Raman Microscopy, a non-invasive, non-destructive and label-free technique, was employed to study the uptake and localization of nanoparticles (NPs) in the Hepatocarcinoma human cell line HepG2 at the level of single cells. Cells were exposed to carbon nanotubes (CNTs) the surface of which was engineered with polyelectrolytes and lipid layers, aluminium oxide and cerium dioxide nanoparticles. Raman spectra deconvolution was applied to obtain the spatial distributions of NPs together with lipids/proteins in cells. The colocalization of the NPs with different intracellular environments, lipid bodies, protein and DNA, was inferred. Lipid coated CNTs associated preferentially with lipid rich regions, whereas polyelectrolyte coated CNTs were excluded from lipid rich regions. Al2O3 NPs were found in the cytoplasm. CeO2 NPs were readily taken up and have been observed all over the cell. Raman z-scans proved the intracellular distribution of the respective NPs.

  6. Multispectral optical tweezers for molecular diagnostics of single biological cells

    Science.gov (United States)

    Butler, Corey; Fardad, Shima; Sincore, Alex; Vangheluwe, Marie; Baudelet, Matthieu; Richardson, Martin

    2012-03-01

    Optical trapping of single biological cells has become an established technique for controlling and studying fundamental behavior of single cells with their environment without having "many-body" interference. The development of such an instrument for optical diagnostics (including Raman and fluorescence for molecular diagnostics) via laser spectroscopy with either the "trapping" beam or secondary beams is still in progress. This paper shows the development of modular multi-spectral imaging optical tweezers combining Raman and Fluorescence diagnostics of biological cells.

  7. Single Cell Confocal Raman Spectroscopy of Human Osteoarthritic Chondrocytes: A Preliminary Study

    Directory of Open Access Journals (Sweden)

    Rajesh Kumar

    2015-04-01

    Full Text Available A great deal of effort has been focused on exploring the underlying molecular mechanism of osteoarthritis (OA especially at the cellular level. We report a confocal Raman spectroscopic investigation on human osteoarthritic chondrocytes. The objective of this investigation is to identify molecular features and the stage of OA based on the spectral signatures corresponding to bio-molecular changes at the cellular level in chondrocytes. In this study, we isolated chondrocytes from human osteoarthritic cartilage and acquired Raman spectra from single cells. Major spectral differences between the cells obtained from different International Cartilage Repair Society (ICRS grades of osteoarthritic cartilage were identified. During progression of OA, a decrease in protein content and an increase in cell death were observed from the vibrational spectra. Principal component analysis and subsequent cross-validation was able to associate osteoarthritic chondrocytes to ICRS Grade I, II and III with specificity 100.0%, 98.1%, and 90.7% respectively, while, sensitivity was 98.6%, 82.8%, and 97.5% respectively. The overall predictive efficiency was 92.2%. Our pilot study encourages further use of Raman spectroscopy as a noninvasive and label free technique for revealing molecular features associated with osteoarthritic chondrocytes.

  8. Condensing Raman spectrum for single-cell phenotype analysis

    KAUST Repository

    Sun, Shiwei

    2015-12-09

    Background In recent years, high throughput and non-invasive Raman spectrometry technique has matured as an effective approach to identification of individual cells by species, even in complex, mixed populations. Raman profiling is an appealing optical microscopic method to achieve this. To fully utilize Raman proling for single-cell analysis, an extensive understanding of Raman spectra is necessary to answer questions such as which filtering methodologies are effective for pre-processing of Raman spectra, what strains can be distinguished by Raman spectra, and what features serve best as Raman-based biomarkers for single-cells, etc. Results In this work, we have proposed an approach called rDisc to discretize the original Raman spectrum into only a few (usually less than 20) representative peaks (Raman shifts). The approach has advantages in removing noises, and condensing the original spectrum. In particular, effective signal processing procedures were designed to eliminate noise, utilising wavelet transform denoising, baseline correction, and signal normalization. In the discretizing process, representative peaks were selected to signicantly decrease the Raman data size. More importantly, the selected peaks are chosen as suitable to serve as key biological markers to differentiate species and other cellular features. Additionally, the classication performance of discretized spectra was found to be comparable to full spectrum having more than 1000 Raman shifts. Overall, the discretized spectrum needs about 5storage space of a full spectrum and the processing speed is considerably faster. This makes rDisc clearly superior to other methods for single-cell classication.

  9. Single-cell photoacoustic thermometry

    Science.gov (United States)

    Gao, Liang; Wang, Lidai; Li, Chiye; Liu, Yan; Ke, Haixin; Zhang, Chi

    2013-01-01

    Abstract. A novel photoacoustic thermometric method is presented for simultaneously imaging cells and sensing their temperature. With three-seconds-per-frame imaging speed, a temperature resolution of 0.2°C was achieved in a photo-thermal cell heating experiment. Compared to other approaches, the photoacoustic thermometric method has the advantage of not requiring custom-developed temperature-sensitive biosensors. This feature should facilitate the conversion of single-cell thermometry into a routine lab tool and make it accessible to a much broader biological research community. PMID:23377004

  10. Compressive Force Spectroscopy: From Living Cells to Single Proteins.

    Science.gov (United States)

    Wang, Jiabin; Liu, Meijun; Shen, Yi; Sun, Jielin; Shao, Zhifeng; Czajkowsky, Daniel Mark

    2018-03-23

    One of the most successful applications of atomic force microscopy (AFM) in biology involves monitoring the effect of force on single biological molecules, often referred to as force spectroscopy. Such studies generally entail the application of pulling forces of different magnitudes and velocities upon individual molecules to resolve individualistic unfolding/separation pathways and the quantification of the force-dependent rate constants. However, a less recognized variation of this method, the application of compressive force, actually pre-dates many of these "tensile" force spectroscopic studies. Further, beyond being limited to the study of single molecules, these compressive force spectroscopic investigations have spanned samples as large as living cells to smaller, multi-molecular complexes such as viruses down to single protein molecules. Correspondingly, these studies have enabled the detailed characterization of individual cell states, subtle differences between seemingly identical viral structures, as well as the quantification of rate constants of functionally important, structural transitions in single proteins. Here, we briefly review some of the recent achievements that have been obtained with compressive force spectroscopy using AFM and highlight exciting areas of its future development.

  11. Cell type discovery using single-cell transcriptomics: implications for ontological representation.

    Science.gov (United States)

    Aevermann, Brian D; Novotny, Mark; Bakken, Trygve; Miller, Jeremy A; Diehl, Alexander D; Osumi-Sutherland, David; Lasken, Roger S; Lein, Ed S; Scheuermann, Richard H

    2018-05-01

    Cells are fundamental function units of multicellular organisms, with different cell types playing distinct physiological roles in the body. The recent advent of single-cell transcriptional profiling using RNA sequencing is producing 'big data', enabling the identification of novel human cell types at an unprecedented rate. In this review, we summarize recent work characterizing cell types in the human central nervous and immune systems using single-cell and single-nuclei RNA sequencing, and discuss the implications that these discoveries are having on the representation of cell types in the reference Cell Ontology (CL). We propose a method, based on random forest machine learning, for identifying sets of necessary and sufficient marker genes, which can be used to assemble consistent and reproducible cell type definitions for incorporation into the CL. The representation of defined cell type classes and their relationships in the CL using this strategy will make the cell type classes being identified by high-throughput/high-content technologies findable, accessible, interoperable and reusable (FAIR), allowing the CL to serve as a reference knowledgebase of information about the role that distinct cellular phenotypes play in human health and disease.

  12. A single-cell resolution map of mouse hematopoietic stem and progenitor cell differentiation.

    Science.gov (United States)

    Nestorowa, Sonia; Hamey, Fiona K; Pijuan Sala, Blanca; Diamanti, Evangelia; Shepherd, Mairi; Laurenti, Elisa; Wilson, Nicola K; Kent, David G; Göttgens, Berthold

    2016-08-25

    Maintenance of the blood system requires balanced cell fate decisions by hematopoietic stem and progenitor cells (HSPCs). Because cell fate choices are executed at the individual cell level, new single-cell profiling technologies offer exciting possibilities for mapping the dynamic molecular changes underlying HSPC differentiation. Here, we have used single-cell RNA sequencing to profile more than 1600 single HSPCs, and deep sequencing has enabled detection of an average of 6558 protein-coding genes per cell. Index sorting, in combination with broad sorting gates, allowed us to retrospectively assign cells to 12 commonly sorted HSPC phenotypes while also capturing intermediate cells typically excluded by conventional gating. We further show that independently generated single-cell data sets can be projected onto the single-cell resolution expression map to directly compare data from multiple groups and to build and refine new hypotheses. Reconstruction of differentiation trajectories reveals dynamic expression changes associated with early lymphoid, erythroid, and granulocyte-macrophage differentiation. The latter two trajectories were characterized by common upregulation of cell cycle and oxidative phosphorylation transcriptional programs. By using external spike-in controls, we estimate absolute messenger RNA (mRNA) levels per cell, showing for the first time that despite a general reduction in total mRNA, a subset of genes shows higher expression levels in immature stem cells consistent with active maintenance of the stem-cell state. Finally, we report the development of an intuitive Web interface as a new community resource to permit visualization of gene expression in HSPCs at single-cell resolution for any gene of choice. © 2016 by The American Society of Hematology.

  13. Single-cell technologies in molecular marine studies

    KAUST Repository

    Kodzius, Rimantas

    2015-01-24

    Middle Eastern countries are experiencing a renaissance, with heavy investment in both in infrastructure and science. King Abdullah University of Science and Technology (KAUST) is a new and modern university in Saudi Arabia. At the Computational Bioscience Research Center (CBRC) we are working on exploring the Red Sea and beyond, collaborating with Japanese and other research centers. We are using the environment to collect and analyze the microorganisms present. The platform being established at CBRC allows to process samples in a pipeline. The pipeline components consist of sample collection, processing and sequencing, following the in silico analysis, determining the gene functions, identifying the organisms. The genomes of microorganisms of interest are targeted modified by genome editing technology such as CRISPR and desired properties are selected by single cell instrumentation. The final output is to identify valuable microorganisms with production of bio-energy, nutrients, the food and fine chemicals.

  14. Protein Expression Analyses at the Single Cell Level

    Directory of Open Access Journals (Sweden)

    Masae Ohno

    2014-09-01

    Full Text Available The central dogma of molecular biology explains how genetic information is converted into its end product, proteins, which are responsible for the phenotypic state of the cell. Along with the protein type, the phenotypic state depends on the protein copy number. Therefore, quantification of the protein expression in a single cell is critical for quantitative characterization of the phenotypic states. Protein expression is typically a dynamic and stochastic phenomenon that cannot be well described by standard experimental methods. As an alternative, fluorescence imaging is being explored for the study of protein expression, because of its high sensitivity and high throughput. Here we review key recent progresses in fluorescence imaging-based methods and discuss their application to proteome analysis at the single cell level.

  15. High-performance analysis of single interphase cells with custom DNA probes spanning translocation break points

    Science.gov (United States)

    Weier, Heinz-Ulli G.; Munne, S.; Lersch, Robert A.; Marquez, C.; Wu, J.; Pedersen, Roger A.; Fung, Jingly

    1999-06-01

    The chromatin organization of interphase cell nuclei, albeit an object of intense investigation, is only poorly understood. In the past, this has hampered the cytogenetic analysis of tissues derived from specimens where only few cells were actively proliferating or a significant number of metaphase cells could be obtained by induction of growth. Typical examples of such hard to analyze cell systems are solid tumors, germ cells and, to a certain extent, fetal cells such as amniocytes, blastomeres or cytotrophoblasts. Balanced reciprocal translocations that do not disrupt essential genes and thus do not led to disease symptoms exit in less than one percent of the general population. Since the presence of translocations interferes with homologue pairing in meiosis, many of these individuals experience problems in their reproduction, such as reduced fertility, infertility or a history of spontaneous abortions. The majority of translocation carriers enrolled in our in vitro fertilization (IVF) programs carry simple translocations involving only two autosomes. While most translocations are relatively easy to spot in metaphase cells, the majority of cells biopsied from embryos produced by IVF are in interphase and thus unsuitable for analysis by chromosome banding or FISH-painting. We therefore set out to analyze single interphase cells for presence or absence of specific translocations. Our assay, based on fluorescence in situ hybridization (FISH) of breakpoint-spanning DNA probes, detects translocations in interphase by visual microscopic inspection of hybridization domains. Probes are prepared so that they span a breakpoint and cover several hundred kb of DNA adjacent to the breakpoint. On normal chromosomes, such probes label a contiguous stretch of DNA and produce a single hybridization domain per chromosome in interphase cells. The translocation disrupts the hybridization domain and the resulting two fragments appear as physically separated hybridization domains in

  16. Single-Cell Quantitative PCR: Advances and Potential in Cancer Diagnostics.

    Science.gov (United States)

    Ok, Chi Young; Singh, Rajesh R; Salim, Alaa A

    2016-01-01

    Tissues are heterogeneous in their components. If cells of interest are a minor population of collected tissue, it would be difficult to obtain genetic or genomic information of the interested cell population with conventional genomic DNA extraction from the collected tissue. Single-cell DNA analysis is important in the analysis of genetics of cell clonality, genetic anticipation, and single-cell DNA polymorphisms. Single-cell PCR using Single Cell Ampligrid/GeXP platform is described in this chapter.

  17. Quantum Dot Platform for Single-Cell Molecular Profiling

    Science.gov (United States)

    Zrazhevskiy, Pavel S.

    In-depth understanding of the nature of cell physiology and ability to diagnose and control the progression of pathological processes heavily rely on untangling the complexity of intracellular molecular mechanisms and pathways. Therefore, comprehensive molecular profiling of individual cells within the context of their natural tissue or cell culture microenvironment is essential. In principle, this goal can be achieved by tagging each molecular target with a unique reporter probe and detecting its localization with high sensitivity at sub-cellular resolution, primarily via microscopy-based imaging. Yet, neither widely used conventional methods nor more advanced nanoparticle-based techniques have been able to address this task up to date. High multiplexing potential of fluorescent probes is heavily restrained by the inability to uniquely match probes with corresponding molecular targets. This issue is especially relevant for quantum dot probes---while simultaneous spectral imaging of up to 10 different probes is possible, only few can be used concurrently for staining with existing methods. To fully utilize multiplexing potential of quantum dots, it is necessary to design a new staining platform featuring unique assignment of each target to a corresponding quantum dot probe. This dissertation presents two complementary versatile approaches towards achieving comprehensive single-cell molecular profiling and describes engineering of quantum dot probes specifically tailored for each staining method. Analysis of expanded molecular profiles is achieved through augmenting parallel multiplexing capacity with performing several staining cycles on the same specimen in sequential manner. In contrast to other methods utilizing quantum dots or other nanoparticles, which often involve sophisticated probe synthesis, the platform technology presented here takes advantage of simple covalent bioconjugation and non-covalent self-assembly mechanisms for straightforward probe

  18. RF Breakdown in Normal Conducting Single-cell Structures

    CERN Document Server

    Dolgashev, Valery A; Higo, Toshiyasu; Nantista, Christopher D; Tantawi, Sami G

    2005-01-01

    Operating accelerating gradient in normal conducting accelerating structures is often limited by rf breakdown. The limit depends on multiple parameters, including input rf power, rf circuit, cavity shape and material. Experimental and theoretical study of the effects of these parameters on the breakdown limit in full scale structures is difficult and costly. We use 11.4 GHz single-cell traveling wave and standing wave accelerating structures for experiments and modeling of rf breakdown behavior. These test structures are designed so that the electromagnetic fields in one cell mimic the fields in prototype multicell structures for the X-band linear collider. Fields elsewhere in the test structures are significantly lower than that of the single cell. The setup uses matched mode converters that launch the circular TM01 mode into short test structures. The test structures are connected to the mode launchers with vacuum rf flanges. This setup allows economic testing of different cell geometries, cell materials an...

  19. Single molecule microscopy in 3D cell cultures and tissues.

    Science.gov (United States)

    Lauer, Florian M; Kaemmerer, Elke; Meckel, Tobias

    2014-12-15

    From the onset of the first microscopic visualization of single fluorescent molecules in living cells at the beginning of this century, to the present, almost routine application of single molecule microscopy, the method has well-proven its ability to contribute unmatched detailed insight into the heterogeneous and dynamic molecular world life is composed of. Except for investigations on bacteria and yeast, almost the entire story of success is based on studies on adherent mammalian 2D cell cultures. However, despite this continuous progress, the technique was not able to keep pace with the move of the cell biology community to adapt 3D cell culture models for basic research, regenerative medicine, or drug development and screening. In this review, we will summarize the progress, which only recently allowed for the application of single molecule microscopy to 3D cell systems and give an overview of the technical advances that led to it. While initially posing a challenge, we finally conclude that relevant 3D cell models will become an integral part of the on-going success of single molecule microscopy. Copyright © 2014 Elsevier B.V. All rights reserved.

  20. Design study of technology requirements for high performance single-propeller-driven business airplanes

    Science.gov (United States)

    Kohlman, D. L.; Hammer, J.

    1985-01-01

    Developments in aerodyamic, structural and propulsion technologies which influence the potential for significant improvements in performance and fuel efficiency of general aviation business airplanes are discussed. The advancements include such technolgies as natural laminar flow, composite materials, and advanced intermittent combustion engines. The design goal for this parameter design study is a range of 1300 nm at 300 knots true airspeed with a payload of 1200lbs at 35,000 ft cruise altitude. The individual and synergistic effects of various advanced technologies on the optimization of this class of high performance, single engine, propeller driven business airplanes are identified.

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

    Science.gov (United States)

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

    2012-06-01

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

  2. Single-cell Hi-C bridges microscopy and genome-wide sequencing approaches to study 3D chromatin organization.

    Science.gov (United States)

    Ulianov, Sergey V; Tachibana-Konwalski, Kikue; Razin, Sergey V

    2017-10-01

    Recent years have witnessed an explosion of the single-cell biochemical toolbox including chromosome conformation capture (3C)-based methods that provide novel insights into chromatin spatial organization in individual cells. The observations made with these techniques revealed that topologically associating domains emerge from cell population averages and do not exist as static structures in individual cells. Stochastic nature of the genome folding is likely to be biologically relevant and may reflect the ability of chromatin fibers to adopt a number of alternative configurations, some of which could be transiently stabilized and serve regulatory purposes. Single-cell Hi-C approaches provide an opportunity to analyze chromatin folding in rare cell types such as stem cells, tumor progenitors, oocytes, and totipotent cells, contributing to a deeper understanding of basic mechanisms in development and disease. Here, we review key findings of single-cell Hi-C and discuss possible biological reasons and consequences of the inferred dynamic chromatin spatial organization. © 2017 WILEY Periodicals, Inc.

  3. Impact of temperature on performance of series and parallel connected mono-crystalline silicon solar cells

    Directory of Open Access Journals (Sweden)

    Subhash Chander

    2015-11-01

    Full Text Available This paper presents a study on impact of temperature on the performance of series and parallel connected mono-crystalline silicon (mono-Si solar cell employing solar simulator. The experiment was carried out at constant light intensity 550 W/m2with cell temperature in the range 25–60 oC for single, series and parallel connected mono-Si solar cells. The performance parameters like open circuit voltage, maximum power, fill factor and efficiency are found to decrease with cell temperature while the short circuit current is observed to increase. The experimental results reveal that silicon solar cells connected in series and parallel combinations follow the Kirchhoff’s laws and the temperature has a significant effect on the performance parameters of solar cell.

  4. Distinct gene expression signatures in human embryonic stem cells differentiated towards definitive endoderm at single-cell level

    DEFF Research Database (Denmark)

    Norrman, Karin; Strömbeck, Anna; Semb, Henrik

    2013-01-01

    for the three activin A based protocols applied. Our data provide novel insights in DE gene expression at the cellular level of in vitro differentiated human embryonic stem cells, and illustrate the power of using single-cell gene expression profiling to study differentiation heterogeneity and to characterize...... of anterior definitive endoderm (DE). Here, we differentiated human embryonic stem cells towards DE using three different activin A based treatments. Differentiation efficiencies were evaluated by gene expression profiling over time at cell population level. A panel of key markers was used to study DE...... formation. Final DE differentiation was also analyzed with immunocytochemistry and single-cell gene expression profiling. We found that cells treated with activin A in combination with sodium butyrate and B27 serum-free supplement medium generated the most mature DE cells. Cell population studies were...

  5. Genomic Sequencing of Single Microbial Cells from Environmental Samples

    Energy Technology Data Exchange (ETDEWEB)

    Ishoey, Thomas; Woyke, Tanja; Stepanauskas, Ramunas; Novotny, Mark; Lasken, Roger S.

    2008-02-01

    Recently developed techniques allow genomic DNA sequencing from single microbial cells [Lasken RS: Single-cell genomic sequencing using multiple displacement amplification, Curr Opin Microbiol 2007, 10:510-516]. Here, we focus on research strategies for putting these methods into practice in the laboratory setting. An immediate consequence of single-cell sequencing is that it provides an alternative to culturing organisms as a prerequisite for genomic sequencing. The microgram amounts of DNA required as template are amplified from a single bacterium by a method called multiple displacement amplification (MDA) avoiding the need to grow cells. The ability to sequence DNA from individual cells will likely have an immense impact on microbiology considering the vast numbers of novel organisms, which have been inaccessible unless culture-independent methods could be used. However, special approaches have been necessary to work with amplified DNA. MDA may not recover the entire genome from the single copy present in most bacteria. Also, some sequence rearrangements can occur during the DNA amplification reaction. Over the past two years many research groups have begun to use MDA, and some practical approaches to single-cell sequencing have been developed. We review the consensus that is emerging on optimum methods, reliability of amplified template, and the proper interpretation of 'composite' genomes which result from the necessity of combining data from several single-cell MDA reactions in order to complete the assembly. Preferred laboratory methods are considered on the basis of experience at several large sequencing centers where >70% of genomes are now often recovered from single cells. Methods are reviewed for preparation of bacterial fractions from environmental samples, single-cell isolation, DNA amplification by MDA, and DNA sequencing.

  6. Image segmentation and dynamic lineage analysis in single-cell fluorescence microscopy.

    Science.gov (United States)

    Wang, Quanli; Niemi, Jarad; Tan, Chee-Meng; You, Lingchong; West, Mike

    2010-01-01

    An increasingly common component of studies in synthetic and systems biology is analysis of dynamics of gene expression at the single-cell level, a context that is heavily dependent on the use of time-lapse movies. Extracting quantitative data on the single-cell temporal dynamics from such movies remains a major challenge. Here, we describe novel methods for automating key steps in the analysis of single-cell, fluorescent images-segmentation and lineage reconstruction-to recognize and track individual cells over time. The automated analysis iteratively combines a set of extended morphological methods for segmentation, and uses a neighborhood-based scoring method for frame-to-frame lineage linking. Our studies with bacteria, budding yeast and human cells, demonstrate the portability and usability of these methods, whether using phase, bright field or fluorescent images. These examples also demonstrate the utility of our integrated approach in facilitating analyses of engineered and natural cellular networks in diverse settings. The automated methods are implemented in freely available, open-source software.

  7. Single cell elemental analysis using nuclear microscopy

    International Nuclear Information System (INIS)

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

    1999-01-01

    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)

  8. Analysis and performance assessment of a new solar-based multigeneration system integrated with ammonia fuel cell and solid oxide fuel cell-gas turbine combined cycle

    Science.gov (United States)

    Siddiqui, Osamah; Dincer, Ibrahim

    2017-12-01

    In the present study, a new solar-based multigeneration system integrated with an ammonia fuel cell and solid oxide fuel cell-gas turbine combined cycle to produce electricity, hydrogen, cooling and hot water is developed for analysis and performance assessment. In this regard, thermodynamic analyses and modeling through both energy and exergy approaches are employed to assess and evaluate the overall system performance. Various parametric studies are conducted to study the effects of varying system parameters and operating conditions on the energy and exergy efficiencies. The results of this study show that the overall multigeneration system energy efficiency is obtained as 39.1% while the overall system exergy efficiency is calculated as 38.7%, respectively. The performance of this multigeneration system results in an increase of 19.3% in energy efficiency as compared to single generation system. Furthermore, the exergy efficiency of the multigeneration system is 17.8% higher than the single generation system. Moreover, both energy and exergy efficiencies of the solid oxide fuel cell-gas turbine combined cycle are determined as 68.5% and 55.9% respectively.

  9. Single Cell HLA Matching Feasibility by Whole Genomic Amplification and Nested PCR

    Institute of Scientific and Technical Information of China (English)

    Xiao-hong Li; Fang-yin Meng

    2004-01-01

    @@ PCR based single-cell DNA analysis has been widely used in forensic science, preimplantation genetic diagnosis and so on. However, the original sample cannot be efficiently retrieved following single cell PCR, consequently the amount of information gained is limited. HLA system is too sophisticated that it is very hard to complete HLA typing by single cell. A Taq polymerase-based method using random primers to amplify whole genome termed as whole genome amplification (WGA) has demonstrated to be a useful method in increasing the copies of minimum sample. We establish a technique in this study to amplify HLA-A and HLA-B loci at same time in a single cell using WGA.

  10. Performance study of sugar-yeast-ethanol bio-hybrid fuel cells

    Science.gov (United States)

    Jahnke, Justin P.; Mackie, David M.; Benyamin, Marcus; Ganguli, Rahul; Sumner, James J.

    2015-05-01

    Renewable alternatives to fossil hydrocarbons for energy generation are of general interest for a variety of political, economic, environmental, and practical reasons. In particular, energy from biomass has many advantages, including safety, sustainability, and the ability to be scavenged from native ecosystems or from waste streams. Microbial fuel cells (MFCs) can take advantage of microorganism metabolism to efficiently use sugar and other biomolecules as fuel, but are limited by low power densities. In contrast, direct alcohol fuel cells (DAFCs) take advantage of proton exchange membranes (PEMs) to generate electricity from alcohols at much higher power densities. Here, we investigate a novel bio-hybrid fuel cell design prepared using commercial off-the-shelf DAFCs. In the bio-hybrid fuel cells, biomass such as sugar is fermented by yeast to ethanol, which can be used to fuel a DAFC. A separation membrane between the fermentation and the DAFC is used to purify the fermentate while avoiding any parasitic power losses. However, shifting the DAFCs from pure alcohol-water solutions to filtered fermented media introduces complications related to how the starting materials, fermentation byproducts, and DAFC waste products affect both the fermentation and the long-term DAFC performance. This study examines the impact of separation membrane pore size, fermentation/fuel cell byproducts, alcohol and salt concentrations, and load resistance on fuel cell performance. Under optimized conditions, the performance obtained is comparable to that of a similar DAFC run with a pure alcohol-water mixture. Additionally, the modified DAFC can provide useable amounts of power for weeks.

  11. Isolation of Kupffer Cells and Hepatocytes from a Single Mouse Liver

    DEFF Research Database (Denmark)

    Aparicio-Vergara, Marcela; Tencerova, Michaela; Morgantini, Cecilia

    2017-01-01

    Liver perfusion is a common technique used to isolate parenchymal and non-parenchymal liver cells for in vitro experiments. This method allows hepatic cells to be separated based on their size and weight, by centrifugation using a density gradient. To date, other methods allow the isolation of only...... one viable hepatic cellular fraction from a single mouse; either parenchymal (hepatocytes) or non-parenchymal cells (i.e., Kupffer cells or hepatic stellate cells). Here, we describe a method to isolate both hepatocytes and Kupffer cells from a single mouse liver, thereby providing the unique...... advantage of studying different liver cell types that have been isolated from the same organism....

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

    Science.gov (United States)

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

    2016-03-01

    Individual cells within the same population show various degrees of heterogeneity, which may be better handled with single-cell analysis to address biological and clinical questions. Single-cell analysis is especially important in developmental biology as subtle spatial and temporal differences in cells have significant associations with cell fate decisions during differentiation and with the description of a particular state of a cell exhibiting an aberrant phenotype. Biotechnological advances, especially in the area of microfluidics, have led to a robust, massively parallel and multi-dimensional capturing, sorting, and lysis of single-cells and amplification of related macromolecules, which have enabled the use of imaging and omics techniques on single cells. There have been improvements in computational single-cell image analysis in developmental biology regarding feature extraction, segmentation, image enhancement and machine learning, handling limitations of optical resolution to gain new perspectives from the raw microscopy images. Omics approaches, such as transcriptomics, genomics and epigenomics, targeting gene and small RNA expression, single nucleotide and structural variations and methylation and histone modifications, rely heavily on high-throughput sequencing technologies. Although there are well-established bioinformatics methods for analysis of sequence data, there are limited bioinformatics approaches which address experimental design, sample size considerations, amplification bias, normalization, differential expression, coverage, clustering and classification issues, specifically applied at the single-cell level. In this review, we summarize biological and technological advancements, discuss challenges faced in the aforementioned data acquisition and analysis issues and present future prospects for application of single-cell analyses to developmental biology. © The Author 2015. Published by Oxford University Press on behalf of the European

  13. Analyzing cell fate control by cytokines through continuous single cell biochemistry.

    Science.gov (United States)

    Rieger, Michael A; Schroeder, Timm

    2009-10-01

    Cytokines are important regulators of cell fates with high clinical and commercial relevance. However, despite decades of intense academic and industrial research, it proved surprisingly difficult to describe the biological functions of cytokines in a precise and comprehensive manner. The exact analysis of cytokine biology is complicated by the fact that individual cytokines control many different cell fates and activate a multitude of intracellular signaling pathways. Moreover, although activating different molecular programs, different cytokines can be redundant in their biological effects. In addition, cytokines with different biological effects can activate overlapping signaling pathways. This prospect article will outline the necessity of continuous single cell biochemistry to unravel the biological functions of molecular cytokine signaling. It focuses on potentials and limitations of recent technical developments in fluorescent time-lapse imaging and single cell tracking allowing constant long-term observation of molecules and behavior of single cells. (c) 2009 Wiley-Liss, Inc.

  14. Microelectromechanical System-Based Sensing Arrays for Comparative in Vitro Nanotoxicity Assessment at Single Cell and Small Cell-Population Using Electrochemical Impedance Spectroscopy.

    Science.gov (United States)

    Shah, Pratikkumar; Zhu, Xuena; Zhang, Xueji; He, Jin; Li, Chen-zhong

    2016-03-09

    The traditional in vitro nanotoxicity assessment approaches are conducted on a monolayer of cell culture. However, to study a cell response without interference from the neighbor cells, a single cell study is necessary; especially in cases of neuronal, cancerous, and stem cells, wherein an individual cell's fate is often not explained by the whole cell population. Nonetheless, a single cell does not mimic the actual in vivo environment and lacks important information regarding cell communication with its microenvironment. Both a single cell and a cell population provide important and complementary information about cells' behaviors. In this research, we explored nanotoxicity assessment on a single cell and a small cell population using electrochemical impedance spectroscopy and a microelectromechanical system (MEMS) device. We demonstrated a controlled capture of PC12 cells in different-sized microwells (to capture a different number of cells) using a combined method of surface functionalization and dielectrophoresis. The present approach provides a rapid nanotoxicity response as compared to other conventional approaches. This is the first study, to our knowledge, which demonstrates a comparative response of a single cell and small cell colonies on the same MEMS platform, when exposed to metaloxide nanoparticles. We demonstrated that the microenvironment of a cell is also accountable for cells' behaviors and their responses to nanomaterials. The results of this experimental study open up a new hypothesis to be tested for identifying the role of cell communication in spreading toxicity in a cell population.

  15. Proximity-based differential single cell analysis of the niche to identify stem/progenitor cell regulators

    Science.gov (United States)

    Silberstein, Lev; Goncalves, Kevin A; Kharchenko, Peter V; Turcotte, Raphael; Kfoury, Youmna; Mercier, Francois; Baryawno, Ninib; Severe, Nicolas; Bachand, Jacqueline; Spencer, Joel; Papazian, Ani; Lee, Dongjun; Chitteti, Brahmananda Reddy; Srour, Edward F; Hoggatt, Jonathan; Tate, Tiffany; Celso, Cristina Lo; Ono, Noriaki; Nutt, Stephen; Heino, Jyrki; Sipilä, Kalle; Shioda, Toshihiro; Osawa, Masatake; Lin, Charles P; Hu, Guo-fu; Scadden, David T

    2016-01-01

    SUMMARY Physiological stem cell function is regulated by secreted factors produced by niche cells. In this study, we describe an unbiased approach based on differential single-cell gene expression analysis of mesenchymal osteolineage cells close to and further removed from hematopoietic stem/progenitor cells to identify candidate niche factors. Mesenchymal cells displayed distinct molecular profiles based on their relative location. Amongst the genes which were preferentially expressed in proximal cells, we functionally examined three secreted or cell surface molecules not previously connected to HSPC biology: the secreted RNase Angiogenin, the cytokine IL18 and the adhesion molecule Embigin and discovered that all of these factors are HSPC quiescence regulators. Our proximity-based differential single cell approach therefore reveals molecular heterogeneity within niche cells and can be used to identify novel extrinsic stem/progenitor cell regulators. Similar approaches could also be applied to other stem cell/niche pairs to advance understanding of microenvironmental regulation of stem cell function. PMID:27524439

  16. Performance of molten carbonate fuel cells with the electrolyte molded at low pressure (IV). Analysis of performance decay factors in MCFC stack

    Energy Technology Data Exchange (ETDEWEB)

    Sonai, Atuo; Ozu, Hideyuki; Murata, Kenji; Shirogami, Tamotsu; Watanabe, Takao; Izaki, Yoshiyuki; Horiuchi, Nagayuki

    1987-09-01

    A 1500-h performance test on a 30 x 30 cm cell stack of 10 molten carbonate fuel cells was performed to evaluate the durability of the stack. Beyond 1000 h, decay of its performance was observed. The result of the study for the cause of the decay is reported. The structures of the single cell and stack are introduced. The effective area of the electrode is 530 m/sup 2/. After 1020 h use, the output voltage decreased. Analysis of the cell characteristics and post-test analysis were performed to study the cause of the decrease. It was found that the main cause for the voltage loss would be the occurrence of slight short circuiting between the edge-seal areas via a corrosion product. However, little transfer of lithium and potassium ions was observed through the manifold seal which had been regarded as the main cause for the decay of stacked cells. It was assumed that this was due to the employment of a sealing material which contained glass of low manifold ion conductivity. (10 figs, 4 refs)

  17. Experimental techniques for single cell and single molecule biomechanics

    International Nuclear Information System (INIS)

    Lim, C.T.; Zhou, E.H.; Li, A.; Vedula, S.R.K.; Fu, H.X.

    2006-01-01

    Stresses and strains that act on the human body can arise either from external physical forces or internal physiological environmental conditions. These biophysical interactions can occur not only at the musculoskeletal but also cellular and molecular levels and can determine the health and function of the human body. Here, we seek to investigate the structure-property-function relationship of cells and biomolecules so as to understand their important physiological functions as well as establish possible connections to human diseases. With the recent advancements in cell and molecular biology, biophysics and nanotechnology, several innovative and state-of-the-art experimental techniques and equipment have been developed to probe the structural and mechanical properties of biostructures from the micro- down to picoscale. Some of these experimental techniques include the optical or laser trap method, micropipette aspiration, step-pressure technique, atomic force microscopy and molecular force spectroscopy. In this article, we will review the basic principles and usage of these techniques to conduct single cell and single molecule biomechanics research

  18. Dynamics of lineage commitment revealed by single-cell transcriptomics of differentiating embryonic stem cells

    NARCIS (Netherlands)

    Semrau, Stefan; Goldmann, Johanna E; Soumillon, Magali; Mikkelsen, Tarjei S; Jaenisch, Rudolf; van Oudenaarden, Alexander

    2017-01-01

    Gene expression heterogeneity in the pluripotent state of mouse embryonic stem cells (mESCs) has been increasingly well-characterized. In contrast, exit from pluripotency and lineage commitment have not been studied systematically at the single-cell level. Here we measure the gene expression

  19. A platform for high-throughput bioenergy production phenotype characterization in single cells

    Science.gov (United States)

    Kelbauskas, Laimonas; Glenn, Honor; Anderson, Clifford; Messner, Jacob; Lee, Kristen B.; Song, Ganquan; Houkal, Jeff; Su, Fengyu; Zhang, Liqiang; Tian, Yanqing; Wang, Hong; Bussey, Kimberly; Johnson, Roger H.; Meldrum, Deirdre R.

    2017-01-01

    Driven by an increasing number of studies demonstrating its relevance to a broad variety of disease states, the bioenergy production phenotype has been widely characterized at the bulk sample level. Its cell-to-cell variability, a key player associated with cancer cell survival and recurrence, however, remains poorly understood due to ensemble averaging of the current approaches. We present a technology platform for performing oxygen consumption and extracellular acidification measurements of several hundreds to 1,000 individual cells per assay, while offering simultaneous analysis of cellular communication effects on the energy production phenotype. The platform comprises two major components: a tandem optical sensor for combined oxygen and pH detection, and a microwell device for isolation and analysis of single and few cells in hermetically sealed sub-nanoliter chambers. Our approach revealed subpopulations of cells with aberrant energy production profiles and enables determination of cellular response variability to electron transfer chain inhibitors and ion uncouplers. PMID:28349963

  20. The Viability of Single Cancer Cells after Exposure to Hydrodynamic Shear Stresses in a Spiral Microchannel: A Canine Cutaneous Mast Cell Tumor Model

    Directory of Open Access Journals (Sweden)

    Dettachai Ketpun

    2017-12-01

    Full Text Available Our laboratory has the fundamental responsibility to study cancer stem cells (CSC in various models of human and animal neoplasms. However, the major impediments that spike our accomplishment are the lack of universal biomarkers and cellular heterogeneity. To cope with these restrictions, we have tried to apply the concept of single cell analysis, which has hitherto been recommended throughout the world as an imperative solution pack for resolving such dilemmas. Accordingly, our first step was to utilize a predesigned spiral microchannel fabricated by our laboratory to perform size-based single cell separation using mast cell tumor (MCT cells as a model. However, the impact of hydrodynamic shear stresses (HSS on mechanical cell injury and viability in a spiral microchannel has not been fully investigated so far. Intuitively, our computational fluid dynamics (CFD simulation has strongly revealed the formations of fluid shear stress (FSS and extensional fluid stress (EFS in the sorting system. The panel of biomedical assays has also disclosed cell degeneration and necrosis in the model. Therefore, we have herein reported the combinatorically detrimental effect of FSS and EFS on the viability of MCT cells after sorting in our spiral microchannel, with discussion on the possibly pathogenic mechanisms of HSS-induced cell injury in the study model.

  1. High-Performance Single-Crystalline Perovskite Thin-Film Photodetector

    KAUST Repository

    Yang, Zhenqian

    2018-01-10

    The best performing modern optoelectronic devices rely on single-crystalline thin-film (SC-TF) semiconductors grown epitaxially. The emerging halide perovskites, which can be synthesized via low-cost solution-based methods, have achieved substantial success in various optoelectronic devices including solar cells, lasers, light-emitting diodes, and photodetectors. However, to date, the performance of these perovskite devices based on polycrystalline thin-film active layers lags behind the epitaxially grown semiconductor devices. Here, a photodetector based on SC-TF perovskite active layer is reported with a record performance of a 50 million gain, 70 GHz gain-bandwidth product, and a 100-photon level detection limit at 180 Hz modulation bandwidth, which as far as we know are the highest values among all the reported perovskite photodetectors. The superior performance of the device originates from replacing polycrystalline thin film by a thickness-optimized SC-TF with much higher mobility and longer recombination time. The results indicate that high-performance perovskite devices based on SC-TF may become competitive in modern optoelectronics.

  2. Single-cell manipulation and DNA delivery technology using atomic force microscopy and nanoneedle.

    Science.gov (United States)

    Han, Sung-Woong; Nakamura, Chikashi; Miyake, Jun; Chang, Sang-Mok; Adachi, Taiji

    2014-01-01

    The recent single-cell manipulation technology using atomic force microscopy (AFM) not only allows high-resolution visualization and probing of biomolecules and cells but also provides spatial and temporal access to the interior of living cells via the nanoneedle technology. Here we review the development and application of single-cell manipulations and the DNA delivery technology using a nanoneedle. We briefly describe various DNA delivery methods and discuss their advantages and disadvantages. Fabrication of the nanoneedle, visualization of nanoneedle insertion into living cells, DNA modification on the nanoneedle surface, and the invasiveness of nanoneedle insertion into living cells are described. Different methods of DNA delivery into a living cell, such as lipofection, microinjection, and nanoneedles, are then compared. Finally, single-cell diagnostics using the nanoneedle and the perspectives of the nanoneedle technology are outlined. The nanoneedle-based DNA delivery technology provides new opportunities for efficient and specific introduction of DNA and other biomolecules into precious living cells with a high spatial resolution within a desired time frame. This technology has the potential to be applied for many basic cellular studies and for clinical studies such as single-cell diagnostics.

  3. Single-hit mechanism of tumour cell killing by radiation.

    Science.gov (United States)

    Chapman, J D

    2003-02-01

    To review the relative importance of the single-hit mechanism of radiation killing for tumour response to 1.8-2.0 Gy day(-1) fractions and to low dose-rate brachytherapy. Tumour cell killing by ionizing radiation is well described by the linear-quadratic equation that contains two independent components distinguished by dose kinetics. Analyses of tumour cell survival curves that contain six or more dose points usually provide good estimates of the alpha- and beta-inactivation coefficients. Superior estimates of tumour cell intrinsic radiosensitivity are obtained when synchronized populations are employed. The characteristics of single-hit inactivation of tumour cells are reviewed and compared with the characteristics of beta-inactivation. Potential molecular targets associated with single-hit inactivation are discussed along with strategies for potentiating cell killing by this mechanism. The single-hit mechanism of tumour cell killing shows no dependence on dose-rate and, consequently, no evidence of sublethal damage repair. It is uniquely potentiated by high linear-energy-transfer radiation, exhibits a smaller oxygen enhancement ratio and exhibits a larger indirect effect by hydroxyl radicals than the beta-mechanism. alpha-inactivation coefficients vary slightly throughout interphase but mitotic cells exhibit extremely high alpha-coefficients in the range of those observed for lymphocytes and some repair-deficient cells. Evidence is accumulating to suggest that chromatin in compacted form could be a radiation-hypersensitive target associated with single-hit radiation killing. Analyses of tumour cell survival curves demonstrate that it is the single-hit mechanism (alpha) that determines the majority of cell killing after doses of 2Gy and that this mechanism is highly variable between tumour cell lines. The characteristics of single-hit inactivation are qualitatively and quantitatively distinct from those of beta-inactivation. Compacted chromatin in tumour cells

  4. A novel single virus infection system reveals that influenza virus preferentially infects cells in g1 phase.

    Directory of Open Access Journals (Sweden)

    Ryuta Ueda

    Full Text Available BACKGROUND: Influenza virus attaches to sialic acid residues on the surface of host cells via the hemagglutinin (HA, a glycoprotein expressed on the viral envelope, and enters into the cytoplasm by receptor-mediated endocytosis. The viral genome is released and transported in to the nucleus, where transcription and replication take place. However, cellular factors affecting the influenza virus infection such as the cell cycle remain uncharacterized. METHODS/RESULTS: To resolve the influence of cell cycle on influenza virus infection, we performed a single-virus infection analysis using optical tweezers. Using this newly developed single-virus infection system, the fluorescence-labeled influenza virus was trapped on a microchip using a laser (1064 nm at 0.6 W, transported, and released onto individual H292 human lung epithelial cells. Interestingly, the influenza virus attached selectively to cells in the G1-phase. To clarify the molecular differences between cells in G1- and S/G2/M-phase, we performed several physical and chemical assays. Results indicated that: 1 the membranes of cells in G1-phase contained greater amounts of sialic acids (glycoproteins than the membranes of cells in S/G2/M-phase; 2 the membrane stiffness of cells in S/G2/M-phase is more rigid than those in G1-phase by measurement using optical tweezers; and 3 S/G2/M-phase cells contained higher content of Gb3, Gb4 and GlcCer than G1-phase cells by an assay for lipid composition. CONCLUSIONS: A novel single-virus infection system was developed to characterize the difference in influenza virus susceptibility between G1- and S/G2/M-phase cells. Differences in virus binding specificity were associated with alterations in the lipid composition, sialic acid content, and membrane stiffness. This single-virus infection system will be useful for studying the infection mechanisms of other viruses.

  5. Factors affecting the performance of a single-chamber microbial fuel cell-type biological oxygen demand sensor.

    Science.gov (United States)

    Yang, Gai-Xiu; Sun, Yong-Ming; Kong, Xiao-Ying; Zhen, Feng; Li, Ying; Li, Lian-Hua; Lei, Ting-Zhou; Yuan, Zhen-Hong; Chen, Guan-Yi

    2013-01-01

    Microbial fuel cells (MFCs) are devices that exploit microorganisms as biocatalysts to degrade organic matter or sludge present in wastewater (WW), and thereby generate electricity. We developed a simple, low-cost single-chamber microbial fuel cell (SCMFC)-type biochemical oxygen demand (BOD) sensor using carbon felt (anode) and activated sludge, and demonstrated its feasibility in the construction of a real-time BOD measurement system. Further, the effects of anodic pH and organic concentration on SCMFC performance were examined, and the correlation between BOD concentration and its response time was analyzed. Our results demonstrated that the SCMFC exhibited a stable voltage after 132 min following the addition of synthetic WW (BOD concentration: 200 mg/L). Notably, the response signal increased with an increase in BOD concentration (range: 5-200 mg/L) and was found to be directly proportional to the substrate concentration. However, at higher BOD concentrations (>120 mg/L) the response signal remained unaltered. Furthermore, we optimized the SCMFC using synthetic WW, and tested it with real WW. Upon feeding real WW, the BOD values exhibited a standard deviation from 2.08 to 8.3% when compared to the standard BOD5 method, thus demonstrating the practical applicability of the developed system to real treatment effluents.

  6. Development of Single Cell Raman Spectroscopy for Cancer Screening and Therapy Monitoring

    Energy Technology Data Exchange (ETDEWEB)

    Chan, James W. [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States)

    2009-02-24

    The overall goal of this project was to develop a new technology for cancer detection based on single cell laser tweezers Raman spectroscopy (LTRS). This method has the potential to improve the detection of cancer characteristics in single cells by acquiring cellular spectral markers that reflect the intrinsic biology of the cell. These spectral biomarkers are a new form of molecular signatures in the field of cancer research that may hold promise in accurately identifying and diagnosing cancer and measuring patient response to treatment. The primary objectives of this proposed work were to perform a full characterization of the Raman spectra of single normal, transformed, and cancer cells to identify cancer spectral signatures, validate the clinical significance of these results by direct correlation to established clinical parameters for assessing cancer, and to develop the optical technology needed for efficient sampling and analysis of cells needed for the practical use of such a system in the clinic. The results indicated that normal T and B lymphocytes could be distinguished from their neoplastic cultured cells and leukemia patient cells with classification sensitivities and specificities routinely exceeding 90% based on multivariate statistical analysis and leave-one-out cross validation. Differences primarily in the Raman peaks associated with DNA and protein were observed between normal and leukemic cells and were consistent for both the cultured and primary cells. Differences between normal and leukemia patient cells were more subtle than between normal and leukemia cultured cells but were still significant to allow for accurate discrimination. Furthermore, it is revealed that the spectral differences are representative of the neoplastic phenotype of the cells and not a reflection of the different metabolic states (resting versus active) of normal and leukemic cells. The effect of different standard cell fixation protocols (i.e. methanol, paraformaledhye

  7. In situ single molecule imaging of cell membranes: linking basic nanotechniques to cell biology, immunology and medicine

    Science.gov (United States)

    Pi, Jiang; Jin, Hua; Yang, Fen; Chen, Zheng W.; Cai, Jiye

    2014-10-01

    The cell membrane, which consists of a viscous phospholipid bilayer, different kinds of proteins and various nano/micrometer-sized domains, plays a very important role in ensuring the stability of the intracellular environment and the order of cellular signal transductions. Exploring the precise cell membrane structure and detailed functions of the biomolecules in a cell membrane would be helpful to understand the underlying mechanisms involved in cell membrane signal transductions, which could further benefit research into cell biology, immunology and medicine. The detection of membrane biomolecules at the single molecule level can provide some subtle information about the molecular structure and the functions of the cell membrane. In particular, information obtained about the molecular mechanisms and other information at the single molecule level are significantly different from that detected from a large amount of biomolecules at the large-scale through traditional techniques, and can thus provide a novel perspective for the study of cell membrane structures and functions. However, the precise investigations of membrane biomolecules prompts researchers to explore cell membranes at the single molecule level by the use of in situ imaging methods, as the exact conformation and functions of biomolecules are highly controlled by the native cellular environment. Recently, the in situ single molecule imaging of cell membranes has attracted increasing attention from cell biologists and immunologists. The size of biomolecules and their clusters on the cell surface are set at the nanoscale, which makes it mandatory to use high- and super-resolution imaging techniques to realize the in situ single molecule imaging of cell membranes. In the past few decades, some amazing imaging techniques and instruments with super resolution have been widely developed for molecule imaging, which can also be further employed for the in situ single molecule imaging of cell membranes. In

  8. Performance of a vanadium redox flow battery with tubular cell design

    Science.gov (United States)

    Ressel, Simon; Laube, Armin; Fischer, Simon; Chica, Antonio; Flower, Thomas; Struckmann, Thorsten

    2017-07-01

    We present a vanadium redox flow battery with a tubular cell design which shall lead to a reduction of cell manufacturing costs and the realization of cell stacks with reduced shunt current losses. Charge/discharge cycling and polarization curve measurements are performed to characterize the single test cell performance. A maximum current density of 70 mAcm-2 and power density of 142 Wl-1 (per cell volume) is achieved and Ohmic overpotential is identified as the dominant portion of the total cell overpotential. Cycling displays Coulomb efficiencies of ≈95% and energy efficiencies of ≈55%. During 113 h of operation a stable Ohmic cell resistance is observed.

  9. RF Breakdown in Normal Conducting Single-Cell Structures

    International Nuclear Information System (INIS)

    Dolgashev, V.A.; Nantista, C.D.; Tantawi, S.G.; Higashi, Y.; Higo, T.

    2006-01-01

    Operating accelerating gradient in normal conducting accelerating structures is often limited by rf breakdown. The limit depends on multiple parameters, including input rf power, rf circuit, cavity shape and material. Experimental and theoretical study of the effects of these parameters on the breakdown limit in full scale structures is difficult and costly. We use 11.4 GHz single-cell traveling wave and standing wave accelerating structures for experiments and modeling of rf breakdown behavior. These test structures are designed so that the electromagnetic fields in one cell mimic the fields in prototype multicell structures for the X-band linear collider. Fields elsewhere in the test structures are significantly lower than that of the single cell. The setup uses matched mode converters that launch the circular TM 01 mode into short test structures. The test structures are connected to the mode launchers with vacuum rf flanges. This setup allows economic testing of different cell geometries, cell materials and preparation techniques with short turn-around time. Simple 2D geometry of the test structures simplifies modeling of the breakdown currents and their thermal effects

  10. STUDY OF PERFORMANCES OF ORGANIC SOLAR CELLS BY ...

    African Journals Online (AJOL)

    30 juin 2011 ... results of analysis of performances of organic solar cells by using what one call the datamining materials. ... Keywords: organic solar cells, gap energie, effiency, PCA. Author Correspondence .... oubli est malencontreux car le type de données disponibles influence toujours la direction de la recherche.

  11. Testing And Performance Analysis Of NASA 5 CM BY 5 CM Bi-Supported Solid Oxide Electrolysis Cells Operated In Both Fuel Cell And Steam Electrolysis Modes

    International Nuclear Information System (INIS)

    O'Brien, R.C.; O'Brien, J.E.; Stoots, C.M.; Zhang, X.; Farmer, S.C.; Cable, T.L.; Setlock, J.A.

    2011-01-01

    A series of 5 cm by 5 cm bi-supported Solid Oxide Electrolysis Cells (SOEC) were produced by NASA for the Idaho National Laboratory (INL) and tested under the INL High Temperature Steam Electrolysis program. The results from the experimental demonstration of cell operation for both hydrogen production and operation as fuel cells is presented. An overview of the cell technology, test apparatus and performance analysis is also provided. The INL High Temperature Steam Electrolysis laboratory has developed significant test infrastructure in support of single cell and stack performance analyses. An overview of the single cell test apparatus is presented. The test data presented in this paper is representative of a first batch of NASA's prototypic 5 cm by 5 cm SOEC single cells. Clearly a significant relationship between the operational current density and cell degradation rate is evident. While the performance of these cells was lower than anticipated, in-house testing at NASA Glenn has yielded significantly higher performance and lower degradation rates with subsequent production batches of cells. Current post-test microstructure analyses of the cells tested at INL will be published in a future paper. Modification to cell compositions and cell reduction techniques will be altered in the next series of cells to be delivered to INL with the aim to decrease the cell degradation rate while allowing for higher operational current densities to be sustained. Results from the testing of new batches of single cells will be presented in a future paper.

  12. Recombinant human albumin supports single cell cloning of CHO cells in chemically defined media.

    Science.gov (United States)

    Zhu, Jiang; Wooh, Jong Wei; Hou, Jeff Jia Cheng; Hughes, Benjamin S; Gray, Peter P; Munro, Trent P

    2012-01-01

    Biologic drugs, such as monoclonal antibodies, are commonly made using mammalian cells in culture. The cell lines used for manufacturing should ideally be clonal, meaning derived from a single cell, which represents a technically challenging process. Fetal bovine serum is often used to support low cell density cultures, however, from a regulatory perspective, it is preferable to avoid animal-derived components to increase process consistency and reduce the risk of contamination from adventitious agents. Chinese hamster ovary (CHO) cells are the most widely used cell line in industry and a large number of serum-free, protein-free, and fully chemically defined growth media are commercially available, although these media alone do not readily support efficient single cell cloning. In this work, we have developed a simple, fully defined, single-cell cloning media, specifically for CHO cells, using commercially available reagents. Our results show that a 1:1 mixture of CD-CHO™ and DMEM/F12 supplemented with 1.5 g/L of recombinant albumin (Albucult®) supports single cell cloning. This formulation can support recovery of single cells in 43% of cultures compared to 62% in the presence of serum. Copyright © 2012 American Institute of Chemical Engineers (AIChE).

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

    Science.gov (United States)

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

    2016-04-01

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

  14. Capillary electrophoretic study of individual exocytotic events in single mast cells

    Energy Technology Data Exchange (ETDEWEB)

    Ho, Andrea Ming-Wei [Iowa State Univ., Ames, IA (United States)

    1999-02-12

    The peak profile of individual degranulation events from the on-column release of serotonin from single rat peritoneal mast cells (RPMCs) was monitored using capillary electrophoresis with laser-induced native fluorescence detection (CE-LINF). Serotonin, an important biogenic amine, is contained in granules (0.25 fL) within RPMCs and is extruded by a process termed exocytosis. The secretagogue, Polymyxin B sulfate, was used as the CE running buffer after injection of a single RPMC into the separation capillary to stimulate the release of the granules. Because the release process occurs on a ms time scale, monitoring individual exocytotic events is possible with the coupling of high-speed CE and LINF detection.

  15. Spatial reconstruction of single-cell gene expression

    Science.gov (United States)

    Satija, Rahul; Farrell, Jeffrey A.; Gennert, David; Schier, Alexander F.; Regev, Aviv

    2015-01-01

    Spatial localization is a key determinant of cellular fate and behavior, but spatial RNA assays traditionally rely on staining for a limited number of RNA species. In contrast, single-cell RNA-seq allows for deep profiling of cellular gene expression, but established methods separate cells from their native spatial context. Here we present Seurat, a computational strategy to infer cellular localization by integrating single-cell RNA-seq data with in situ RNA patterns. We applied Seurat to spatially map 851 single cells from dissociated zebrafish (Danio rerio) embryos, inferring a transcriptome-wide map of spatial patterning. We confirmed Seurat’s accuracy using several experimental approaches, and used it to identify a set of archetypal expression patterns and spatial markers. Additionally, Seurat correctly localizes rare subpopulations, accurately mapping both spatially restricted and scattered groups. Seurat will be applicable to mapping cellular localization within complex patterned tissues in diverse systems. PMID:25867923

  16. Experimental study on the performance of single screw expanders by gap adjustment

    International Nuclear Information System (INIS)

    Wang, Wei; Wu, Yu-ting; Ma, Chong-fang; Xia, Guo-dong; Wang, Jing-fu

    2013-01-01

    Improving thermodynamic efficiency of prime movers is the key issue for efficient utilization of low-temperature heat resources. Single screw expander may be a good candidate because of its many good characteristics. Precisions in manufacture and assembly are very important factors to the performance of single screw expanders. In this paper, the shaft efficiency, volumetric efficiency and gas consumption rate of the single screw expander prototypes were tested and discussed. We have manufactured three prototypes with different gaps to investigate their performance. The first prototype (A) has the largest gap, and the second one (B) has the smallest gap, the third prototype (C) has the medium gap configuration. Experimental result of the prototypes was obtained. From the experimental data of prototype A, the power output was about 5 kW, the gas consumption rate was above 105 kg/kWh and the volumetric efficiency was below 20%, the shaft efficiency was only 34%. From experimental data of prototype B, the mass flow rate was significantly decreased. The power output was only 1.4 kW and the volumetric efficiency was slightly lower than prototype A. The gas consumption rate was much more than that of prototype A. From the experimental data of prototype C, the power output was about 4.5 kW, but the mass flow rate was sharply decreased. The gas consumption rate was about 65 kg/kWh, the maximum volumetric efficiency was about 66%, and the shaft efficiency was about 60%. The experimental results indicated that prototype C of single screw expanders had the best overall performance, which may be further improved by optimizing its configuration. - Highlights: • The experiment for three single screw expander prototypes at different gaps was carried out. • Maximum shaft efficiency is about 60%. • Minimum gas consumption rate is about 65 kg/kWh. • Maximum volumetric efficiency is about 66%. • Leaked gas still has part of working capability

  17. Single chamber microbial fuel cell with spiral anode for dairy wastewater treatment.

    Science.gov (United States)

    Mardanpour, Mohammad Mahdi; Nasr Esfahany, Mohsen; Behzad, Tayebeh; Sedaqatvand, Ramin

    2012-01-01

    This study reports on the fabrication of a novel annular single chamber microbial fuel cell (ASCMFC) with spiral anode. The stainless steel mesh anode with graphite coating was used as anode. Dairy wastewater, containing complex organic matter, was used as substrate. ASCMFC had been operated for 450 h and results indicated a high open circuit voltage (about 810 mV) compared with previously published results. The maximum power density of 20.2 W/m(3) obtained in this study is significantly greater than the power densities reported in previous studies. Besides, a maximum coulombic efficiency of 26.87% with 91% COD removal was achieved. Good bacterial adhesion on the spiral anode is clearly shown in SEM micrographs. High power density and a successful performance in wastewater treatment in ASCMFC suggest it as a promising alternative to conventional MFCs for power generation and wastewater treatment. ASCMFC performance as a power generator was characterized based on polarization behavior and cell potentials. Copyright © 2012 Elsevier B.V. All rights reserved.

  18. Raman tweezers spectroscopy of live, single red and white blood cells.

    Directory of Open Access Journals (Sweden)

    Aseefhali Bankapur

    Full Text Available An optical trap has been combined with a Raman spectrometer to make high-resolution measurements of Raman spectra of optically-immobilized, single, live red (RBC and white blood cells (WBC under physiological conditions. Tightly-focused, near infrared wavelength light (1064 nm is utilized for trapping of single cells and 785 nm light is used for Raman excitation at low levels of incident power (few mW. Raman spectra of RBC recorded using this high-sensitivity, dual-wavelength apparatus has enabled identification of several additional lines; the hitherto-unreported lines originate purely from hemoglobin molecules. Raman spectra of single granulocytes and lymphocytes are interpreted on the basis of standard protein and nucleic acid vibrational spectroscopy data. The richness of the measured spectrum illustrates that Raman studies of live cells in suspension are more informative than conventional micro-Raman studies where the cells are chemically bound to a glass cover slip.

  19. Assessing T cell differentiation at the single-cell level

    NARCIS (Netherlands)

    Gerlach, Carmen

    2012-01-01

    This thesis describes the development and use of a novel technology for single-cell fate mapping, called cellular barcoding. With this technology, unique and heritable genetic tags (barcodes) are introduced into naïve T cells. Using cellular barcoding, we investigated I) how different

  20. Voltage controlled nano-injection system for single-cell surgery

    Science.gov (United States)

    Seger, R. Adam; Actis, Paolo; Penfold, Catherine; Maalouf, Michelle; Vilozny, Boaz; Pourmand, Nader

    2015-01-01

    Manipulation and analysis of single cells is the next frontier in understanding processes that control the function and fate of cells. Herein we describe a single-cell injection platform based on nanopipettes. The system uses scanning microscopy techniques to detect cell surfaces, and voltage pulses to deliver molecules into individual cells. As a proof of concept, we injected adherent mammalian cells with fluorescent dyes. PMID:22899383

  1. Experimental and Computer Modelling Studies of Metastability of Amorphous Silicon Based Solar Cells

    NARCIS (Netherlands)

    Munyeme, Geoffrey

    2003-01-01

    We present a combination of experimental and computer modelling studies of the light induced degradation in the performance of amorphous silicon based single junction solar cells. Of particular interest in this study is the degradation kinetics of different types of amorphous silicon single junction

  2. Amrubicin therapy improves patients with refractory small-cell lung cancer: A single-arm confirmatory Chinese clinical study

    Directory of Open Access Journals (Sweden)

    Mengli Zheng

    2016-09-01

    Full Text Available Our objective was to evaluate an open-label, multicenter, single-arm study to appraise whether amrubicin therapy improves patients with refractory small-cell lung cancer in Chinese clinical study. Patients (n=95 with refractory small-cell lung cancer received 3 consecutive days amrubicin therapy for 21 days. Overall response rate of response to amrubicin was 39%. Anemia, febrile neutropenia, thrombocytopenia, hyperglycemia, hyponatremia, infection, elevated serum transaminases levels were appeared, but the incidences of adverse events were very few. Our results suggest amrubicin therapy can improve patients with refractory small-cell lung cancer and may be an effective and safe treatment option.

  3. High-pressure raman study on single crystalline methane hydrate surrounded by methane in a diamond anvil cell

    International Nuclear Information System (INIS)

    Ohno, Y; Sasaki, S; Kume, T; Shimizu, H

    2008-01-01

    High-pressure Raman measurements have been performed for single crystalline methane hydrate (MH) surrounded by fluid or solid methane in a diamond anvil cell. We successfully obtained the pure O-H stretching and lattice vibration spectra in MH-sI and MH-II phases. In these Raman spectra, there is no Raman band from water or ice-VI. The observed pressure of phase transformation from MH-sI to MH-II is 0.9 GPa, which is the same result as methane hydrate surrounded by water

  4. Single-cell force spectroscopy as a technique to quantify human red blood cell adhesion to subendothelial laminin.

    Science.gov (United States)

    Maciaszek, Jamie L; Partola, Kostyantyn; Zhang, Jing; Andemariam, Biree; Lykotrafitis, George

    2014-12-18

    Single-cell force spectroscopy (SCFS), an atomic force microscopy (AFM)-based assay, enables quantitative study of cell adhesion while maintaining the native state of surface receptors in physiological conditions. Human healthy and pathological red blood cells (RBCs) express a large number of surface proteins which mediate cell-cell interactions, or cell adhesion to the extracellular matrix. In particular, RBCs adhere with high affinity to subendothelial matrix laminin via the basal cell adhesion molecule and Lutheran protein (BCAM/Lu). Here, we established SCFS as an in vitro technique to study human RBC adhesion at baseline and following biochemical treatment. Using blood obtained from healthy human subjects, we recorded adhesion forces from single RBCs attached to AFM cantilevers as the cell was pulled-off of substrates coated with laminin protein. We found that an increase in the overall cell adhesion measured via SCFS is correlated with an increase in the resultant total force measured on 1 µm(2) areas of the RBC membrane. Further, we showed that SCFS can detect significant changes in the adhesive response of RBCs to modulation of the cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) pathway. Lastly, we identified variability in the RBC adhesion force to laminin amongst the human subjects, suggesting that RBCs maintain diverse levels of active BCAM/Lu adhesion receptors. By using single-cell measurements, we established a powerful new method for the quantitative measurement of single RBC adhesion with specific receptor-mediated binding. Copyright © 2014 Elsevier Ltd. All rights reserved.

  5. The role of nanotechnology in single-cell detection: a review.

    Science.gov (United States)

    Wang, Changling; Zhang, Yuxiang; Xia, Mingdian; Zhu, Xingxi; Qi, Shitao; Shen, Huaqiang; Liu, Tiebing; Tang, Liming

    2014-10-01

    Biological processes in single cells, such as signal transduction, DNA duplication, and protein synthesis and trafficking, occur in subcellular compartments at nanoscale level. Achieving high spatial-temporal resolution, high sensitivity, and high specificity in single-cell detection poses a great challenge. Nanotechnology, which has been widely applied in the fields of medicine, electronics, biomaterials, and energy production, has the potential to provide solutions for single-cell detection. Here we present a review of the use of nanotechnology in single-cell detection over the past two decades. First, we review the main areas of scientific interest, including morphology, ion concentration, DNA, RNA, protein, intracellular temperature, elements, and mechanical properties. Second, four categories of application of nanotechnology to single-cell detection are described: nanomanipulation, nanodevices, nanomaterials as labels, and nano Secondary ion mass spectrometry. Finally, the prospects and future trends in single-cell detection and analysis are discussed.

  6. Microfluidic device for continuous single cells analysis via Raman spectroscopy enhanced by integrated plasmonic nanodimers

    DEFF Research Database (Denmark)

    Perozziello, Gerardo; Candeloro, Patrizio; De Grazia, Antonio

    2016-01-01

    In this work a Raman flow cytometer is presented. It consists of a microfluidic device that takes advantages of the basic principles of Raman spectroscopy and flow cytometry. The microfluidic device integrates calibrated microfluidic channels-where the cells can flow one-by-one -, allowing single...... cell Raman analysis. The microfluidic channel integrates plasmonic nanodimers in a fluidic trapping region. In this way it is possible to perform Enhanced Raman Spectroscopy on single cell. These allow a label-free analysis, providing information about the biochemical content of membrane and cytoplasm...

  7. Proximity-Based Differential Single-Cell Analysis of the Niche to Identify Stem/Progenitor Cell Regulators.

    Science.gov (United States)

    Silberstein, Lev; Goncalves, Kevin A; Kharchenko, Peter V; Turcotte, Raphael; Kfoury, Youmna; Mercier, Francois; Baryawno, Ninib; Severe, Nicolas; Bachand, Jacqueline; Spencer, Joel A; Papazian, Ani; Lee, Dongjun; Chitteti, Brahmananda Reddy; Srour, Edward F; Hoggatt, Jonathan; Tate, Tiffany; Lo Celso, Cristina; Ono, Noriaki; Nutt, Stephen; Heino, Jyrki; Sipilä, Kalle; Shioda, Toshihiro; Osawa, Masatake; Lin, Charles P; Hu, Guo-Fu; Scadden, David T

    2016-10-06

    Physiological stem cell function is regulated by secreted factors produced by niche cells. In this study, we describe an unbiased approach based on the differential single-cell gene expression analysis of mesenchymal osteolineage cells close to, and further removed from, hematopoietic stem/progenitor cells (HSPCs) to identify candidate niche factors. Mesenchymal cells displayed distinct molecular profiles based on their relative location. We functionally examined, among the genes that were preferentially expressed in proximal cells, three secreted or cell-surface molecules not previously connected to HSPC biology-the secreted RNase angiogenin, the cytokine IL18, and the adhesion molecule Embigin-and discovered that all of these factors are HSPC quiescence regulators. Therefore, our proximity-based differential single-cell approach reveals molecular heterogeneity within niche cells and can be used to identify novel extrinsic stem/progenitor cell regulators. Similar approaches could also be applied to other stem cell/niche pairs to advance the understanding of microenvironmental regulation of stem cell function. Copyright © 2016 Elsevier Inc. All rights reserved.

  8. The comparison between gallium arsenide and indium gallium arsenide as materials for solar cell performance using Silvaco application

    Science.gov (United States)

    Zahari, Suhaila Mohd; Norizan, Mohd Natashah; Mohamad, Ili Salwani; Osman, Rozana Aina Maulat; Taking, Sanna

    2015-05-01

    The work presented in this paper is about the development of single and multilayer solar cells using GaAs and InGaAs in AM1.5 condition. The study includes the modeling structure and simulation of the device using Silvaco applications. The performance in term of efficiency of Indium Gallium Arsenide (InGaAs) and GaAs material was studied by modification of the doping concentration and thickness of material in solar cells. The efficiency of the GaAs solar cell was higher than InGaAs solar cell for single layer solar cell. Single layer GaAs achieved an efficiency about 25% compared to InGaAs which is only 2.65% of efficiency. For multilayer which includes both GaAs and InGaAs, the output power, Pmax was 8.91nW/cm² with the efficiency only 8.51%. GaAs is one of the best materials to be used in solar cell as a based compared to InGaAs.

  9. The comparison between gallium arsenide and indium gallium arsenide as materials for solar cell performance using Silvaco application

    International Nuclear Information System (INIS)

    Zahari, Suhaila Mohd; Norizan, Mohd Natashah; Mohamad, Ili Salwani; Osman, Rozana Aina Maulat; Taking, Sanna

    2015-01-01

    The work presented in this paper is about the development of single and multilayer solar cells using GaAs and InGaAs in AM1.5 condition. The study includes the modeling structure and simulation of the device using Silvaco applications. The performance in term of efficiency of Indium Gallium Arsenide (InGaAs) and GaAs material was studied by modification of the doping concentration and thickness of material in solar cells. The efficiency of the GaAs solar cell was higher than InGaAs solar cell for single layer solar cell. Single layer GaAs achieved an efficiency about 25% compared to InGaAs which is only 2.65% of efficiency. For multilayer which includes both GaAs and InGaAs, the output power, P max was 8.91nW/cm² with the efficiency only 8.51%. GaAs is one of the best materials to be used in solar cell as a based compared to InGaAs

  10. Single-cell atomic quantum memory for light

    International Nuclear Information System (INIS)

    Opatrny, Tomas

    2006-01-01

    Recent experiments demonstrating atomic quantum memory for light [B. Julsgaard et al., Nature 432, 482 (2004)] involve two macroscopic samples of atoms, each with opposite spin polarization. It is shown here that a single atomic cell is enough for the memory function if the atoms are optically pumped with suitable linearly polarized light, and quadratic Zeeman shift and/or ac Stark shift are used to manipulate rotations of the quadratures. This should enhance the performance of our quantum memory devices since less resources are needed and losses of light in crossing different media boundaries are avoided

  11. Response of Listeria monocytogenes to disinfection stress at the single-cell and population levels as monitored by intracellular pH measurements and viable-cell counts

    DEFF Research Database (Denmark)

    Kastbjerg, Vicky Gaedt; Nielsen, Dennis S.; Arneborg, Nils

    2009-01-01

    of the bacterium. In situ analyses of Listeria monocytogenes single cells were performed during exposure to different concentrations of the disinfectant Incimaxx DES to study a possible population subdivision. Bacterial survival was quantified with plate counting and disinfection stress at the single-cell level...... by measuring intracellular pH (pHi) over time by fluorescence ratio imaging microscopy. pHi values were initially 7 to 7.5 and decreased in both attached and planktonic L. monocytogenes cells during exposure to sublethal and lethal concentrations of Incimaxx DES. The response of the bacterial population...... was homogenous; hence, subpopulations were not detected. However, pregrowth with NaCl protected the planktonic bacterial cells during disinfection with Incimaxx (0.0015%) since pHi was higher (6 to 6.5) for the bacterial population pregrown with NaCl than for cells grown without NaCl (pHi 5 to 5.5) (P

  12. Design and analysis of single- ended robust low power 8T SRAM cell

    Directory of Open Access Journals (Sweden)

    Gupta Neha

    2016-01-01

    Full Text Available This paper is based on the observation of 8T single ended static random access memory (SRAM and two techniques for reducing the sub threshold leakage current, power consumption are examined. In the first technique, effective supply voltage and ground node voltages are changed using a dynamic variable voltage level technique(VVL. In the second technique power supply is scaled down. This 8T SRAM cell uses one word line, two bitlinesand a transmission gate. Simulations and analytical results show that when the two techniques combine the new SRAM cell has correct read and write operation and also the cell contains 55.6% less leakage and the dynamic power is 98.8% less than the 8T single ended SRAM cell. Simulations are performed using cadence virtuoso tool at 45nm technology.

  13. Long Term Performance Study of a Direct Methanol Fuel Cell Fed with Alcohol Blends

    Directory of Open Access Journals (Sweden)

    Eleuterio Mora

    2013-01-01

    Full Text Available The use of alcohol blends in direct alcohol fuel cells may be a more environmentally friendly and less toxic alternative to the use of methanol alone in direct methanol fuel cells. This paper assesses the behaviour of a direct methanol fuel cell fed with aqueous methanol, aqueous ethanol and aqueous methanol/ethanol blends in a long term experimental study followed by modelling of polarization curves. Fuel cell performance is seen to decrease as the ethanol content rises, and subsequent operation with aqueous methanol only partly reverts this loss of performance. It seems that the difference in the oxidation rate of these alcohols may not be the only factor affecting fuel cell performance.

  14. Temporal dynamics and transcriptional control using single-cell gene expression analysis.

    Science.gov (United States)

    Kouno, Tsukasa; de Hoon, Michiel; Mar, Jessica C; Tomaru, Yasuhiro; Kawano, Mitsuoki; Carninci, Piero; Suzuki, Harukazu; Hayashizaki, Yoshihide; Shin, Jay W

    2013-01-01

    Changes in environmental conditions lead to expression variation that manifest at the level of gene regulatory networks. Despite a strong understanding of the role noise plays in synthetic biological systems, it remains unclear how propagation of expression heterogeneity in an endogenous regulatory network is distributed and utilized by cells transitioning through a key developmental event. Here we investigate the temporal dynamics of a single-cell transcriptional network of 45 transcription factors in THP-1 human myeloid monocytic leukemia cells undergoing differentiation to macrophages. We systematically measure temporal regulation of expression and variation by profiling 120 single cells at eight distinct time points, and infer highly controlled regulatory modules through which signaling operates with stochastic effects. This reveals dynamic and specific rewiring as a cellular strategy for differentiation. The integration of both positive and negative co-expression networks further identifies the proto-oncogene MYB as a network hinge to modulate both the pro- and anti-differentiation pathways. Compared to averaged cell populations, temporal single-cell expression profiling provides a much more powerful technique to probe for mechanistic insights underlying cellular differentiation. We believe that our approach will form the basis of novel strategies to study the regulation of transcription at a single-cell level.

  15. TESTING AND PERFORMANCE ANALYSIS OF NASA 5 CM BY 5 CM BI-SUPPORTED SOLID OXIDE ELECTROLYSIS CELLS OPERATED IN BOTH FUEL CELL AND STEAM ELECTROLYSIS MODES

    Energy Technology Data Exchange (ETDEWEB)

    R. C. O' Brien; J. E. O' Brien; C. M. Stoots; X. Zhang; S. C. Farmer; T. L. Cable; J. A. Setlock

    2011-11-01

    A series of 5 cm by 5 cm bi-supported Solid Oxide Electrolysis Cells (SOEC) were produced by NASA for the Idaho National Laboratory (INL) and tested under the INL High Temperature Steam Electrolysis program. The results from the experimental demonstration of cell operation for both hydrogen production and operation as fuel cells is presented. An overview of the cell technology, test apparatus and performance analysis is also provided. The INL High Temperature Steam Electrolysis laboratory has developed significant test infrastructure in support of single cell and stack performance analyses. An overview of the single cell test apparatus is presented. The test data presented in this paper is representative of a first batch of NASA's prototypic 5 cm by 5 cm SOEC single cells. Clearly a significant relationship between the operational current density and cell degradation rate is evident. While the performance of these cells was lower than anticipated, in-house testing at NASA Glenn has yielded significantly higher performance and lower degradation rates with subsequent production batches of cells. Current post-test microstructure analyses of the cells tested at INL will be published in a future paper. Modification to cell compositions and cell reduction techniques will be altered in the next series of cells to be delivered to INL with the aim to decrease the cell degradation rate while allowing for higher operational current densities to be sustained. Results from the testing of new batches of single cells will be presented in a future paper.

  16. In situ probing of cholesterol in astrocytes at the single-cell level using laser desorption ionization mass spectrometric imaging with colloidal silver.

    Science.gov (United States)

    Perdian, D C; Cha, Sangwon; Oh, Jisun; Sakaguchi, Donald S; Yeung, Edward S; Lee, Young Jin

    2010-04-30

    Mass spectrometric imaging has been utilized to localize individual astrocytes and to obtain cholesterol populations at the single-cell level in laser desorption ionization (LDI) with colloidal silver. The silver ion adduct of membrane-bound cholesterol was monitored to detect individual cells. Good correlation between mass spectrometric and optical images at different cell densities indicates the ability to perform single-cell studies of cholesterol abundance. The feasibility of quantification is confirmed by the agreement between the LDI-MS ion signals and the results from a traditional enzymatic fluorometric assay. We propose that this approach could be an effective tool to study chemical populations at the cellular level. Published in 2010 by John Wiley & Sons, Ltd.

  17. Massively parallel whole genome amplification for single-cell sequencing using droplet microfluidics.

    Science.gov (United States)

    Hosokawa, Masahito; Nishikawa, Yohei; Kogawa, Masato; Takeyama, Haruko

    2017-07-12

    Massively parallel single-cell genome sequencing is required to further understand genetic diversities in complex biological systems. Whole genome amplification (WGA) is the first step for single-cell sequencing, but its throughput and accuracy are insufficient in conventional reaction platforms. Here, we introduce single droplet multiple displacement amplification (sd-MDA), a method that enables massively parallel amplification of single cell genomes while maintaining sequence accuracy and specificity. Tens of thousands of single cells are compartmentalized in millions of picoliter droplets and then subjected to lysis and WGA by passive droplet fusion in microfluidic channels. Because single cells are isolated in compartments, their genomes are amplified to saturation without contamination. This enables the high-throughput acquisition of contamination-free and cell specific sequence reads from single cells (21,000 single-cells/h), resulting in enhancement of the sequence data quality compared to conventional methods. This method allowed WGA of both single bacterial cells and human cancer cells. The obtained sequencing coverage rivals those of conventional techniques with superior sequence quality. In addition, we also demonstrate de novo assembly of uncultured soil bacteria and obtain draft genomes from single cell sequencing. This sd-MDA is promising for flexible and scalable use in single-cell sequencing.

  18. Distribution of inorganic elements in single cells of Chara corallina

    International Nuclear Information System (INIS)

    Li Zijie; Zhang Zhiyong; Chai Zhifang; Yu Ming; Zhou Yunlong

    2005-01-01

    There are actually 20 chemical elements necessary or beneficial for plant growth. Carbon, hydrogen, and oxygen are supplied by air and water. The six macronutrients, nitrogen, phosphorus, potassium., calcium, magnesium, and sulfur are required by plants in large amounts. The rest of the elements are required in trace amounts (micronutrients). Essential trace elements include boron, chlorine, copper, iron, manganese, sodium, zinc, molybdenum, and nickel. Beneficial mineral elements include silicon and cobalt. The functions of the inorganic elements closely related to their destinations in plant cells. Plant cells have unique structures, including a central vacuole, plastids, and a thick cell wall that surrounds the cell membrane. Generally, it is very difficult to determine concentrations of inorganic elements in a single plant cell. Chara corallina is a freshwater plant that inhabits temperate zone ponds and lakes. It consists of alternating nodes and internodes. Each internodal segment is a single large cell, up to 10 cm in length, and 1 mm in diameter. With this species it was possible to isolate subcellular fractions with surgical methods with minimal risk of cross contamination. In this study, concentrations of magnesium, calcium, manganese, iron, copper, zinc, and molybdenum in the cell wall, cytoplasm, and vacuole of single cells of Chara corallina were determined by inductively coupled plasma mass spectrometry (ICP-MS). The distribution characteristics of these elements in the cell components were discussed.

  19. Sampling strategies to capture single-cell heterogeneity

    OpenAIRE

    Satwik Rajaram; Louise E. Heinrich; John D. Gordan; Jayant Avva; Kathy M. Bonness; Agnieszka K. Witkiewicz; James S. Malter; Chloe E. Atreya; Robert S. Warren; Lani F. Wu; Steven J. Altschuler

    2017-01-01

    Advances in single-cell technologies have highlighted the prevalence and biological significance of cellular heterogeneity. A critical question is how to design experiments that faithfully capture the true range of heterogeneity from samples of cellular populations. Here, we develop a data-driven approach, illustrated in the context of image data, that estimates the sampling depth required for prospective investigations of single-cell heterogeneity from an existing collection of samples. ...

  20. Accurately tracking single-cell movement trajectories in microfluidic cell sorting devices.

    Science.gov (United States)

    Jeong, Jenny; Frohberg, Nicholas J; Zhou, Enlu; Sulchek, Todd; Qiu, Peng

    2018-01-01

    Microfluidics are routinely used to study cellular properties, including the efficient quantification of single-cell biomechanics and label-free cell sorting based on the biomechanical properties, such as elasticity, viscosity, stiffness, and adhesion. Both quantification and sorting applications require optimal design of the microfluidic devices and mathematical modeling of the interactions between cells, fluid, and the channel of the device. As a first step toward building such a mathematical model, we collected video recordings of cells moving through a ridged microfluidic channel designed to compress and redirect cells according to cell biomechanics. We developed an efficient algorithm that automatically and accurately tracked the cell trajectories in the recordings. We tested the algorithm on recordings of cells with different stiffness, and showed the correlation between cell stiffness and the tracked trajectories. Moreover, the tracking algorithm successfully picked up subtle differences of cell motion when passing through consecutive ridges. The algorithm for accurately tracking cell trajectories paves the way for future efforts of modeling the flow, forces, and dynamics of cell properties in microfluidics applications.

  1. Accurately tracking single-cell movement trajectories in microfluidic cell sorting devices.

    Directory of Open Access Journals (Sweden)

    Jenny Jeong

    Full Text Available Microfluidics are routinely used to study cellular properties, including the efficient quantification of single-cell biomechanics and label-free cell sorting based on the biomechanical properties, such as elasticity, viscosity, stiffness, and adhesion. Both quantification and sorting applications require optimal design of the microfluidic devices and mathematical modeling of the interactions between cells, fluid, and the channel of the device. As a first step toward building such a mathematical model, we collected video recordings of cells moving through a ridged microfluidic channel designed to compress and redirect cells according to cell biomechanics. We developed an efficient algorithm that automatically and accurately tracked the cell trajectories in the recordings. We tested the algorithm on recordings of cells with different stiffness, and showed the correlation between cell stiffness and the tracked trajectories. Moreover, the tracking algorithm successfully picked up subtle differences of cell motion when passing through consecutive ridges. The algorithm for accurately tracking cell trajectories paves the way for future efforts of modeling the flow, forces, and dynamics of cell properties in microfluidics applications.

  2. Dissecting Transcriptional Heterogeneity in Pluripotency: Single Cell Analysis of Mouse Embryonic Stem Cells.

    Science.gov (United States)

    Guedes, Ana M V; Henrique, Domingos; Abranches, Elsa

    2016-01-01

    Mouse Embryonic Stem cells (mESCs) show heterogeneous and dynamic expression of important pluripotency regulatory factors. Single-cell analysis has revealed the existence of cell-to-cell variability in the expression of individual genes in mESCs. Understanding how these heterogeneities are regulated and what their functional consequences are is crucial to obtain a more comprehensive view of the pluripotent state.In this chapter we describe how to analyze transcriptional heterogeneity by monitoring gene expression of Nanog, Oct4, and Sox2, using single-molecule RNA FISH in single mESCs grown in different cell culture medium. We describe in detail all the steps involved in the protocol, from RNA detection to image acquisition and processing, as well as exploratory data analysis.

  3. Characterizing human stem cell-derived sensory neurons at the single-cell level reveals their ion channel expression and utility in pain research.

    Science.gov (United States)

    Young, Gareth T; Gutteridge, Alex; Fox, Heather DE; Wilbrey, Anna L; Cao, Lishuang; Cho, Lily T; Brown, Adam R; Benn, Caroline L; Kammonen, Laura R; Friedman, Julia H; Bictash, Magda; Whiting, Paul; Bilsland, James G; Stevens, Edward B

    2014-08-01

    The generation of human sensory neurons by directed differentiation of pluripotent stem cells opens new opportunities for investigating the biology of pain. The inability to generate this cell type has meant that up until now their study has been reliant on the use of rodent models. Here, we use a combination of population and single-cell techniques to perform a detailed molecular, electrophysiological, and pharmacological phenotyping of sensory neurons derived from human embryonic stem cells. We describe the evolution of cell populations over 6 weeks of directed differentiation; a process that results in the generation of a largely homogeneous population of neurons that are both molecularly and functionally comparable to human sensory neurons derived from mature dorsal root ganglia. This work opens the prospect of using pluripotent stem-cell-derived sensory neurons to study human neuronal physiology and as in vitro models for drug discovery in pain and sensory disorders.

  4. Ciliary heterogeneity within a single cell: the Paramecium model.

    Science.gov (United States)

    Aubusson-Fleury, Anne; Cohen, Jean; Lemullois, Michel

    2015-01-01

    Paramecium is a single cell able to divide in its morphologically differentiated stage that has many cilia anchored at its cell surface. Many thousands of cilia are thus assembled in a short period of time during division to duplicate the cell pattern while the cell continues swimming. Most, but not all, of these sensory cilia are motile and involved in two main functions: prey capture and cell locomotion. These cilia display heterogeneity, both in their length and their biochemical properties. Thanks to these properties, as well as to the availability of many postgenomic tools and the possibility to follow the regrowth of cilia after deciliation, Paramecium offers a nice opportunity to study the assembly of the cilia, as well as the genesis of their diversity within a single cell. In this paper, after a brief survey of Paramecium morphology and cilia properties, we describe the tools and the protocols currently used for immunofluorescence, transmission electron microscopy, and ultrastructural immunocytochemistry to analyze cilia, with special recommendations to overcome the problem raised by cilium diversity. Copyright © 2015. Published by Elsevier Inc.

  5. Simultaneous Measurement of Multiple Mechanical Properties of Single Cells Using AFM by Indentation and Vibration.

    Science.gov (United States)

    Zhang, Chuang; Shi, Jialin; Wang, Wenxue; Xi, Ning; Wang, Yuechao; Liu, Lianqing

    2017-12-01

    The mechanical properties of cells, which are the main characteristics determining their physical performance and physiological functions, have been actively studied in the fields of cytobiology and biomedical engineering and for the development of medicines. In this study, an indentation-vibration-based method is proposed to simultaneously measure the mechanical properties of cells in situ, including cellular mass (m), elasticity (k), and viscosity (c). The proposed measurement method is implemented based on the principle of forced vibration stimulated by simple harmonic force using an atomic force microscope (AFM) system integrated with a piezoelectric transducer as the substrate vibrator. The corresponding theoretical model containing the three mechanical properties is derived and used to perform simulations and calculations. Living and fixed human embryonic kidney 293 (HEK 293) cells were subjected to indentation and vibration to measure and compare their mechanical parameters and verify the proposed approach. The results that the fixed sample cells are more viscous and elastic than the living sample cells and the measured mechanical properties of cell are consistent within, but not outside of the central region of the cell, are in accordance with the previous studies. This work provides an approach to simultaneous measurement of the multiple mechanical properties of single cells using an integrated AFM system based on the principle force vibration and thickness-corrected Hertz model. This study should contribute to progress in biomedical engineering, cytobiology, medicine, early diagnosis, specific therapy and cell-powered robots.

  6. Early dynamic fate changes in haemogenic endothelium characterized at the single-cell level

    Science.gov (United States)

    Swiers, Gemma; Baumann, Claudia; O'Rourke, John; Giannoulatou, Eleni; Taylor, Stephen; Joshi, Anagha; Moignard, Victoria; Pina, Cristina; Bee, Thomas; Kokkaliaris, Konstantinos D.; Yoshimoto, Momoko; Yoder, Mervin C.; Frampton, Jon; Schroeder, Timm; Enver, Tariq; Göttgens, Berthold; de Bruijn, Marella F. T. R.

    2013-12-01

    Haematopoietic stem cells (HSCs) are the founding cells of the adult haematopoietic system, born during ontogeny from a specialized subset of endothelium, the haemogenic endothelium (HE) via an endothelial-to-haematopoietic transition (EHT). Although recently imaged in real time, the underlying mechanism of EHT is still poorly understood. We have generated a Runx1 +23 enhancer-reporter transgenic mouse (23GFP) for the prospective isolation of HE throughout embryonic development. Here we perform functional analysis of over 1,800 and transcriptional analysis of 268 single 23GFP+ HE cells to explore the onset of EHT at the single-cell level. We show that initiation of the haematopoietic programme occurs in cells still embedded in the endothelial layer, and is accompanied by a previously unrecognized early loss of endothelial potential before HSCs emerge. Our data therefore provide important insights on the timeline of early haematopoietic commitment.

  7. Single-cell transcriptomics uncovers distinct molecular signatures of stem cells in chronic myeloid leukemia.

    Science.gov (United States)

    Giustacchini, Alice; Thongjuea, Supat; Barkas, Nikolaos; Woll, Petter S; Povinelli, Benjamin J; Booth, Christopher A G; Sopp, Paul; Norfo, Ruggiero; Rodriguez-Meira, Alba; Ashley, Neil; Jamieson, Lauren; Vyas, Paresh; Anderson, Kristina; Segerstolpe, Åsa; Qian, Hong; Olsson-Strömberg, Ulla; Mustjoki, Satu; Sandberg, Rickard; Jacobsen, Sten Eirik W; Mead, Adam J

    2017-06-01

    Recent advances in single-cell transcriptomics are ideally placed to unravel intratumoral heterogeneity and selective resistance of cancer stem cell (SC) subpopulations to molecularly targeted cancer therapies. However, current single-cell RNA-sequencing approaches lack the sensitivity required to reliably detect somatic mutations. We developed a method that combines high-sensitivity mutation detection with whole-transcriptome analysis of the same single cell. We applied this technique to analyze more than 2,000 SCs from patients with chronic myeloid leukemia (CML) throughout the disease course, revealing heterogeneity of CML-SCs, including the identification of a subgroup of CML-SCs with a distinct molecular signature that selectively persisted during prolonged therapy. Analysis of nonleukemic SCs from patients with CML also provided new insights into cell-extrinsic disruption of hematopoiesis in CML associated with clinical outcome. Furthermore, we used this single-cell approach to identify a blast-crisis-specific SC population, which was also present in a subclone of CML-SCs during the chronic phase in a patient who subsequently developed blast crisis. This approach, which might be broadly applied to any malignancy, illustrates how single-cell analysis can identify subpopulations of therapy-resistant SCs that are not apparent through cell-population analysis.

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

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Daojing; Bodovitz, Steven

    2010-01-14

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

  9. Performance study on the east-west oriented single-axis tracked panel

    International Nuclear Information System (INIS)

    Chang, Tian Pau

    2009-01-01

    A theoretical study on the performance of an east-west oriented single-axis tracked panel was originally proposed in this paper. Mathematic expressions applicable for calculating the angle that the tracked panel should rotate by to follow the Sun are derived. The incident angle of sunlight upon the panel as well as the instantaneous increments of solar energy captured by the panel relative to a fixed horizontal surface are then demonstrated graphically. To simulate different operation environments, three kinds of radiation sources will be considered, i.e. the extraterrestrial radiation, global radiation predicted by empirical models under clear sky situation and global radiation observed in Taiwan. Simulation results show that the yearly gains correlate positively with the radiation level, i.e. 21.2%, 13.5% and 7.4% for the extraterrestrial, predicted and observed radiations, respectively, which are far less than those obtained from a north-south oriented single-axis tracked panel. The irradiation increases with the maximum rotation angle of the panel, the benefit of increasing the rotation in overcast environment is not as good as in clear sky, for annual energy collection 45 o is recommended. The irradiation received decreases with latitude, but it has a greater gain in higher latitude zone.

  10. Dissecting stem cell differentiation using single cell expression profiling

    OpenAIRE

    Moignard, Victoria Rachel; Göttgens, Berthold

    2016-01-01

    Many assumptions about the way cells behave are based on analyses of populations. However, it is now widely recognized that even apparently pure populations can display a remarkable level of heterogeneity. This is particularly true in stem cell biology where it hinders our understanding of normal development and the development of strategies for regenerative medicine. Over the past decade technologies facilitating gene expression analysis at the single cell level have become widespread, provi...

  11. Microfluidic platform for multiplexed detection in single cells and methods thereof

    Science.gov (United States)

    Wu, Meiye; Singh, Anup K.

    2018-05-01

    The present invention relates to a microfluidic device and platform configured to conduct multiplexed analysis within the device. In particular, the device allows multiple targets to be detected on a single-cell level. Also provided are methods of performing multiplexed analyses to detect one or more target nucleic acids, proteins, and post-translational modifications.

  12. Cell shunt resistance and photovoltaic module performance

    Energy Technology Data Exchange (ETDEWEB)

    McMahon, T.J.; Basso, T.S.; Rummel, S.R. [National Renewable Energy Lab., Golden, CO (United States)

    1996-05-01

    Shunt resistance of cells in photovoltaic modules can affect module power output and could indicate flawed manufacturing processes and reliability problems. The authors describe a two-terminal diagnostic method to directly measure the shunt resistance of individual cells in a series-connected module non-intrusively, without deencapsulation. Peak power efficiency vs. light intensity was measured on a 12-cell, series-connected, single crystalline module having relatively high cell shunt resistances. The module was remeasured with 0.5-, 1-, and 2-ohm resistors attached across each cell to simulate shunt resistances of several emerging technologies. Peak power efficiencies decreased dramatically at lower light levels. Using the PSpice circuit simulator, the authors verified that cell shunt and series resistances can indeed be responsible for the observed peak power efficiency vs. intensity behavior. The authors discuss the effect of basic cell diode parameters, i.e., shunt resistance, series resistance, and recombination losses, on PV module performance as a function of light intensity.

  13. Single cell amperometry reveals curcuminoids modulate the release of neurotransmitters during exocytosis from PC12 cells

    Science.gov (United States)

    Li, Xianchan; Mohammadi, Amir Saeid; Ewing, Andrew G.

    2016-01-01

    We used single cell amperometry to examine whether curcumin and bisdemethoxycurcumin (BDMC), substances that are suggested to affect learning and memory, can modulate monoamine release from PC12 cells. Our results indicate both curcumin and BDMC need long-term treatment (72 h in this study) to influence exocytosis effectively. By analyzing the parameters calculated from single exocytosis events, it can be concluded that curcumin and BDMC affect exocytosis through different mechanisms. Curcumin accelerates the event dynamics with no significant change of the monoamine amount released from single exocytotic events, whereas BDMC attenuates the amount from single exocytotic event with no significant change of the event dynamics. This comparison of the effect of curcumin and BDMC on exocytosis at the single cell level brings insight into their different mechanisms, which might lead to different biological actions. The effect of curcumin and BDMC on the opening and closing of the exocytotic fusion pore were also investigated. These results might be helpful for understanding the improvement of learning and memory and the anti-depression properties of curcuminoids. PMID:28579928

  14. Effect of time-varying humidity on the performance of a polymer electrolyte membrane fuel cells

    Energy Technology Data Exchange (ETDEWEB)

    Noorani, Shamsuddin [Department of Mechanical Engineering, University of Michigan-Dearborn (United States); Shamim, Tariq [Mechanical Engineering, Masdar Institute of Science and Technology (United Arab Emirates)], E-mail: tshamim@masdar.ac.ae

    2011-07-01

    In the energy sector, fuel cells constitute a promising solution for the future due to their energy-efficient and environment-friendly characteristics. However, the performance of fuel cells is very much affected by the humidification level of the reactants, particularly in hot regions. The aim of this paper is to develop a better understanding of the effect of driving conditions on the performance of fuel cells. A macroscopic single-fuel-cell-based, one dimensional, isothermal model was used on a polymer electrolyte membrane fuel cell to carry out a computational study of the impact of humidity conditions which vary over time. It was found that the variation of humidity has a significant effect on water distribution but a much lower impact on power and current densities. This paper provided useful information on fuel cells' performance under varying conditions which could be used to improve their design for mobile applications.

  15. Single-Cell-Based Platform for Copy Number Variation Profiling through Digital Counting of Amplified Genomic DNA Fragments.

    Science.gov (United States)

    Li, Chunmei; Yu, Zhilong; Fu, Yusi; Pang, Yuhong; Huang, Yanyi

    2017-04-26

    We develop a novel single-cell-based platform through digital counting of amplified genomic DNA fragments, named multifraction amplification (mfA), to detect the copy number variations (CNVs) in a single cell. Amplification is required to acquire genomic information from a single cell, while introducing unavoidable bias. Unlike prevalent methods that directly infer CNV profiles from the pattern of sequencing depth, our mfA platform denatures and separates the DNA molecules from a single cell into multiple fractions of a reaction mix before amplification. By examining the sequencing result of each fraction for a specific fragment and applying a segment-merge maximum likelihood algorithm to the calculation of copy number, we digitize the sequencing-depth-based CNV identification and thus provide a method that is less sensitive to the amplification bias. In this paper, we demonstrate a mfA platform through multiple displacement amplification (MDA) chemistry. When performing the mfA platform, the noise of MDA is reduced; therefore, the resolution of single-cell CNV identification can be improved to 100 kb. We can also determine the genomic region free of allelic drop-out with mfA platform, which is impossible for conventional single-cell amplification methods.

  16. Single-cell transcriptomic reconstruction reveals cell cycle and multi-lineage differentiation defects in Bcl11a-deficient hematopoietic stem cells.

    Science.gov (United States)

    Tsang, Jason C H; Yu, Yong; Burke, Shannon; Buettner, Florian; Wang, Cui; Kolodziejczyk, Aleksandra A; Teichmann, Sarah A; Lu, Liming; Liu, Pentao

    2015-09-21

    Hematopoietic stem cells (HSCs) are a rare cell type with the ability of long-term self-renewal and multipotency to reconstitute all blood lineages. HSCs are typically purified from the bone marrow using cell surface markers. Recent studies have identified significant cellular heterogeneities in the HSC compartment with subsets of HSCs displaying lineage bias. We previously discovered that the transcription factor Bcl11a has critical functions in the lymphoid development of the HSC compartment. In this report, we employ single-cell transcriptomic analysis to dissect the molecular heterogeneities in HSCs. We profile the transcriptomes of 180 highly purified HSCs (Bcl11a (+/+) and Bcl11a (-/-)). Detailed analysis of the RNA-seq data identifies cell cycle activity as the major source of transcriptomic variation in the HSC compartment, which allows reconstruction of HSC cell cycle progression in silico. Single-cell RNA-seq profiling of Bcl11a (-/-) HSCs reveals abnormal proliferative phenotypes. Analysis of lineage gene expression suggests that the Bcl11a (-/-) HSCs are constituted of two distinct myeloerythroid-restricted subpopulations. Remarkably, similar myeloid-restricted cells could also be detected in the wild-type HSC compartment, suggesting selective elimination of lymphoid-competent HSCs after Bcl11a deletion. These defects are experimentally validated in serial transplantation experiments where Bcl11a (-/-) HSCs are myeloerythroid-restricted and defective in self-renewal. Our study demonstrates the power of single-cell transcriptomics in dissecting cellular process and lineage heterogeneities in stem cell compartments, and further reveals the molecular and cellular defects in the Bcl11a-deficient HSC compartment.

  17. Single-cell Analysis of Lambda Immunity Regulation

    DEFF Research Database (Denmark)

    Bæk, Kristoffer Torbjørn; Svenningsen, Sine Lo; Eisen, Harvey

    2003-01-01

    We have examined expression of the ¿cI operon in single cells via a rexgfp substitution. Although average fluorescence agreed with expectations for expression of ¿-repressor, fluorescence fluctuated greatly from cell-to-cell. Fluctuations in repressor concentration are not predicted by previous m...

  18. FogBank: a single cell segmentation across multiple cell lines and image modalities.

    Science.gov (United States)

    Chalfoun, Joe; Majurski, Michael; Dima, Alden; Stuelten, Christina; Peskin, Adele; Brady, Mary

    2014-12-30

    Many cell lines currently used in medical research, such as cancer cells or stem cells, grow in confluent sheets or colonies. The biology of individual cells provide valuable information, thus the separation of touching cells in these microscopy images is critical for counting, identification and measurement of individual cells. Over-segmentation of single cells continues to be a major problem for methods based on morphological watershed due to the high level of noise in microscopy cell images. There is a need for a new segmentation method that is robust over a wide variety of biological images and can accurately separate individual cells even in challenging datasets such as confluent sheets or colonies. We present a new automated segmentation method called FogBank that accurately separates cells when confluent and touching each other. This technique is successfully applied to phase contrast, bright field, fluorescence microscopy and binary images. The method is based on morphological watershed principles with two new features to improve accuracy and minimize over-segmentation. First, FogBank uses histogram binning to quantize pixel intensities which minimizes the image noise that causes over-segmentation. Second, FogBank uses a geodesic distance mask derived from raw images to detect the shapes of individual cells, in contrast to the more linear cell edges that other watershed-like algorithms produce. We evaluated the segmentation accuracy against manually segmented datasets using two metrics. FogBank achieved segmentation accuracy on the order of 0.75 (1 being a perfect match). We compared our method with other available segmentation techniques in term of achieved performance over the reference data sets. FogBank outperformed all related algorithms. The accuracy has also been visually verified on data sets with 14 cell lines across 3 imaging modalities leading to 876 segmentation evaluation images. FogBank produces single cell segmentation from confluent cell

  19. Chasing the precursor of functional hematopoietic stem cells at the single cell levels in mouse embryos.

    Science.gov (United States)

    Wang, Xiaochen; Gong, Yuemin; Ema, Hideo

    2016-07-22

    Adult hematopoietic stem cells (HSCs), the ideal system for regenerative research, were isolated at single cell levels decades ago, whereas studies on embryonic HSCs are much more difficult. Zhou et al identified a new pre-HSC cell surface marker, CD201, by which they isolated pre-HSCs at single cell levels for further analyses. The novel expression pattern of HSC development is revealed, including the fundamental role of mammalian targets of rapamycin (mTOR) signaling pathway in HSCs emergence, and the repopulation potential of S/G2/M phase pre-HSCs. Deeper understandings of the cellular origin and developmental regulatory network of HSCs are essential to develop new strategies of generating HSCs in vitro for clinical application.

  20. MeV single-ion beam irradiation of mammalian cells using the Surrey vertical nanobeam, compared with broad proton beam and X-ray irradiations

    Energy Technology Data Exchange (ETDEWEB)

    Prakrajang, K. [Plasma and Beam Physics Research Facility, Department of Physics and Materials Science, Faculty of Science, Chiang Mai University, Chiang Mai 50200 (Thailand); Faculty of Science, Maejo University, Chiang Mai 50290 (Thailand); Jeynes, J.C.G.; Merchant, M.J.; Kirkby, K.; Kirkby, N. [Surrey Ion Beam Center, Faculty of Engineering and Physical Science, University of Surrey, Guildford Surrey, GU2 7XH (United Kingdom); Thopan, P. [Plasma and Beam Physics Research Facility, Department of Physics and Materials Science, Faculty of Science, Chiang Mai University, Chiang Mai 50200 (Thailand); Yu, L.D., E-mail: yuld@fnrf.science.cmu.ac.th [Plasma and Beam Physics Research Facility, Department of Physics and Materials Science, Faculty of Science, Chiang Mai University, Chiang Mai 50200 (Thailand); Thailand Center of Excellence in Physics, Commission on Higher Education, 328 Si Ayutthaya Road, Bangkok 10400 (Thailand)

    2013-07-15

    Highlights: •Recently completed nanobeam at the Surrey Ion Beam Centre was used. •3.8-MeV single and broad proton beams irradiated Chinese hamster cells. •Cell survival curves were measured and compared with 300-kV X-ray irradiation. •Single ion irradiation had a lower survival part at ultra-low dose. •It implies hypersensitivity, bystander effect and cell cycle phase of cell death. -- Abstract: As a part of a systematic study on mechanisms involved in physical cancer therapies, this work investigated response of mammalian cells to ultra-low-dose ion beam irradiation. The ion beam irradiation was performed using the recently completed nanobeam facility at the Surrey Ion Beam Centre. A scanning focused vertical ion nano-beam was applied to irradiate Chinese hamster V79 cells. The V79 cells were irradiated in two different beam modes, namely, focused single ion beam and defocused scanning broad ion beam of 3.8-MeV protons. The single ion beam was capable of irradiating a single cell with a precisely controlled number of the ions to extremely low doses. After irradiation and cell incubation, the number of surviving colonies as a function of the number of the irradiating ions was measured for the cell survival fraction curve. A lower survival for the single ion beam irradiation than that of the broad beam case implied the hypersensitivity and bystander effect. The ion-beam-induced cell survival curves were compared with that from 300-kV X-ray irradiation. Theoretical studies indicated that the cell death in single ion irradiation mainly occurred in the cell cycle phases of cell division and intervals between the cell division and the DNA replication. The success in the experiment demonstrated the Surrey vertical nanobeam successfully completed.

  1. Single-cell-type quantitative proteomic and ionomic analysis of epidermal bladder cells from the halophyte model plant Mesembryanthemum crystallinum to identify salt-responsive proteins.

    Science.gov (United States)

    Barkla, Bronwyn J; Vera-Estrella, Rosario; Raymond, Carolyn

    2016-05-10

    Epidermal bladder cells (EBC) are large single-celled, specialized, and modified trichomes found on the aerial parts of the halophyte Mesembryanthemum crystallinum. Recent development of a simple but high throughput technique to extract the contents from these cells has provided an opportunity to conduct detailed single-cell-type analyses of their molecular characteristics at high resolution to gain insight into the role of these cells in the salt tolerance of the plant. In this study, we carry out large-scale complementary quantitative proteomic studies using both a label (DIGE) and label-free (GeLC-MS) approach to identify salt-responsive proteins in the EBC extract. Additionally we perform an ionomics analysis (ICP-MS) to follow changes in the amounts of 27 different elements. Using these methods, we were able to identify 54 proteins and nine elements that showed statistically significant changes in the EBC from salt-treated plants. GO enrichment analysis identified a large number of transport proteins but also proteins involved in photosynthesis, primary metabolism and Crassulacean acid metabolism (CAM). Validation of results by western blot, confocal microscopy and enzyme analysis helped to strengthen findings and further our understanding into the role of these specialized cells. As expected EBC accumulated large quantities of sodium, however, the most abundant element was chloride suggesting the sequestration of this ion into the EBC vacuole is just as important for salt tolerance. This single-cell type omics approach shows that epidermal bladder cells of M. crystallinum are metabolically active modified trichomes, with primary metabolism supporting cell growth, ion accumulation, compatible solute synthesis and CAM. Data are available via ProteomeXchange with identifier PXD004045.

  2. QSpec: online control and data analysis system for single-cell Raman spectroscopy

    Directory of Open Access Journals (Sweden)

    Lihui Ren

    2014-06-01

    Full Text Available Single-cell phenotyping is critical to the success of biological reductionism. Raman-activated cell sorting (RACS has shown promise in resolving the dynamics of living cells at the individual level and to uncover population heterogeneities in comparison to established approaches such as fluorescence-activated cell sorting (FACS. Given that the number of single-cells would be massive in any experiment, the power of Raman profiling technique for single-cell analysis would be fully utilized only when coupled with a high-throughput and intelligent process control and data analysis system. In this work, we established QSpec, an automatic system that supports high-throughput Raman-based single-cell phenotyping. Additionally, a single-cell Raman profile database has been established upon which data-mining could be applied to discover the heterogeneity among single-cells under different conditions. To test the effectiveness of this control and data analysis system, a sub-system was also developed to simulate the phenotypes of single-cells as well as the device features.

  3. CEL-Seq: Single-Cell RNA-Seq by Multiplexed Linear Amplification

    Directory of Open Access Journals (Sweden)

    Tamar Hashimshony

    2012-09-01

    Full Text Available High-throughput sequencing has allowed for unprecedented detail in gene expression analyses, yet its efficient application to single cells is challenged by the small starting amounts of RNA. We have developed CEL-Seq, a method for overcoming this limitation by barcoding and pooling samples before linearly amplifying mRNA with the use of one round of in vitro transcription. We show that CEL-Seq gives more reproducible, linear, and sensitive results than a PCR-based amplification method. We demonstrate the power of this method by studying early C. elegans embryonic development at single-cell resolution. Differential distribution of transcripts between sister cells is seen as early as the two-cell stage embryo, and zygotic expression in the somatic cell lineages is enriched for transcription factors. The robust transcriptome quantifications enabled by CEL-Seq will be useful for transcriptomic analyses of complex tissues containing populations of diverse cell types.

  4. Performance study of a MegaPixel single photon position sensitive photodetector EBCMOS

    International Nuclear Information System (INIS)

    Barbier, Remi; Baudot, J.; Chabanat, E.; Depasse, P.; Dulinski, W.; Estre, N.; Kaiser, C.T.; Laurent, N.; Winter, M.

    2009-01-01

    This development is related to the design and the integration of a Monolithic Active Pixel Sensor (MAPS) into a photosensitive proximity focusing vacuum-based tube. This EBCMOS project is dedicated to the fluorescent and the bioluminescent high speed imaging. The results of the full characterization of the first prototype are presented. Comparative tests with different fluorescent dyes have been performed in biology laboratories. Preliminary conclusions on the ability of EBCMOS to perform fast single-molecule tracking will be given.

  5. Scientist, Single Cell Analysis Facility | Center for Cancer Research

    Science.gov (United States)

    The Cancer Research Technology Program (CRTP) develops and implements emerging technology, cancer biology expertise and research capabilities to accomplish NCI research objectives.  The CRTP is an outward-facing, multi-disciplinary hub purposed to enable the external cancer research community and provides dedicated support to NCI’s intramural Center for Cancer Research (CCR).  The dedicated units provide electron microscopy, protein characterization, protein expression, optical microscopy and nextGen sequencing. These research efforts are an integral part of CCR at the Frederick National Laboratory for Cancer Research (FNLCR).  CRTP scientists also work collaboratively with intramural NCI investigators to provide research technologies and expertise. KEY ROLES AND RESPONSIBILITIES We are seeking a highly motivated Scientist II to join the newly established Single Cell Analysis Facility (SCAF) of the Center for Cancer Research (CCR) at NCI. The SCAF will house state of the art single cell sequencing technologies including 10xGenomics Chromium, BD Genomics Rhapsody, DEPPArray, and other emerging single cell technologies. The Scientist: Will interact with close to 200 laboratories within the CCR to design and carry out single cell experiments for cancer research Will work on single cell isolation/preparation from various tissues and cells and related NexGen sequencing library preparation Is expected to author publications in peer reviewed scientific journals

  6. A Robust Single Primate Neuroepithelial Cell Clonal Expansion System for Neural Tube Development and Disease Studies

    Directory of Open Access Journals (Sweden)

    Xiaoqing Zhu

    2016-02-01

    Full Text Available Developing a model of primate neural tube (NT development is important to promote many NT disorder studies in model organisms. Here, we report a robust and stable system to allow for clonal expansion of single monkey neuroepithelial stem cells (NESCs to develop into miniature NT-like structures. Single NESCs can produce functional neurons in vitro, survive, and extensively regenerate neuron axons in monkey brain. NT formation and NESC maintenance depend on high metabolism activity and Wnt signaling. NESCs are regionally restricted to a telencephalic fate. Moreover, single NESCs can turn into radial glial progenitors (RGPCs. The transition is accurately regulated by Wnt signaling through regulation of Notch signaling and adhesion molecules. Finally, using the “NESC-TO-NTs” system, we model the functions of folic acid (FA on NT closure and demonstrate that FA can regulate multiple mechanisms to prevent NT defects. Our system is ideal for studying NT development and diseases.

  7. Review of cell performance in anion exchange membrane fuel cells

    Science.gov (United States)

    Dekel, Dario R.

    2018-01-01

    Anion exchange membrane fuel cells (AEMFCs) have recently received increasing attention since in principle they allow for the use of non-precious metal catalysts, which dramatically reduces the cost per kilowatt of power in fuel cell devices. Until not long ago, the main barrier in the development of AEMFCs was the availability of highly conductive anion exchange membranes (AEMs); however, improvements on this front in the past decade show that newly developed AEMs have already reached high levels of conductivity, leading to satisfactory cell performance. In recent years, a growing number of research studies have reported AEMFC performance results. In the last three years, new records in performance were achieved. Most of the literature reporting cell performance is based on hydrogen-AEMFCs, although an increasing number of studies have also reported the use of fuels others than hydrogen - such as alcohols, non-alcohol C-based fuels, as well as N-based fuels. This article reviews the cell performance and performance stability achieved in AEMFCs through the years since the first reports in the early 2000s.

  8. Atomic force microscopy imaging and 3-D reconstructions of serial thin sections of a single cell and its interior structures

    International Nuclear Information System (INIS)

    Chen Yong; Cai Jiye; Zhao Tao; Wang Chenxi; Dong Shuo; Luo Shuqian; Chen, Zheng W.

    2005-01-01

    The thin sectioning has been widely applied in electron microscopy (EM), and successfully used for an in situ observation of inner ultrastructure of cells. This powerful technique has recently been extended to the research field of atomic force microscopy (AFM). However, there have been no reports describing AFM imaging of serial thin sections and three-dimensional (3-D) reconstruction of cells and their inner structures. In the present study, we used AFM to scan serial thin sections approximately 60 nm thick of a mouse embryonic stem (ES) cell, and to observe the in situ inner ultrastructure including cell membrane, cytoplasm, mitochondria, nucleus membrane, and linear chromatin. The high-magnification AFM imaging of single mitochondria clearly demonstrated the outer membrane, inner boundary membrane and cristal membrane of mitochondria in the cellular compartment. Importantly, AFM imaging on six serial thin sections of a single mouse ES cell showed that mitochondria underwent sequential changes in the number, morphology and distribution. These nanoscale images allowed us to perform 3-D surface reconstruction of interested interior structures in cells. Based on the serial in situ images, 3-D models of morphological characteristics, numbers and distributions of interior structures of the single ES cells were validated and reconstructed. Our results suggest that the combined AFM and serial-thin-section technique is useful for the nanoscale imaging and 3-D reconstruction of single cells and their inner structures. This technique may facilitate studies of proliferating and differentiating stages of stem cells or somatic cells at a nanoscale

  9. The comparison between gallium arsenide and indium gallium arsenide as materials for solar cell performance using Silvaco application

    Energy Technology Data Exchange (ETDEWEB)

    Zahari, Suhaila Mohd; Norizan, Mohd Natashah; Mohamad, Ili Salwani; Osman, Rozana Aina Maulat; Taking, Sanna [School of Microelectronic Engineering, Universiti Malaysia Perlis, Kampus Pauh Putra, 02600 Arau, Perlis (Malaysia)

    2015-05-15

    The work presented in this paper is about the development of single and multilayer solar cells using GaAs and InGaAs in AM1.5 condition. The study includes the modeling structure and simulation of the device using Silvaco applications. The performance in term of efficiency of Indium Gallium Arsenide (InGaAs) and GaAs material was studied by modification of the doping concentration and thickness of material in solar cells. The efficiency of the GaAs solar cell was higher than InGaAs solar cell for single layer solar cell. Single layer GaAs achieved an efficiency about 25% compared to InGaAs which is only 2.65% of efficiency. For multilayer which includes both GaAs and InGaAs, the output power, P{sub max} was 8.91nW/cm² with the efficiency only 8.51%. GaAs is one of the best materials to be used in solar cell as a based compared to InGaAs.

  10. Central dogma at the single-molecule level in living cells.

    Science.gov (United States)

    Li, Gene-Wei; Xie, X Sunney

    2011-07-20

    Gene expression originates from individual DNA molecules within living cells. Like many single-molecule processes, gene expression and regulation are stochastic, that is, sporadic in time. This leads to heterogeneity in the messenger-RNA and protein copy numbers in a population of cells with identical genomes. With advanced single-cell fluorescence microscopy, it is now possible to quantify transcriptomes and proteomes with single-molecule sensitivity. Dynamic processes such as transcription-factor binding, transcription and translation can be monitored in real time, providing quantitative descriptions of the central dogma of molecular biology and the demonstration that a stochastic single-molecule event can determine the phenotype of a cell.

  11. Ramifications of single-port laparoscopic surgery: measuring differences in task performance using simulation.

    Science.gov (United States)

    Conway, Nathan E; Romanelli, John R; Bush, Ron W; Seymour, Neal E

    2014-02-01

    Single-port laparoscopic surgery imposes unique psychomotor challenges. We used surgical simulation to define performance differences between surgeons with and without single-port clinical experience and examined whether a short course of training resulted in improved performance. Study participants were assigned to 3 groups: resident group (RES), experienced laparoscopic surgeons with (SP) and without (LAP) prior single-port laparoscopic experience. Participants performed the Fundamentals of Laparoscopic Surgery precision cutting task on a ProMIS trainer through conventional ports or with articulating instruments via a SILS Port (Covidien, Inc). Two iterations of each method were performed. Then, 6 residents performed 10 successive single-port iterations to assess the effect of practice on task performance. The SP group had faster task times for both laparoscopic (P = .0486) and single-port (P = .0238) methods. The LAP group had longer path lengths for the single-port task than for the laparoscopic task (P = .03). The RES group was slower (P = .0019), with longer path length (P = .0010) but with greater smoothness (P = .0186) on the single-port task than the conventional laparoscopic task. Resident performance task time (P = .005) and smoothness (P = .045) improved with successive iterations. Our data show that surgeons with clinical single-port surgery experience perform a simulated single-port surgical task better than inexperienced single-port surgeons. Furthermore, this performance is comparable to that achieved with conventional laparoscopic techniques. Performance of residents declined dramatically when confronted with the challenges of the single-port task but improved with practice. These results suggest a role for lab-based single-port training.

  12. Performance analysis of high-concentrated multi-junction solar cells in hot climate

    Science.gov (United States)

    Ghoneim, Adel A.; Kandil, Kandil M.; Alzanki, Talal H.; Alenezi, Mohammad R.

    2018-03-01

    Multi-junction concentrator solar cells are a promising technology as they can fulfill the increasing energy demand with renewable sources. Focusing sunlight upon the aperture of multi-junction photovoltaic (PV) cells can generate much greater power densities than conventional PV cells. So, concentrated PV multi-junction solar cells offer a promising way towards achieving minimum cost per kilowatt-hour. However, these cells have many aspects that must be fixed to be feasible for large-scale energy generation. In this work, a model is developed to analyze the impact of various atmospheric factors on concentrator PV performance. A single-diode equivalent circuit model is developed to examine multi-junction cells performance in hot weather conditions, considering the impacts of both temperature and concentration ratio. The impacts of spectral variations of irradiance on annual performance of various high-concentrated photovoltaic (HCPV) panels are examined, adapting spectra simulations using the SMARTS model. Also, the diode shunt resistance neglected in the existing models is considered in the present model. The present results are efficiently validated against measurements from published data to within 2% accuracy. Present predictions show that the single-diode model considering the shunt resistance gives accurate and reliable results. Also, aerosol optical depth (AOD) and air mass are most important atmospheric parameters having a significant impact on HCPV cell performance. In addition, the electrical efficiency (η) is noticed to increase with concentration to a certain concentration degree after which it decreases. Finally, based on the model predictions, let us conclude that the present model could be adapted properly to examine HCPV cells' performance over a broad range of operating conditions.

  13. Opto-injection into single living cells by femtosecond near-infrared laser

    Science.gov (United States)

    Peng, Cheng

    This dissertation presents a novel technique to deliver membrane impermeable molecules into single living cells with the assistance of femtosecond (fs) near-infrared (NIR) laser pulses. This approach merges ultrafast laser technology with key biological, biomedical, and medical applications, such as gene transfection, gene therapy and drug delivery. This technique promises several major advantages, namely, very high transfection efficiency, high cell survival rate (≈100%) and fully preserved cell viabilities. It is also a promising method to deliver molecules into cells that are difficult or even completely resistant to established physical methods, such as microinjection by glass pipettes, electroporation, and biolistics. In this work, the system for fs NIR opto-injection was designed and built. Successful fs NIR opto-injection has been performed on several cell systems including single mammalian cells (bovine aortic endothelial cells), marine animal eggs (Spisula solidissima oocytes), and human cancer cells (fibrosarcoma HT1080) cultured in a tissue-like environment. The connections between laser parameters and cell responses were explored through further experiments and in-depth analyses, especially the relationship between dye uptake rate and incident laser intensity, and the relationship between pore size created on cell membranes and incident laser intensity. Dye uptake rate of the target cells was observed to depend on incident laser intensity. Pore size was found dependent on incident laser intensity. The conclusion was made that laser-induced breakdown and plasma-induced ablation in cell membrane are the physical principles that govern the process of fs NIR opto-injection.

  14. Single-Cell Analysis of Human Pancreas Reveals Transcriptional Signatures of Aging and Somatic Mutation Patterns.

    Science.gov (United States)

    Enge, Martin; Arda, H Efsun; Mignardi, Marco; Beausang, John; Bottino, Rita; Kim, Seung K; Quake, Stephen R

    2017-10-05

    As organisms age, cells accumulate genetic and epigenetic errors that eventually lead to impaired organ function or catastrophic transformation such as cancer. Because aging reflects a stochastic process of increasing disorder, cells in an organ will be individually affected in different ways, thus rendering bulk analyses of postmitotic adult cells difficult to interpret. Here, we directly measure the effects of aging in human tissue by performing single-cell transcriptome analysis of 2,544 human pancreas cells from eight donors spanning six decades of life. We find that islet endocrine cells from older donors display increased levels of transcriptional noise and potential fate drift. By determining the mutational history of individual cells, we uncover a novel mutational signature in healthy aging endocrine cells. Our results demonstrate the feasibility of using single-cell RNA sequencing (RNA-seq) data from primary cells to derive insights into genetic and transcriptional processes that operate on aging human tissue. Copyright © 2017 Elsevier Inc. All rights reserved.

  15. High-throughput deterministic single-cell encapsulation and droplet pairing, fusion, and shrinkage in a single microfluidic device

    NARCIS (Netherlands)

    Schoeman, R.M.; Kemna, Evelien; Wolbers, F.; van den Berg, Albert

    In this article, we present a microfluidic device capable of successive high-yield single-cell encapsulation in droplets, with additional droplet pairing, fusion, and shrinkage. Deterministic single-cell encapsulation is realized using Dean-coupled inertial ordering of cells in a Yin-Yang-shaped

  16. A portable system powered with hydrogen and one single air-breathing PEM fuel cell

    International Nuclear Information System (INIS)

    Fernández-Moreno, J.; Guelbenzu, G.; Martín, A.J.; Folgado, M.A.; Ferreira-Aparicio, P.; Chaparro, A.M.

    2013-01-01

    Highlights: • A portable system based on hydrogen and single air breathing PEM fuel cell. • Control electronics designed for low single cell voltage (0.5–0.8 V). • Forced air convection and anode purging required to help water management. • Application consisting of a propeller able to display a luminous message. • Up to 20 h autonomy with continuous 1.1 W consumption, using 1 g H 2 . - Abstract: A portable system for power generation based on hydrogen and a single proton exchange membrane fuel cell (PEMFC) has been built and operated. The fuel cell is fed in the anode with hydrogen stored in a metal hydrides cartridge, and in the cathode with oxygen from quiescent ambient air (‘air breathing’). The control electronics of the system performs DC–DC conversion from the low voltage (0.5–0.8 V) and high current output (200–300 mA cm −2 ) of the single fuel cell, up to 3.3 V to power an electronic application. System components assist fuel cell operation, including an electronic valve for anode purging, a fan in front of the open cathode, two supercapacitors for auxiliary power requirements, four LED lights, and a display screen. The influence of the system components on fuel cell behaviour is analyzed. The cathode fan and anodic purging help excess water removal from the electrodes leading to steadier cell response at the expense of extra power consumption. The power system is able to provide above 1 W DC electricity to an external application during 20 h using 1 g of H 2 . An application consisting of a propeller able to display a luminous message is chosen to test system. It is shown that one single air breathing PEM fuel cell powered with hydrogen may provide high energy density and autonomy for portable applications

  17. Spatial reconstruction of single-cell gene expression data.

    Science.gov (United States)

    Satija, Rahul; Farrell, Jeffrey A; Gennert, David; Schier, Alexander F; Regev, Aviv

    2015-05-01

    Spatial localization is a key determinant of cellular fate and behavior, but methods for spatially resolved, transcriptome-wide gene expression profiling across complex tissues are lacking. RNA staining methods assay only a small number of transcripts, whereas single-cell RNA-seq, which measures global gene expression, separates cells from their native spatial context. Here we present Seurat, a computational strategy to infer cellular localization by integrating single-cell RNA-seq data with in situ RNA patterns. We applied Seurat to spatially map 851 single cells from dissociated zebrafish (Danio rerio) embryos and generated a transcriptome-wide map of spatial patterning. We confirmed Seurat's accuracy using several experimental approaches, then used the strategy to identify a set of archetypal expression patterns and spatial markers. Seurat correctly localizes rare subpopulations, accurately mapping both spatially restricted and scattered groups. Seurat will be applicable to mapping cellular localization within complex patterned tissues in diverse systems.

  18. Single-Cell Gene Expression Analysis of a Human ESC Model of Pancreatic Endocrine Development Reveals Different Paths to β-Cell Differentiation.

    Science.gov (United States)

    Petersen, Maja Borup Kjær; Azad, Ajuna; Ingvorsen, Camilla; Hess, Katja; Hansson, Mattias; Grapin-Botton, Anne; Honoré, Christian

    2017-10-10

    The production of insulin-producing β cells from human embryonic stem cells (hESCs) in vitro represents a promising strategy for a cell-based therapy for type 1 diabetes mellitus. To explore the cellular heterogeneity and temporal progression of endocrine progenitors and their progeny, we performed single-cell qPCR on more than 500 cells across several stages of in vitro differentiation of hESCs and compared them with human islets. We reveal distinct subpopulations along the endocrine differentiation path and an early lineage bifurcation toward either polyhormonal cells or β-like cells. We uncover several similarities and differences with mouse development and reveal that cells can take multiple paths to the same differentiation state, a principle that could be relevant to other systems. Notably, activation of the key β-cell transcription factor NKX6.1 can be initiated before or after endocrine commitment. The single-cell temporal resolution we provide can be used to improve the production of functional β cells. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  19. Connecting single cell to collective cell behavior in a unified theoretical framework

    Science.gov (United States)

    George, Mishel; Bullo, Francesco; Campàs, Otger

    Collective cell behavior is an essential part of tissue and organ morphogenesis during embryonic development, as well as of various disease processes, such as cancer. In contrast to many in vitro studies of collective cell migration, most cases of in vivo collective cell migration involve rather small groups of cells, with large sheets of migrating cells being less common. The vast majority of theoretical descriptions of collective cell behavior focus on large numbers of cells, but fail to accurately capture the dynamics of small groups of cells. Here we introduce a low-dimensional theoretical description that successfully captures single cell migration, cell collisions, collective dynamics in small groups of cells, and force propagation during sheet expansion, all within a common theoretical framework. Our description is derived from first principles and also includes key phenomenological aspects of cell migration that control the dynamics of traction forces. Among other results, we explain the counter-intuitive observations that pairs of cells repel each other upon collision while they behave in a coordinated manner within larger clusters.

  20. Performance Analysis of Air Breathing Proton Exchange Membrane Fuel Cell Stack (PEMFCS) At Different Operating Condition

    Science.gov (United States)

    Sunil, V.; Venkata siva, G.; Yoganjaneyulu, G.; Ravikumar, V. V.

    2017-08-01

    The answer for an emission free power source in future is in the form of fuel cells which combine hydrogen and oxygen producing electricity and a harmless by product-water. A proton exchange membrane (PEM) fuel cell is ideal for automotive applications. A single cell cannot supply the essential power for any application. Hence PEM fuel cell stacks are used. The effect of different operating parameters namely: type of convection, type of draught, hydrogen flow rate, hydrogen inlet pressure, ambient temperature and humidity, hydrogen humidity, cell orientation on the performance of air breathing PEM fuel cell stack was analyzed using a computerized fuel cell test station. Then, the fuel cell stack was subjected to different load conditions. It was found that the stack performs very poorly at full capacity (runs only for 30 min. but runs for 3 hours at 50% capacity). Hence, a detailed study was undertaken to maximize the duration of the stack’s performance at peak load.

  1. Automated assembling of single fuel cell units for use in a fuel cell stack

    Science.gov (United States)

    Jalba, C. K.; Muminovic, A.; Barz, C.; Nasui, V.

    2017-05-01

    The manufacturing of PEMFC stacks (POLYMER ELEKTROLYT MEMBRAN Fuel Cell) is nowadays still done by hand. Over hundreds of identical single components have to be placed accurate together for the construction of a fuel cell stack. Beside logistic problems, higher total costs and disadvantages in weight the high number of components produce a higher statistic interference because of faulty erection or material defects and summation of manufacturing tolerances. The saving of costs is about 20 - 25 %. Furthermore, the total weight of the fuel cells will be reduced because of a new sealing technology. Overall a one minute cycle time has to be aimed per cell at the manufacturing of these single components. The change of the existing sealing concept to a bonded sealing is one of the important requisites to get an automated manufacturing of single cell units. One of the important steps for an automated gluing process is the checking of the glue application by using of an image processing system. After bonding the single fuel cell the sealing and electrical function can be checked, so that only functional and high qualitative cells can get into further manufacturing processes.

  2. Repair of γ-irradiation-induced DNA single-strand breaks in human bone marrow cells. Analysis of unfractionated and CD34+ cells using single-cell gel electrophoresis

    International Nuclear Information System (INIS)

    Lankinen, Maarit H.; Vilpo, Juhani A.

    1997-01-01

    Human bone marrow mononuclear cells (BMMNCs) were separated by density gradient centrifugation, and a subpopulation of progenitor cells was further isolated using anti-CD34-coated magnetic beads. The cells were irradiated with γ-rays (0.93-5.43 Gy) from a 137 Cs source. The extent of DNA damage, i.e., single-strand breaks (SSBs) and alkali-labile lesions of individual cells, was investigated using the alkaline single-cell gel electrophoresis technique. The irradiation resulted in a dose-dependent increase in DNA migration, reflecting the number of detectable DNA lesions. An approximately similar extent of SSB formation was observed in BMMNCs and CD34+ cells. Damage was repaired when the cells were incubated at 37C: a fast initial repair phase was followed by a slower rejoining of SSBs in both BMMNC and CD34+ cell populations. A significantly longer time was required to repair the lesions caused by 5.43 Gy than those caused by 0.93 Gy. In the present work we report, for the first time, the induction and repair of DNA SSBs at the level of single human bone marrow cells when exposed to ionizing radiation at clinically relevant doses. These data, together with our previous results with human blood granulocytes and lymphocytes, indicate an approximately similar extent of formation and repair of γ-irradiation-induced DNA SSBs in immature and mature human hematopoietic cells

  3. Single-cell analyses identify bioengineered niches for enhanced maintenance of hematopoietic stem cells.

    Science.gov (United States)

    Roch, Aline; Giger, Sonja; Girotra, Mukul; Campos, Vasco; Vannini, Nicola; Naveiras, Olaia; Gobaa, Samy; Lutolf, Matthias P

    2017-08-09

    The in vitro expansion of long-term hematopoietic stem cells (HSCs) remains a substantial challenge, largely because of our limited understanding of the mechanisms that control HSC fate choices. Using single-cell multigene expression analysis and time-lapse microscopy, here we define gene expression signatures and cell cycle hallmarks of murine HSCs and the earliest multipotent progenitors (MPPs), and analyze systematically single HSC fate choices in culture. Our analysis revealed twelve differentially expressed genes marking the quiescent HSC state, including four genes encoding cell-cell interaction signals in the niche. Under basal culture conditions, most HSCs rapidly commit to become early MPPs. In contrast, when we present ligands of the identified niche components such as JamC or Esam within artificial niches, HSC cycling is reduced and long-term multipotency in vivo is maintained. Our approach to bioengineer artificial niches should be useful in other stem cell systems.Haematopoietic stem cell (HSC) self-renewal is not sufficiently understood to recapitulate in vitro. Here, the authors generate gene signature and cell cycle hallmarks of single murine HSCs, and use identified endothelial receptors Esam and JamC as substrates to enhance HSC growth in engineered niches.

  4. Centrosome Amplification Increases Single-Cell Branching in Post-mitotic Cells.

    Science.gov (United States)

    Ricolo, Delia; Deligiannaki, Myrto; Casanova, Jordi; Araújo, Sofia J

    2016-10-24

    Centrosome amplification is a hallmark of cancer, although we are still far from understanding how this process affects tumorigenesis [1, 2]. Besides the contribution of supernumerary centrosomes to mitotic defects, their biological effects in the post-mitotic cell are not well known. Here, we exploit the effects of centrosome amplification in post-mitotic cells during single-cell branching. We show that Drosophila tracheal cells with extra centrosomes branch more than wild-type cells. We found that mutations in Rca1 and CycA affect subcellular branching, causing tracheal tip cells to form more than one subcellular lumen. We show that Rca1 and CycA post-mitotic cells have supernumerary centrosomes and that other mutant conditions that increase centrosome number also show excess of subcellular lumen branching. Furthermore, we show that de novo lumen formation is impaired in mutant embryos with fewer centrioles. The data presented here define a requirement for the centrosome as a microtubule-organizing center (MTOC) for the initiation of subcellular lumen formation. We propose that centrosomes are necessary to drive subcellular lumen formation. In addition, centrosome amplification increases single-cell branching, a process parallel to capillary sprouting in blood vessels [3]. These results shed new light on how centrosomes can contribute to pathology independently of mitotic defects. Copyright © 2016 Elsevier Ltd. All rights reserved.

  5. What Population Reveals about Individual Cell Identity: Single-Cell Parameter Estimation of Models of Gene Expression in Yeast.

    Directory of Open Access Journals (Sweden)

    Artémis Llamosi

    2016-02-01

    Full Text Available Significant cell-to-cell heterogeneity is ubiquitously observed in isogenic cell populations. Consequently, parameters of models of intracellular processes, usually fitted to population-averaged data, should rather be fitted to individual cells to obtain a population of models of similar but non-identical individuals. Here, we propose a quantitative modeling framework that attributes specific parameter values to single cells for a standard model of gene expression. We combine high quality single-cell measurements of the response of yeast cells to repeated hyperosmotic shocks and state-of-the-art statistical inference approaches for mixed-effects models to infer multidimensional parameter distributions describing the population, and then derive specific parameters for individual cells. The analysis of single-cell parameters shows that single-cell identity (e.g. gene expression dynamics, cell size, growth rate, mother-daughter relationships is, at least partially, captured by the parameter values of gene expression models (e.g. rates of transcription, translation and degradation. Our approach shows how to use the rich information contained into longitudinal single-cell data to infer parameters that can faithfully represent single-cell identity.

  6. Irradiation of single cells with individual high-LET particles

    International Nuclear Information System (INIS)

    Nelson, J.M.; Braby, L.A.

    1993-01-01

    The dose-limiting normal tissue of concern when irradiating head and neck lesions is often the vascular endothelium within the treatment field. Consequently, the response of capillary endothelial cells exposed to moderate doses of high LET particles is essential for establishing exposure limits for neutron-capture therapy. In an effort to characterize the high-LET radiation biology of cultured endothelial cells, the authors are attempting to measure cellular response to single particles. The single-particle irradiation apparatus, described below, allows them to expose individual cells to known numbers of high-LET particles and follow these cells for extended periods, in order to assess the impact of individual particles on cell growth kinetics. Preliminary cell irradiation experiments have revealed complications related to the smooth and efficient operation of the equipment; these are being resolved. Therefore, the following paragraphs deal primarily with the manner by which high LET particles deposit energy, the requirements for single-cell irradiation, construction and assembly of such apparatus, and testing of experimental procedures, rather than with the radiation biology of endothelial cells

  7. A microfluidic system for studying ageing and dynamic single-cell responses in budding yeast.

    Directory of Open Access Journals (Sweden)

    Matthew M Crane

    Full Text Available Recognition of the importance of cell-to-cell variability in cellular decision-making and a growing interest in stochastic modeling of cellular processes has led to an increased demand for high density, reproducible, single-cell measurements in time-varying surroundings. We present ALCATRAS (A Long-term Culturing And TRApping System, a microfluidic device that can quantitatively monitor up to 1000 cells of budding yeast in a well-defined and controlled environment. Daughter cells are removed by fluid flow to avoid crowding allowing experiments to run for over 60 hours, and the extracellular media may be changed repeatedly and in seconds. We illustrate use of the device by measuring ageing through replicative life span curves, following the dynamics of the cell cycle, and examining history-dependent behaviour in the general stress response.

  8. Single-cell proteomics: potential implications for cancer diagnostics.

    Science.gov (United States)

    Gavasso, Sonia; Gullaksen, Stein-Erik; Skavland, Jørn; Gjertsen, Bjørn T

    2016-01-01

    Single-cell proteomics in cancer is evolving and promises to provide more accurate diagnoses based on detailed molecular features of cells within tumors. This review focuses on technologies that allow for collection of complex data from single cells, but also highlights methods that are adaptable to routine cancer diagnostics. Current diagnostics rely on histopathological analysis, complemented by mutational detection and clinical imaging. Though crucial, the information gained is often not directly transferable to defined therapeutic strategies, and predicting therapy response in a patient is difficult. In cancer, cellular states revealed through perturbed intracellular signaling pathways can identify functional mutations recurrent in cancer subsets. Single-cell proteomics remains to be validated in clinical trials where serial samples before and during treatment can reveal excessive clonal evolution and therapy failure; its use in clinical trials is anticipated to ignite a diagnostic revolution that will better align diagnostics with the current biological understanding of cancer.

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

    Science.gov (United States)

    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.

  10. The effect of Cytochalasin D on F-Actin behavior of single-cell electroendocytosis using multi-chamber micro cell chip

    KAUST Repository

    Lin, Ran

    2012-03-01

    Electroendocytosis (EED) is a pulsed-electric-field (PEF) induced endocytosis, facilitating cells uptake molecules through nanometer-sized EED vesicles. We herein investigate the effect of a chemical inhibitor, Cytochalasin D (CD) on the actin-filaments (F-Actin) behavior of single-cell EED. The CD concentration (C CD) can control the depolymerization of F-actin. A multi-chamber micro cell chip was fabricated to study the EED under different conditions. Large-scale single-cell data demonstrated EED highly depends on both electric field and C CD. © 2012 IEEE.

  11. The effect of Cytochalasin D on F-Actin behavior of single-cell electroendocytosis using multi-chamber micro cell chip

    KAUST Repository

    Lin, Ran; Chang, Donald C.; Lee, Yi Kuen

    2012-01-01

    Electroendocytosis (EED) is a pulsed-electric-field (PEF) induced endocytosis, facilitating cells uptake molecules through nanometer-sized EED vesicles. We herein investigate the effect of a chemical inhibitor, Cytochalasin D (CD) on the actin-filaments (F-Actin) behavior of single-cell EED. The CD concentration (C CD) can control the depolymerization of F-actin. A multi-chamber micro cell chip was fabricated to study the EED under different conditions. Large-scale single-cell data demonstrated EED highly depends on both electric field and C CD. © 2012 IEEE.

  12. Magnetic domain wall conduits for single cell applications

    DEFF Research Database (Denmark)

    Donolato, Marco; Torti, A.; Kostesha, Natalie

    2011-01-01

    The ability to trap, manipulate and release single cells on a surface is important both for fundamental studies of cellular processes and for the development of novel lab-on-chip miniaturized tools for biological and medical applications. In this paper we demonstrate how magnetic domain walls...... walls over 16 hours. Moreover, we demonstrate the controlled transport and release of individual yeast cells via displacement and annihilation of individual domain walls in micro- and nano-sized magnetic structures. These results pave the way to the implementation of magnetic devices based on domain...... walls technology in lab-on-chip systems devoted to accurate individual cell trapping and manipulation....

  13. Mutation dynamics and fitness effects followed in single cells.

    Science.gov (United States)

    Robert, Lydia; Ollion, Jean; Robert, Jerome; Song, Xiaohu; Matic, Ivan; Elez, Marina

    2018-03-16

    Mutations have been investigated for more than a century but remain difficult to observe directly in single cells, which limits the characterization of their dynamics and fitness effects. By combining microfluidics, time-lapse imaging, and a fluorescent tag of the mismatch repair system in Escherichia coli , we visualized the emergence of mutations in single cells, revealing Poissonian dynamics. Concomitantly, we tracked the growth and life span of single cells, accumulating ~20,000 mutations genome-wide over hundreds of generations. This analysis revealed that 1% of mutations were lethal; nonlethal mutations displayed a heavy-tailed distribution of fitness effects and were dominated by quasi-neutral mutations with an average cost of 0.3%. Our approach has enabled the investigation of single-cell individuality in mutation rate, mutation fitness costs, and mutation interactions. Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

  14. Micropillar arrays enabling single microbial cell encapsulation in hydrogels.

    Science.gov (United States)

    Park, Kyun Joo; Lee, Kyoung G; Seok, Seunghwan; Choi, Bong Gill; Lee, Moon-Keun; Park, Tae Jung; Park, Jung Youn; Kim, Do Hyun; Lee, Seok Jae

    2014-06-07

    Single microbial cell encapsulation in hydrogels is an important task to find valuable biological resources for human welfare. The conventional microfluidic designs are mainly targeted only for highly dispersed spherical bioparticles. Advanced structures should be taken into consideration for handling such aggregated and non-spherical microorganisms. Here, to address the challenge, we propose a new type of cylindrical-shaped micropillar array in a microfluidic device for enhancing the dispersion of cell clusters and the isolation of individual cells into individual micro-hydrogels for potential practical applications. The incorporated micropillars act as a sieve for the breaking of Escherichia coli (E. coli) clusters into single cells in a polymer mixture. Furthermore, the combination of hydrodynamic forces and a flow-focusing technique will improve the probability of encapsulation of a single cell into each hydrogel with a broad range of cell concentrations. This proposed strategy and device would be a useful platform for genetically modified microorganisms for practical applications.

  15. Differentiation of a bipotential glial progenitor cell in a single cell microculture.

    Science.gov (United States)

    Temple, S; Raff, M C

    Although it is known that most cells of the vertebrate central nervous system (CNS) are derived from the neuroepithelial cells of the neural tube, the factors determining whether an individual neuroepithelial cell develops into a particular type of neurone or glial cell remain unknown. A promising model for studying this problem is the bipotential glial progenitor cell in the developing rat optic nerve; this cell differentiates into a particular type of astrocyte (a type-2 astrocyte) if cultured in 10% fetal calf serum (FCS) and into an oligodendrocyte if cultured in serum-free medium. As the oligodendrocyte-type-2 astrocyte (0-2A) progenitor cell can differentiate along either glial pathway in neurone-free cultures, living axons clearly are not required for its differentiation, at least in vitro. However, the studies on 0-2A progenitor cells were carried out in bulk cultures of optic nerve, and so it was possible that other cell-cell interactions were required for differentiation in culture. We show here that 0-2A progenitor cells can differentiate into type-2 astrocytes or oligodendrocytes when grown as isolated cells in microculture, indicating that differentiation along either glial pathway in vitro does not require signals from other CNS cells, apart from the signals provided by components of the culture medium. We also show that single 0-2A progenitor cells can differentiate along either pathway without dividing, supporting our previous studies using 3H-thymidine and suggesting that DNA replication is not required for these cells to choose between the two differentiation programmes.

  16. Bridging the Timescales of Single-Cell and Population Dynamics

    Science.gov (United States)

    Jafarpour, Farshid; Wright, Charles S.; Gudjonson, Herman; Riebling, Jedidiah; Dawson, Emma; Lo, Klevin; Fiebig, Aretha; Crosson, Sean; Dinner, Aaron R.; Iyer-Biswas, Srividya

    2018-04-01

    How are granular details of stochastic growth and division of individual cells reflected in smooth deterministic growth of population numbers? We provide an integrated, multiscale perspective of microbial growth dynamics by formulating a data-validated theoretical framework that accounts for observables at both single-cell and population scales. We derive exact analytical complete time-dependent solutions to cell-age distributions and population growth rates as functionals of the underlying interdivision time distributions, for symmetric and asymmetric cell division. These results provide insights into the surprising implications of stochastic single-cell dynamics for population growth. Using our results for asymmetric division, we deduce the time to transition from the reproductively quiescent (swarmer) to the replication-competent (stalked) stage of the Caulobacter crescentus life cycle. Remarkably, population numbers can spontaneously oscillate with time. We elucidate the physics leading to these population oscillations. For C. crescentus cells, we show that a simple measurement of the population growth rate, for a given growth condition, is sufficient to characterize the condition-specific cellular unit of time and, thus, yields the mean (single-cell) growth and division timescales, fluctuations in cell division times, the cell-age distribution, and the quiescence timescale.

  17. Allogeneic cell therapy bioprocess economics and optimization: single-use cell expansion technologies.

    Science.gov (United States)

    Simaria, Ana S; Hassan, Sally; Varadaraju, Hemanthram; Rowley, Jon; Warren, Kim; Vanek, Philip; Farid, Suzanne S

    2014-01-01

    For allogeneic cell therapies to reach their therapeutic potential, challenges related to achieving scalable and robust manufacturing processes will need to be addressed. A particular challenge is producing lot-sizes capable of meeting commercial demands of up to 10(9) cells/dose for large patient numbers due to the current limitations of expansion technologies. This article describes the application of a decisional tool to identify the most cost-effective expansion technologies for different scales of production as well as current gaps in the technology capabilities for allogeneic cell therapy manufacture. The tool integrates bioprocess economics with optimization to assess the economic competitiveness of planar and microcarrier-based cell expansion technologies. Visualization methods were used to identify the production scales where planar technologies will cease to be cost-effective and where microcarrier-based bioreactors become the only option. The tool outputs also predict that for the industry to be sustainable for high demand scenarios, significant increases will likely be needed in the performance capabilities of microcarrier-based systems. These data are presented using a technology S-curve as well as windows of operation to identify the combination of cell productivities and scale of single-use bioreactors required to meet future lot sizes. The modeling insights can be used to identify where future R&D investment should be focused to improve the performance of the most promising technologies so that they become a robust and scalable option that enables the cell therapy industry reach commercially relevant lot sizes. The tool outputs can facilitate decision-making very early on in development and be used to predict, and better manage, the risk of process changes needed as products proceed through the development pathway. © 2013 Wiley Periodicals, Inc.

  18. Single wall carbon nanotube supports for portable direct methanol fuel cells.

    Science.gov (United States)

    Girishkumar, G; Hall, Timothy D; Vinodgopal, K; Kamat, Prashant V

    2006-01-12

    Single-wall and multiwall carbon nanotubes are employed as carbon supports in direct methanol fuel cells (DMFC). The morphology and electrochemical activity of single-wall and multiwall carbon nanotubes obtained from different sources have been examined to probe the influence of carbon support on the overall performance of DMFC. The improved activity of the Pt-Ru catalyst dispersed on carbon nanotubes toward methanol oxidation is reflected as a shift in the onset potential and a lower charge transfer resistance at the electrode/electrolyte interface. The evaluation of carbon supports in a passive air breathing DMFC indicates that the observed power density depends on the nature and source of carbon nanostructures. The intrinsic property of the nanotubes, dispersion of the electrocatalyst and the electrochemically active surface area collectively influence the performance of the membrane electrode assembly (MEA). As compared to the commercial carbon black support, single wall carbon nanotubes when employed as the support for anchoring the electrocatalyst particles in the anode and cathode sides of MEA exhibited a approximately 30% enhancement in the power density of a single stack DMFC operating at 70 degrees C.

  19. Unravelling biology and shifting paradigms in cancer with single-cell sequencing.

    Science.gov (United States)

    Baslan, Timour; Hicks, James

    2017-08-24

    The fundamental operative unit of a cancer is the genetically and epigenetically innovative single cell. Whether proliferating or quiescent, in the primary tumour mass or disseminated elsewhere, single cells govern the parameters that dictate all facets of the biology of cancer. Thus, single-cell analyses provide the ultimate level of resolution in our quest for a fundamental understanding of this disease. Historically, this quest has been hampered by technological shortcomings. In this Opinion article, we argue that the rapidly evolving field of single-cell sequencing has unshackled the cancer research community of these shortcomings. From furthering an elemental understanding of intra-tumoural genetic heterogeneity and cancer genome evolution to illuminating the governing principles of disease relapse and metastasis, we posit that single-cell sequencing promises to unravel the biology of all facets of this disease.

  20. Few single nucleotide variations in exomes of human cord blood induced pluripotent stem cells.

    Directory of Open Access Journals (Sweden)

    Rui-Jun Su

    Full Text Available The effect of the cellular reprogramming process per se on mutation load remains unclear. To address this issue, we performed whole exome sequencing analysis of induced pluripotent stem cells (iPSCs reprogrammed from human cord blood (CB CD34(+ cells. Cells from a single donor and improved lentiviral vectors for high-efficiency (2-14% reprogramming were used to examine the effects of three different combinations of reprogramming factors: OCT4 and SOX2 (OS, OS and ZSCAN4 (OSZ, OS and MYC and KLF4 (OSMK. Five clones from each group were subject to whole exome sequencing analysis. We identified 14, 11, and 9 single nucleotide variations (SNVs, in exomes, including untranslated regions (UTR, in the five clones of OSMK, OS, and OSZ iPSC lines. Only 8, 7, and 4 of these, respectively, were protein-coding mutations. An average of 1.3 coding mutations per CB iPSC line is remarkably lower than previous studies using fibroblasts and low-efficiency reprogramming approaches. These data demonstrate that point nucleotide mutations during cord blood reprogramming are negligible and that the inclusion of genome stabilizers like ZSCAN4 during reprogramming may further decrease reprogramming-associated mutations. Our findings provide evidence that CB is a superior source of cells for iPSC banking.

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

    Science.gov (United States)

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

    2013-01-01

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

  2. Single and multi-frequency impedance characterization of symmetric activated carbon single capacitor cells

    Directory of Open Access Journals (Sweden)

    Suzana Sopčić

    2018-05-01

    Full Text Available Electrochemical impedance spectroscopy (EIS technique is used for characterization of single cell symmetric capacitors having different mass loadings of activated carbon (AC. Relevant values of charge storage capacitance (CT and internal resistance (ESR were evaluated by the single frequency and multi-frequency analyses of measured impedance spectra. Curve fittings were based on the non-ideal R-C model that takes into account the parasitic inductance, contributions from electrode materials/contacts and the effects of AC porosity. Higher CT and lower ESR values were obtained not only for the cell with higher mass of AC, but also using the single vs. multi-frequency approach. Lower CT and higher values of ESR that are generally obtained using the multi-frequency method and curve fittings should be related to the not ideal capacitive response of porous AC material and too high frequency chosen in applying the single frequency analysis.

  3. Performance of Electrolyte Supported Solid Oxide Fuel Cells with STN Anodes

    DEFF Research Database (Denmark)

    Veltzé, Sune; Reddy Sudireddy, Bhaskar; Jørgensen, Peter Stanley

    2013-01-01

    In order to replace the state of the art Ni-cermet as SOFC anode, electrolyte supported cells comprising CGO/Ni infiltrated Nbdoped SrTiO3 anodes, and LSM/YSZ cathodes have been developed and tested as single 5 x 5 cm2 cells. The initial performance reached 0.4 W/cm2 at 850 C. Further tests under...

  4. Gravisensing in single-celled systems

    Science.gov (United States)

    Braun, M.; Limbach, C.

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

  5. Single-cell tracking reveals antibiotic-induced changes in mycobacterial energy metabolism.

    Science.gov (United States)

    Maglica, Željka; Özdemir, Emre; McKinney, John D

    2015-02-17

    ATP is a key molecule of cell physiology, but despite its importance, there are currently no methods for monitoring single-cell ATP fluctuations in live bacteria. This is a major obstacle in studies of bacterial energy metabolism, because there is a growing awareness that bacteria respond to stressors such as antibiotics in a highly individualistic manner. Here, we present a method for long-term single-cell tracking of ATP levels in Mycobacterium smegmatis based on a combination of microfluidics, time-lapse microscopy, and Förster resonance energy transfer (FRET)-based ATP biosensors. Upon treating cells with antibiotics, we observed that individual cells undergo an abrupt and irreversible switch from high to low intracellular ATP levels. The kinetics and extent of ATP switching clearly discriminate between an inhibitor of ATP synthesis and other classes of antibiotics. Cells that resume growth after 24 h of antibiotic treatment maintain high ATP levels throughout the exposure period. In contrast, antibiotic-treated cells that switch from ATP-high to ATP-low states never resume growth after antibiotic washout. Surprisingly, only a subset of these nongrowing ATP-low cells stains with propidium iodide (PI), a widely used live/dead cell marker. These experiments also reveal a cryptic subset of cells that do not resume growth after antibiotic washout despite remaining ATP high and PI negative. We conclude that ATP tracking is a more dynamic, sensitive, reliable, and discriminating marker of cell viability than staining with PI. This method could be used in studies to evaluate antimicrobial effectiveness and mechanism of action, as well as for high-throughput screening. New antimicrobials are urgently needed to stem the rising tide of antibiotic-resistant bacteria. All antibiotics are expected to affect bacterial energy metabolism, directly or indirectly, yet tools to assess the impact of antibiotics on the ATP content of individual bacterial cells are lacking. The

  6. Each cell counts: Hematopoiesis and immunity research in the era of single cell genomics.

    Science.gov (United States)

    Jaitin, Diego Adhemar; Keren-Shaul, Hadas; Elefant, Naama; Amit, Ido

    2015-02-01

    Hematopoiesis and immunity are mediated through complex interactions between multiple cell types and states. This complexity is currently addressed following a reductionist approach of characterizing cell types by a small number of cell surface molecular features and gross functions. While the introduction of global transcriptional profiling technologies enabled a more comprehensive view, heterogeneity within sampled populations remained unaddressed, obscuring the true picture of hematopoiesis and immune system function. A critical mass of technological advances in molecular biology and genomics has enabled genome-wide measurements of single cells - the fundamental unit of immunity. These new advances are expected to boost detection of less frequent cell types and fuzzy intermediate cell states, greatly expanding the resolution of current available classifications. This new era of single-cell genomics in immunology research holds great promise for further understanding of the mechanisms and circuits regulating hematopoiesis and immunity in both health and disease. In the near future, the accuracy of single-cell genomics will ultimately enable precise diagnostics and treatment of multiple hematopoietic and immune related diseases. Copyright © 2015 Elsevier Ltd. All rights reserved.

  7. Targeted sorting of single virus-infected cells of the coccolithophore Emiliania huxleyi.

    Science.gov (United States)

    Martínez Martínez, Joaquín; Poulton, Nicole J; Stepanauskas, Ramunas; Sieracki, Michael E; Wilson, William H

    2011-01-01

    Discriminating infected from healthy cells is the first step to understanding the mechanisms and ecological implications of viral infection. We have developed a method for detecting, sorting, and performing molecular analysis of individual, infected cells of the important microalga Emiliania huxleyi, based on known physiological responses to viral infection. Of three fluorescent dyes tested, FM 1-43 (for detecting membrane blebbing) gave the most unequivocal and earliest separation of cells. Furthermore, we were able to amplify the genomes of single infected cells using Multiple Displacement Amplification. This novel method to reliably discriminate infected from healthy cells in cultures will allow researchers to answer numerous questions regarding the mechanisms and implications of viral infection of E. huxleyi. The method may be transferable to other virus-host systems.

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

    Science.gov (United States)

    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.

  9. Single-cell 5hmC sequencing reveals chromosome-wide cell-to-cell variability and enables lineage reconstruction

    NARCIS (Netherlands)

    Mooijman, Dylan; Dey, Siddharth S; Boisset, Jean-Charles; Crosetto, Nicola; van Oudenaarden, Alexander

    2016-01-01

    The epigenetic DNA modification 5-hydroxymethylcytosine (5hmC) has crucial roles in development and gene regulation. Quantifying the abundance of this epigenetic mark at the single-cell level could enable us to understand its roles. We present a single-cell, genome-wide and strand-specific 5hmC

  10. Single-cell protein secretomic signatures as potential correlates to tumor cell lineage evolution and cell-cell interaction

    Directory of Open Access Journals (Sweden)

    Minsuk eKwak

    2013-02-01

    Full Text Available Secreted proteins including cytokines, chemokines and growth factors represent important functional regulators mediating a range of cellular behavior and cell-cell paracrine/autocrine signaling, e.g. in the immunological system, tumor microenvironment or stem cell niche. Detection of these proteins is of great value not only in basic cell biology but also for diagnosis and therapeutic monitoring of human diseases such as cancer. However, due to co-production of multiple effector proteins from a single cell, referred to as polyfunctionality, it is biologically informative to measure a panel of secreted proteins, or secretomic signature, at the level of single cells. Recent evidence further indicates that a genetically-identical cell population can give rise to diverse phenotypic differences. It is known that cytokines, for example, in the immune system define the effector functions and lineage differentiation of immune cells. In this Perspective Article, we hypothesize that protein secretion profile may represent a universal measure to identify the definitive correlate in the larger context of cellular functions to dissect cellular heterogeneity and evolutionary lineage relationship in human cancer.

  11. Experimental study of commercial size proton exchange membrane fuel cell performance

    International Nuclear Information System (INIS)

    Yan, Wei-Mon; Wang, Xiao-Dong; Lee, Duu-Jong; Zhang, Xin-Xin; Guo, Yi-Fan; Su, Ay

    2011-01-01

    Commercial sized (16 x 16 cm 2 active surface area) proton exchange membrane (PEM) fuel cells with serpentine flow chambers are fabricated. The GORE-TEX (registered) PRIMEA 5621 was used with a 35-μm-thick PEM with an anode catalyst layer with 0.45 mg cm -2 Pt and cathode catalyst layer with 0.6 mg cm -2 Pt and Ru or GORE-TEX (registered) PRIMEA 57 was used with an 18-μm-thick PEM with an anode catalyst layer at 0.2 mg cm -2 Pt and cathode catalyst layer at 0.4 mg cm -2 of Pt and Ru. At the specified cell and humidification temperatures, the thin PRIMEA 57 membrane yields better cell performance than the thick PRIMEA 5621 membrane, since hydration of the former is more easily maintained with the limited amount of produced water. Sufficient humidification at both the cathode and anode sides is essential to achieve high cell performance with a thick membrane, like the PRIMEA 5621. The optimal cell temperature to produce the best cell performance with PRIMEA 5621 is close to the humidification temperature. For PRIMEA 57, however, optimal cell temperature exceeds the humidification temperature.

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

    Science.gov (United States)

    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

    2016-01-12

    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. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  13. Real-time Bacterial Detection by Single Cell Based Sensors UsingSynchrotron FTIR Spectromicroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Veiseh, Mandana; Veiseh, Omid; Martin, Michael C.; Bertozzi,Carolyn; Zhang, Miqin

    2005-08-10

    Microarrays of single macrophage cell based sensors weredeveloped and demonstrated for real time bacterium detection bysynchrotron FTIR microscopy. The cells were patterned on gold-SiO2substrates via a surface engineering technique by which the goldelectrodes were immobilized with fibronectin to mediate cell adhesion andthe silicon oxide background were passivated with PEG to resist proteinadsorption and cell adhesion. Cellular morphology and IR spectra ofsingle, double, and triple cells on gold electrodes exposed tolipopolysaccharide (LPS) of different concentrations were compared toreveal the detection capabilities of these biosensors. The single-cellbased sensors were found to generate the most significant IR wave numbervariation and thus provide the highest detection sensitivity. Changes inmorphology and IR spectrum for single cells exposed to LPS were found tobe time- and concentration-dependent and correlated with each other verywell. FTIR spectra from single cell arrays of gold electrodes withsurface area of 25 mu-m2, 100 mu-m2, and 400 mu-m2 were acquired usingboth synchrotron and conventional FTIR spectromicroscopes to study thesensitivity of detection. The results indicated that the developedsingle-cell platform can be used with conventional FTIRspectromicroscopy. This technique provides real-time, label-free, andrapid bacterial detection, and may allow for statistic and highthroughput analyses, and portability.

  14. Correlated receptor transport processes buffer single-cell heterogeneity.

    Directory of Open Access Journals (Sweden)

    Stefan M Kallenberger

    2017-09-01

    Full Text Available Cells typically vary in their response to extracellular ligands. Receptor transport processes modulate ligand-receptor induced signal transduction and impact the variability in cellular responses. Here, we quantitatively characterized cellular variability in erythropoietin receptor (EpoR trafficking at the single-cell level based on live-cell imaging and mathematical modeling. Using ensembles of single-cell mathematical models reduced parameter uncertainties and showed that rapid EpoR turnover, transport of internalized EpoR back to the plasma membrane, and degradation of Epo-EpoR complexes were essential for receptor trafficking. EpoR trafficking dynamics in adherent H838 lung cancer cells closely resembled the dynamics previously characterized by mathematical modeling in suspension cells, indicating that dynamic properties of the EpoR system are widely conserved. Receptor transport processes differed by one order of magnitude between individual cells. However, the concentration of activated Epo-EpoR complexes was less variable due to the correlated kinetics of opposing transport processes acting as a buffering system.

  15. Performance requirements for the single-shell tank

    International Nuclear Information System (INIS)

    GRENARD, C.E.

    1999-01-01

    This document provides performance requirements for the waste storage and waste feed delivery functions of the Single-Shell Tank (SST) System. The requirements presented here in will be used as a basis for evaluating the ability of the system to complete the single-shell tank waste feed delivery mission. They will also be used to select the technology or technologies for retrieving waste from the tanks selected for the single-shell tank waste feed delivery mission, assumed to be 241-C-102 and 241-C-104. This revision of the Performance Requirements for the SST is based on the findings of the SST Functional Analysis, and are reflected in the current System Specification for the SST System

  16. Effect of single-dose radiation on cell survival and growth hormone secretion by rat anterior pituitary cells

    International Nuclear Information System (INIS)

    Hochberg, Z.; Kuten, A.; Hertz, P.; Tatcher, M.; Kedar, A.; Benderly, A.

    1983-01-01

    Cranial irradiation has been shown to impair growth hormone secretion in children. In this study a cell culture of dispersed rat anterior pituitary cells was exposed to single doses of radiation in the range of 100 to 1500 rad. Survival curves were obtained for the different anterior pituitary cell lines, and growth hormone secretion was measured in the tissue culture medium. Both survival and growth hormone secretion curves showed an initial shoulder in the range of 0 to 300 rad, followed by a decline between 300 to 750 rad. It is concluded that growth hormone secreting acidophilic pituicytes are sensitive to radiation at single doses greater than 300 rad

  17. Single-Cell Memory Regulates a Neural Circuit for Sensory Behavior.

    Science.gov (United States)

    Kobayashi, Kyogo; Nakano, Shunji; Amano, Mutsuki; Tsuboi, Daisuke; Nishioka, Tomoki; Ikeda, Shingo; Yokoyama, Genta; Kaibuchi, Kozo; Mori, Ikue

    2016-01-05

    Unveiling the molecular and cellular mechanisms underlying memory has been a challenge for the past few decades. Although synaptic plasticity is proven to be essential for memory formation, the significance of "single-cell memory" still remains elusive. Here, we exploited a primary culture system for the analysis of C. elegans neurons and show that a single thermosensory neuron has an ability to form, retain, and reset a temperature memory. Genetic and proteomic analyses found that the expression of the single-cell memory exhibits inter-individual variability, which is controlled by the evolutionarily conserved CaMKI/IV and Raf pathway. The variable responses of a sensory neuron influenced the neural activity of downstream interneurons, suggesting that modulation of the sensory neurons ultimately determines the behavioral output in C. elegans. Our results provide proof of single-cell memory and suggest that the individual differences in neural responses at the single-cell level can confer individuality. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  18. High-throughput deterministic single-cell encapsulation and droplet pairing, fusion, and shrinkage in a single microfluidic device.

    Science.gov (United States)

    Schoeman, Rogier M; Kemna, Evelien W M; Wolbers, Floor; van den Berg, Albert

    2014-02-01

    In this article, we present a microfluidic device capable of successive high-yield single-cell encapsulation in droplets, with additional droplet pairing, fusion, and shrinkage. Deterministic single-cell encapsulation is realized using Dean-coupled inertial ordering of cells in a Yin-Yang-shaped curved microchannel using a double T-junction, with a frequency over 2000 Hz, followed by controlled droplet pairing with a 100% success rate. Subsequently, droplet fusion is realized using electrical actuation resulting in electro-coalescence of two droplets, each containing a single HL60 cell, with 95% efficiency. Finally, volume reduction of the fused droplet up to 75% is achieved by a triple pitchfork structure. This droplet volume reduction is necessary to obtain close cell-cell membrane contact necessary for final cell electrofusion, leading to hybridoma formation, which is the ultimate aim of this research. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Dissecting human cerebral organoids and fetal neocortex using single-cell RNAseq

    Science.gov (United States)

    Treutlein, Barbara

    Cerebral organoids - three-dimensional cultures of human cerebral tissue derived from pluripotent stem cells - have emerged as models of human cortical development. However, the extent to which in vitro organoid systems recapitulate neural progenitor cell proliferation and neuronal differentiation programs observed in vivo remains unclear. Here we use single-cell RNA sequencing (scRNA-seq) to dissect and compare cell composition and progenitor-to-neuron lineage relationships in human cerebral organoids and fetal neocortex. Covariation network analysis using the fetal neocortex data reveals known and novel interactions among genes central to neural progenitor proliferation and neuronal differentiation. In the organoid, we detect diverse progenitors and differentiated cell types of neuronal and mesenchymal lineages, and identify cells that derived from regions resembling the fetal neocortex. We find that these organoid cortical cells use gene expression programs remarkably similar to those of the fetal tissue in order to organize into cerebral cortex-like regions. Our comparison of in vivo and in vitro cortical single cell transcriptomes illuminates the genetic features underlying human cortical development that can be studied in organoid cultures.

  20. Single cell FRET analysis for the identification of optimal FRET-pairs in Bacillus subtilis using a prototype MEM-FLIM system.

    Directory of Open Access Journals (Sweden)

    Ruud G J Detert Oude Weme

    Full Text Available Protein-protein interactions can be studied in vitro, e.g. with bacterial or yeast two-hybrid systems or surface plasmon resonance. In contrast to in vitro techniques, in vivo studies of protein-protein interactions allow examination of spatial and temporal behavior of such interactions in their native environment. One approach to study protein-protein interactions in vivo is via Förster Resonance Energy Transfer (FRET. Here, FRET efficiency of selected FRET-pairs was studied at the single cell level using sensitized emission and Frequency Domain-Fluorescence Lifetime Imaging Microscopy (FD-FLIM. For FRET-FLIM, a prototype Modulated Electron-Multiplied FLIM system was used, which is, to the best of our knowledge, the first account of Frequency Domain FLIM to analyze FRET in single bacterial cells. To perform FRET-FLIM, we first determined and benchmarked the best fluorescent protein-pair for FRET in Bacillus subtilis using a novel BglBrick-compatible integration vector. We show that GFP-tagRFP is an excellent donor-acceptor pair for B. subtilis in vivo FRET studies. As a proof of concept, selected donor and acceptor fluorescent proteins were fused using a linker that contained a tobacco etch virus (TEV-protease recognition sequence. Induction of TEV-protease results in loss of FRET efficiency and increase in fluorescence lifetime. The loss of FRET efficiency after TEV induction can be followed in time in single cells via time-lapse microscopy. This work will facilitate future studies of in vivo dynamics of protein complexes in single B. subtilis cells.

  1. MicroBioRobots for single cell manipulation

    Science.gov (United States)

    Sakar, Mahmut Selman

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

  2. Unit-cell design for two-dimensional phase-field simulation of microstructure evolution in single-crystal Ni-based superalloys during solidification

    Directory of Open Access Journals (Sweden)

    Dongjia Cao

    2017-12-01

    Full Text Available Phase-field simulation serves as an effective tool for quantitative characterization of microstructure evolution in single-crystal Ni-based superalloys during solidification nowadays. The classic unit cell is either limited to γ dendrites along crystal orientation or too ideal to cover complex morphologies for γ dendrites. An attempt to design the unit cell for two-dimensional (2-D phase-field simulations of microstructure evolution in single-crystal Ni-based superalloys during solidification was thus performed by using the MICRESS (MICRostructure Evolution Simulation Software in the framework of the multi-phase-field (MPF model, and demonstrated in a commercial TMS-113 superalloy. The coupling to CALPHAD (CALculation of PHAse Diagram thermodynamic database was realized via the TQ interface and the experimental diffusion coefficients were utilized in the simulation. Firstly, the classic unit cell with a single γ dendrite along crystal orientation was employed for the phase-field simulation in order to reproduce the microstructure features. Then, such simple unit cell was extended into the cases with two other different crystal orientations, i.e., and . Thirdly, for crystal orientations, the effect of γ dendritic orientations and unit cell sizes on microstructure and microsegregation was comprehensively studied, from which a new unit cell with multiple γ dendrites was proposed. The phase-field simulation with the newly proposed unit cell was further performed in the TMS-113 superalloy, and the microstructure features including the competitive growth of γ dendrites, microsegregation of different solutes and distribution of γ′ grains, can be nicely reproduced.

  3. A simple optical fiber device for quantitative fluorescence microscopy of single living cells

    NARCIS (Netherlands)

    van Graft, M.; van Graft, Marja; Oosterhuis, B.; Oosterhuis, Bernard; van der Werf, Kees; de Grooth, B.G.; Greve, Jan

    1993-01-01

    simple and relatively inexpensive system is described for obtaining quantitative fluorescence measurements on single living cells loaded with a fluorescent probe to study cell physiological processes. The light emitted from the fluorescent cells is captured by and transported through an optical

  4. Bubble Jet agent release cartridge for chemical single cell stimulation.

    Science.gov (United States)

    Wangler, N; Welsche, M; Blazek, M; Blessing, M; Vervliet-Scheebaum, M; Reski, R; Müller, C; Reinecke, H; Steigert, J; Roth, G; Zengerle, R; Paust, N

    2013-02-01

    We present a new method for the distinct specific chemical stimulation of single cells and small cell clusters within their natural environment. By single-drop release of chemical agents with droplets in size of typical cell diameters (d agent release cartridge with integrated fluidic structures and integrated agent reservoirs are shown, tested, and compared in this publication. The single channel setup features a fluidic structure fabricated by anisotropic etching of silicon. To allow for simultaneous release of different agents even though maintaining the same device size, the second type comprises a double channel fluidic structure, fabricated by photolithographic patterning of TMMF. Dispensed droplet volumes are V = 15 pl and V = 10 pl for the silicon and the TMMF based setups, respectively. Utilizing the agent release cartridges, the application in biological assays was demonstrated by hormone-stimulated premature bud formation in Physcomitrella patens and the individual staining of one single L 929 cell within a confluent grown cell culture.

  5. Tip chip : Subcellular sampling from single cancer cells

    NARCIS (Netherlands)

    Quist, Jos; Sarajlic, Edin; Lai, Stanley C.S.; Lemay, Serge G.

    2016-01-01

    To analyze the molecular content of single cells, cell lysis is typically required, yielding a snapshot of cell behavior only. To follow complex molecular profiles over time, subcellular sampling methods potentially can be used, but to date these methods involve laborious offline analysis. Here we

  6. Non-invasive optoacoustic probing of the density and stiffness of single biological cells

    Science.gov (United States)

    Dehoux, T.; Audoin, B.

    2012-12-01

    Recently, the coherent generation of GHz acoustic waves using ultrashort laser pulses has demonstrated the ability to probe the sound velocity in vegetal cells and in cell-mimicking soft micro-objects with micrometer resolution, opening tremendous potentialities for single-cell biology. However, manipulating biological media in physiological conditions is often a technical challenge when using a laser-based setup. In this article, we present a new opto-acoustic bio-transducer composed of a thin metal film sputtered on a transparent heat sink that allows reducing importantly the laser-induced cellular stresses, and offers a wide variety of optical configurations. In particular, by exploiting the acoustic reflection coefficient at the sample-transducer interface and the photoacoustic interaction inside the transparent sample, the density and compressibility of the sample can be probed simultaneously. Using an ad hoc signal analysis based on Hilbert and wavelet transforms, these quantities are measured accurately for a reference fluid. Similar analysis performed in a single vegetal cell also suggests high sensitivity to the state of the transducer-cell interface, and notably to the presence of the plasma membrane that encloses the cell vacuole.

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

    Directory of Open Access Journals (Sweden)

    Spyros Darmanis

    2016-01-01

    Full Text Available 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.

  8. Specific single-cell isolation and genomic amplification of uncultured microorganisms

    DEFF Research Database (Denmark)

    Kvist, Thomas; Ahring, Birgitte Kiær; Lasken, R.S.

    2007-01-01

    We in this study describe a new method for genomic studies of individual uncultured prokaryotic organisms, which was used for the isolation and partial genome sequencing of a soil archaeon. The diversity of Archaea in a soil sample was mapped by generating a clone library using group-specific pri......We in this study describe a new method for genomic studies of individual uncultured prokaryotic organisms, which was used for the isolation and partial genome sequencing of a soil archaeon. The diversity of Archaea in a soil sample was mapped by generating a clone library using group......-specific primers in combination with a terminal restriction fragment length polymorphism profile. Intact cells were extracted from the environmental sample, and fluorescent in situ hybridization probing with Cy3-labeled probes designed from the clone library was subsequently used to detect the organisms...... of interest. Single cells with a bright fluorescent signal were isolated using a micromanipulator and the genome of the single isolated cells served as a template for multiple displacement amplification (MDA) using the Phi29 DNA polymerase. The generated MDA product was afterwards used for 16S rRNA gene...

  9. Effects of Taiwan Roselle anthocyanin treatment and single-walled carbon nanotube addition on the performance of dye-sensitized solar cells

    International Nuclear Information System (INIS)

    Chou, C S; Huang, Y H; Chen, Y S; Tsai, P J; Wu, P; Shu, G G

    2014-01-01

    This study investigates the relationship between the performance of a dye-sensitized solar cell (DSSC) sensitized by a natural sensitizer of Taiwan Roselle anthocyanin (TRA) and fabrication process conditions of the DSSC. A set of systematic experiments has been carried out at various soaking temperatures, soaking periods, sensitizer concentrations, pH values, and additions of single-walled carbon nanotube (SWCNT). An absorption peak (520 nm) is found for TRA, and it is close to that of the N719 dye (518 nm). At a fixed concentration of TRA and a fixed soaking period, a lower pH of the extract or a lower soaking temperature is found favorable to the formation of pigment cations, which leads to an enhanced power conversion efficiency (η) of DSSC. For instance, by applying 17.53 mg/100ml TRA at 30 for 10 h, as the pH of the extract decreases to 2.00 from 2.33 (the original pH of TRA), the η of DSSC with TiO 2 +SWCNT electrode increases to 0.67% from 0.11% of a traditional DSSC with TiO 2 electrode. This performance improvement can be explained by the combined effect of the pH of sensitizer and the additions of SWCNT, a first investigation in DSSC using the natural sensitizer with SWCNT.

  10. Effects of Taiwan Roselle anthocyanin treatment and single-walled carbon nanotube addition on the performance of dye-sensitized solar cells

    Science.gov (United States)

    Chou, C. S.; Tsai, P. J.; Wu, P.; Shu, G. G.; Huang, Y. H.; Chen, Y. S.

    2014-04-01

    This study investigates the relationship between the performance of a dye-sensitized solar cell (DSSC) sensitized by a natural sensitizer of Taiwan Roselle anthocyanin (TRA) and fabrication process conditions of the DSSC. A set of systematic experiments has been carried out at various soaking temperatures, soaking periods, sensitizer concentrations, pH values, and additions of single-walled carbon nanotube (SWCNT). An absorption peak (520 nm) is found for TRA, and it is close to that of the N719 dye (518 nm). At a fixed concentration of TRA and a fixed soaking period, a lower pH of the extract or a lower soaking temperature is found favorable to the formation of pigment cations, which leads to an enhanced power conversion efficiency (η) of DSSC. For instance, by applying 17.53 mg/100ml TRA at 30 for 10 h, as the pH of the extract decreases to 2.00 from 2.33 (the original pH of TRA), the η of DSSC with TiO2+SWCNT electrode increases to 0.67% from 0.11% of a traditional DSSC with TiO2 electrode. This performance improvement can be explained by the combined effect of the pH of sensitizer and the additions of SWCNT, a first investigation in DSSC using the natural sensitizer with SWCNT.

  11. Chromosomal aberrations and deoxyribonucleic acid single-strand breaks in adipose-derived stem cells during long-term expansion in vitro.

    Science.gov (United States)

    Froelich, Katrin; Mickler, Johannes; Steusloff, Gudrun; Technau, Antje; Ramos Tirado, Mario; Scherzed, Agmal; Hackenberg, Stephan; Radeloff, Andreas; Hagen, Rudolf; Kleinsasser, Norbert

    2013-07-01

    Adipose-derived stem cells (ASCs) are a promising mesenchymal cell source for tissue engineering approaches. To obtain an adequate cell amount, in vitro expansion of the cells may be required in some cases. To monitor potential contraindications for therapeutic applications in humans, DNA strand breaks and chromosomal aberrations in ASCs during in vitro expansion were examined. After isolation of ASC from human lipoaspirates of seven patients, in vitro expansion over 10 passages was performed. Cells from passages 1, 2, 3, 5 and 10 were used for the alkaline single-cell microgel electrophoresis (comet) assay to detect DNA single-strand breaks and alkali labile as well as incomplete excision repair sites. Chromosomal changes were examined by means of the chromosomal aberration test. During in vitro expansion, ASC showed no DNA single-strand breaks in the comet assay. With the chromosomal aberration test, however, a significant increase in chromosomal aberrations were detected. The study showed that although no DNA fragmentation could be determined, the safety of ASC cannot be ensured with respect to chromosome stability during in vitro expansion. Thus, reliable analyses for detecting ASC populations, which accumulate chromosomal aberrations or even undergo malignant transformation during extensive in vitro expansion, must be implemented as part of the safety evaluation of these cells for stem cell-based therapy. Copyright © 2013 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  12. Quantifying the Traction Force of a Single Cell by Aligned Silicon Nanowire Array

    KAUST Repository

    Li, Zhou

    2009-10-14

    The physical behaviors of stationary cells, such as the morphology, motility, adhesion, anchorage, invasion and metastasis, are likely to be important for governing their biological characteristics. A change in the physical properties of mammalian cells could be an indication of disease. In this paper, we present a silicon-nanowire-array based technique for quantifying the mechanical behavior of single cells representing three distinct groups: normal mammalian cells, benign cells (L929), and malignant cells (HeLa). By culturing the cells on top of NW arrays, the maximum traction forces of two different tumor cells (HeLa, L929) have been measured by quantitatively analyzing the bending of the nanowires. The cancer cell exhibits a larger traction force than the normal cell by ∼20% for a HeLa cell and ∼50% for a L929 cell. The traction forces have been measured for the L929 cells and mechanocytes as a function of culture time. The relationship between cells extending area and their traction force has been investigated. Our study is likely important for studying the mechanical properties of single cells and their migration characteristics, possibly providing a new cellular level diagnostic technique. © 2009 American Chemical Society.

  13. Prognostic significance of tumor budding and single cell invasion in gastric adenocarcinoma

    Directory of Open Access Journals (Sweden)

    Che K

    2017-02-01

    Full Text Available Keying Che,1,* Yang Zhao,2,3,* Xiao Qu,1 Zhaofei Pang,1 Yang Ni,4 Tiehong Zhang,4 Jiajun Du,1,5 Hongchang Shen4 1Institute of Oncology, Shandong Provincial Hospital Affiliated to Shandong University, Jinan, 2Department of Breast Surgery, Key Laboratory of Breast Cancer in Shanghai, Collaborative Innovation Center of Cancer Medicine, Fudan University Shanghai Cancer Center, 3Department of Oncology, Shanghai Medical College, Fudan University, Shanghai, 4Department of Oncology, Shandong Provincial Hospital Affiliated to Shandong University, 5Department of Thoracic Surgery, Shandong Provincial Hospital Affiliated to Shandong University, Jinan, People’s Republic of China *These authors contributed equally to this work Purpose: Gastric carcinoma (GC is a highly aggressive cancer and one of the leading causes of cancer-related deaths worldwide. Histopathological evaluation pertaining to invasiveness is likely to provide additional information in relation to patient outcome. In this study, we aimed to evaluate the prognostic significance of tumor budding and single cell invasion in gastric adenocarcinoma.Materials and methods: Hematoxylin and eosin-stained slides generated from 296 gastric adenocarcinoma patients with full clinical and pathological and follow-up information were systematically reviewed. The patients were grouped on the basis of tumor budding, single cell invasion, large cell invasion, mitotic count, and fibrosis. The association between histopathological parameters, different classification systems, and overall survival (OS was statistically analyzed.Results: Among the 296 cases that were analyzed, high-grade tumor budding was observed in 49.0% (145 of them. Single cell invasion and large cell invasion were observed in 62.8% (186 and 16.9% (50 of the cases, respectively. Following univariate analysis, patients with high-grade tumor budding had shorter OS than those with low-grade tumor budding (hazard ratio [HR]: 2.260, P<0

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

    Energy Technology Data Exchange (ETDEWEB)

    Gross, Benjamin J. [Department of Physics and Astronomy, University of Southern California, 920 Bloom Walk, Los Angeles, California 90089-0484 (United States); El-Naggar, Mohamed Y., E-mail: mnaggar@usc.edu [Department of Physics and Astronomy, University of Southern California, 920 Bloom Walk, Los Angeles, California 90089-0484 (United States); Molecular and Computational Biology Section, Department of Biological Sciences, University of Southern California, Los Angeles, California 90089-0484 (United States); Department of Chemistry, University of Southern California, Los Angeles, California 90089-0484 (United States)

    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.

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

    Science.gov (United States)

    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.

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

    International Nuclear Information System (INIS)

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

    2015-01-01

    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

  17. Targeted sorting of single virus-infected cells of the coccolithophore Emiliania huxleyi.

    Directory of Open Access Journals (Sweden)

    Joaquín Martínez Martínez

    Full Text Available Discriminating infected from healthy cells is the first step to understanding the mechanisms and ecological implications of viral infection. We have developed a method for detecting, sorting, and performing molecular analysis of individual, infected cells of the important microalga Emiliania huxleyi, based on known physiological responses to viral infection. Of three fluorescent dyes tested, FM 1-43 (for detecting membrane blebbing gave the most unequivocal and earliest separation of cells. Furthermore, we were able to amplify the genomes of single infected cells using Multiple Displacement Amplification. This novel method to reliably discriminate infected from healthy cells in cultures will allow researchers to answer numerous questions regarding the mechanisms and implications of viral infection of E. huxleyi. The method may be transferable to other virus-host systems.

  18. Precision toxicology based on single cell sequencing: an evolving trend in toxicological evaluations and mechanism exploration.

    Science.gov (United States)

    Zhang, Boyang; Huang, Kunlun; Zhu, Liye; Luo, Yunbo; Xu, Wentao

    2017-07-01

    In this review, we introduce a new concept, precision toxicology: the mode of action of chemical- or drug-induced toxicity can be sensitively and specifically investigated by isolating a small group of cells or even a single cell with typical phenotype of interest followed by a single cell sequencing-based analysis. Precision toxicology can contribute to the better detection of subtle intracellular changes in response to exogenous substrates, and thus help researchers find solutions to control or relieve the toxicological effects that are serious threats to human health. We give examples for single cell isolation and recommend laser capture microdissection for in vivo studies and flow cytometric sorting for in vitro studies. In addition, we introduce the procedures for single cell sequencing and describe the expected application of these techniques to toxicological evaluations and mechanism exploration, which we believe will become a trend in toxicology.

  19. Single cell adhesion assay using computer controlled micropipette.

    Directory of Open Access Journals (Sweden)

    Rita Salánki

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

  20. An Optogenetic Platform for Real-Time, Single-Cell Interrogation of Stochastic Transcriptional Regulation.

    Science.gov (United States)

    Rullan, Marc; Benzinger, Dirk; Schmidt, Gregor W; Milias-Argeitis, Andreas; Khammash, Mustafa

    2018-05-17

    Transcription is a highly regulated and inherently stochastic process. The complexity of signal transduction and gene regulation makes it challenging to analyze how the dynamic activity of transcriptional regulators affects stochastic transcription. By combining a fast-acting, photo-regulatable transcription factor with nascent RNA quantification in live cells and an experimental setup for precise spatiotemporal delivery of light inputs, we constructed a platform for the real-time, single-cell interrogation of transcription in Saccharomyces cerevisiae. We show that transcriptional activation and deactivation are fast and memoryless. By analyzing the temporal activity of individual cells, we found that transcription occurs in bursts, whose duration and timing are modulated by transcription factor activity. Using our platform, we regulated transcription via light-driven feedback loops at the single-cell level. Feedback markedly reduced cell-to-cell variability and led to qualitative differences in cellular transcriptional dynamics. Our platform establishes a flexible method for studying transcriptional dynamics in single cells. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

  1. Influence of Electrodes Characteristics on The Performance of a Microbial Fuel Cell

    Directory of Open Access Journals (Sweden)

    Muhammad Hadi Radi

    2017-07-01

    Full Text Available A single chamber microbial fuel cell is designed incorporating microorganism as catalyst with Escherichia coli, Staphylococcus, Kelbssila bacteria as an electrolyte at pH =7 and an operating temperature of 30 C0 in batch mode. The electrodes are made of three different types of materials, namely; aluminum, copper and zinc. Each material is configurated at three different shape (circle, rectangle and square in three different cross sectional areas of (3.14,7.065and 12.56cm2. The distance between anode and cathode is fixed at different values of 0.5,1,2,4 and 6cm. Results indicate that electrodes of circular shape show the best performance among other shapes investigated in this study, however the area of the anode is found to affect the cell performance more than its shape. Using zinc as an anode material and copper as a cathode in circular shape with cross sectional area of 12.56 cm2 and a 2 cm distance between them output the best performance in comparison to other combinations investigated in this study.

  2. Preimplantation genetic diagnosis guided by single-cell genomics

    Science.gov (United States)

    2013-01-01

    Preimplantation genetic diagnosis (PGD) aims to help couples with heritable genetic disorders to avoid the birth of diseased offspring or the recurrence of loss of conception. Following in vitro fertilization, one or a few cells are biopsied from each human preimplantation embryo for genetic testing, allowing diagnosis and selection of healthy embryos for uterine transfer. Although classical methods, including single-cell PCR and fluorescent in situ hybridization, enable PGD for many genetic disorders, they have limitations. They often require family-specific designs and can be labor intensive, resulting in long waiting lists. Furthermore, certain types of genetic anomalies are not easy to diagnose using these classical approaches, and healthy offspring carrying the parental mutant allele(s) can result. Recently, state-of-the-art methods for single-cell genomics have flourished, which may overcome the limitations associated with classical PGD, and these underpin the development of generic assays for PGD that enable selection of embryos not only for the familial genetic disorder in question, but also for various other genetic aberrations and traits at once. Here, we discuss the latest single-cell genomics methodologies based on DNA microarrays, single-nucleotide polymorphism arrays or next-generation sequence analysis. We focus on their strengths, their validation status, their weaknesses and the challenges for implementing them in PGD. PMID:23998893

  3. CMEIAS color segmentation: an improved computing technology to process color images for quantitative microbial ecology studies at single-cell resolution.

    Science.gov (United States)

    Gross, Colin A; Reddy, Chandan K; Dazzo, Frank B

    2010-02-01

    Quantitative microscopy and digital image analysis are underutilized in microbial ecology largely because of the laborious task to segment foreground object pixels from background, especially in complex color micrographs of environmental samples. In this paper, we describe an improved computing technology developed to alleviate this limitation. The system's uniqueness is its ability to edit digital images accurately when presented with the difficult yet commonplace challenge of removing background pixels whose three-dimensional color space overlaps the range that defines foreground objects. Image segmentation is accomplished by utilizing algorithms that address color and spatial relationships of user-selected foreground object pixels. Performance of the color segmentation algorithm evaluated on 26 complex micrographs at single pixel resolution had an overall pixel classification accuracy of 99+%. Several applications illustrate how this improved computing technology can successfully resolve numerous challenges of complex color segmentation in order to produce images from which quantitative information can be accurately extracted, thereby gain new perspectives on the in situ ecology of microorganisms. Examples include improvements in the quantitative analysis of (1) microbial abundance and phylotype diversity of single cells classified by their discriminating color within heterogeneous communities, (2) cell viability, (3) spatial relationships and intensity of bacterial gene expression involved in cellular communication between individual cells within rhizoplane biofilms, and (4) biofilm ecophysiology based on ribotype-differentiated radioactive substrate utilization. The stand-alone executable file plus user manual and tutorial images for this color segmentation computing application are freely available at http://cme.msu.edu/cmeias/ . This improved computing technology opens new opportunities of imaging applications where discriminating colors really matter most

  4. A mixed-pH dual-electrolyte microfluidic aluminum–air cell with high performance

    International Nuclear Information System (INIS)

    Chen, Binbin; Leung, Dennis Y.C.; Xuan, Jin; Wang, Huizhi

    2017-01-01

    Highlights: • A mix-pH dual-electrolyte Al–air cell is proposed. • Cells with dual-electrolyte exhibit higher performance. • Cell performance increases with increasing electrolyte concentration and flow rate. • Optimized channel thickness is 0.3 mm. • A restriction of reaction activation on the Al side is observed. - Abstract: Energy storage capacity has been a major limiting factor in pursuit of increasing functionality and mobility for portable devices. To increase capacity limits, novel battery designs with multi-electron redox couples and increased voltages have been listed as a priority research direction by the US Department of Energy. This study leverages the benefits of microfluidics technology to develop a novel mixed-pH media aluminum–air cell which incorporates the advantages of the trivalence of aluminum and mixed-pH thermodynamics. Experimentally, the new cell exhibited an open circuit potential of 2.2 V and a maximum power density of 176 mW cm −2 , which are respectively 37.5% and 104.6% higher than conventional single alkaline aluminum–air cell under similar conditions. With further optimization of channel thickness, a power density of 216 mW cm −2 was achieved in the present study.

  5. Mass-charge-heat coupled transfers in a single cell of a proton exchange membrane fuel cell; Transferts couples masse-charge-chaleur dans une cellule de pile a combustible a membrane polymere

    Energy Technology Data Exchange (ETDEWEB)

    Ramousse, J

    2005-11-15

    Understanding and modelling of coupled mass, charges and heat transfers phenomena are fundamental to analyze the electrical behaviour of the system. The aim of the present model is to describe electrical performances of a PEFMC according to the fluidic and thermal operating conditions. The water content of the membrane and the water distribution in the single cell are estimated according to the coupled simulations of mass transport in the thickness of the single cell and in the feeding channels of the bipolar plates. A microscopic model of a Gas Diffusion Electrode is built up to describe charges transfer phenomena occurring at the electrodes. Completed by a study of heat transfer in the Membrane Electrode Assembly, conditions and preferential sites of water vapor condensation can be highlighted. A set of measurements of the effective thermal conductivity of carbon felts used in fuel cells as porous backing layers have also been performed. Although the value of this parameter is essential for the study of heat transfer, it is still under investigation because of the strong thermal anisotropy of the medium. (author)

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

    Science.gov (United States)

    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. © 2015 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

  7. Nano-scale microfluidics to study 3D chemotaxis at the single cell level.

    Directory of Open Access Journals (Sweden)

    Corina Frick

    Full Text Available Directed migration of cells relies on their ability to sense directional guidance cues and to interact with pericellular structures in order to transduce contractile cytoskeletal- into mechanical forces. These biomechanical processes depend highly on microenvironmental factors such as exposure to 2D surfaces or 3D matrices. In vivo, the majority of cells are exposed to 3D environments. Data on 3D cell migration are mostly derived from intravital microscopy or collagen-based in vitro assays. Both approaches offer only limited controllability of experimental conditions. Here, we developed an automated microfluidic system that allows positioning of cells in 3D microenvironments containing highly controlled diffusion-based chemokine gradients. Tracking migration in such gradients was feasible in real time at the single cell level. Moreover, the setup allowed on-chip immunocytochemistry and thus linking of functional with phenotypical properties in individual cells. Spatially defined retrieval of cells from the device allows down-stream off-chip analysis. Using dendritic cells as a model, our setup specifically allowed us for the first time to quantitate key migration characteristics of cells exposed to identical gradients of the chemokine CCL19 yet placed on 2D vs in 3D environments. Migration properties between 2D and 3D migration were distinct. Morphological features of cells migrating in an in vitro 3D environment were similar to those of cells migrating in animal tissues, but different from cells migrating on a surface. Our system thus offers a highly controllable in vitro-mimic of a 3D environment that cells traffic in vivo.

  8. A single-cell scraper based on an atomic force microscope for detaching a living cell from a substrate

    Energy Technology Data Exchange (ETDEWEB)

    Iwata, Futoshi, E-mail: iwata.futoshi@shizuoka.ac.jp [Department of Mechanical Engineering, Faculty of Engineering, Shizuoka University, Johoku, Naka-ku, Hamamatsu 432-8561 (Japan); Research Institute of Electronics, Shizuoka University, Johoku, Naka-ku, Hamamatsu 432-8011 (Japan); Adachi, Makoto; Hashimoto, Shigetaka [Department of Mechanical Engineering, Faculty of Engineering, Shizuoka University, Johoku, Naka-ku, Hamamatsu 432-8561 (Japan)

    2015-10-07

    We describe an atomic force microscope (AFM) manipulator that can detach a single, living adhesion cell from its substrate without compromising the cell's viability. The micrometer-scale cell scraper designed for this purpose was fabricated from an AFM micro cantilever using focused ion beam milling. The homemade AFM equipped with the scraper was compact and standalone and could be mounted on a sample stage of an inverted optical microscope. It was possible to move the scraper using selectable modes of operation, either a manual mode with a haptic device or a computer-controlled mode. The viability of the scraped single cells was evaluated using a fluorescence dye of calcein-acetoxymethl ester. Single cells detached from the substrate were collected by aspiration into a micropipette capillary glass using an electro-osmotic pump. As a demonstration, single HeLa cells were selectively detached from the substrate and collected by the micropipette. It was possible to recultivate HeLa cells from the single cells collected using the system.

  9. Design and Implementation of 8051 Single-Chip Microcontroller for Stationary 1.0 kW PEM Fuel Cell System

    Directory of Open Access Journals (Sweden)

    Pei-Hsing Huang

    2014-01-01

    Full Text Available Proton exchange membrane fuel cells (PEMFCs have attracted significant interest as a potential green energy source. However, if the performance of such systems is to be enhanced, appropriate control strategies must be applied. Accordingly, the present study proposes a sophisticated control system for a 1.0 kW PEMFC system comprising a fuel cell stack, an auxiliary power supply, a DC-DC buck converter, and a DC-AC inverter. The control system is implemented using an 8051 single-chip microcontroller and is designed to optimize the system performance and safety in both the startup phase and the long-term operation phase. The major features of the proposed control system are described and the circuit diagrams required for its implementation introduced. In addition, the touch-sensitive, intuitive human-machine interface is introduced and typical screens are presented. Finally, the electrical characteristics of the PEMFC system are briefly examined. Overall, the results confirm that the single-chip microcontroller presented in this study has significant potential for commercialization in the near future.

  10. Characterization of nanostructures in the live cell plasma membrane utilizing advanced single molecule fluorescence techniques

    International Nuclear Information System (INIS)

    Brameshuber, M.

    2009-01-01

    lipid-lipid or protein-lipid interactions, protein-protein interactions play of mayor role for the regulation of cell metabolism and function. In this thesis I further characterized the interaction between human CD4, the major co-receptor in T cell activation, and human Lck, the protein tyrosine kinase essential for early T cell signaling using an ultra-sensitive fluorescence-based method. Interaction dynamics were studied in detail by performing photobleaching experiments and single molecule brightness analysis. This enabled a combined mobility and stoichiometry analysis of Lck-molecules interacting with the captured CD4 protein. In the last part of my thesis I present a single molecule fluorescence study using a variant of an oxidized phospholipid - which is known to induce apoptosis - to probe the structure of the cellular plasmamembrane. The cells were illuminated using a recently introduced technique which utilizes a highly inclined and laminated optical sheet (HILO) to reduce background signal arising from intracellular fluorophores or from cellular autofluorescence. Our data demonstrate the relevance of plasma membrane properties for uptake of oxidized phospholipids, and indicate a novel indirect mechanism for the control of endocytosis. (author) [de

  11. Radiosensitivity evaluation of Human tumor cell lines by single cell gel electrophoresis

    International Nuclear Information System (INIS)

    Zhang Yipei; Cao Jia; Wang Yan; Du Liqing; Li Jin; Wang Qin; Fan Feiyue; Liu Qiang

    2011-01-01

    Objective: To explore the feasibility of determining radiosensitivity of human tumor cell lines in vitro using single cell gel electrophoresis (SCGE). Methods: Three human tumor cell lines were selected in this study, HepG 2 , EC-9706 and MCF-7. The surviving fraction (SF) and DNA damage were detected by MTT assay, nested PCR technique and comet assay respectively. Results: MTT assay: The SF of HepG 2 and EC-9706 after irradiated by 2, 4 and 8 Gy was lower significantly than that of MCF-7, which showed that the radiosensitivity of HepG 2 and EC-9706 was higher than that of MCF-7. But there was no statistical difference of SF between HepG 2 and EC-9706. SCGE: The difference of radiosensitivity among these three tumor cell lines was significant after 8 Gy γ-ray irradiation. Conclusion: The multi-utilization of many biological parameter is hopeful to evaluate the radiosensitivity of tumor cells more objectively and exactly. (authors)

  12. Single cell-type comparative metabolomics of epidermal bladder cells from the halophyte Mesembryanthemum crystallinum

    OpenAIRE

    Barkla, Bronwyn J.; Vera-Estrella, Rosario

    2015-01-01

    One of the remarkable adaptive features of the halophyte and facultative CAM plant Mesembryathemum crystallinum are the specialized modified trichomes called epidermal bladder cells (EBC) which cover the leaves, stems, and peduncle of the plant. They are present from an early developmental stage but upon salt stress rapidly expand due to the accumulation of water and sodium. This particular plant feature makes it an attractive system for single cell type studies, with recent proteomics and tr...

  13. Co-extrusion of electrolyte/anode functional layer/anode triple-layer ceramic hollow fibres for micro-tubular solid oxide fuel cells-electrochemical performance study

    Science.gov (United States)

    Li, Tao; Wu, Zhentao; Li, K.

    2015-01-01

    In this study, the effects of an anode functional layer (AFL) with controlled thickness on physical and electrochemical properties of a micro-tubular SOFC have been systematically studied. A series of electrolyte/AFL/anode triple-layer hollow fibres with controllable AFL thicknesses (16.9-52.7 μm) have been fabricated via a single-step phase-inversion assisted co-extrusion technique. Both robustness of the cell and gas-tightness of the electrolyte layer are considerably improved by introducing the AFL of this type. The fracture force of the sample with the thickest AFL (9.67 N) almost doubles when compared to the electrolyte/anode dual-layer counterpart (5.24 N). Gas-tightness of the electrolyte layer is also considerably increased as AFL contributes to better-matched sintering behaviours between different components. Moreover, the formation of an AFL simultaneously with electrolyte and anode significantly improves the cell performances. The sample with the thinnest AFL (approximately 16.9 μm, 6% of the total anode thickness) leads to a 30% (from 0.89 to 1.21 W cm-2) increase in maximum power density, due to increased triple-phase boundaries (TPB). However, further increase in TPB from a thicker AFL is less effective for improving the cell performance, due to the substantially increased fuel diffusion resistance and subsequently higher concentration polarization. This indicates that the control over the AFL thickness is critically important in avoiding offsetting the benefits of extended TPB and consequently decreased cell performances.

  14. Red blood cell alloimmunization in patients with sickle cell disease in Turkey: a single center retrospective cohort study

    Directory of Open Access Journals (Sweden)

    Soner Solmaz

    2016-12-01

    Results: Of 216 SCD patients included in the study. Alloimmunization was detected in 67 (31.0% out of 216 patients who underwent transfusion, and in 17 (30.4% out of 56 patients in Group 1 and in 50 (31.3% out of 160 patients in Group 2. When the patients were analyzed according to alloimmunization development, our study revealed that neither SCD complications are a risk factor for alloimmunization nor alloimmunization increases mortality rates. Conclusion: High alloimmunization frequency found in our study suggests the insufficient adherence of alloimmunization-prevention policies in RBC transfusions performed except experienced institutions. Therefore alloimmunization may be reduced or prevented through performing extended red cell typing among SCD patients. [Cukurova Med J 2016; 41(4.000: 622-627

  15. Enhancing hybrid direct carbon fuel cell anode performance using Ag2O

    DEFF Research Database (Denmark)

    Deleebeeck, Lisa; Ippolito, Davide; Kammer Hansen, Kent

    2015-01-01

    A hybrid-direct carbon fuel cell (HDCFC), consisting of a molten slurry of solid carbon black and (Li-K)2CO3 added to the anode chamber of a solid oxide fuel cell, was characterized using current-potential-power density curves, electrochemical impedance spectroscopy, and cyclic voltammetry. Two...... types of experimental setups were employed in this study, an anode-supported full cell configuration (two electrodes, two atmospheres setup) and a 3-electrode electrolyte-supported half-cell setup (single atmosphere). Anode processes with and without catalysts were investigated as a function...... of temperature (700-800 °C) and anode sweep gas (N2, 4-100% CO2 in N2-CO2). It was shown that the addition of silver based catalysts (Ag, Ag2O, Ag2CO3) into the carbon-carbonate slurry enhanced the performance of the HDCFC....

  16. Radiobiological study on DNA strand breaks and repair using single cell gel electrophoresis

    International Nuclear Information System (INIS)

    Ikushima, Takaji

    1994-01-01

    Single cell gel electrophoresis (SCGE) provides a novel method to measure DNA damage in individual cells and more importantly, to assess heterogeneity in response within a mixed population of cells. Cells embedded in agarose are lysed, subjected to electrophoresis, stained with a fluorescent DNA-specific dye, and viewed under a fluorescence microscope. Damaged cells display 'comets', broken DNA migrating farther to the anode in the electric field. We have previously used this technique to quantify DNA damage induced by moderate doses of low and high LET radiations in cultured Chinese hamster cells. The assay has been optimized in terms of lysing and electrophoresis conditions, and applied to analyse the DNA strand breaks, their repair kinetics and heterogeneity in response in individual Chinese hamster cells exposed to gamma-rays, and to KUR thermal neutrons with and without 10 B or to KEK PF monochromatic soft X-rays as well as to a radio-mimetic agent, neocarzinostatin. The DNA double-strand breaks induced by boron-neutron captured reactions were repaired at a slower rate, but a heterogeneity in response might not contribute to the difference. The neocarzinostatin-induced DNA damage were efficiently repaired in a dose-dependent fashion. The initial amount of gamma-ray induced DNA double-strand breaks was not significantly altered in cells pre-exposed to very low adapting dose. (author)

  17. Single cells from human primary colorectal tumors exhibit polyfunctional heterogeneity in secretions of ELR+ CXC chemokines.

    Science.gov (United States)

    Adalsteinsson, Viktor A; Tahirova, Narmin; Tallapragada, Naren; Yao, Xiaosai; Campion, Liam; Angelini, Alessandro; Douce, Thomas B; Huang, Cindy; Bowman, Brittany; Williamson, Christina A; Kwon, Douglas S; Wittrup, K Dane; Love, J Christopher

    2013-10-01

    Cancer is an inflammatory disease of tissue that is largely influenced by the interactions between multiple cell types, secreted factors, and signal transduction pathways. While single-cell sequencing continues to refine our understanding of the clonotypic heterogeneity within tumors, the complex interplay between genetic variations and non-genetic factors ultimately affects therapeutic outcome. Much has been learned through bulk studies of secreted factors in the tumor microenvironment, but the secretory behavior of single cells has been largely uncharacterized. Here we directly profiled the secretions of ELR+ CXC chemokines from thousands of single colorectal tumor and stromal cells, using an array of subnanoliter wells and a technique called microengraving to characterize both the rates of secretion of several factors at once and the numbers of cells secreting each chemokine. The ELR+ CXC chemokines are highly redundant, pro-angiogenic cytokines that signal via the CXCR1 and CXCR2 receptors, influencing tumor growth and progression. We find that human primary colorectal tumor and stromal cells exhibit polyfunctional heterogeneity in the combinations and magnitudes of secretions for these chemokines. In cell lines, we observe similar variance: phenotypes observed in bulk can be largely absent among the majority of single cells, and discordances exist between secretory states measured and gene expression for these chemokines among single cells. Together, these measures suggest secretory states among tumor cells are complex and can evolve dynamically. Most importantly, this study reveals new insight into the intratumoral phenotypic heterogeneity of human primary tumors.

  18. Abseq: Ultrahigh-throughput single cell protein profiling with droplet microfluidic barcoding

    Science.gov (United States)

    Shahi, Payam; Kim, Samuel C.; Haliburton, John R.; Gartner, Zev J.; Abate, Adam R.

    2017-03-01

    Proteins are the primary effectors of cellular function, including cellular metabolism, structural dynamics, and information processing. However, quantitative characterization of proteins at the single-cell level is challenging due to the tiny amount of protein available. Here, we present Abseq, a method to detect and quantitate proteins in single cells at ultrahigh throughput. Like flow and mass cytometry, Abseq uses specific antibodies to detect epitopes of interest; however, unlike these methods, antibodies are labeled with sequence tags that can be read out with microfluidic barcoding and DNA sequencing. We demonstrate this novel approach by characterizing surface proteins of different cell types at the single-cell level and distinguishing between the cells by their protein expression profiles. DNA-tagged antibodies provide multiple advantages for profiling proteins in single cells, including the ability to amplify low-abundance tags to make them detectable with sequencing, to use molecular indices for quantitative results, and essentially limitless multiplexing.

  19. Long Term Performance Study of a Direct Methanol Fuel Cell Fed with Alcohol Blends

    OpenAIRE

    Teresa J. Leo; Miguel A. Raso; Emilio Navarro; Eleuterio Mora

    2013-01-01

    The use of alcohol blends in direct alcohol fuel cells may be a more environmentally friendly and less toxic alternative to the use of methanol alone in direct methanol fuel cells. This paper assesses the behaviour of a direct methanol fuel cell fed with aqueous methanol, aqueous ethanol and aqueous methanol/ethanol blends in a long term experimental study followed by modelling of polarization curves. Fuel cell performance is seen to decrease as the ethanol content rises, and subsequent opera...

  20. Limited angle STIM and PIXE tomography of single cells

    International Nuclear Information System (INIS)

    Andrea, T.; Rothermel, M.; Werner, R.; Butz, T.; Reinert, T.

    2010-01-01

    STIM (scanning transmission ion microscopy) tomography has been shown to be a valuable method for the three-dimensional characterization of microsamples. It has, however, rarely been employed for the study of single cells, since a free-standing sample is needed for an ordinary tomography experiment. This requirement places high demands on sample preparation techniques. In this study cells fixated on a substrate rather than free-standing were used for tomography. Since the substrate prevented a full rotation of the sample an algorithm for limited-angle tomography was devised. STIM projections covering only a limited angular range of ca. 120 o were supplemented with simulated projections generated from a back and forth iteration between real space and Radon space. The energy loss caused by the substrate was subtracted from each projection. The cells were reconstructed using filtered backprojection. The surface of the cells as well as some interior structures could be reconstructed. Following the STIM projections a lesser number of PIXE (particle induced X-ray emission) projections were taken in order to obtain information about the elemental distribution of the sample. From the PIXE projections the three-dimensional phosphorus distribution within the cell was reconstructed using limited-angle tomography. Superimposition of the STIM and PIXE tomograms revealed the location of intracellular structures. Whereas STIM tomography is sensitive to density contrast, which are greatest at the surface, PIXE tomography is sensitive to changes in elemental concentration. Hence, the combination of the two methods can be very fruitful, while the limited angle approach can compensate some of the difficulties associated with tomography of single cells, namely preparation difficulties and excessive sample damage.

  1. Impact of cell-voltage on energy and power performance of supercapacitors with single-walled carbon nanotube electrodes

    Energy Technology Data Exchange (ETDEWEB)

    Izadi-Najafabadi, Ali; Yamada, Takeo; Futaba, Don N.; Iijima, Sumio [Nanotube Research Center, National Institute of Advanced Industrial Science and Technology, Tsukuba (Japan); Hatori, Hiroaki [Project Headquarters, National Institute of Advanced Industrial Science and Technology, Tsukuba (Japan); Hata, Kenji [Japan Science and Technology Agency JST, Kawaguchi (Japan)

    2010-12-15

    We report the energy and power voltage-dependencies of supercapacitors using single-walled carbon nanotube electrodes. The energy density was dependent on the cell-voltage cubed (up to 4 V: E = 1.43 x V{sup 3}). The cubic relationship was attributed to the linear increase of the capacitance as a function of voltage, enabled by electrochemical doping. Furthermore, while up to 3.5 V, the maximum power rating of the nanotube electrodes increased as a function of the cell-voltage squared, beyond 3.5 V, a decline in power was observed as a result of depletion of the electrolyte's ions. (author)

  2. Performance evaluation and parametric optimum design of a molten carbonate fuel cell-thermophotovoltaic cell hybrid system

    International Nuclear Information System (INIS)

    Yang, Zhimin; Liao, Tianjun; Zhou, Yinghui; Lin, Guoxing; Chen, Jincan

    2016-01-01

    Highlights: • A molten carbonate fuel cell-thermophotovoltaic cell hybrid system is established. • The performance characteristics of the hybrid system are systematically evaluated. • The optimal regions of the power output density and efficiency are determined. • The values of key parameters at the maximum power output density are calculated. • The proposed system is proved to have advantages over other hybrid systems. - Abstract: A new model of the hybrid system composed of a molten carbonate fuel cell (MCFC) and a thermophotovoltaic cell (TPVC) is proposed to recovery the waste heat produced by the MCFC. Expressions for the power output and the efficiency of the hybrid system are analytically derived. The performance characteristics of the hybrid system are evaluated. It is found that when the current density of the MCFC, voltage output of the TPVC, electrode area ratio of the MCFC to the TPVC, and energy gap of the material in the photovoltaic cell are optimally chosen, the maximum power output density of the hybrid system is obviously larger than that of the single MCFC. Moreover, the improved percentages of the maximum power output density of the proposed model relative to that of the single MCFC are calculated for differently operating temperatures of the MCFC and are compared with those of some MCFC-based hybrid systems reported in the literature, and consequently, the advantages of the MCFC-TPVC hybrid system are revealed.

  3. Quantification of DNA damage by single-cell electrophoresis

    International Nuclear Information System (INIS)

    Ikushima, Takaji

    1990-01-01

    A simple technique of micro-agarose gel electrophoresis has been developed to quantify DNA damage in individual cells. Cells are embedded in agarose gel on microscope slides, lysed by detergents and then electrophoresed for a short time under neutral or alkaline condition. In irradiated cells, DNA migrates from the nucleus toward the anode, displaying commet-like pattern by staining with DNA-specific fluorescence dye. DNA damage is evaluated by measuring the distance of DNA migration. The technique was applied for measuring DNA damage in single cells exposed to 60 Co γ-rays, or to KUR radiation in the presence or absence of 10 B-enriched boric acid. The enhanced production of double-stranded DNA breaks by 10 B(n,α) 7 Li reaction was demonstrated here. The significant increase in the length of DNA migration was observed in single cells exposed to such a low dose as 20 cGy after alkaline micro electrophoresis. (author)

  4. Cryo-imaging of fluorescently labeled single cells in a mouse

    Science.gov (United States)

    Steyer, Grant J.; Roy, Debashish; Salvado, Olivier; Stone, Meredith E.; Wilson, David L.

    2009-02-01

    We developed a cryo-imaging system to provide single-cell detection of fluorescently labeled cells in mouse, with particular applicability to stem cells and metastatic cancer. The Case cryoimaging system consists of a fluorescence microscope, robotic imaging positioner, customized cryostat, PC-based control system, and visualization/analysis software. The system alternates between sectioning (10-40 μm) and imaging, collecting color brightfield and fluorescent blockface image volumes >60GB. In mouse experiments, we imaged quantum-dot labeled stem cells, GFP-labeled cancer and stem cells, and cell-size fluorescent microspheres. To remove subsurface fluorescence, we used a simplified model of light-tissue interaction whereby the next image was scaled, blurred, and subtracted from the current image. We estimated scaling and blurring parameters by minimizing entropy of subtracted images. Tissue specific attenuation parameters were found [uT : heart (267 +/- 47.6 μm), liver (218 +/- 27.1 μm), brain (161 +/- 27.4 μm)] to be within the range of estimates in the literature. "Next image" processing removed subsurface fluorescence equally well across multiple tissues (brain, kidney, liver, adipose tissue, etc.), and analysis of 200 microsphere images in the brain gave 97+/-2% reduction of subsurface fluorescence. Fluorescent signals were determined to arise from single cells based upon geometric and integrated intensity measurements. Next image processing greatly improved axial resolution, enabled high quality 3D volume renderings, and improved enumeration of single cells with connected component analysis by up to 24%. Analysis of image volumes identified metastatic cancer sites, found homing of stem cells to injury sites, and showed microsphere distribution correlated with blood flow patterns. We developed and evaluated cryo-imaging to provide single-cell detection of fluorescently labeled cells in mouse. Our cryo-imaging system provides extreme (>60GB), micron

  5. Single-Cell Transcriptomics Reveals a Population of Dormant Neural Stem Cells that Become Activated upon Brain Injury.

    Science.gov (United States)

    Llorens-Bobadilla, Enric; Zhao, Sheng; Baser, Avni; Saiz-Castro, Gonzalo; Zwadlo, Klara; Martin-Villalba, Ana

    2015-09-03

    Heterogeneous pools of adult neural stem cells (NSCs) contribute to brain maintenance and regeneration after injury. The balance of NSC activation and quiescence, as well as the induction of lineage-specific transcription factors, may contribute to diversity of neuronal and glial fates. To identify molecular hallmarks governing these characteristics, we performed single-cell sequencing of an unbiased pool of adult subventricular zone NSCs. This analysis identified a discrete, dormant NSC subpopulation that already expresses distinct combinations of lineage-specific transcription factors during homeostasis. Dormant NSCs enter a primed-quiescent state before activation, which is accompanied by downregulation of glycolytic metabolism, Notch, and BMP signaling and a concomitant upregulation of lineage-specific transcription factors and protein synthesis. In response to brain ischemia, interferon gamma signaling induces dormant NSC subpopulations to enter the primed-quiescent state. This study unveils general principles underlying NSC activation and lineage priming and opens potential avenues for regenerative medicine in the brain. Copyright © 2015 Elsevier Inc. All rights reserved.

  6. Microbial Challenge Testing of Single Liquid Cathode Feed Water Electrolysis Cells for the International Space Station (ISS) Oxygen Generator Assembly (OGA)

    Science.gov (United States)

    Roy, Robert J.; Wilson, Mark E.; Diderich, Greg S.; Steele, John W.

    2011-01-01

    The International Space Station (ISS) Oxygen Generator Assembly (OGA) operational performance may be adversely impacted by microbiological growth and biofilm formation over the electrolysis cell membranes. Biofilms could hinder the transport of water from the bulk fluid stream to the membranes and increase the cell concentration overpotential resulting in higher cell voltages and a shorter cell life. A microbial challenge test was performed on duplicate single liquid-cathode feed water electrolysis cells to evaluate operational performance with increasing levels of a mixture of five bacteria isolated from ISS and Space Shuttle potable water systems. Baseline performance of the single water electrolysis cells was determined for approximately one month with deionized water. Monthly performance was also determined following each inoculation of the feed tank with 100, 1000, 10,000 and 100,000 cells/ml of the mixed suspension of test bacteria. Water samples from the feed tank and recirculating water loops for each cell were periodically analyzed for enumeration and speciation of bacteria and total organic carbon. While initially a concern, this test program has demonstrated that the performance of the electrolysis cell is not adversely impacted by feed water containing the five species of bacteria tested at a concentration measured as high as 1,000,000 colony forming units (CFU)/ml. This paper presents the methodologies used in the conduct of this test program along with the performance test results at each level of bacteria concentration.

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

    Science.gov (United States)

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

    2013-12-01

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

  8. Comparison of whole genome amplification techniques for human single cell exome sequencing.

    Science.gov (United States)

    Borgström, Erik; Paterlini, Marta; Mold, Jeff E; Frisen, Jonas; Lundeberg, Joakim

    2017-01-01

    Whole genome amplification (WGA) is currently a prerequisite for single cell whole genome or exome sequencing. Depending on the method used the rate of artifact formation, allelic dropout and sequence coverage over the genome may differ significantly. The largest difference between the evaluated protocols was observed when analyzing the target coverage and read depth distribution. These differences also had impact on the downstream variant calling. Conclusively, the products from the AMPLI1 and MALBAC kits were shown to be most similar to the bulk samples and are therefore recommended for WGA of single cells. In this study four commercial kits for WGA (AMPLI1, MALBAC, Repli-G and PicoPlex) were used to amplify human single cells. The WGA products were exome sequenced together with non-amplified bulk samples from the same source. The resulting data was evaluated in terms of genomic coverage, allelic dropout and SNP calling.

  9. Probing living bacterial adhesion by single cell force spectroscopy using atomic force microscopy

    DEFF Research Database (Denmark)

    Zeng, Guanghong; Ogaki, Ryosuke; Regina, Viduthalai R.

    be considered. We have therefore developed a simple and versatile method to make single-cell bacterial probes for measuring single cell adhesion with atomic force microscopy (AFM).[1] A single-cell probe was readily made by picking up a bacterial cell from a glass surface using a tipless AFM cantilever coated...... random immobilization is obtained by submerging the cantilever in a bacterial suspension. The reported method provides a general platform for investigating single cell interactions of bacteria with different surfaces and other cells by AFM force spectroscopy, thus improving our understanding....... The strain-dependent susceptibility to bacterial colonization on conventional PLL-g-PEG illustrates how bacterial diversity challenges development of “universal” antifouling coatings, and AFM single-cell force spectroscopy was proven to be a powerful tool to provide insights into the molecular mechanisms...

  10. Dynamic single-cell NAD(P)H measurement reveals oscillatory metabolism throughout the E. coli cell division cycle.

    Science.gov (United States)

    Zhang, Zheng; Milias-Argeitis, Andreas; Heinemann, Matthias

    2018-02-01

    Recent work has shown that metabolism between individual bacterial cells in an otherwise isogenetic population can be different. To investigate such heterogeneity, experimental methods to zoom into the metabolism of individual cells are required. To this end, the autofluoresence of the redox cofactors NADH and NADPH offers great potential for single-cell dynamic NAD(P)H measurements. However, NAD(P)H excitation requires UV light, which can cause cell damage. In this work, we developed a method for time-lapse NAD(P)H imaging in single E. coli cells. Our method combines a setup with reduced background emission, UV-enhanced microscopy equipment and optimized exposure settings, overall generating acceptable NAD(P)H signals from single cells, with minimal negative effect on cell growth. Through different experiments, in which we perturb E. coli's redox metabolism, we demonstrated that the acquired fluorescence signal indeed corresponds to NAD(P)H. Using this new method, for the first time, we report that intracellular NAD(P)H levels oscillate along the bacterial cell division cycle. The developed method for dynamic measurement of NAD(P)H in single bacterial cells will be an important tool to zoom into metabolism of individual cells.

  11. Precise mass determination of single cell with cantilever-based microbiosensor system.

    Directory of Open Access Journals (Sweden)

    Bogdan Łabędź

    Full Text Available Having determined the mass of a single cell of brewer yeast Saccharomyces cerevisiae by means of a microcantilever-based biosensor Cantisens CSR-801 (Concentris, Basel, Switzerland, it was found that its dry mass is 47,65 ± 1,05 pg. Found to be crucial in this mass determination was the cell position along the length of the cantilever. Moreover, calculations including cells positions on the cantilever provide a threefold better degree of accuracy than those which assume uniform mass distribution. We have also examined the influence of storage time on the single cell mass. Our results show that after 6 months there is an increase in the average mass of a single yeast cell.

  12. Precise mass determination of single cell with cantilever-based microbiosensor system.

    Science.gov (United States)

    Łabędź, Bogdan; Wańczyk, Aleksandra; Rajfur, Zenon

    2017-01-01

    Having determined the mass of a single cell of brewer yeast Saccharomyces cerevisiae by means of a microcantilever-based biosensor Cantisens CSR-801 (Concentris, Basel, Switzerland), it was found that its dry mass is 47,65 ± 1,05 pg. Found to be crucial in this mass determination was the cell position along the length of the cantilever. Moreover, calculations including cells positions on the cantilever provide a threefold better degree of accuracy than those which assume uniform mass distribution. We have also examined the influence of storage time on the single cell mass. Our results show that after 6 months there is an increase in the average mass of a single yeast cell.

  13. Performance equations of proton exchange membrane fuel cells with feeds of varying degrees of humidification

    International Nuclear Information System (INIS)

    Hsuen, Hsiao-Kuo; Yin, Ken-Ming

    2012-01-01

    Performance equations that describe the dependence of cell potential on current density for proton exchange membrane fuel cells (PEMFCs) with feeds of varying degrees of humidification have been formulated in algebraic form. The equations are developed by the reduction of a one-dimensional multi-domain model that takes into account, in details, the transport limitations of gas species, proton migration and electron conduction, electrochemical kinetics, as well as liquid water flow within the cathode, anode, and membrane. The model equations for the anode and membrane were integrated with those of the cathode developed in the previous studies to form a complete set of equations for one-dimensional single cell model. Because the transport equations for the anode diffuser can be solved analytically, calculations of integrals are only needed in the membrane and the two-phase region of cathode diffuser. The proposed approach greatly reduces the complexity of the model equations, and only iterations of a single algebraic equation are required to obtain final solutions. Since the performance equations are originated from a mechanistic one-dimensional model, all the parameters appearing in the equations are endowed with a precise physical significance.

  14. Study on performance and emission characteristics of a single cylinder diesel engine using exhaust gas recirculation

    Directory of Open Access Journals (Sweden)

    Anantha Raman Lakshmipathi

    2017-01-01

    Full Text Available Exhaust gas re-circulation is a method used in compression ignition engines to control and reduce NOx emission. These emissions are controlled by reducing the oxygen concentration inside the cylinder and thereby reducing the flame temperature of the charge mixture inside the combustion chamber. In the present investigation, experiments were performed to study the effect of exhaust gas re-circulation on performance and emission characteristics in a four stroke single cylinder, water cooled and constant speed diesel engine. The experiments were performed to study the performance and emissions for different exhaust gas re-circulation ratios of the engine. Performance parameters such as brake thermal efficiency, indicated thermal efficiency, specific fuel consumption, total fuel consumption and emission parameters such as oxides of nitrogen, unburned hydrocarbons, carbon monoxide, carbon dioxide and smoke opacity were measured. Reductions in NOx and CO2 were observed but other emissions like HC, CO, and smoke opacity were found to have increased with the usage of exhaust gas re-circulation. The 15% exhaust gas re-circulation was found optimum for the engine in the aspects of performance and emission.

  15. Time-resolved, single-cell analysis of induced and programmed cell death via non-invasive propidium iodide and counterstain perfusion.

    Science.gov (United States)

    Krämer, Christina E M; Wiechert, Wolfgang; Kohlheyer, Dietrich

    2016-09-01

    Conventional propidium iodide (PI) staining requires the execution of multiple steps prior to analysis, potentially affecting assay results as well as cell vitality. In this study, this multistep analysis method has been transformed into a single-step, non-toxic, real-time method via live-cell imaging during perfusion with 0.1 μM PI inside a microfluidic cultivation device. Dynamic PI staining was an effective live/dead analytical tool and demonstrated consistent results for single-cell death initiated by direct or indirect triggers. Application of this method for the first time revealed the apparent antibiotic tolerance of wild-type Corynebacterium glutamicum cells, as indicated by the conversion of violet fluorogenic calcein acetoxymethyl ester (CvAM). Additional implementation of this method provided insight into the induced cell lysis of Escherichia coli cells expressing a lytic toxin-antitoxin module, providing evidence for non-lytic cell death and cell resistance to toxin production. Finally, our dynamic PI staining method distinguished necrotic-like and apoptotic-like cell death phenotypes in Saccharomyces cerevisiae among predisposed descendants of nutrient-deprived ancestor cells using PO-PRO-1 or green fluorogenic calcein acetoxymethyl ester (CgAM) as counterstains. The combination of single-cell cultivation, fluorescent time-lapse imaging, and PI perfusion facilitates spatiotemporally resolved observations that deliver new insights into the dynamics of cellular behaviour.

  16. Granatum: a graphical single-cell RNA-Seq analysis pipeline for genomics scientists.

    Science.gov (United States)

    Zhu, Xun; Wolfgruber, Thomas K; Tasato, Austin; Arisdakessian, Cédric; Garmire, David G; Garmire, Lana X

    2017-12-05

    Single-cell RNA sequencing (scRNA-Seq) is an increasingly popular platform to study heterogeneity at the single-cell level. Computational methods to process scRNA-Seq data are not very accessible to bench scientists as they require a significant amount of bioinformatic skills. We have developed Granatum, a web-based scRNA-Seq analysis pipeline to make analysis more broadly accessible to researchers. Without a single line of programming code, users can click through the pipeline, setting parameters and visualizing results via the interactive graphical interface. Granatum conveniently walks users through various steps of scRNA-Seq analysis. It has a comprehensive list of modules, including plate merging and batch-effect removal, outlier-sample removal, gene-expression normalization, imputation, gene filtering, cell clustering, differential gene expression analysis, pathway/ontology enrichment analysis, protein network interaction visualization, and pseudo-time cell series construction. Granatum enables broad adoption of scRNA-Seq technology by empowering bench scientists with an easy-to-use graphical interface for scRNA-Seq data analysis. The package is freely available for research use at http://garmiregroup.org/granatum/app.

  17. Effects of sample treatments on genome recovery via single-cell genomics

    Energy Technology Data Exchange (ETDEWEB)

    Clingenpeel, Scott [USDOE Joint Genome Institute (JGI), Walnut Creek, CA (United States); Schwientek, Patrick [USDOE Joint Genome Institute (JGI), Walnut Creek, CA (United States); Hugenholtz, Philip [Univ. of Queensland, Brisbane (Australia); Woyke, Tanja [USDOE Joint Genome Institute (JGI), Walnut Creek, CA (United States)

    2014-06-13

    It is known that single-cell genomics is a powerful tool for accessing genetic information from uncultivated microorganisms. Methods of handling samples before single-cell genomic amplification may affect the quality of the genomes obtained. Using three bacterial strains we demonstrate that, compared to cryopreservation, lower-quality single-cell genomes are recovered when the sample is preserved in ethanol or if the sample undergoes fluorescence in situ hybridization, while sample preservation in paraformaldehyde renders it completely unsuitable for sequencing.

  18. An ATP sensitive light addressable biosensor for extracellular monitoring of single taste receptor cell.

    Science.gov (United States)

    Wu, Chunsheng; Du, Liping; Zou, Ling; Zhao, Luhang; Wang, Ping

    2012-12-01

    Adenosine triphosphate (ATP) is considered as the key neurotransmitter in taste buds for taste signal transmission and processing. Measurements of ATP secreted from single taste receptor cell (TRC) with high sensitivity and specificity are essential for investigating mechanisms underlying taste cell-to-cell communications. In this study, we presented an aptamer-based biosensor for the detection of ATP locally secreted from single TRC. ATP sensitive DNA aptamer was used as recognition element and its DNA competitor was served as signal transduction element that was covalently immobilized on the surface of light addressable potentiometric sensor (LAPS). Due to the light addressable capability of LAPS, local ATP secretion from single TRC can be detected by monitoring the working potential shifts of LAPS. The results show this biosensor can detect ATP with high sensitivity and specificity. It is demonstrated this biosensor can effectively detect the local ATP secretion from single TRC responding to tastant mixture. This biosensor could provide a promising new tool for the research of taste cell-to-cell communications as well as for the detection of local ATP secretion from other types of ATP secreting individual cells.

  19. Single-cell multiple gene expression analysis based on single-molecule-detection microarray assay for multi-DNA determination

    Energy Technology Data Exchange (ETDEWEB)

    Li, Lu [School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100 (China); Wang, Xianwei [School of Life Sciences, Shandong University, Jinan 250100 (China); Zhang, Xiaoli [School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100 (China); Wang, Jinxing [School of Life Sciences, Shandong University, Jinan 250100 (China); Jin, Wenrui, E-mail: jwr@sdu.edu.cn [School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100 (China)

    2015-01-07

    Highlights: • A single-molecule-detection (SMD) microarray for 10 samples is fabricated. • The based-SMD microarray assay (SMA) can determine 8 DNAs for each sample. • The limit of detection of SMA is as low as 1.3 × 10{sup −16} mol L{sup −1}. • The SMA can be applied in single-cell multiple gene expression analysis. - Abstract: We report a novel ultra-sensitive and high-selective single-molecule-detection microarray assay (SMA) for multiple DNA determination. In the SMA, a capture DNA (DNAc) microarray consisting of 10 subarrays with 9 spots for each subarray is fabricated on a silanized glass coverslip as the substrate. On the subarrays, the spot-to-spot spacing is 500 μm and each spot has a diameter of ∼300 μm. The sequence of the DNAcs on the 9 spots of a subarray is different, to determine 8 types of target DNAs (DNAts). Thus, 8 types of DNAts are captured to their complementary DNAcs at 8 spots of a subarray, respectively, and then labeled with quantum dots (QDs) attached to 8 types of detection DNAs (DNAds) with different sequences. The ninth spot is used to detect the blank value. In order to determine the same 8 types of DNAts in 10 samples, the 10 DNAc-modified subarrays on the microarray are identical. Fluorescence single-molecule images of the QD-labeled DNAts on each spot of the subarray are acquired using a home-made single-molecule microarray reader. The amounts of the DNAts are quantified by counting the bright dots from the QDs. For a microarray, 8 types of DNAts in 10 samples can be quantified in parallel. The limit of detection of the SMA for DNA determination is as low as 1.3 × 10{sup −16} mol L{sup −1}. The SMA for multi-DNA determination can also be applied in single-cell multiple gene expression analysis through quantification of complementary DNAs (cDNAs) corresponding to multiple messenger RNAs (mRNAs) in single cells. To do so, total RNA in single cells is extracted and reversely transcribed into their cDNAs. Three

  20. High resolution imaging of surface patterns of single bacterial cells

    International Nuclear Information System (INIS)

    Greif, Dominik; Wesner, Daniel; Regtmeier, Jan; Anselmetti, Dario

    2010-01-01

    We systematically studied the origin of surface patterns observed on single Sinorhizobium meliloti bacterial cells by comparing the complementary techniques atomic force microscopy (AFM) and scanning electron microscopy (SEM). Conditions ranged from living bacteria in liquid to fixed bacteria in high vacuum. Stepwise, we applied different sample modifications (fixation, drying, metal coating, etc.) and characterized the observed surface patterns. A detailed analysis revealed that the surface structure with wrinkled protrusions in SEM images were not generated de novo but most likely evolved from similar and naturally present structures on the surface of living bacteria. The influence of osmotic stress to the surface structure of living cells was evaluated and also the contribution of exopolysaccharide and lipopolysaccharide (LPS) by imaging two mutant strains of the bacterium under native conditions. AFM images of living bacteria in culture medium exhibited surface structures of the size of single proteins emphasizing the usefulness of AFM for high resolution cell imaging.

  1. Direct Correlation between Motile Behavior and Protein Abundance in Single Cells.

    Directory of Open Access Journals (Sweden)

    Yann S Dufour

    2016-09-01

    Full Text Available Understanding how stochastic molecular fluctuations affect cell behavior requires the quantification of both behavior and protein numbers in the same cells. Here, we combine automated microscopy with in situ hydrogel polymerization to measure single-cell protein expression after tracking swimming behavior. We characterized the distribution of non-genetic phenotypic diversity in Escherichia coli motility, which affects single-cell exploration. By expressing fluorescently tagged chemotaxis proteins (CheR and CheB at different levels, we quantitatively mapped motile phenotype (tumble bias to protein numbers using thousands of single-cell measurements. Our results disagreed with established models until we incorporated the role of CheB in receptor deamidation and the slow fluctuations in receptor methylation. Beyond refining models, our central finding is that changes in numbers of CheR and CheB affect the population mean tumble bias and its variance independently. Therefore, it is possible to adjust the degree of phenotypic diversity of a population by adjusting the global level of expression of CheR and CheB while keeping their ratio constant, which, as shown in previous studies, confers functional robustness to the system. Since genetic control of protein expression is heritable, our results suggest that non-genetic diversity in motile behavior is selectable, supporting earlier hypotheses that such diversity confers a selective advantage.

  2. Do bacterial cell numbers follow a theoretical Poisson distribution? Comparison of experimentally obtained numbers of single cells with random number generation via computer simulation.

    Science.gov (United States)

    Koyama, Kento; Hokunan, Hidekazu; Hasegawa, Mayumi; Kawamura, Shuso; Koseki, Shigenobu

    2016-12-01

    We investigated a bacterial sample preparation procedure for single-cell studies. In the present study, we examined whether single bacterial cells obtained via 10-fold dilution followed a theoretical Poisson distribution. Four serotypes of Salmonella enterica, three serotypes of enterohaemorrhagic Escherichia coli and one serotype of Listeria monocytogenes were used as sample bacteria. An inoculum of each serotype was prepared via a 10-fold dilution series to obtain bacterial cell counts with mean values of one or two. To determine whether the experimentally obtained bacterial cell counts follow a theoretical Poisson distribution, a likelihood ratio test between the experimentally obtained cell counts and Poisson distribution which parameter estimated by maximum likelihood estimation (MLE) was conducted. The bacterial cell counts of each serotype sufficiently followed a Poisson distribution. Furthermore, to examine the validity of the parameters of Poisson distribution from experimentally obtained bacterial cell counts, we compared these with the parameters of a Poisson distribution that were estimated using random number generation via computer simulation. The Poisson distribution parameters experimentally obtained from bacterial cell counts were within the range of the parameters estimated using a computer simulation. These results demonstrate that the bacterial cell counts of each serotype obtained via 10-fold dilution followed a Poisson distribution. The fact that the frequency of bacterial cell counts follows a Poisson distribution at low number would be applied to some single-cell studies with a few bacterial cells. In particular, the procedure presented in this study enables us to develop an inactivation model at the single-cell level that can estimate the variability of survival bacterial numbers during the bacterial death process. Copyright © 2016 Elsevier Ltd. All rights reserved.

  3. Whey utilization for single-cell protein production

    Energy Technology Data Exchange (ETDEWEB)

    Barraquio, V; Silverio, L G; Revilleza, R P; Fernadez, W L

    1980-01-01

    The production of single-cell protein by yeast assimilation of lactose in soft cheese whey was studied using Candida pseudotropicalis as a test organism. Under shake-flask cultivation conditions with deproteinized whey as the medium, lactose (initially 4.20%) was completely assimilated in 48h; cell mass was 5.56 mg/mL after 72h; and average protein content of the dried mass was approximately 11.8%. Batch cultivation using undeproteinized whey resulted in a faster lactose utilization rate from an initial 3.93% to a residual 0.56% in 12 h; cell mass was 8.41 mg/mL in 10 h; and average protein was approximately 37.7%. In a semicontinuous culture with 10 to the power of 7 viable cells/mL as initial cell concentration, 15.69 mg/mL cell mass with a mean protein content of approximately 21.4% could be produced and lactose could be considerably consumed (from an initial 4.75% to a residual 0.42%) within 13-14 h. Supplementation with (NH/sub 4/)/sub 2/S0/sub 4/ and KH/sub 2/P0/sub 4/ did not increase cell mass (12.47 mg/mL in 12 h) and hasten lactose assimulation (from initial 4.49% to residual 0.3% in 12 h). Average protein content was approximately 31%. Cell mass yield was established as 0.29 mg yeast cell/mg lactose consumed. Factors that might have affected protein content are also discussed.

  4. Quantitative analysis of circadian single cell oscillations in response to temperature.

    Science.gov (United States)

    Abraham, Ute; Schlichting, Julia Katharina; Kramer, Achim; Herzel, Hanspeter

    2018-01-01

    Body temperature rhythms synchronize circadian oscillations in different tissues, depending on the degree of cellular coupling: the responsiveness to temperature is higher when single circadian oscillators are uncoupled. So far, the role of coupling in temperature responsiveness has only been studied in organotypic tissue slices of the central circadian pacemaker, because it has been assumed that peripheral target organs behave like uncoupled multicellular oscillators. Since recent studies indicate that some peripheral tissues may exhibit cellular coupling as well, we asked whether peripheral network dynamics also influence temperature responsiveness. Using a novel technique for long-term, high-resolution bioluminescence imaging of primary cultured cells, exposed to repeated temperature cycles, we were able to quantitatively measure period, phase, and amplitude of central (suprachiasmatic nuclei neuron dispersals) and peripheral (mouse ear fibroblasts) single cell oscillations in response to temperature. Employing temperature cycles of different lengths, and different cell densities, we found that some circadian characteristics appear cell-autonomous, e.g. period responses, while others seem to depend on the quality/degree of cellular communication, e.g. phase relationships, robustness of the oscillation, and amplitude. Overall, our findings indicate a strong dependence on the cell's ability for intercellular communication, which is not only true for neuronal pacemakers, but, importantly, also for cells in peripheral tissues. Hence, they stress the importance of comparative studies that evaluate the degree of coupling in a given tissue, before it may be used effectively as a target for meaningful circadian manipulation.

  5. A precise pointing nanopipette for single-cell imaging via electroosmotic injection.

    Science.gov (United States)

    Lv, Jian; Qian, Ruo-Can; Hu, Yong-Xu; Liu, Shao-Chuang; Cao, Yue; Zheng, Yong-Jie; Long, Yi-Tao

    2016-11-24

    The precise transportation of fluorescent probes to the designated location in living cells is still a challenge. Here, we present a new addition to nanopipettes as a powerful tool to deliver fluorescent molecules to a given place in a single cell by electroosmotic flow, indicating favorable potential for further application in single-cell imaging.

  6. Effect of short-term alkaline intervention on the performance of buffer-free single-chamber microbial fuel cell.

    Science.gov (United States)

    Yang, Na; Ren, Yueping; Li, Xiufen; Wang, Xinhua

    2017-06-01

    Anolyte acidification is a drawback restricting the electricity generation performance of the buffer-free microbial fuel cells (MFC). In this paper, a small amount of alkali-treated anion exchange resin (AER) was placed in front of the anode in the KCl mediated single-chamber MFC to slowly release hydroxyl ions (OH - ) and neutralize the H + ions that are generated by the anodic reaction in two running cycles. This short-term alkaline intervention to the KCl anolyte has promoted the proliferation of electroactive Geobacter sp. and enhanced the self-buffering capacity of the KCl-AER-MFC. The pH of the KCl anolyte in the KCl-AER-MFC increased and became more stable in each running cycle compared with that of the KCl-MFC after the short-term alkaline intervention. The maximum power density (P max ) of the KCl-AER-MFC increased from 307.5mW·m -2 to 542.8mW·m -2 , slightly lower than that of the PBS-MFC (640.7mW·m -2 ). The coulombic efficiency (CE) of the KCl-AER-MFC increased from 54.1% to 61.2% which is already very close to that of the PBS-MFC (61.9%). The results in this paper indicate that short-term alkaline intervention to the anolyte is an effective strategy to further promote the performance of buffer-free MFCs. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. Cell growth regulation studies on our Biophotonics Workstation

    DEFF Research Database (Denmark)

    Chouliara, Manto; Engay, Einstom; Bañas, Andrew

    2018-01-01

    The past several years have seen an accelerated development of technologies and methods that enable the non-invasive analysis of single cells. These are vital as single cell studies provide important evidence and deepen our understanding of how networks of cells work and evolve. Exploring the ful...

  8. Efficient generation of monoclonal antibodies from single rhesus macaque antibody secreting cells.

    Science.gov (United States)

    Meng, Weixu; Li, Leike; Xiong, Wei; Fan, Xuejun; Deng, Hui; Bett, Andrew J; Chen, Zhifeng; Tang, Aimin; Cox, Kara S; Joyce, Joseph G; Freed, Daniel C; Thoryk, Elizabeth; Fu, Tong-Ming; Casimiro, Danilo R; Zhang, Ningyan; A Vora, Kalpit; An, Zhiqiang

    2015-01-01

    Nonhuman primates (NHPs) are used as a preclinical model for vaccine development, and the antibody profiles to experimental vaccines in NHPs can provide critical information for both vaccine design and translation to clinical efficacy. However, an efficient protocol for generating monoclonal antibodies from single antibody secreting cells of NHPs is currently lacking. In this study we established a robust protocol for cloning immunoglobulin (IG) variable domain genes from single rhesus macaque (Macaca mulatta) antibody secreting cells. A sorting strategy was developed using a panel of molecular markers (CD3, CD19, CD20, surface IgG, intracellular IgG, CD27, Ki67 and CD38) to identify the kinetics of B cell response after vaccination. Specific primers for the rhesus macaque IG genes were designed and validated using cDNA isolated from macaque peripheral blood mononuclear cells. Cloning efficiency was averaged at 90% for variable heavy (VH) and light (VL) domains, and 78.5% of the clones (n = 335) were matched VH and VL pairs. Sequence analysis revealed that diverse IGHV subgroups (for VH) and IGKV and IGLV subgroups (for VL) were represented in the cloned antibodies. The protocol was tested in a study using an experimental dengue vaccine candidate. About 26.6% of the monoclonal antibodies cloned from the vaccinated rhesus macaques react with the dengue vaccine antigens. These results validate the protocol for cloning monoclonal antibodies in response to vaccination from single macaque antibody secreting cells, which have general applicability for determining monoclonal antibody profiles in response to other immunogens or vaccine studies of interest in NHPs.

  9. Microfluidic Impedance Flow Cytometry Enabling High-Throughput Single-Cell Electrical Property Characterization

    Science.gov (United States)

    Chen, Jian; Xue, Chengcheng; Zhao, Yang; Chen, Deyong; Wu, Min-Hsien; Wang, Junbo

    2015-01-01

    This article reviews recent developments in microfluidic impedance flow cytometry for high-throughput electrical property characterization of single cells. Four major perspectives of microfluidic impedance flow cytometry for single-cell characterization are included in this review: (1) early developments of microfluidic impedance flow cytometry for single-cell electrical property characterization; (2) microfluidic impedance flow cytometry with enhanced sensitivity; (3) microfluidic impedance and optical flow cytometry for single-cell analysis and (4) integrated point of care system based on microfluidic impedance flow cytometry. We examine the advantages and limitations of each technique and discuss future research opportunities from the perspectives of both technical innovation and clinical applications. PMID:25938973

  10. Design of coated standing nanowire array solar cell performing beyond the planar efficiency limits

    Energy Technology Data Exchange (ETDEWEB)

    Zeng, Yang; Ye, Qinghao; Shen, Wenzhong, E-mail: wzshen@sjtu.edu.cn [Institute of Solar Energy, and Key Laboratory of Artificial Structures and Quantum Control (Ministry of Education), Department of Physics and Astronomy, Shanghai Jiao Tong University, Shanghai 200240 (China)

    2016-05-28

    The single standing nanowire (SNW) solar cells have been proven to perform beyond the planar efficiency limits in both open-circuit voltage and internal quantum efficiency due to the built-in concentration and the shifting of the absorption front. However, the expandability of these nano-scale units to a macro-scale photovoltaic device remains unsolved. The main difficulty lies in the simultaneous preservation of an effective built-in concentration in each unit cell and a broadband high absorption capability of their array. Here, we have provided a detailed theoretical guideline for realizing a macro-scale solar cell that performs furthest beyond the planar limits. The key lies in a complementary design between the light-trapping of the single SNWs and that of the photonic crystal slab formed by the array. By tuning the hybrid HE modes of the SNWs through the thickness of a coaxial dielectric coating, the optimized coated SNW array can sustain an absorption rate over 97.5% for a period as large as 425 nm, which, together with the inherited carrier extraction advantage, leads to a cell efficiency increment of 30% over the planar limit. This work has demonstrated the viability of a large-size solar cell that performs beyond the planar limits.

  11. An automated approach for single-cell tracking in epifluorescence microscopy applied to E. coli growth analysis on microfluidics biochips

    Science.gov (United States)

    Fetita, Catalin; Kirov, Boris; Jaramillo, Alfonso; Lefevre, Christophe

    2012-03-01

    With the accumulation of knowledge for the intimate molecular mechanisms governing the processes inside the living cells in the later years, the ability to characterize the performance of elementary genetic circuits and parts at the single-cell level is becoming of crucial importance. Biological science is arriving to the point where it can develop hypothesis for the action of each molecule participating in the biochemical reactions and need proper techniques to test those hypothesis. Microfluidics is emerging as the technology that combined with high-magnification microscopy will allow for the long-term single-cell level observation of bacterial physiology. In this study we design, build and characterize the gene dynamics of genetic circuits as one of the basic parts governing programmed cell behavior. We use E. coli as model organism and grow it in microfluidics chips, which we observe with epifluorescence microscopy. One of the most invaluable segments of this technology is the consequent image processing, since it allows for the automated analysis of vast amount of single-cell observation and the fast and easy derivation of conclusions based on that data. Specifically, we are interested in promoter activity as function of time. We expect it to be oscillatory and for that we use GFP (green fluorescent protein) as a reporter in our genetic circuits. In this paper, an automated framework for single-cell tracking in phase-contrast microscopy is developed, combining 2D segmentation of cell time frames and graph-based reconstruction of their spatiotemporal evolution with fast tracking of the associated fluorescence signal. The results obtained on the investigated biological database are presented and discussed.

  12. Considerably improved photovoltaic performance of carbon nanotube-based solar cells using metal oxide layers

    Science.gov (United States)

    Wang, Feijiu; Kozawa, Daichi; Miyauchi, Yuhei; Hiraoka, Kazushi; Mouri, Shinichiro; Ohno, Yutaka; Matsuda, Kazunari

    2015-02-01

    Carbon nanotube-based solar cells have been extensively studied from the perspective of potential application. Here we demonstrated a significant improvement of the carbon nanotube solar cells by the use of metal oxide layers for efficient carrier transport. The metal oxides also serve as an antireflection layer and an efficient carrier dopant, leading to a reduction in the loss of the incident solar light and an increase in the photocurrent, respectively. As a consequence, the photovoltaic performance of both p-single-walled carbon nanotube (SWNT)/n-Si and n-SWNT/p-Si heterojunction solar cells using MoOx and ZnO layers is improved, resulting in very high photovoltaic conversion efficiencies of 17.0 and 4.0%, respectively. These findings regarding the use of metal oxides as multifunctional layers suggest that metal oxide layers could improve the performance of various electronic devices based on carbon nanotubes.

  13. Considerably improved photovoltaic performance of carbon nanotube-based solar cells using metal oxide layers.

    Science.gov (United States)

    Wang, Feijiu; Kozawa, Daichi; Miyauchi, Yuhei; Hiraoka, Kazushi; Mouri, Shinichiro; Ohno, Yutaka; Matsuda, Kazunari

    2015-02-18

    Carbon nanotube-based solar cells have been extensively studied from the perspective of potential application. Here we demonstrated a significant improvement of the carbon nanotube solar cells by the use of metal oxide layers for efficient carrier transport. The metal oxides also serve as an antireflection layer and an efficient carrier dopant, leading to a reduction in the loss of the incident solar light and an increase in the photocurrent, respectively. As a consequence, the photovoltaic performance of both p-single-walled carbon nanotube (SWNT)/n-Si and n-SWNT/p-Si heterojunction solar cells using MoOx and ZnO layers is improved, resulting in very high photovoltaic conversion efficiencies of 17.0 and 4.0%, respectively. These findings regarding the use of metal oxides as multifunctional layers suggest that metal oxide layers could improve the performance of various electronic devices based on carbon nanotubes.

  14. Single cell wound generates electric current circuit and cell membrane potential variations that requires calcium influx.

    Science.gov (United States)

    Luxardi, Guillaume; Reid, Brian; Maillard, Pauline; Zhao, Min

    2014-07-24

    Breaching of the cell membrane is one of the earliest and most common causes of cell injury, tissue damage, and disease. If the compromise in cell membrane is not repaired quickly, irreversible cell damage, cell death and defective organ functions will result. It is therefore fundamentally important to efficiently repair damage to the cell membrane. While the molecular aspects of single cell wound healing are starting to be deciphered, its bio-physical counterpart has been poorly investigated. Using Xenopus laevis oocytes as a model for single cell wound healing, we describe the temporal and spatial dynamics of the wound electric current circuitry and the temporal dynamics of cell membrane potential variation. In addition, we show the role of calcium influx in controlling electric current circuitry and cell membrane potential variations. (i) Upon wounding a single cell: an inward electric current appears at the wound center while an outward electric current is observed at its sides, illustrating the wound electric current circuitry; the cell membrane is depolarized; calcium flows into the cell. (ii) During cell membrane re-sealing: the wound center current density is maintained for a few minutes before decreasing; the cell membrane gradually re-polarizes; calcium flow into the cell drops. (iii) In conclusion, calcium influx is required for the formation and maintenance of the wound electric current circuitry, for cell membrane re-polarization and for wound healing.

  15. An Inert Continuous Microreactor for the Isolation and Analysis of a Single Microbial Cell

    Directory of Open Access Journals (Sweden)

    Katrin Rosenthal

    2015-11-01

    Full Text Available Studying biological phenomena of individual cells is enabled by matching the scales of microbes and cultivation devices. We present a versatile, chemically inert microfluidic lab-on-a-chip (LOC device for biological and chemical analyses of isolated microorganisms. It is based on the Envirostat concept and guarantees constant environmental conditions. A new manufacturing process for direct fusion bonding chips with functional microelectrodes for selective and gentle cell manipulation via negative dielectrophoresis (nDEP was generated. The resulting LOC system offered a defined surface chemistry and exceptional operational stability, maintaining its structural integrity even after harsh chemical treatment. The microelectrode structures remained fully functional after thermal bonding and were proven to be efficient for single-cell trapping via nDEP. The microfluidic network consisted solely of glass, which led to enhanced chip reusability and minimized interaction of the material with chemical and biological compounds. We validated the LOC for single-cell studies with the amino acid secreting bacterium Corynebacterium glutamicum. Intracellular l-lysine production dynamics of individual bacteria were monitored based on a genetically encoded fluorescent nanosensor. The results demonstrate the applicability of the presented LOC for pioneering chemical and biological studies, where robustness and chemically inert surfaces are crucial parameters for approaching fundamental biological questions at a single-cell level.

  16. Mass sensors with mechanical traps for weighing single cells in different fluids.

    Science.gov (United States)

    Weng, Yaochung; Delgado, Francisco Feijó; Son, Sungmin; Burg, Thomas P; Wasserman, Steven C; Manalis, Scott R

    2011-12-21

    We present two methods by which single cells can be mechanically trapped and continuously monitored within the suspended microchannel resonator (SMR) mass sensor. Since the fluid surrounding the trapped cell can be quickly and completely replaced on demand, our methods are well suited for measuring changes in cell size and growth in response to drugs or other chemical stimuli. We validate our methods by measuring the density of single polystyrene beads and Saccharomyces cerevisiae yeast cells with a precision of approximately 10(-3) g cm(-3), and by monitoring the growth of single mouse lymphoblast cells before and after drug treatment.

  17. Single-cell cloning and expansion of human induced pluripotent stem cells by a microfluidic culture device.

    Science.gov (United States)

    Matsumura, Taku; Tatsumi, Kazuya; Noda, Yuichiro; Nakanishi, Naoyuki; Okonogi, Atsuhito; Hirano, Kunio; Li, Liu; Osumi, Takashi; Tada, Takashi; Kotera, Hidetoshi

    2014-10-10

    The microenvironment of cells, which includes basement proteins, shear stress, and extracellular stimuli, should be taken into consideration when examining physiological cell behavior. Although microfluidic devices allow cellular responses to be analyzed with ease at the single-cell level, few have been designed to recover cells. We herein demonstrated that a newly developed microfluidic device helped to improve culture conditions and establish a clonality-validated human pluripotent stem cell line after tracing its growth at the single-cell level. The device will be a helpful tool for capturing various cell types in the human body that have not yet been established in vitro. Copyright © 2014 Elsevier Inc. All rights reserved.

  18. An electrochemical impedance spectroscopy study of polymer electrolyte membrane fuel cells electrocatalyst single wall carbon nanohorns-supported.

    Science.gov (United States)

    Brandão, Lúcia; Boaventura, Marta; Passeira, Carolina; Gattia, Daniele Mirabile; Marazzi, Renzo; Antisari, Marco Vittori; Mendes, Adélio

    2011-10-01

    Electrochemical impedance spectroscopy (EIS) was used to study the polymer electrolyte membrane fuel cells (PEMFC) performance when using single wall carbon nanohorns (SWNH) to support Pt nanoparticles. Additionally, as-prepared and oxidized SWNH Pt-supports were compared with conventional carbon black. Two different oxidizing treatments were considered: oxygen flow at 500 degrees C and reflux in an acid solution at 85 degrees C. Both oxidizing treatments increased SWNH surface area; oxygen treatment increased surface area 4 times while acid treatment increased 2.6 times. The increase in surface area should be related to the opening access to the inner tube of SWNH. Acid treatment of SWNH increased chemical fragility and decreased electrocatalyst load in comparison with as-prepared SWNH. On the other hand, the oxygen treated SWNH sample allowed to obtain the highest electrocatalyst load. The use of as-prepared and oxygen treated SWNH showed in both cases catalytic activities 60% higher than using conventional carbon black as electrocatalyst support in PEMFC. Moreover, EIS analysis indicated that the major improvement in performance is related to the cathode kinetics in the as-prepared SWNH sample, while concerning the oxidized SWNH sample, the improvements are related to the electrokinetics in both anode and cathode electrodes. These improvements should be related with differences in the hydrophobic character between SWNH and carbon black.

  19. Micromagnetic Cancer Cell Immobilization and Release for Real-Time Single Cell Analysis

    Energy Technology Data Exchange (ETDEWEB)

    Jaiswal, Devina; Rad, Armin Tahmasbi [Department of Biomedical Engineering, University of Connecticut, Storrs, CT, 06269 (United States); Nieh, Mu-Ping [Department of Biomedical Engineering, University of Connecticut, Storrs, CT, 06269 (United States); Department of Chemical and Biomolecular Engineering, University of Connecticut, Storrs, CT 06269 (United States); Polymer Program, Institute of Materials Science, University of Connecticut, Storrs, CT 06269 (United States); Claffey, Kevin P. [Department of Cell Biology, University of Connecticut Health Center, Farmington, CT 06030 (United States); Hoshino, Kazunori, E-mail: hoshino@engr.uconn.edu [Department of Biomedical Engineering, University of Connecticut, Storrs, CT, 06269 (United States)

    2017-04-01

    Understanding the interaction of live cells with macromolecules is crucial for designing efficient therapies. Considering the functional heterogeneity found in cancer cells, real-time single cell analysis is necessary to characterize responses. In this study, we have designed and fabricated a microfluidic channel with patterned micromagnets which can temporarily immobilize the cells during analysis and release them after measurements. The microchannel is composed of plain coverslip top and bottom panels to facilitate easy microscopic observation and undisturbed application of analytes to the cells. Cells labeled with functionalized magnetic beads were immobilized in the device with an efficiency of 90.8±3.6%. Since the micromagnets are made of soft magnetic material (Ni), they released cells when external magnetic field was turned off from the channel. This allows the reuse of the channel for a new sample. As a model drug analysis, the immobilized breast cancer cells (MCF7) were exposed to fluorescent lipid nanoparticles and association and dissociation were measured through fluorescence analysis. Two concentrations of nanoparticles, 0.06 µg/ml and 0.08 µg/ml were tested and time lapse images were recorded and analyzed. The microfluidic device was able to provide a microenvironment for sample analysis, making it an efficient platform for real-time analysis.

  20. Linnorm: improved statistical analysis for single cell RNA-seq expression data.

    Science.gov (United States)

    Yip, Shun H; Wang, Panwen; Kocher, Jean-Pierre A; Sham, Pak Chung; Wang, Junwen

    2017-12-15

    Linnorm is a novel normalization and transformation method for the analysis of single cell RNA sequencing (scRNA-seq) data. Linnorm is developed to remove technical noises and simultaneously preserve biological variations in scRNA-seq data, such that existing statistical methods can be improved. Using real scRNA-seq data, we compared Linnorm with existing normalization methods, including NODES, SAMstrt, SCnorm, scran, DESeq and TMM. Linnorm shows advantages in speed, technical noise removal and preservation of cell heterogeneity, which can improve existing methods in the discovery of novel subtypes, pseudo-temporal ordering of cells, clustering analysis, etc. Linnorm also performs better than existing DEG analysis methods, including BASiCS, NODES, SAMstrt, Seurat and DESeq2, in false positive rate control and accuracy. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  1. Effect of compressive force on PEM fuel cell performance

    Science.gov (United States)

    MacDonald, Colin Stephen

    Polymer electrolyte membrane (PEM) fuel cells possess the potential, as a zero-emission power source, to replace the internal combustion engine as the primary option for transportation applications. Though there are a number of obstacles to vast PEM fuel cell commercialization, such as high cost and limited durability, there has been significant progress in the field to achieve this goal. Experimental testing and analysis of fuel cell performance has been an important tool in this advancement. Experimental studies of the PEM fuel cell not only identify unfiltered performance response to manipulation of variables, but also aid in the advancement of fuel cell modelling, by allowing for validation of computational schemes. Compressive force used to contain a fuel cell assembly can play a significant role in how effectively the cell functions, the most obvious example being to ensure proper sealing within the cell. Compression can have a considerable impact on cell performance beyond the sealing aspects. The force can manipulate the ability to deliver reactants and the electrochemical functions of the cell, by altering the layers in the cell susceptible to this force. For these reasons an experimental study was undertaken, presented in this thesis, with specific focus placed on cell compression; in order to study its effect on reactant flow fields and performance response. The goal of the thesis was to develop a consistent and accurate general test procedure for the experimental analysis of a PEM fuel cell in order to analyse the effects of compression on performance. The factors potentially affecting cell performance, which were a function of compression, were identified as: (1) Sealing and surface contact; (2) Pressure drop across the flow channel; (3) Porosity of the GDL. Each factor was analysed independently in order to determine the individual contribution to changes in performance. An optimal degree of compression was identified for the cell configuration in

  2. Single-dose-response curves of murine gastrointestinal crypt stem cells

    International Nuclear Information System (INIS)

    Masuda, K.; Withers, H.R.; Mason, K.A.; Chen, K.Y.

    1977-01-01

    Dose-response curves for the reproductive capacity of crypt stem cells of murine colonic, jejunal, and gastric mucosae exposed in situ to multifractionated gamma ray exposures were analyzed and single-dose-survival curves of these cells were constructed. The following conclusions were drawn: (1) The single-dose-response curves bend downward over a dose range of approximately 200 to 1500 rad; (2) cell death seems to be due to nonrepairable damage at doses less than 250 rad for colon, and 220 rad for jejunum; (3) there are 21, 110, and 140 stem cells per crypt of gastric, colonic, and jejunal mucosa, respectively; and (4) jejunal stem cells are the most radiosensitive and gastric mucosal stem cells are the most resistant

  3. A simple method for encapsulating single cells in alginate microspheres allows for direct PCR and whole genome amplification.

    Directory of Open Access Journals (Sweden)

    Saharnaz Bigdeli

    Full Text Available Microdroplets are an effective platform for segregating individual cells and amplifying DNA. However, a key challenge is to recover the contents of individual droplets for downstream analysis. This paper offers a method for embedding cells in alginate microspheres and performing multiple serial operations on the isolated cells. Rhodobacter sphaeroides cells were diluted in alginate polymer and sprayed into microdroplets using a fingertip aerosol sprayer. The encapsulated cells were lysed and subjected either to conventional PCR, or whole genome amplification using either multiple displacement amplification (MDA or a two-step PCR protocol. Microscopic examination after PCR showed that the lumen of the occupied microspheres contained fluorescently stained DNA product, but multiple displacement amplification with phi29 produced only a small number of polymerase colonies. The 2-step WGA protocol was successful in generating fluorescent material, and quantitative PCR from DNA extracted from aliquots of microspheres suggested that the copy number inside the microspheres was amplified up to 3 orders of magnitude. Microspheres containing fluorescent material were sorted by a dilution series and screened with a fluorescent plate reader to identify single microspheres. The DNA was extracted from individual isolates, re-amplified with full-length sequencing adapters, and then a single isolate was sequenced using the Illumina MiSeq platform. After filtering the reads, the only sequences that collectively matched a genome in the NCBI nucleotide database belonged to R. sphaeroides. This demonstrated that sequencing-ready DNA could be generated from the contents of a single microsphere without culturing. However, the 2-step WGA strategy showed limitations in terms of low genome coverage and an uneven frequency distribution of reads across the genome. This paper offers a simple method for embedding cells in alginate microspheres and performing PCR on isolated

  4. Single-crystalline self-branched anatase titania nanowires for dye-sensitized solar cells

    Science.gov (United States)

    Li, Zhenquan; Yang, Huang; Wu, Fei; Fu, Jianxun; Wang, Linjun; Yang, Weiguang

    2017-03-01

    The morphology of the anatase titania plays an important role in improving the photovoltaic performance in dye-sensitized solar cells. In this work, single-crystalline self-branched anatase TiO2 nanowires have been synthesized by hydrothermal method using TBAH and CTAB as morphology controlling agents. The obtained self-branched TiO2 nanowires dominated by a large percentage of (010) facets. The photovoltaic conversion efficiency (6.37%) of dye-sensitized solar cell (DSSC) based on the self-branched TiO2 nanowires shows a significant improvement (26.6%) compared to that of P25 TiO2 (5.03%). The enhanced performance of the self-branched TiO2 nanowires-based DSSC is due to heir large percent of exposed (010) facets which have strong dye adsorption capacity and effective charge transport of the self-branched 1D nanostructures.

  5. Performance optimization studies of solution processed bulk-heterojunction solar cells

    Science.gov (United States)

    Ali, Bakhtyar

    2011-12-01

    Organic Solar Cells (OSCs), which rely on the concept of bulk-heterojunction, stand out due primarily to their simple construction, mechanical flexibility and exceptional ease of processing. These characteristics make them potential candidates to substitute for the expensive photovoltaic counterparts. Among other OSCs, devices containing poly(3-hexylthiophene) (P3HT) and phenyl C61 butaric acid methyl ester (PCBM) as photo-active layer have shown promising results. However, the power conversion efficiency (PCE) is still lower than the required commercialization mark (˜10%). Devices with structure glass/ITO/PEDOT:PSS/P3HT:PCBM/LiF/Al, annealed and un-annealed with device area ˜0.4 cm2 (unless otherwise stated), have been studied. An investigation of the device processing variables has led to the conclusion that the optimum loading of PCBM in the blend for optimum performance is in the range of 1:1 to 1:2. Characterization of the active layer with UV-vis absorption, PL spectra and XRD reveal that the addition of PCBM to P3HT matrix is detrimental for the self-organization of P3HT chains (crystallinity) and it also increases the resistivity. Similarly, 1,2 dichlorobenzene (DCB) has been found to be the best solvent among other solvents such as chloroform (CF) and chlorobenzene (CB), for optimum PCE. The rho(T) data from the samples (pristine P3HT and P3HT/PCBM blends) exhibit anisotropy in conduction where it follows the variable range hoping (VRH) in the lateral (parallel to film) and polaronic behavior in vertical (perpendicular to film) transport. The activation energy obtained from the fit to polaronic model is 329 meV for P3HT/ PCBM blend (1:1). Furthermore, the photovoltaic parameters extracted from a lumped circuit analysis of voltage and temperature dependence of photocurrent, JL(V), in P3HT/PCBM OSCs, completely describe the illuminated J-V data from far reverse bias to beyond the open circuit voltage (Voc). A simple model for carrier collection has been

  6. Impaired laparoscopic performance of novice surgeons due to phone call distraction: a single-centre, prospective study.

    Science.gov (United States)

    Yang, Cui; Heinze, Julia; Helmert, Jens; Weitz, Juergen; Reissfelder, Christoph; Mees, Soeren Torge

    2017-12-01

    Distractions such as phone calls during laparoscopic surgery play an important role in many operating rooms. The aim of this single-centre, prospective study was to assess if laparoscopic performance is impaired by intraoperative phone calls in novice surgeons. From October 2015 to June 2016, 30 novice surgeons (medical students) underwent a laparoscopic surgery training curriculum including two validated tasks (peg transfer, precision cutting) until achieving a defined level of proficiency. For testing, participants were required to perform these tasks under three conditions: no distraction (control) and two standardised distractions in terms of phone calls requiring response (mild and strong distraction). Task performance was evaluated by analysing time and accuracy of the tasks and response of the phone call. In peg transfer (easy task), mild distraction did not worsen the performance significantly, while strong distraction was linked to error and inefficiency with significantly deteriorated performance (P phone call distractions result in impaired laparoscopic performance under certain circumstances. To ensure patient safety, phone calls should be avoided as far as possible in operating rooms.

  7. Development of the IFJ single ion hit facility for cells irradiation

    International Nuclear Information System (INIS)

    Veselov, O.; Polak, W.; Ugenskiene, R.; Hajduk, R.; Lebed, K.; Lekki, J.; Horwacik, T.; Dutkiewicz, E.M.; Maranda, S.; Pieprzyca, T.; Sarnecki, C.; Stachura, Z.; Szklarz, Z.; Styczen, J.

    2005-12-01

    In recent years a single ion hit facility (SIHF) has been constructed at the IFJ ion microprobe. The setup is used for the precise irradiations of living cells by a controlled number of ions. The facility allows investigations in various aspects of biomedical research, such as adaptive response, bystander effect, inverse dose-rate effect, low-dose hypersensitivity, etc. Those investigations have two very important requirements: (i) cells must be examined in their natural state and environment, i.e. without previously being killed, and preferentially, neither fixed nor stained, and (ii) a possibility of automatic irradiation of large number of cells with a computer recognition of their positions must be provided. This work presents some of the crucial features of the off-line and on-line optical systems, including self-developed software responsible for the automatic cell recognition. We also show several tests carried out to determine the efficiency of the whole setup and some segments. In conclusion, the results of our first irradiation measurements performed with living cells are demonstrated. (author)

  8. Cryogenic motion performances of a piezoelectric single crystal micromotor

    Science.gov (United States)

    Li, Xiaotian; Wu, Yuting; Chen, Zhijiang; Wei, Xiaoyong; Luo, Haosu; Dong, Shuxiang

    2014-04-01

    This study investigates the cryogenic performances of a millimeter-size piezoelectric ultrasonic linear micromotor. The piezoelectric vibrator of the micromotor is made of Pb(In1/2Nb1/2)O3 -Pb(Mg1/3Nb2/3)-PbTiO3 single crystal and operated in first-bending wobbling mode. Experiments show that the piezoelectric single crystal micromotor works effectively even at extremely low temperature of -175 °C, although its resonance peaks vary with temperature significantly. This work confirms the feasibility of cryogenic operation of the piezo-micromotor, which is meaningful for aerospace or superconducting microwave application.

  9. Mass Spectrometric Method for Analyzing Metabolites in Yeast with Single Cell Sensitivity

    NARCIS (Netherlands)

    Amantonico, Andrea; Oh, Joo Yeon; Sobek, Jens; Heinemann, Matthias; Zenobi, Renato

    2008-01-01

    Getting a look-in: An optimized MALDI-MS procedure has been developed to detect endogenous primary metabolites directly in the cell extract. A detection limit corresponding to metabolites from less than a single cell has been attained, opening the door to single-cell metabolomics by mass

  10. High-performance ternary blend polymer solar cells involving both energy transfer and hole relay processes.

    Science.gov (United States)

    Lu, Luyao; Chen, Wei; Xu, Tao; Yu, Luping

    2015-06-04

    The integration of multiple materials with complementary absorptions into a single junction device is regarded as an efficient way to enhance the power conversion efficiency (PCE) of organic solar cells (OSCs). However, because of increased complexity with one more component, only limited high-performance ternary systems have been demonstrated previously. Here we report an efficient ternary blend OSC with a PCE of 9.2%. We show that the third component can reduce surface trap densities in the ternary blend. Detailed studies unravel that the improved performance results from synergistic effects of enlarged open circuit voltage, suppressed trap-assisted recombination, enhanced light absorption, increased hole extraction, efficient energy transfer and better morphology. The working mechanism and high device performance demonstrate new insights and design guidelines for high-performance ternary blend solar cells and suggest that ternary structure is a promising platform to boost the efficiency of OSCs.

  11. A single-cell analysis platform for electrochemiluminescent detection of platelets adhesion to endothelial cells based on Au@DL-ZnCQDs nanoprobes.

    Science.gov (United States)

    Long, Dongping; Shang, Yunfei; Qiu, Youyi; Zhou, Bin; Yang, Peihui

    2018-04-15

    A novel single-cell analysis platform (SCA) was developed for the investigation of platelets adhesion to single human umbilical vein endothelial cell (HUVEC) via using the adhesion molecule (E-selectin) on the damaged HUVEC as the marker site, and integrating electrochemiluminescence (ECL) with the ultrasensitive Au@DL-ZnCQDs nanoprobes. The Au@DL-ZnCQDs nanocomposite, a kind of double layer zinc-coadsorbed carbon quantum dot (ZnCQDs) core-shell nanoprobe, was firstly constructed by using gold nanoparticles (AuNPs) as the core to load with ZnCQDs and then the citrate-modified silver nanoparticles (AgNPs) as the bridge to link AuNPs-ZnCQDs with ZnCQDs to form the core-shell with double layer ZnCQDs (DL-ZnCQDs) nanoprobe, revealed a 10-fold signal amplification. The H 2 O 2 -induced oxidative damage HUVECs were utilized as the cellular model on which anti-E-selectin functionalized nanoprobes specially recognized E-selectin, the SCA showed that the ECL signals decreased with platelets adhesion to single HUVEC. The proposed SCA could effectively and dynamically monitor the adhesion between single HUVEC and platelets in the absence and presence of collagen activation, moreover, be able to quantitatively detect the number of platelets adhesion to single HUVEC, and show a good analytical performance with linear range from 1 to 15 platelets. In contrast, the HUVEC was down-regulated the expression of adhesion molecules by treating with quercetin inhibitor, and the SCA also exhibited the feasibility for analysis of platelets adhesion to single HUVEC. Therefore, the single-cell analysis platform provided a novel and promising protocol for analysis of the single intercellular adhesion, and it will be beneficial to elucidate the pathogenesis of cardiovascular diseases. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. Performance of proton exchange membrane fuel cells at elevated temperature

    International Nuclear Information System (INIS)

    Shyu, Jin-Cherng; Hsueh, Kan-Lin; Tsau, Fanghei

    2011-01-01

    Highlights: → At 1 atm, cell has best performance (∼1300 mA/cm at 0.6 V) at 100 deg. C and RH = 100%. → The A value in Eq. increased with increases in the back pressure and RH. →R i dramatically decreased at back pressure of 1 atm. → At each RH, R i decreased and then increased as cell temperature increased at 1 atm. - Abstract: The polarization curves of a single PEMFC having a Nafion membrane fed with H 2 /O 2 with relative humidity (RH) of 35%, 70% and 100% were measured at cell temperatures ranging from 65 deg. C to 120 deg. C at back pressures of 0 atm and 1 atm, respectively. Measured results showed that the best cell performance at 0.6 V operated within 65-120 deg. C at zero back pressure was 1000 mA cm -2 at 65 deg. C and RH = 100%, while the best cell performance at 1 atm back pressure was 1300 mA cm -2 at 100 deg. C and RH = 100%. Based on the analysis of impedance data measured at anode and cathode humidification temperatures of 90 deg. C and cell temperature of 100 deg. C at back pressures of 0 and 1 atm (90-100p0 and 90-100p1), it could be found that the membrane resistance was reduced and the catalyst became more active as the back pressure increases. The present results showed that increasing back pressure was able to dramatically improve cell performance and the effect of the back pressure surpassed that of humidification in the internal resistance of cell.

  13. The effect of oxygen transfer mechanism on the cathode performance based on proton-conducting solid oxide fuel cells

    KAUST Repository

    Hou, Jie

    2015-01-01

    Two types of proton-blocking composites, La2NiO4+δ-LaNi0.6Fe0.4O3-δ (LNO-LNF) and Sm0.2Ce0.8O2-δ-LaNi0.6Fe0.4O3-δ (SDC-LNF), were evaluated as cathode materials for proton-conducting solid oxide fuel cells (H-SOFCs) based on the BaZr0.1Ce0.7Y0.2O3-δ (BZCY) electrolyte, in order to compare and investigate the influence of two different oxygen transfer mechanism on the performance of the cathode for H-SOFCs. The X-ray diffraction (XRD) results showed that the chemical compatibility of the components in both compounds was excellent up to 1000°C. Electrochemical studies revealed that LNO-LNF showed lower area specific polarization resistances in symmetrical cells and better electrochemical performance in single cell tests. The single cell with LNO-LNF cathode generated remarkable higher maximum power densities (MPDs) and lower interfacial polarization resistances (Rp) than that with SDC-LNF cathode. Correspondingly, the MPDs of the single cell with the LNO-LNF cathode were 490, 364, 266, 180 mW cm-2 and the Rp were 0.103, 0.279, 0.587, 1.367 Ω cm2 at 700, 650, 600 and 550°C, respectively. Moreover, after the single cell with LNO-LNF cathode optimized with an anode functional layer (AFL) between the anode and electrolyte, the power outputs reached 708 mW cm-2 at 700°C. These results demonstrate that the LNO-LNF composite cathode with the interstitial oxygen transfer mechanism is a more preferable alternative for H-SOFCs than SDC-LNF composite cathode with the oxygen vacancy transfer mechanism.

  14. Single-cell mRNA cytometry via sequence-specific nanoparticle clustering and trapping

    Science.gov (United States)

    Labib, Mahmoud; Mohamadi, Reza M.; Poudineh, Mahla; Ahmed, Sharif U.; Ivanov, Ivaylo; Huang, Ching-Lung; Moosavi, Maral; Sargent, Edward H.; Kelley, Shana O.

    2018-05-01

    Cell-to-cell variation in gene expression creates a need for techniques that can characterize expression at the level of individual cells. This is particularly true for rare circulating tumour cells, in which subtyping and drug resistance are of intense interest. Here we describe a method for cell analysis—single-cell mRNA cytometry—that enables the isolation of rare cells from whole blood as a function of target mRNA sequences. This approach uses two classes of magnetic particles that are labelled to selectively hybridize with different regions of the target mRNA. Hybridization leads to the formation of large magnetic clusters that remain localized within the cells of interest, thereby enabling the cells to be magnetically separated. Targeting specific intracellular mRNAs enablescirculating tumour cells to be distinguished from normal haematopoietic cells. No polymerase chain reaction amplification is required to determine RNA expression levels and genotype at the single-cell level, and minimal cell manipulation is required. To demonstrate this approach we use single-cell mRNA cytometry to detect clinically important sequences in prostate cancer specimens.

  15. Mechanical control of mitotic progression in single animal cells

    OpenAIRE

    Cattin, Cedric J.; Düggelin, Marcel; Martinez-Martin, David; Gerber, Christoph; Müller, Daniel J.; Stewart, Martin P.

    2015-01-01

    Despite the importance of mitotic cell rounding in tissue development and cell proliferation, there remains a paucity of approaches to investigate the mechanical robustness of cell rounding. Here we introduce ion beam-sculpted microcantilevers that enable precise force-feedback-controlled confinement of single cells while characterizing their progression through mitosis. We identify three force regimes according to the cell response: small forces (∼5 nN) that accelerate mitotic progression, i...

  16. Through the Looking Glass: Time-lapse Microscopy and Longitudinal Tracking of Single Cells to Study Anti-cancer Therapeutics.

    Science.gov (United States)

    Burke, Russell T; Orth, James D

    2016-05-14

    The response of single cells to anti-cancer drugs contributes significantly in determining the population response, and therefore is a major contributing factor in the overall outcome. Immunoblotting, flow cytometry and fixed cell experiments are often used to study how cells respond to anti-cancer drugs. These methods are important, but they have several shortcomings. Variability in drug responses between cancer and normal cells, and between cells of different cancer origin, and transient and rare responses are difficult to understand using population averaging assays and without being able to directly track and analyze them longitudinally. The microscope is particularly well suited to image live cells. Advancements in technology enable us to routinely image cells at a resolution that enables not only cell tracking, but also the observation of a variety of cellular responses. We describe an approach in detail that allows for the continuous time-lapse imaging of cells during the drug response for essentially as long as desired, typically up to 96 hr. Using variations of the approach, cells can be monitored for weeks. With the employment of genetically encoded fluorescent biosensors numerous processes, pathways and responses can be followed. We show examples that include tracking and quantification of cell growth and cell cycle progression, chromosome dynamics, DNA damage, and cell death. We also discuss variations of the technique and its flexibility, and highlight some common pitfalls.

  17. Single-cell network profiling of peripheral blood mononuclear cells from healthy donors reveals age- and race-associated differences in immune signaling pathway activation.

    Science.gov (United States)

    Longo, Diane M; Louie, Brent; Putta, Santosh; Evensen, Erik; Ptacek, Jason; Cordeiro, James; Wang, Ena; Pos, Zoltan; Hawtin, Rachael E; Marincola, Francesco M; Cesano, Alessandra

    2012-02-15

    A greater understanding of the function of the human immune system at the single-cell level in healthy individuals is critical for discerning aberrant cellular behavior that occurs in settings such as autoimmunity, immunosenescence, and cancer. To achieve this goal, a systems-level approach capable of capturing the response of the interdependent immune cell types to external stimuli is required. In this study, an extensive characterization of signaling responses in multiple immune cell subpopulations within PBMCs from a cohort of 60 healthy donors was performed using single-cell network profiling (SCNP). SCNP is a multiparametric flow cytometry-based approach that enables the simultaneous measurement of basal and evoked signaling in multiple cell subsets within heterogeneous populations. In addition to establishing the interindividual degree of variation within a broad panel of immune signaling responses, the possible association of any observed variation with demographic variables including age and race was investigated. Using half of the donors as a training set, multiple age- and race-associated variations in signaling responses in discrete cell subsets were identified, and several were subsequently confirmed in the remaining samples (test set). Such associations may provide insight into age-related immune alterations associated with high infection rates and diminished protection following vaccination and into the basis for ethnic differences in autoimmune disease incidence and treatment response. SCNP allowed for the generation of a functional map of healthy immune cell signaling responses that can provide clinically relevant information regarding both the mechanisms underlying immune pathological conditions and the selection and effect of therapeutics.

  18. Long-term in vivo imaging of multiple organs at the single cell level.

    Directory of Open Access Journals (Sweden)

    Benny J Chen

    Full Text Available Two-photon microscopy has enabled the study of individual cell behavior in live animals. Many organs and tissues cannot be studied, especially longitudinally, because they are located too deep, behind bony structures or too close to the lung and heart. Here we report a novel mouse model that allows long-term single cell imaging of many organs. A wide variety of live tissues were successfully engrafted in the pinna of the mouse ear. Many of these engrafted tissues maintained the normal tissue histology. Using the heart and thymus as models, we further demonstrated that the engrafted tissues functioned as would be expected. Combining two-photon microscopy with fluorescent tracers, we successfully visualized the engrafted tissues at the single cell level in live mice over several months. Four dimensional (three-dimensional (3D plus time information of individual cells was obtained from this imaging. This model makes long-term high resolution 4D imaging of multiple organs possible.

  19. Expression of membrane-associated proteins within single emulsion cell facsimiles.

    Science.gov (United States)

    Chanasakulniyom, Mayuree; Martino, Chiara; Paterson, David; Horsfall, Louise; Rosser, Susan; Cooper, Jonathan M

    2012-07-07

    MreB is a structural membrane-associated protein which is one of the key components of the bacterial cytoskeleton. Although it plays an important role in shape maintenance of rod-like bacteria, the understanding of its mechanism of action is still not fully understood. This study shows how segmented flow and microdroplet technology can be used as a new tool for biological in vitro investigation of this protein. In this paper, we demonstrate cell-free expression in a single emulsion system to express red fluorescence protein (RFP) and MreB linked RFP (MreB-RFP). We follow the aggregation and localisation of the fusion protein MreB-RFP in this artificial cell-like environment. The expression of MreB-RFP in single emulsion droplets leads to the formation of micrometer-scale protein patches distributed at the water/oil interface.

  20. Intracellular Drug Uptake-A Comparison of Single Cell Measurements Using ToF-SIMS Imaging and Quantification from Cell Populations with LC/MS/MS.

    Science.gov (United States)

    Newman, Carla F; Havelund, Rasmus; Passarelli, Melissa K; Marshall, Peter S; Francis, Ian; West, Andy; Alexander, Morgan R; Gilmore, Ian S; Dollery, Colin T

    2017-11-21

    ToF-SIMS is a label-free imaging method that has been shown to enable imaging of amiodarone in single rat macrophage (NR8383) cells. In this study, we show that the method extends to three other cell lines relevant to drug discovery: human embryonic kidney (HEK293), cervical cancer (HeLa), and liver cancer (HepG2). There is significant interest in the variation of drug uptake at the single cell level, and we use ToF-SIMS to show that there is great diversity between individual cells and when comparing each of the cell types. These single cell measurements are compared to quantitative measurements of cell-associated amiodarone for the population using LC/MS/MS and cell counting with flow cytometry. NR8383 and HepG2 cells uptake the greatest amount of amiodarone with an average of 2.38 and 2.60 pg per cell, respectively, and HeLa and Hek 293 have a significantly lower amount of amiodarone at 0.43 and 0.36 pg per cell, respectively. The amount of cell-associated drug for the ensemble population measurement (LC/MS/MS) is compared with the ToF-SIMS single cell data: a similar amount of drug was detected per cell for the NR8383, and HepG2 cells at a greater level than that for the HEK293 cells. However, the two techniques did not agree for the HeLa cells, and we postulate potential reasons for this.

  1. RT-qPCR work-flow for single-cell data analysis

    Czech Academy of Sciences Publication Activity Database

    Stahlberg, A.; Rusňáková, Vendula; Forootan, A.; Anděrová, Miroslava; Kubista, Mikael

    2013-01-01

    Roč. 59, č. 1 (2013), s. 80-88 ISSN 1046-2023 R&D Projects: GA MŠk(CZ) ME10052; GA ČR GAP303/10/1338 Institutional research plan: CEZ:AV0Z50520701 Institutional support: RVO:68378041 Keywords : RT-qPCR * Single-cell biology * Single-cell data analysis Subject RIV: EB - Genetics ; Molecular Biology; FH - Neurology (UEM-P) Impact factor: 3.221, year: 2013

  2. Single-leg squats can predict leg alignment in dancers performing ballet movements in "turnout".

    Science.gov (United States)

    Hopper, Luke S; Sato, Nahoko; Weidemann, Andries L

    2016-01-01

    The physical assessments used in dance injury surveillance programs are often adapted from the sports and exercise domain. Bespoke physical assessments may be required for dance, particularly when ballet movements involve "turning out" or external rotation of the legs beyond that typically used in sports. This study evaluated the ability of the traditional single-leg squat to predict the leg alignment of dancers performing ballet movements with turnout. Three-dimensional kinematic data of dancers performing the single-leg squat and five ballet movements were recorded and analyzed. Reduction of the three-dimensional data into a one-dimensional variable incorporating the ankle, knee, and hip joint center positions provided the strongest predictive model between the single-leg squat and the ballet movements. The single-leg squat can predict leg alignment in dancers performing ballet movements, even in "turned out" postures. Clinicians should pay careful attention to observational positioning and rating criteria when assessing dancers performing the single-leg squat.

  3. Fabrication and performance analysis of 4-sq cm indium tin oxide/InP photovoltaic solar cells

    Science.gov (United States)

    Gessert, T. A.; Li, X.; Phelps, P. W.; Coutts, T. J.; Tzafaras, N.

    1991-01-01

    Large-area photovoltaic solar cells based on direct current magnetron sputter deposition of indium tin oxide (ITO) into single-crystal p-InP substrates demonstrated both the radiation hardness and high performance necessary for extraterrestrial applications. A small-scale production project was initiated in which approximately 50 ITO/InP cells are being produced. The procedures used in this small-scale production of 4-sq cm ITO/InP cells are presented and discussed. The discussion includes analyses of performance range of all available production cells, and device performance data of the best cells thus far produced. Additionally, processing experience gained from the production of these cells is discussed, indicating other issues that may be encountered when large-scale productions are begun.

  4. Micromechanical and surface adhesive properties of single saccharomyces cerevisiae cells

    Science.gov (United States)

    Farzi, Bahman; Cetinkaya, Cetin

    2017-09-01

    The adhesion and mechanical properties of a biological cell (e.g. cell membrane elasticity and adhesiveness) are often strong indicators for the state of its health. Many existing techniques for determining mechanical properties of cells require direct physical contact with a single cell or a group of cells. Physical contact with the cell can trigger complex mechanotransduction mechanisms, leading to cellular responses, and consequently interfering with measurement accuracy. In the current work, based on ultrasonic excitation and interferometric (optical) motion detection, a non-contact method for characterizing the adhesion and mechanical properties of single cells is presented. It is experimentally demonstrated that the rocking (rigid body) motion and internal vibrational resonance frequencies of a single saccharomyces cerevisiae (SC) (baker’s yeast) cell can be acquired with the current approach, and the Young’s modulus and surface tension of the cell membrane as well as surface adhesion energy can be extracted from the values of these acquired resonance frequencies. The detected resonance frequency ranges for single SC cells include a rocking (rigid body) frequency of 330  ±  70 kHz and two breathing resonance frequencies of 1.53  ±  0.12 and 2.02  ±  0.31 MHz. Based on these values, the average work-of-adhesion of SC cells on a silicon substrate in aqueous medium is extracted, for the first time, as WASC-Si=16.2+/- 3.8 mJ {{m}-2} . Similarly, the surface tension and the Young’s modulus of the SC cell wall are predicted as {{σ }SC}=0.16+/- 0.02 N {{m}-1} and {{E}SC}= 9.20  ±  2.80 MPa, respectively. These results are compared to those reported in the literature by utilizing various methods, and good agreements are found. The current approach eliminates the measurement inaccuracies associated with the physical contact. Exciting and detecting cell dynamics at micro-second time-scales is significantly faster than the

  5. Mass Cytometry for Detection of Silver at the Bacterial Single Cell Level

    Directory of Open Access Journals (Sweden)

    Yuting Guo

    2017-07-01

    Full Text Available Background: Mass cytometry (Cytometry by Time of Flight, CyTOF allows single-cell characterization on the basis of specific metal-based cell markers. In addition, other metals in the mass range such as silver can be detected per cell. Bacteria are known to be sensible to silver and a protocol was developed to measure both the number of affected cells per population and the quantities of silver per cell.Methods: For mass cytometry ruthenium red was used as a marker for all cells of a population while parallel application of cisplatin discriminated live from dead cells. Silver quantities per cell and frequencies of silver containing cells in a population were measured by mass cytometry. In addition, live/dead subpopulations were analyzed by flow cytometry and distinguished by cell sorting based on ruthenium red and propidium iodide double staining. Verification of the cells’ silver load was performed on the bulk level by using ICP-MS in combination with cell sorting. The protocol was developed by conveying both, fast and non-growing Pseudomonas putida cells as test organisms.Results: A workflow for labeling bacteria in order to be analyzed by mass cytometry was developed. Three different parameters were tested: ruthenium red provided counts for all bacterial cells in a population while consecutively applied cisplatin marked the frequency of dead cells. Apparent population heterogeneity was detected by different frequencies of silver containing cells. Silver quantities per cell were also well measurable. Generally, AgNP-10 treatment caused higher frequencies of dead cells, higher frequencies of silver containing cells and higher per-cell silver quantities. Due to an assumed chemical equilibrium of free and bound silver ions live and dead cells were associated with silver in equal quantities and this preferably during exponential growth. With ICP-MS up to 1.5 fg silver per bacterial cell were detected.Conclusion: An effective mass cytometry

  6. Wireless data performance in multi-cell scenarios

    NARCIS (Netherlands)

    Bonald, T.; Borst, S.C.; Hegde, N.; Proutière, A.

    2004-01-01

    The performance of wireless data systems has been extensively studied in the context of a single base station. In the present paper we investigate the flow-level performance in networks with multiple base stations. We specifically examine the complex, dynamic interaction of the number of active

  7. A single sensor and single actuator approach to performance tailoring over a prescribed frequency band.

    Science.gov (United States)

    Wang, Jiqiang

    2016-03-01

    Restricted sensing and actuation control represents an important area of research that has been overlooked in most of the design methodologies. In many practical control engineering problems, it is necessitated to implement the design through a single sensor and single actuator for multivariate performance variables. In this paper, a novel approach is proposed for the solution to the single sensor and single actuator control problem where performance over any prescribed frequency band can also be tailored. The results are obtained for the broad band control design based on the formulation for discrete frequency control. It is shown that the single sensor and single actuator control problem over a frequency band can be cast into a Nevanlinna-Pick interpolation problem. An optimal controller can then be obtained via the convex optimization over LMIs. Even remarkable is that robustness issues can also be tackled in this framework. A numerical example is provided for the broad band attenuation of rotor blade vibration to illustrate the proposed design procedures. Copyright © 2016 ISA. Published by Elsevier Ltd. All rights reserved.

  8. Performance of direct methanol fuel cell with a palladium–silica nanofibre/Nafion composite membrane

    International Nuclear Information System (INIS)

    Thiam, H.S.; Daud, W.R.W.; Kamarudin, S.K.; Mohamad, A.B.; Kadhum, A.A.H.; Loh, K.S.; Majlan, E.H.

    2013-01-01

    Highlights: • This study introduces Pd–SiO 2 Carbon Nano Fibre as an additive to Nafion membrane. • It investigates the effects of membrane annealing temperature and casting solvent. • Results show that Pd–SiO 2 fibre/Nafion performs lower methanol permeability. • This could effectively reduces methanol crossover in direct methanol fuel cell. - Abstract: Palladium–silica nanofibres (Pd–SiO 2 fibre) were adopted as an additive to Nafion recast membranes in order to reduce methanol crossover and improve the cell performance. The performance of a membrane electrode assembly (MEA) with fabricated composite membrane was evaluated through a passive air-breathing single cell direct methanol fuel cell (DMFC). The limiting crossover current density was measured to determine the methanol permeation in the DMFC. The effects of membrane annealing temperature and casting solvent of composite membrane on the cell performance were investigated and are discussed here. Compared to recast Nafion with the same thickness (150 μm), the Pd–SiO 2 fibre/Nafion composite membrane exhibited higher performance and lower methanol permeability. A maximum power density of 10.4 mW cm −2 was obtained with a 2 M methanol feed, outperforming the much thicker commercial Nafion 117 with a power density of 7.95 mW cm −2 under the same operating conditions. The experimental results showed that the Pd–SiO 2 fibre as inorganic fillers for Nafion could effectively reduce methanol crossover and improve the membrane performance in DMFC applications

  9. Single Cell Analysis of Dystrophin and SRY Gene by Using Whole Genome Amplification

    Institute of Scientific and Technical Information of China (English)

    徐晨明; 金帆; 黄荷凤; 陶冶; 叶英辉

    2001-01-01

    Objective To develop a reliable and sensitive method for detection of sex and multiloci of Duchenne muscular dystrophy (DMD) gene in single cell Materials & methods Whole genome of single cell were amplified by using 15-base random primers (primer extension preamplification, PEP), then a small aliquot of PEP product were analyzed by using locus-specific nest PCR amplification. The procedure was evaluated by detection dystrophin exons 8, 17, 19, 44, 45, 48 and human testis-determining gene (SRY)in single lymphocytes from known sources and single blastomeres from the couples with no family history of DMD.Results The amplification efficiency rate of six dystrophin exons from single lymphocytes and single blastomeres were 97. 2% (175/180) and 100% (60/60) respectively.Results of SRY showed that 100% (15/15) amplification in single male-derived lymphocytes and 0% (0/15) amplification in single female-derived lymphocytes. Conclusion The technique of single cell PEP-nest PCR for dystrophin exons 8, 17,19, 44, 45, 48 and SRY is highly specifc. PEP-nest PCR is suitable for Preimplantation genetic diagnosis (PGD) of DMD at single cell level.

  10. Integrin and glycocalyx mediated contributions to cell adhesion identified by single cell force spectroscopy

    International Nuclear Information System (INIS)

    Boettiger, D; Wehrle-Haller, B

    2010-01-01

    The measurement of cell adhesion using single cell force spectroscopy methods was compared with earlier methods for measuring cell adhesion. This comparison provided a means and rationale for separating components of the measurement retract curve that were due to interactions between the substrate and the glycocalyx, and interactions that were due to cell surface integrins binding to a substrate-bound ligand. The glycocalyx adhesion was characterized by multiple jumps with dispersed jump sizes that extended from 5 to 30 μm from the origin. The integrin mediated adhesion was represented by the F max (maximum detachment force), was generally within the first 5 μm and commonly detached with a single rupture cascade. The integrin peak (F max ) increases with time and the rate of increase shows large cell to cell variability with a peak ∼ 50 nN s -1 and an average rate of increase of 75 pN s -1 . This is a measure of the rate of increase in the number of adhesive integrin-ligand bonds/cell as a function of contact time.

  11. Modifying infrared scattering effects of single yeast cells with plasmonic metal mesh

    Science.gov (United States)

    Malone, Marvin A.; Prakash, Suraj; Heer, Joseph M.; Corwin, Lloyd D.; Cilwa, Katherine E.; Coe, James V.

    2010-11-01

    The scattering effects in the infrared (IR) spectra of single, isolated bread yeast cells (Saccharomyces cerevisiae) on a ZnSe substrate and in metal microchannels have been probed by Fourier transform infrared imaging microspectroscopy. Absolute extinction [(3.4±0.6)×10-7 cm2 at 3178 cm-1], scattering, and absorption cross sections for a single yeast cell and a vibrational absorption spectrum have been determined by comparing it to the scattering properties of single, isolated, latex microspheres (polystyrene, 5.0 μm in diameter) on ZnSe, which are well modeled by the Mie scattering theory. Single yeast cells were then placed into the holes of the IR plasmonic mesh, i.e., metal films with arrays of subwavelength holes, yielding "scatter-free" IR absorption spectra, which have undistorted vibrational lineshapes and a rising generic IR absorption baseline. Absolute extinction, scattering, and absorption spectral profiles were determined for a single, ellipsoidal yeast cell to characterize the interplay of these effects.

  12. Production of oncolytic adenovirus and human mesenchymal stem cells in a single-use, Vertical-Wheel bioreactor system: Impact of bioreactor design on performance of microcarrier-based cell culture processes.

    Science.gov (United States)

    Sousa, Marcos F Q; Silva, Marta M; Giroux, Daniel; Hashimura, Yas; Wesselschmidt, Robin; Lee, Brian; Roldão, António; Carrondo, Manuel J T; Alves, Paula M; Serra, Margarida

    2015-01-01

    Anchorage-dependent cell cultures are used for the production of viruses, viral vectors, and vaccines, as well as for various cell therapies and tissue engineering applications. Most of these applications currently rely on planar technologies for the generation of biological products. However, as new cell therapy product candidates move from clinical trials towards potential commercialization, planar platforms have proven to be inadequate to meet large-scale manufacturing demand. Therefore, a new scalable platform for culturing anchorage-dependent cells at high cell volumetric concentrations is urgently needed. One promising solution is to grow cells on microcarriers suspended in single-use bioreactors. Toward this goal, a novel bioreactor system utilizing an innovative Vertical-Wheel™ technology was evaluated for its potential to support scalable cell culture process development. Two anchorage-dependent human cell types were used: human lung carcinoma cells (A549 cell line) and human bone marrow-derived mesenchymal stem cells (hMSC). Key hydrodynamic parameters such as power input, mixing time, Kolmogorov length scale, and shear stress were estimated. The performance of Vertical-Wheel bioreactors (PBS-VW) was then evaluated for A549 cell growth and oncolytic adenovirus type 5 production as well as for hMSC expansion. Regarding the first cell model, higher cell growth and number of infectious viruses per cell were achieved when compared with stirred tank (ST) bioreactors. For the hMSC model, although higher percentages of proliferative cells could be reached in the PBS-VW compared with ST bioreactors, no significant differences in the cell volumetric concentration and expansion factor were observed. Noteworthy, the hMSC population generated in the PBS-VW showed a significantly lower percentage of apoptotic cells as well as reduced levels of HLA-DR positive cells. Overall, these results showed that process transfer from ST bioreactor to PBS-VW, and scale-up was

  13. Basal CD34+ Cell Count Predicts Peripheral Blood Stem Cell Mobilization in Healthy Donors after Administration of Granulocyte Colony-Stimulating Factor: A Longitudinal, Prospective, Observational, Single-Center, Cohort Study.

    Science.gov (United States)

    Martino, Massimo; Gori, Mercedes; Pitino, Annalisa; Gentile, Massimo; Dattola, Antonia; Pontari, Antonella; Vigna, Ernesto; Moscato, Tiziana; Recchia, Anna Grazia; Barilla', Santina; Tripepi, Giovanni; Morabito, Fortunato

    2017-07-01

    A longitudinal, prospective, observational, single-center, cohort study on healthy donors (HDs) was designed to identify predictors of CD34 + cells on day 5 with emphasis on the predictive value of the basal CD34 + cell count. As potential predictors of mobilization, age, sex, body weight, height, blood volume as well as white blood cell count, peripheral blood (PB) mononuclear cells, platelet count, hematocrit, and hemoglobin levels were considered. Two different evaluations of CD34 + cell counts were determined for each donor: baseline (before granulocyte colony-stimulating factor [G-CSF] administration) and in PB after G-CSF administration on the morning of the fifth day (day 5). A total of 128 consecutive HDs (66 males) with a median age of 43 years were enrolled. CD34 + levels on day 5 displayed a non-normal distribution, with a median value of 75.5 cells/µL. To account for the non-normal distribution of the dependent variable, a quantile regression analysis to predict CD34 + on day 5 using the baseline value of CD34 + as the key predictor was performed. On crude analysis, a baseline value of CD34 + ranging from .5 cells/µL to 1 cells/µL predicts a median value of 50 cells/µL on day 5; a value of 2 cells/µL predicts a median value of 70.7 cells/µL; a value of 3 cells/µL to 4 cells/µL predicts a median value of 91.3 cells/µL, and a value ≥ 5 predicts a median value of 112 cells/µL. In conclusion, the baseline PB CD34 + cell count correlates with the effectiveness of allogeneic PB stem cell mobilization and could be useful to plan the collection. Copyright © 2017 The American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.

  14. Stochasticity in the enterococcal sex pheromone response revealed by quantitative analysis of transcription in single cells.

    Science.gov (United States)

    Breuer, Rebecca J; Bandyopadhyay, Arpan; O'Brien, Sofie A; Barnes, Aaron M T; Hunter, Ryan C; Hu, Wei-Shou; Dunny, Gary M

    2017-07-01

    In Enterococcus faecalis, sex pheromone-mediated transfer of antibiotic resistance plasmids can occur under unfavorable conditions, for example, when inducing pheromone concentrations are low and inhibiting pheromone concentrations are high. To better understand this paradox, we adapted fluorescence in situ hybridization chain reaction (HCR) methodology for simultaneous quantification of multiple E. faecalis transcripts at the single cell level. We present direct evidence for variability in the minimum period, maximum response level, and duration of response of individual cells to a specific inducing condition. Tracking of induction patterns of single cells temporally using a fluorescent reporter supported HCR findings. It also revealed subpopulations of rapid responders, even under low inducing pheromone concentrations where the overall response of the entire population was slow. The strong, rapid induction of small numbers of cells in cultures exposed to low pheromone concentrations is in agreement with predictions of a stochastic model of the enterococcal pheromone response. The previously documented complex regulatory circuitry controlling the pheromone response likely contributes to stochastic variation in this system. In addition to increasing our basic understanding of the biology of a horizontal gene transfer system regulated by cell-cell signaling, demonstration of the stochastic nature of the pheromone response also impacts any future efforts to develop therapeutic agents targeting the system. Quantitative single cell analysis using HCR also has great potential to elucidate important bacterial regulatory mechanisms not previously amenable to study at the single cell level, and to accelerate the pace of functional genomic studies.

  15. Affinity of antibody secreted by a single cell

    International Nuclear Information System (INIS)

    Doran, D.M.

    1978-01-01

    It was the intention of this research to measure the affinity of antibody secreted by a single cell, and to describe the spectrum of affinities displayed in response to antigenic stimulation. The single cell secreting specific antibody was isolated by means of the hemolytic plaque assay. The amount of antibody secreted by the cell was to be measured through the use of a solid phase radioimmunoassay. The affinity of the antibody would be estimated by comparing the diameter of the plaque, and the amount of antibody secreted, with a mathematical theory of the formation of a plaque in agar. As a test system, a solid phase radioimmunoassay was developed for human serum albumin using antibody coupled to Sephadex. A sensitivity of 1 nanogram was attained with this assay. A solid phase radioimmunoassay for mouse immunoglobulin M was developed, using antibody coupled to Sepharose. The sensitivity attained with this assay was only on the order of 10 micrograms. The mouse immunoglobulin M radioimmunoassay was not sensitive enough to measure the amount of antibody secreted by a single cell. From a theoretical equation, the relationship between antibody affinity, plaque diameter and antibody secretion rate was calculated for the experimental conditions used in this research. By assuming a constant antibody secretion rate, an effective binding constant for the antibody was estimated from the average plaque diameters. This effective binding constant was observed to increase during the immune response

  16. Sex chromosomes and germline transcriptomics explored by single-cell sequencing and RNA-tomography

    NARCIS (Netherlands)

    Vértesy, Ábel

    2018-01-01

    In our study of germ cell differentiation, we applied two recently developed technologies on the germline of various model organisms: single-cell mRNA sequencing and RNA-tomography. For the first time we could look at gene expression with such a high resolution, and this led us to discover the

  17. Single cell analysis contemporary research and clinical applications

    CERN Document Server

    Cossarizza, Andrea

    2017-01-01

    This book highlights the current state of the art in single cell analysis, an area that involves many fields of science – from clinical hematology, functional analysis and drug screening, to platelet and microparticle analysis, marine biology and fundamental cancer research. This book brings together an eclectic group of current applications, all of which have a significant impact on our current state of knowledge. The authors of these chapters are all pioneering researchers in the field of single cell analysis. The book will not only appeal to those readers more focused on clinical applications, but also those interested in highly technical aspects of the technologies. All of the technologies identified utilize unique applications of photon detection systems.

  18. A study of pile-up in integrated time-correlated single photon counting systems.

    Science.gov (United States)

    Arlt, Jochen; Tyndall, David; Rae, Bruce R; Li, David D-U; Richardson, Justin A; Henderson, Robert K

    2013-10-01

    Recent demonstration of highly integrated, solid-state, time-correlated single photon counting (TCSPC) systems in CMOS technology is set to provide significant increases in performance over existing bulky, expensive hardware. Arrays of single photon single photon avalanche diode (SPAD) detectors, timing channels, and signal processing can be integrated on a single silicon chip with a degree of parallelism and computational speed that is unattainable by discrete photomultiplier tube and photon counting card solutions. New multi-channel, multi-detector TCSPC sensor architectures with greatly enhanced throughput due to minimal detector transit (dead) time or timing channel dead time are now feasible. In this paper, we study the potential for future integrated, solid-state TCSPC sensors to exceed the photon pile-up limit through analytic formula and simulation. The results are validated using a 10% fill factor SPAD array and an 8-channel, 52 ps resolution time-to-digital conversion architecture with embedded lifetime estimation. It is demonstrated that pile-up insensitive acquisition is attainable at greater than 10 times the pulse repetition rate providing over 60 dB of extended dynamic range to the TCSPC technique. Our results predict fut