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Sample records for single mesophyll cells

  1. Metabolomic Responses of Guard Cells and Mesophyll Cells to Bicarbonate

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    Misra, Biswapriya B.; de Armas, Evaldo; Tong, Zhaohui; Chen, Sixue

    2015-01-01

    Anthropogenic CO2 presently at 400 ppm is expected to reach 550 ppm in 2050, an increment expected to affect plant growth and productivity. Paired stomatal guard cells (GCs) are the gate-way for water, CO2, and pathogen, while mesophyll cells (MCs) represent the bulk cell-type of green leaves mainly for photosynthesis. We used the two different cell types, i.e., GCs and MCs from canola (Brassica napus) to profile metabolomic changes upon increased CO2 through supplementation with bicarbonate (HCO3 -). Two metabolomics platforms enabled quantification of 268 metabolites in a time-course study to reveal short-term responses. The HCO3 - responsive metabolomes of the cell types differed in their responsiveness. The MCs demonstrated increased amino acids, phenylpropanoids, redox metabolites, auxins and cytokinins, all of which were decreased in GCs in response to HCO3 -. In addition, the GCs showed differential increases of primary C-metabolites, N-metabolites (e.g., purines and amino acids), and defense-responsive pathways (e.g., alkaloids, phenolics, and flavonoids) as compared to the MCs, indicating differential C/N homeostasis in the cell-types. The metabolomics results provide insights into plant responses and crop productivity under future climatic changes where elevated CO2 conditions are to take center-stage. PMID:26641455

  2. Programmed Cell Death Progresses Differentially in Epidermal and Mesophyll Cells of Lily Petals.

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    Hiroko Mochizuki-Kawai

    Full Text Available In the petals of some species of flowers, programmed cell death (PCD begins earlier in mesophyll cells than in epidermal cells. However, PCD progression in each cell type has not been characterized in detail. We separately constructed a time course of biochemical signs and expression patterns of PCD-associated genes in epidermal and mesophyll cells in Lilium cv. Yelloween petals. Before visible signs of senescence could be observed, we found signs of PCD, including DNA degradation and decreased protein content in mesophyll cells only. In these cells, the total proteinase activity increased on the day after anthesis. Within 3 days after anthesis, the protein content decreased by 61.8%, and 22.8% of mesophyll cells was lost. A second peak of proteinase activity was observed on day 6, and the number of mesophyll cells decreased again from days 4 to 7. These biochemical and morphological results suggest that PCD progressed in steps during flower life in the mesophyll cells. PCD began in epidermal cells on day 5, in temporal synchrony with the time course of visible senescence. In the mesophyll cells, the KDEL-tailed cysteine proteinase (LoCYP and S1/P1 nuclease (LoNUC genes were upregulated before petal wilting, earlier than in epidermal cells. In contrast, relative to that in the mesophyll cells, the expression of the SAG12 cysteine proteinase homolog (LoSAG12 drastically increased in epidermal cells in the final stage of senescence. These results suggest that multiple PCD-associated genes differentially contribute to the time lag of PCD progression between epidermal and mesophyll cells of lily petals.

  3. Intracellular position of mitochondria in mesophyll cells differs between C3and C4grasses.

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    Hatakeyama, Yuto; Ueno, Osamu

    2017-09-01

    In C 3 plants, part of the CO 2 fixed during photosynthesis in chloroplasts is released from mitochondria during photorespiration by decarboxylation of glycine via glycine decarboxylase (GDC), thereby reducing photosynthetic efficiency. The apparent positioning of most mitochondria in the interior (vacuole side of chloroplasts) of mesophyll cells in C 3 grasses would increase the efficiency of refixation of CO 2 released from mitochondria by ribulose 1,5-bisphosphate carboxylase/​oxygenase (Rubisco) in chloroplasts. Therefore, in mesophyll cells of C 4 grasses, which lack both GDC and Rubisco, the mitochondria ought not to be positioned the same way as in C 3 mesophyll cells. To test this hypothesis, we investigated the intracellular position of mitochondria in mesophyll cells of 14 C 4 grasses of different C 4 subtypes and subfamilies (Chloridoideae, Micrairoideae, and Panicoideae) and a C 3 -C 4 intermediate grass, Steinchisma hians, under an electron microscope. In C 4 mesophyll cells, most mitochondria were positioned adjacent to the cell wall, which clearly differs from the positioning in C 3 mesophyll cells. In S. hians mesophyll cells, the positioning was similar to that in C 3 cells. These results suggest that the mitochondrial positioning in C 4 mesophyll cells reflects the absence of both GDC and Rubisco in the mesophyll cells and the high activity of phosphoenolpyruvate carboxylase. In contrast, the relationship between the mitochondrial positioning and enzyme distribution in S. hians is complex, but the positioning may be related to the capture of respiratory CO 2 by Rubisco. Our study provides new possible insight into the physiological role of mitochondrial positioning in photosynthetic cells.

  4. Ultrastructural analyses of somatic embryo initiation, development and polarity establishment from mesophyll cells of Dactylis glomerata

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    Vasilenko, A.; McDaniel, J. K.; Conger, B. V.

    2000-01-01

    Somatic embryos initiate and develop directly from single mesophyll cells in in vitro-cultured leaf segments of orchardgrass (Dactylis glomerata L.). Embryogenic cells establish themselves in the predivision stage by formation of thicker cell walls and dense cytoplasm. Electron microscopy observations for embryos ranging from the pre-cell-division stage to 20-cell proembryos confirm previous light microscopy studies showing a single cell origin. They also confirm that the first division is predominantly periclinal and that this division plane is important in establishing embryo polarity and in determining the embryo axis. If the first division is anticlinal or if divisions are in random planes after the first division, divisions may not continue to produce an embryo. This result may produce an embryogenic cell mass, callus formation, or no structure at all. Grant numbers: NAGW-3141, NAG10-0221.

  5. FUNCTION OF MALATDEHYDROGENASE COMPLEX OF MAIZE MESOPHYLL AND BUNDLE SHEATH CELLS UNDER SALT STRESS CONDITION

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    Еprintsev А.Т.

    2006-12-01

    Full Text Available Salt-induced changes in malatdehydrogenase system activity make the essential contribution to cell adaptation to stress condition. The enzyme systems of C4-plants are most interesting due to their ability for adaptation to environment conditions. The role of separate components of malatdehydrogenase complex of mesophyll and bundle sheath cells of corn in formation of adaptive reaction in stressful conditions is investigated in presented work.The activation of all enzymes of malatdehydrogenase system and the subsequent decrease in their activity was observed in mesophyll durring the first stage of adaptation to salt influence. In bundle sheath cells such parameters are differed from control less essentially. Fast accumulation of piruvate in cells and malate in both investigated tissues was induced. The further salinity led to falling of concentration this intermediate. The concentration of piruvate was below control level, and it was raised by the end of an exposition.The results show that sodium chloride causes induction of Krebs-cycle in mesophyll and bundle sheath cells of corn and intensification of Hatch-Slack cycle. The described differences in function malatdehydrogenase systems of mesophyll and bundle sheath cells of leaves of corn under salinity mainly consist of the activity of enzymes of a studied complex in bundle sheath cells is subject to the minimal changes in comparison with mesophyll. Role of this enzymesystem in mechanisms of adaptive reaction of various tissues of corn to salt stress is discussed.

  6. Three-dimensional intracellular structure of a whole rice mesophyll cell observed with FIB-SEM.

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    Oi, Takao; Enomoto, Sakiko; Nakao, Tomoyo; Arai, Shigeo; Yamane, Koji; Taniguchi, Mitsutaka

    2017-07-01

    Ultrathin sections of rice leaf blades observed two-dimensionally using a transmission electron microscope (TEM) show that the chlorenchyma is composed of lobed mesophyll cells, with intricate cell boundaries, and lined with chloroplasts. The lobed cell shape and chloroplast positioning are believed to enhance the area available for the gas exchange surface for photosynthesis in rice leaves. However, a cell image revealing the three-dimensional (3-D) ultrastructure of rice mesophyll cells has not been visualized. In this study, a whole rice mesophyll cell was observed using a focused ion beam scanning electron microscope (FIB-SEM), which provides many serial sections automatically, rapidly and correctly, thereby enabling 3-D cell structure reconstruction. Rice leaf blades were fixed chemically using the method for conventional TEM observation, embedded in resin and subsequently set in the FIB-SEM chamber. Specimen blocks were sectioned transversely using the FIB, and block-face images were captured using the SEM. The sectioning and imaging were repeated overnight for 200-500 slices (each 50 nm thick). The resultant large-volume image stacks ( x = 25 μm, y = 25 μm, z = 10-25 μm) contained one or two whole mesophyll cells. The 3-D models of whole mesophyll cells were reconstructed using image processing software. The reconstructed cell models were discoid shaped with several lobes around the cell periphery. The cell shape increased the surface area, and the ratio of surface area to volume was twice that of a cylinder having the same volume. The chloroplasts occupied half the cell volume and spread as sheets along the cell lobes, covering most of the inner cell surface, with adjacent chloroplasts in close contact with each other. Cellular and sub-cellular ultrastructures of a whole mesophyll cell in a rice leaf blade are demonstrated three-dimensionally using a FIB-SEM. The 3-D models and numerical information support the hypothesis that rice mesophyll

  7. Relative quantification of membrane-associated calcium in red spruce mesophyll cells

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    Catherine H. Borer; Paul Schaberg; Jonathan R. Cumming

    1997-01-01

    We describe a method for localizing and comparing relative amounts of plasma membrane-associated calcium ions (mCa) in complex tissues and verify the procedure for mesophyll cells of red spruce (Picea rubens Sarg.) needles. This technique incorporates epifluorescence microscopy using the fluorescent probe chlorotetracycline (CTC) with computer image...

  8. Factors affecting polyhydroxybutyrate accumulation in mesophyll cells of sugarcane and switchgrass

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    2014-01-01

    Background Polyhydroxyalkanoates are linear biodegradable polyesters produced by bacteria as a carbon store and used to produce a range of bioplastics. Widespread polyhydroxyalkanoate production in C4 crops would decrease petroleum dependency by producing a renewable supply of biodegradable plastics along with residual biomass that could be converted into biofuels or energy. Increasing yields to commercial levels in biomass crops however remains a challenge. Previously, lower accumulation levels of the short side chain polyhydroxyalkanoate, polyhydroxybutyrate (PHB), were observed in the chloroplasts of mesophyll (M) cells compared to bundle sheath (BS) cells in transgenic maize (Zea mays), sugarcane (Saccharum sp.), and switchgrass (Panicum virgatum L.) leading to a significant decrease in the theoretical yield potential. Here we explore various factors which might affect polymer accumulation in mesophyll cells, including targeting of the PHB pathway enzymes to the mesophyll plastid and their access to substrate. Results The small subunit of Rubisco from pea effectively targeted the PHB biosynthesis enzymes to both M and BS chloroplasts of sugarcane and switchgrass. PHB enzyme activity was retained following targeting to M plastids and was equivalent to that found in the BS plastids. Leaf total fatty acid content was not affected by PHB production. However, when fatty acid synthesis was chemically inhibited, polymer accumulated in M cells. Conclusions In this study, we provide evidence that access to substrate and neither poor targeting nor insufficient activity of the PHB biosynthetic enzymes may be the limiting factor for polymer production in mesophyll chloroplasts of C4 plants. PMID:25209261

  9. Signal function of cytokinin 6-benzylaminopurine in the reaction of Triticum aestivum L. mesophyll cells to hyperthermia

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    M. M. Musienko

    2014-12-01

    Full Text Available The signaling effect of 6-benzylaminopurine (BAP on leaf mesophyll cells of Triticum aestivum L. under hyperthermic conditions was studied­. It was found that BAP regulated photosynthetic pigment, hydrogen peroxide content and activity of antioxidant enzymes, namely superoxide dismutase, ascorbate peroxidase and catalase under high-temperature conditions. The additive effect of BAP and high temperature on the activation of cell antioxidant systems was demonstrated. BAP regulated reducing processes in mesophyll leaf cells under high-temperature conditions.

  10. Evidence for a specific glutamate/H+ cotransport in isolated mesophyll cells

    International Nuclear Information System (INIS)

    McCutcheon, S.L.; Bown, A.W.

    1987-01-01

    Mechanically isolated Asparagus sprengeri Regel mesophyll cells were suspended in 1 millimolar CaSO 4 . Immediate alkalinization of the medium occurred on the addition of 1 millimolar concentrations of L-glutamate (Glu) and its analog L-methionine-D,L-sulfoximine (L-MSO). D-Glu and the L isomers of the protein amino acids did not elicit alkalinization. L-Glu dependent alkalinization was transient and acidification resumed after approximately 30 to 45 minutes. At pH 6.0, 5 millimolar L-Glu stimulated initial rates of alkalinization that varied between 1.3 to 4.1 nmol H + /10 6 cells minute. L-Glu dependent alkalinization was saturable, increased with decreasing pH, was inhibited by carbonyl cyanide-p-trichloromethoxyphenyl hydrazone (CCCP), and was not stimulated by light. Uptake of L-[U- 14 C]glutamate increased as the pH decreased from 6.5 to 5.5, and was inhibited by L-MSO. L-Glu had no influence on K + efflux. Although evidence for multiple amino acid/proton cotransport systems has been found in other tissues, the present report indicates that a highly specific L-Glu/proton uptake process is present in Asparagus mesophyll cells

  11. Air pollution effects on the ultrastructure of Phlomis fruticosa mesophyll cells

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    Psaras, G.K.; Christodoulakis, N.S.

    1987-04-01

    Plant physiologists and environmental scientists suggest that a basic effect of air pollution on plants leads towards the minimization of their productivity. On the other hand the action of individual pollutants on intact plants has been studied from biochemical as well as structural viewpoint. Thus the study of plant responses to SO/sub 2/ exposure revealed that this agent causes acute and chronic injury. Chronic injury results in chlorosis and subsequent necrosis due to destruction of chlorophylls and final chloroplast lysis. It has been documented that ultrastructural characteristics of leaves are affected prior to any visible injury. Electron microscope examination of SO/sub 2/ fumigated plant-attached leaves of Vicia faba revealed chloroplast thylakoids starting to swell whilst photosynthesis rate was drastically reduced. The first light microscope-detected effects of air pollution on the leaf structure of plants common in natural ecosystems of Athens metropolitan area, have been reported. A chlorosis phenomenon in Urginea maritima leaves as well as an indication of detrimental effects of Phlomis fruticosa mesophyll chloroplasts were documented. In this work further investigation has been undertaken in order to elucidate the precise effects of air pollution on the ultrastructure of the photosynthesizing mesophyll cells.

  12. Starch Biosynthesis in Guard Cells But Not in Mesophyll Cells Is Involved in CO2-Induced Stomatal Closing1[OPEN

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    Stephan, Aaron B.; Schroeder, Julian I.

    2016-01-01

    Starch metabolism is involved in stomatal movement regulation. However, it remains unknown whether starch-deficient mutants affect CO2-induced stomatal closing and whether starch biosynthesis in guard cells and/or mesophyll cells is rate limiting for high CO2-induced stomatal closing. Stomatal responses to [CO2] shifts and CO2 assimilation rates were compared in Arabidopsis (Arabidopsis thaliana) mutants that were either starch deficient in all plant tissues (ADP-Glc-pyrophosphorylase [ADGase]) or retain starch accumulation in guard cells but are starch deficient in mesophyll cells (plastidial phosphoglucose isomerase [pPGI]). ADGase mutants exhibited impaired CO2-induced stomatal closure, but pPGI mutants did not, showing that starch biosynthesis in guard cells but not mesophyll functions in CO2-induced stomatal closing. Nevertheless, starch-deficient ADGase mutant alleles exhibited partial CO2 responses, pointing toward a starch biosynthesis-independent component of the response that is likely mediated by anion channels. Furthermore, whole-leaf CO2 assimilation rates of both ADGase and pPGI mutants were lower upon shifts to high [CO2], but only ADGase mutants caused impairments in CO2-induced stomatal closing. These genetic analyses determine the roles of starch biosynthesis for high CO2-induced stomatal closing. PMID:27208296

  13. Effects of herbicides on 14CO2 fixation in isolated mesophyll cells from Beta vulgaris (sugar beet) and Chenopodium album

    International Nuclear Information System (INIS)

    Baumann, G.; Guenther, G.

    1979-01-01

    10 -4 - 10 -6 molar solutions of herbicides (atrazine, 2,4-D, desmetryne, diallate, diquat, feuron, lenacil, NaTa, paraquat, phenmedipham, prometryne, propham, pyrazone, and simazine) cause similar inhibitory effects on the photosynthetic 14 CO 2 fixation in isolated mesophyll cells from Chenopodium album and Beta vulgaris. Correlatdion between inhibition and herbicide resistance of the whole plants could be realized for lenacil only

  14. Ultrastructural response of cabbage outer leaf mesophyll cells (Brassica oleracea L. to excess of nickel

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    Jolanta Molas

    2014-01-01

    Full Text Available Changes in the structure and in the ultrastructure of cabbage outer leaf mesophyll cells [Brassica oleracea L.] cv. Sława from Enkhouizen were examined by means of light and electron microscopy. The examined plants were grown on the basic Murashige and Skoog medium with addition of excesive concentrations of nickel (added as NiSO4 x 7H2O,i.e. Ni 5, Ni 10 and Ni 20 mg/dm3. In Ni 5 mg samples mainly adaptation changes to the conditions of stress were observed. These changes were manifested by the increase of cytoplasm content and by cytoplasm vacuolization, by the increase of nucleus and nucleous volume, nucleolus vacuolization, the increase of plasmalemma invaginations and of the amount of rough ER, by the central arrangement of smooth ER and of the thylakoids of chloroplasts; it was also shown by the growth of the number of mitochondria and of peroxisomes in the cell. In Ni 10 mg samples, apart from adaptation changes, such as the increase of the nucleus volume, increase of plasmalemma invaginations, cytoplasm and nucleolus vacuolization, degeneration changes were also observed. They concerned mainly the nucleus (the increasing amount of condensed chromatin, ER (swelling and fragmentation of rER and sER, mitochondrium (swelling and reduction of cristae, Golgi apparatus (disintegration and decay and chloroplasts (changes of shape, swelling and reduction of thylakoids, disappearance of starch and presence of big plastoglobuli. In Ni 20 mg samples cell protoplasts were in different stages of degeneration and the cell organelles that were identifiable, were usually damaged.

  15. Inorganic carbon uptake during photosynthesis. II. Uptake by isolated Asparagus mesophyll cells during isotopic disequilibrium

    International Nuclear Information System (INIS)

    Espie, G.S.; Owttrim, G.W.; Colman, B.

    1986-01-01

    The species of inorganic carbon (CO 2 or HCO 3 - ) taken up as a source of substrate for photosynthetic fixation by isolated Asparagus sprengeri mesophyll cells is investigated. Discrimination between CO 2 or HCO 3 - transport, during steady state photosynthesis, is achieved by monitoring the changes (by 14 C fixation) which occur in the specific activity of the intracellular pool of inorganic carbon when the inorganic carbon present in the suspending medium is in a state of isotopic disequilibrium. Quantitative comparisons between theoretical (CO 2 or HCO 3 - transport) and experimental time-courses of 14 C incorporation, over the pH range of 5.2 to 7.5, indicate that the specific activity of extracellular CO 2 , rather than HCO 3 - , is the appropriate predictor of the intracellular specific activity. It is concluded, therefore, that CO 2 is the major source of exogenous inorganic carbon taken up by Asparagus cells. However, at high pH (8.5), a component of net DIC uptake may be attributable to HCO 3 - transport, as the incorporation of 14 C during isotopic disequilibrium exceeds the maximum possible incorporation predicted on the basis of CO 2 uptake alone. The contribution of HCO 3 - to net inorganic carbon uptake (pH 8.5) is variable, ranging from 5 to 16%, but is independent of the extracellular HCO 3 - concentration. The evidence for direct HCO 3 - transport is subject to alternative explanations and must, therefore, be regarded as equivocal. Nonlinear regression analysis of the rate of 14 C incorporation as a function of time indicates the presence of a small extracellular resistance to the diffusion of CO 2 , which is partially alleviated by a high extracellular concentration of HCO 3 -

  16. Unbiased estimation of chloroplast number in mesophyll cells: advantage of a genuine three-dimensional approach

    Czech Academy of Sciences Publication Activity Database

    Kubínová, Z.; Janáček, Jiří; Lhotáková, Z.; Kubínová, Lucie; Albrechtová, J.

    2014-01-01

    Roč. 65, č. 2 (2014), s. 609-620 ISSN 0022-0957 R&D Projects: GA ČR(CZ) GAP501/10/0340 Grant - others:Univerzita Karlova(CZ) SVV265203 Institutional research plan: CEZ:AV0Z50110509 Institutional support: RVO:67985823 Keywords : chloroplast counting * confocal microscopy * disector method * mesophyll * coniferous needle structure * Norway spruce (Picea abies L. Karst.), * profile counting * stereology Subject RIV: GK - Forestry Impact factor: 5.526, year: 2014

  17. Early local differentiation of the cell wall matrix defines the contact sites in lobed mesophyll cells of Zea mays.

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    Giannoutsou, E; Sotiriou, P; Apostolakos, P; Galatis, B

    2013-10-01

    The morphogenesis of lobed mesophyll cells (MCs) is highly controlled and coupled with intercellular space formation. Cortical microtubule rings define the number and the position of MC isthmi. This work investigated early events of MC morphogenesis, especially the mechanism defining the position of contacts between MCs. The distributions of plasmodesmata, the hemicelluloses callose and (1 → 3,1 → 4)-β-d-glucans (MLGs) and the pectin epitopes recognized by the 2F4, JIM5, JIM7 and LM6 antibodies were studied in the cell walls of Zea mays MCs. Matrix cell wall polysaccharides were immunolocalized in hand-made sections and in sections of material embedded in LR White resin. Callose was also localized using aniline blue in hand-made sections. Plasmodesmata distribution was examined by transmission electron microscopy. Before reorganization of the dispersed cortical microtubules into microtubule rings, particular bands of the longitudinal MC walls, where the MC contacts will form, locally differentiate by selective (1) deposition of callose and the pectin epitopes recognized by the 2F4, LM6, JIM5 and JIM7 antibodies, (2) degradation of MLGs and (3) formation of secondary plasmodesmata clusterings. This cell wall matrix differentiation persists in cell contacts of mature MCs. Simultaneously, the wall bands between those of future cell contacts differentiate with (1) deposition of local cell wall thickenings including cellulose microfibrils, (2) preferential presence of MLGs, (3) absence of callose and (4) transient presence of the pectins identified by the JIM5 and JIM7 antibodies. The wall areas between cell contacts expand determinately to form the cell isthmi and the cell lobes. The morphogenesis of lobed MCs is characterized by the early patterned differentiation of two distinct cell wall subdomains, defining the sites of the future MC contacts and of the future MC isthmi respectively. This patterned cell wall differentiation precedes cortical microtubule

  18. Blue light-dependent changes in loosely bound calcium in Arabidopsis mesophyll cells: an X-ray microanalysis study.

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    Łabuz, Justyna; Samardakiewicz, Sławomir; Hermanowicz, Paweł; Wyroba, Elżbieta; Pilarska, Maria; Gabryś, Halina

    2016-06-01

    Calcium is involved in the signal transduction pathway from phototropins, the blue light photoreceptor kinases which mediate chloroplast movements. The chloroplast accumulation response in low light is controlled by both phot1 and phot2, while only phot2 is involved in avoidance movement induced by strong light. Phototropins elevate cytosolic Ca(2+) after activation by blue light. In higher plants, both types of chloroplast responses depend on Ca(2+), and internal calcium stores seem to be crucial for these processes. Yet, the calcium signatures generated after the perception of blue light by phototropins are not well understood. To characterize the localization of calcium in Arabidopsis mesophyll cells, loosely bound (exchangeable) Ca(2+) was precipitated with potassium pyroantimonate and analyzed by transmission electron microscopy followed by energy-dispersive X-ray microanalysis. In dark-adapted wild-type Arabidopsis leaves, calcium precipitates were observed at the cell wall, where they formed spherical structures. After strong blue light irradiation, calcium at the apoplast prevailed, and bigger, multilayer precipitates were found. Spherical calcium precipitates were also detected at the tonoplast. After red light treatment as a control, the precipitates at the cell wall were smaller and less numerous. In the phot2 and phot1phot2 mutants, calcium patterns were different from those of wild-type plants. In both mutants, no elevation of calcium after blue light treatment was observed at the cell periphery (including the cell wall and a fragment of cytoplasm). This result confirms the involvement of phototropin2 in the regulation of Ca(2+) homeostasis in mesophyll cells. © The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  19. Blue light-dependent changes in loosely bound calcium in Arabidopsis mesophyll cells: an X-ray microanalysis study

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    Łabuz, Justyna; Samardakiewicz, Sławomir; Hermanowicz, Paweł; Wyroba, Elżbieta; Pilarska, Maria; Gabryś, Halina

    2016-01-01

    Calcium is involved in the signal transduction pathway from phototropins, the blue light photoreceptor kinases which mediate chloroplast movements. The chloroplast accumulation response in low light is controlled by both phot1 and phot2, while only phot2 is involved in avoidance movement induced by strong light. Phototropins elevate cytosolic Ca2+ after activation by blue light. In higher plants, both types of chloroplast responses depend on Ca2+, and internal calcium stores seem to be crucial for these processes. Yet, the calcium signatures generated after the perception of blue light by phototropins are not well understood. To characterize the localization of calcium in Arabidopsis mesophyll cells, loosely bound (exchangeable) Ca2+ was precipitated with potassium pyroantimonate and analyzed by transmission electron microscopy followed by energy-dispersive X-ray microanalysis. In dark-adapted wild-type Arabidopsis leaves, calcium precipitates were observed at the cell wall, where they formed spherical structures. After strong blue light irradiation, calcium at the apoplast prevailed, and bigger, multilayer precipitates were found. Spherical calcium precipitates were also detected at the tonoplast. After red light treatment as a control, the precipitates at the cell wall were smaller and less numerous. In the phot2 and phot1phot2 mutants, calcium patterns were different from those of wild-type plants. In both mutants, no elevation of calcium after blue light treatment was observed at the cell periphery (including the cell wall and a fragment of cytoplasm). This result confirms the involvement of phototropin2 in the regulation of Ca2+ homeostasis in mesophyll cells. PMID:26957564

  20. Intracellular position of mitochondria and chloroplasts in bundle sheath and mesophyll cells of C3 grasses in relation to photorespiratory CO2 loss

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    Yuto Hatakeyama

    2016-10-01

    Full Text Available In C3 plants, photosynthetic efficiency is reduced by photorespiration. A part of CO2 fixed during photosynthesis in chloroplasts is lost from mitochondria during photorespiration by decarboxylation of glycine by glycine decarboxylase (GDC. Thus, the intracellular position of mitochondria in photosynthetic cells is critical to the rate of photorespiratory CO2 loss. We investigated the intracellular position of mitochondria in parenchyma sheath (PS and mesophyll cells of 10 C3 grasses from 3 subfamilies (Ehrhartoideae, Panicoideae, and Pooideae by immunostaining for GDC and light and electron microscopic observation. Immunostaining suggested that many mitochondria were located in the inner half of PS cells and on the vacuole side of chloroplasts in mesophyll cells. Organelle quantification showed that 62–75% of PS mitochondria were located in the inner half of cells, and 62–78% of PS chloroplasts were in the outer half. In mesophyll cells, 61–92% of mitochondria were positioned on the vacuole side of chloroplasts and stromules. In PS cells, such location would reduce the loss of photorespiratory CO2 by lengthening the path of CO2 diffusion and allow more efficient fixation of CO2 from intercellular spaces. In mesophyll cells, it would facilitate scavenging by chloroplasts of photorespiratory CO2 released from mitochondria. Our data suggest that the PS cells of C3 grasses have already acquired an initial structure leading to proto-Kranz and further C3–C4 intermediate anatomy. We also found that in the Pooideae, organelle positioning in PS cells on the phloem side resembles that in mesophyll cells.

  1. Cyst(e)ine Is the Transport Metabolite of Assimilated Sulfur from Bundle-Sheath to Mesophyll Cells in Maize Leaves1

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    Burgener, Marta; Suter, Marianne; Jones, Stephanie; Brunold, Christian

    1998-01-01

    The intercellular distribution of the enzymes and metabolites of assimilatory sulfate reduction and glutathione synthesis was analyzed in maize (Zea mays L. cv LG 9) leaves. Mesophyll cells and strands of bundle-sheath cells from second leaves of 11-d-old maize seedlings were obtained by two different mechanical-isolation methods. Cross-contamination of cell preparations was determined using ribulose bisphosphate carboxylase (EC 4.1.1.39) and nitrate reductase (EC 1.6.6.1) as marker enzymes for bundle-sheath and mesophyll cells, respectively. ATP sulfurylase (EC 2.7.7.4) and adenosine 5′-phosphosulfate sulfotransferase activities were detected almost exclusively in the bundle-sheath cells, whereas GSH synthetase (EC 6.3.2.3) and cyst(e)ine, γ-glutamylcysteine, and glutathione were located predominantly in the mesophyll cells. Feeding experiments using [35S]sulfate with intact leaves indicated that cyst(e)ine was the transport metabolite of reduced sulfur from bundle-sheath to mesophyll cells. This result was corroborated by tracer experiments, which showed that isolated bundle-sheath strands fed with [35S]sulfate secreted radioactive cyst(e)ine as the sole thiol into the resuspending medium. The results presented in this paper show that assimilatory sulfate reduction is restricted to the bundle-sheath cells, whereas the formation of glutathione takes place predominantly in the mesophyll cells, with cyst(e)ine functioning as a transport metabolite between the two cell types. PMID:9536048

  2. Establishment of transient gene expression systems in protoplasts from Liriodendron hybrid mesophyll cells.

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    Ailing Huo

    Full Text Available Liriodendron is a genus of the magnolia family comprised of two flowering tree species that produce hardwoods of great ecological and economic value. However, only a limited amount of genetic research has been performed on the Liriodendron genus partly because transient or stable transgenic trees have been difficult to produce. In general, transient expression systems are indispensable for rapid, high-throughput screening and systematic characterization of gene functions at a low cost; therefore, development of such a system for Liriodendron would provide a necessary step forward for research on Magnoliaceae and other woody trees. Herein, we describe an efficient and rapid protocol for preparing protoplasts from the leaf mesophyll tissue of a Liriodendron hybrid and an optimized system for polyethylene glycol-mediated transient transfection of the protoplasts. Because the leaves of the Liriodendron hybrid are waxy, we formulated an enzyme mix containing 1.5% (w/v Cellulase R-10, 0.5% (w/v Macerozyme R-10, and 0.1% (w/v Pectolyase Y-23 to efficiently isolate protoplasts from the Liriodendron hybrid leaf mesophyll tissue in 3 h. We optimized Liriodendron protoplast transfection efficiency by including 20 μg plasmid DNA per 104 protoplasts, a transformation time of 20 min, and inclusion of 20% (w/v polyethylene glycol 4000. After integrating the Liriodendron WOX1 gene into pJIT166-GFP to produce a WOX1-GFP fusion product and transfecting it into isolated protoplasts, LhWOX1-GFP was found to localize to the nucleus according to its green fluorescence.

  3. Early H2O2 Accumulation in Mesophyll Cells Leads to Induction of Glutathione during the Hyper-Sensitive Response in the Barley-Powdery Mildew Interaction1

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    Vanacker, Helene; Carver, Tim L.W.; Foyer, Christine H.

    2000-01-01

    H2O2 production and changes in glutathione, catalase, and peroxidase were followed in whole-leaf extracts from the susceptible (AlgS [Algerian/4* (F14) Man.(S)]; ml-a1 allele) and resistant (AlgR [Algerian/4* (F14) Man.(R)]; Ml-a1 allele) barley (Hordeum vulgare) isolines between 12 and 24 h after inoculation with powdery mildew (Blumeria graminis [DC]. Speer [syn. Erysiphe graminis DC] f.sp hordei Marchal). Localized papilla responses and cell death hypersensitive responses were not observed within the same cell. In hypersensitive response sites, H2O2 accumulation first occurred in the mesophyll underlying the attacked epidermal cell. Subsequently, H2O2 disappeared from the mesophyll and accumulated around attacked epidermal cells. In AlgR, transient glutathione oxidation coincided with H2O2 accumulation in the mesophyll. Subsequently, total foliar glutathione and catalase activities transiently increased in AlgR. These changes, absent from AlgS, preceded inoculation-dependent increases in peroxidase activity that were observed in both AlgR and AlgS at 18 h. An early intercellular signal precedes H2O2, and this elicits anti-oxidant responses in leaves prior to events leading to death of attacked cells. PMID:10938348

  4. Structural characterization of a mixed-linkage glucan deficient mutant reveals alteration in cellulose microfibril orientation in rice coleoptile mesophyll cell walls

    Directory of Open Access Journals (Sweden)

    Andreia Michelle Smith-Moritz

    2015-08-01

    Full Text Available The CELLULOSE SYNTHASE-LIKE F6 (CslF6 gene was previously shown to mediate the biosynthesis of mixed-linkage glucan (MLG, a cell wall polysaccharide that is hypothesized to be a tightly associated with cellulose and also have a role in cell expansion in the primary cell wall of young seedlings in grass species. We have recently shown that loss-of-function cslf6 rice mutants do not accumulate MLG in most vegetative tissues. Despite the absence of a structurally important polymer, MLG, these mutants are unexpectedly viable and only show a moderate growth compromise compared to wild type. Therefore these mutants are ideal biological systems to test the current grass cell wall model. In order to gain a better understanding of the role of MLG in the primary wall, we performed in-depth compositional and structural analyses of the cell walls of three day-old rice seedlings using various biochemical and novel microspectroscopic approaches. We found that cellulose content as well as matrix polysaccharide composition was not significantly altered in the MLG deficient mutant. However, we observed a significant change in cellulose microfibril bundle organization in mesophyll cell walls of the cslf6 mutant. Using synchrotron source Fourier Transform Mid-Infrared Spectromicroscopy for high-resolution imaging, we determined that the bonds associated with cellulose and arabinoxylan, another major component of the primary cell was of grasses, were in a lower energy configuration compared to wild type, suggesting a slightly weaker primary wall in MLG deficient mesophyll cells. Taken together, these results suggest that MLG may influence cellulose deposition in mesophyll cell walls without significantly affecting anisotropic growth thus challenging MLG importance in cell wall expansion.

  5. Effect of Hf on the fine structure of mesophyll cells from Glycine max, Merr

    Energy Technology Data Exchange (ETDEWEB)

    Wei, L.; Miller, G.W.

    1972-04-01

    A series of ultrastructural changes were observed in soybean leaves fumigated with 40 to 50 ppb of hydrogen fluoride. In the cytoplasm the presence of small vacuoles was the first noticeable initial change. The fragmentation of the vacuolar membrane occurred either simultaneously or followed immediately. Lipid-droplet-like globules and numerous vesicles occurred subsequently in the cytoplasm and increased as the injury became more severe. There was a decrease in polysomes and a detachment of ribosome from the rough endoplasmic reticulum. Free ribosome concentration also decreased as the injury became severe. Mitochondrial modifications involving dilation of outer and cristae membranes followed by reduction of both cristae number and matrix electron density and the disappearance of mitochondrial granules were observed in the chlorotic leaves. Electron dense inclusions accumulated in some mitochondria as well. The first noticeable change observed in the chloroplast was the presence of clusters of phytoferritin granules within the stoma after only 2 days of fumigation. Alterations in nuclear structures were observed in later stages of injury. Numerous small electron dense particles were found on various types of membranes in cells of severely chlorotic leaves. They were distributed on outer mitochondrial membranes, endoplasmic reticula, dictyosomes, tonoplasts, plasmalemma, nuclear envelopes, and disintegrating organelles and vesicles, but were never observed on membranes of chloroplasts and microbodies. The presence of fluoride has attracted the attention of many workers primarily in certain industrial areas where the emitted atmospheric fluoride concentrates and is accumulated by plants initiating injury. 6 references.

  6. Plastidic phosphoglucose isomerase is an important determinant of starch accumulation in mesophyll cells, growth, photosynthetic capacity, and biosynthesis of plastidic cytokinins in Arabidopsis.

    Directory of Open Access Journals (Sweden)

    Abdellatif Bahaji

    Full Text Available Phosphoglucose isomerase (PGI catalyzes the reversible isomerization of glucose-6-phosphate and fructose-6-phosphate. It is involved in glycolysis and in the regeneration of glucose-6-P molecules in the oxidative pentose phosphate pathway (OPPP. In chloroplasts of illuminated mesophyll cells PGI also connects the Calvin-Benson cycle with the starch biosynthetic pathway. In this work we isolated pgi1-3, a mutant totally lacking pPGI activity as a consequence of aberrant intron splicing of the pPGI encoding gene, PGI1. Starch content in pgi1-3 source leaves was ca. 10-15% of that of wild type (WT leaves, which was similar to that of leaves of pgi1-2, a T-DNA insertion pPGI null mutant. Starch deficiency of pgi1 leaves could be reverted by the introduction of a sex1 null mutation impeding β-amylolytic starch breakdown. Although previous studies showed that starch granules of pgi1-2 leaves are restricted to both bundle sheath cells adjacent to the mesophyll and stomata guard cells, microscopy analyses carried out in this work revealed the presence of starch granules in the chloroplasts of pgi1-2 and pgi1-3 mesophyll cells. RT-PCR analyses showed high expression levels of plastidic and extra-plastidic β-amylase encoding genes in pgi1 leaves, which was accompanied by increased β-amylase activity. Both pgi1-2 and pgi1-3 mutants displayed slow growth and reduced photosynthetic capacity phenotypes even under continuous light conditions. Metabolic analyses revealed that the adenylate energy charge and the NAD(PH/NAD(P ratios in pgi1 leaves were lower than those of WT leaves. These analyses also revealed that the content of plastidic 2-C-methyl-D-erythritol 4-phosphate (MEP-pathway derived cytokinins (CKs in pgi1 leaves were exceedingly lower than in WT leaves. Noteworthy, exogenous application of CKs largely reverted the low starch content phenotype of pgi1 leaves. The overall data show that pPGI is an important determinant of photosynthesis, energy

  7. IRON UPTAKE BY LEAF MESOPHYLL-CELLS - THE ROLE OF THE PLASMA MEMBRANE-BOUND FERRIC-CHELATE REDUCTASE

    NARCIS (Netherlands)

    BRUGGEMANN, W; MAASKANTEL, K; MOOG, PR

    The uptake of Fe-59 from FeCl3, ferric (Fe3+) citrate (FeCitr) and Fe3+-EDTA (FeEDTA) was studied in leaf mesophyll of Vigna unguiculata (L.) Walp. Uptake rates decreased in the order FeCl3>FeCitr much greater than FeEDTA, and uptake depended on an obligatory reduction step of Fe3+ to Fe2+, after

  8. High-contrast three-dimensional imaging of the Arabidopsis leaf enables the analysis of cell dimensions in the epidermis and mesophyll

    Directory of Open Access Journals (Sweden)

    Granier Christine

    2010-07-01

    Full Text Available Abstract Background Despite the wide spread application of confocal and multiphoton laser scanning microscopy in plant biology, leaf phenotype assessment still relies on two-dimensional imaging with a limited appreciation of the cells' structural context and an inherent inaccuracy of cell measurements. Here, a successful procedure for the three-dimensional imaging and analysis of plant leaves is presented. Results The procedure was developed based on a range of developmental stages, from leaf initiation to senescence, of soil-grown Arabidopsis thaliana (L. Heynh. Rigorous clearing of tissues, made possible by enhanced leaf permeability to clearing agents, allowed the optical sectioning of the entire leaf thickness by both confocal and multiphoton microscopy. The superior image quality, in resolution and contrast, obtained by the latter technique enabled the three-dimensional visualisation of leaf morphology at the individual cell level, cell segmentation and the construction of structural models. Image analysis macros were developed to measure leaf thickness and tissue proportions, as well as to determine for the epidermis and all layers of mesophyll tissue, cell density, volume, length and width. For mesophyll tissue, the proportion of intercellular spaces and the surface areas of cells were also estimated. The performance of the procedure was demonstrated for the expanding 6th leaf of the Arabidopsis rosette. Furthermore, it was proven to be effective for leaves of another dicotyledon, apple (Malus domestica Borkh., which has a very different cellular organisation. Conclusions The pipeline for the three-dimensional imaging and analysis of plant leaves provides the means to include variables on internal tissues in leaf growth studies and the assessment of leaf phenotypes. It also allows the visualisation and quantification of alterations in leaf structure alongside changes in leaf functioning observed under environmental constraints. Data

  9. The relationship between turgor pressure and titratable acidity in mesophyll cells of intact leaves of a Crassulacean-acid-metabolism plant, Kalanchoe daigremontiana Hamet et Perr.

    Science.gov (United States)

    Rygol, J; Winter, K; Zimmermann, U

    1987-12-01

    Day/night changes in turgor pressure (P) and titratable acidity content were investigated in the (Crassulacean-acid-metabolism (CAM) plant Kalanchoe daigremontiana. Measurements of P were made on individual mesophyll cells of intact attached leaves using the pressure-probe technique. Under conditions of high relative humidity, when transpiration rates were minimal, changes in P correlated well with changes in the level of titratable acidity. During the standard 12 h light/12 h dark cycle, maximum turgor pressure (0.15 MPa) occurred at the end of the dark period when the level of titratable acidity was highest (about 300 μeq H(+)·g(-1) fresh weight). A close relationship between P and titratable acidity was also seen in leaves exposed to perturbations of the standard light/dark cycle. (The dark period was either prolonged, or else only CO2-free air was supplied in this period). In plants deprived of irrigation for five weeks, diurnal changes in titratable acidity of the leaves were reduced (ΔH=160 μeq H(+)·g(-1) fresh weight) and P increased from essentially zero at the end of the light period to 0.02 MPa at the end of the dark period. Following more severe water stress (experiments were made on leaves which had been detached for five weeks), P was zero throughout day and night, yet small diurnal changes in titratable acidity were still measured. These findings are discussed in relation to a hypothesis by Lüttge et al. 1975 (Plant Physiol. 56,613-616) for the role of P in the regulation of acidification/de-acidification cycles of plants exhibiting CAM.

  10. Plant, cell, and molecular mechanisms of abscisic-acid regulation of stomatal apertures. A new mechanism for the regulation of stomatal-aperture size in intact leaves: Accumulation of mesophyll-derived sucrose in the guard-cell wall of Vicia faba L.

    Energy Technology Data Exchange (ETDEWEB)

    Lu, P.; Outlaw, W.H. Jr.; Smith, B.G.; Freed, G.A.

    1996-12-31

    At various times after pulse labeling Vicia faba L. leaflets with {sup 14}CO{sub 2}, whole-leaf pieces and rinsed epidermal peels were harvested and subsequently processed for histochemical analysis. Cells dissected from whole leaf retained apoplastic contents whereas those from rinsed peels contained only cytoplastic contents. Sucrose specific radioactivity peaked in palisade cells, 111 GBq{center_dot}mol{sup {minus}1}, at 20 min. In contrast, the {sup 14}C content and sucrose specific radioactivity were very low in guard cells for 20 min, implying little CO{sub 2} incorporation; both then peaked at 40 min. The guard-cell apoplast had a high maximum sucrose specific radioactivity and a high sucrose influx rate. These and other comparisons implied the presence of (a) multiple sucrose pools in mesophyll cells, (b) a localized mesophyll-apoplast region that exchanges with phloem and stomata, and (c) mesophyll-derived sucrose in guard-cell walls sufficient to diminish stomatal opening by {approximately} 4 {micro}m. Factors expected to enhance sucrose accumulation in guard-cell walls are (a) high transpiration rate, which closes stomata, and (b) high apoplastic sucrose concentration, which is elevated when mesophyll-sucrose efflux exceeds translocation. Therefore, multiple physiological factors are integrated in the attenuation of stomatal-aperture size by this previously unrecognized mechanism.

  11. Carbon isotope ratios of epidermal and mesophyll tissues from leaves of C3 and CAM plants

    International Nuclear Information System (INIS)

    Nishida, K.; Roksandic, Z.; Osmond, B.

    1981-01-01

    The δ 13 C values for epidermal and mesophyll tissues of two C 3 plants, Commelina communis and Tulipa gesneriana, and a CAM plant, Kalanchoē daigremontiana, were measured. The values for the tissues of both C 3 plants were similar. In young leaves of Kalanchoē, the epidermis and the mesophyll showed S 13 C values which were nearly identical, and similar to those found in C 3 plants. However, markedly more negative values for epidermal compared to mesophyll tissue, were obtained in the mature Kalanchoē leaf. This is consistent with the facts that the epidermis in a CAM leaf is formed when leaves engage in C 3 photosynthesis and that subsequent dark CO 2 fixation in guard cells or mesophyll cells makes only a small contribution to total epidermal carbon

  12. Increased leaf mesophyll porosity following transient retinoblastoma-related protein silencing is revealed by microcomputed tomography imaging and leads to a system-level physiological response to the altered cell division pattern.

    Science.gov (United States)

    Dorca-Fornell, Carmen; Pajor, Radoslaw; Lehmeier, Christoph; Pérez-Bueno, Marísa; Bauch, Marion; Sloan, Jen; Osborne, Colin; Rolfe, Stephen; Sturrock, Craig; Mooney, Sacha; Fleming, Andrew

    2013-12-01

    The causal relationship between cell division and growth in plants is complex. Although altered expression of cell-cycle genes frequently leads to altered organ growth, there are many examples where manipulation of the division machinery leads to a limited outcome at the level of organ form, despite changes in constituent cell size. One possibility, which has been under-explored, is that altered division patterns resulting from manipulation of cell-cycle gene expression alter the physiology of the organ, and that this has an effect on growth. We performed a series of experiments on retinoblastoma-related protein (RBR), a well characterized regulator of the cell cycle, to investigate the outcome of altered cell division on leaf physiology. Our approach involved combination of high-resolution microCT imaging and physiological analysis with a transient gene induction system, providing a powerful approach for the study of developmental physiology. Our investigation identifies a new role for RBR in mesophyll differentiation that affects tissue porosity and the distribution of air space within the leaf. The data demonstrate the importance of RBR in early leaf development and the extent to which physiology adapts to modified cellular architecture resulting from altered cell-cycle gene expression. © 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.

  13. 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...

  14. Analysis of single biological cells

    International Nuclear Information System (INIS)

    Watt, Frank

    2002-01-01

    The extraction of elemental information from single cultured cells using nuclear microscopy is an area of great potential because it can provide both quantitative information on the uptake of elements by the cell, and also its elemental response to a wide variety of external stimuli. A recent technique based on nuclear physics technology enables the analysis of single cells down to the parts per million level to be achieved

  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. Single cell electroporation on chip

    NARCIS (Netherlands)

    Valero, Ana

    2006-01-01

    In this thesis the results of the development of microfluidic cell trap devices for single cell electroporation are described, which are to be used for gene transfection. The performance of two types of Lab-on-a-Chip trapping devices was tested using beads and cells, whereas the functionality for

  17. [Effect of fucoidan on the ultrastructure of mesophyll cells of Datura stramonium L. and accumulation of potato virus X in them].

    Science.gov (United States)

    Lapshina, L A; Reunov, A V; Nagorskaia, V P; Zviagintseva, T N; Shevchenko, N M

    2009-01-01

    Influence of fucoidan from brown alga Fucus evanescens C. Ag. on the development of infection induced by potato virus X (PVX) in Datura stramonium leaves was studied. It as been shown that 24 h after the treatment of the leaves with fucoidan and following infection of them with PVX the accumulation of virus particles in infected cells during early infection period was substantially less than that in untreated control. Using ultrastructure-morphometric analysis, it has been established that fucoidan treatment increases at protein-synthesizing capability of cells (nucleolus dimension, amount of mitochondria and rough endoplasmic reticulum membranes become increased). At the same time, the fucoidan treatment causes some activation of lytic compartment which leads to destruction of virus particles and, therefore, might be considered as one of fucoidan-dependent protective mechanisms limiting virus accumulation in cells. Fucoidan stimulation of the formation of PVX-specific laminated structures capable of virus particles binding is possibly another induced antiviral cell mechanism, preventing from virus reproduction and transposition.

  18. Single-cell western blotting.

    Science.gov (United States)

    Quadri, Syed M S

    2015-01-01

    Cell heterogeneity is a variation in cellular processes in functionally similar cells. Cells from the same tissue which are considered genetically identical may have difference in size, structure, and level of protein expression which can lead to major impact on the functions of cell leading to difference in physiological consequences. Single-cell proteome-wide studies are used to detect cell heterogeneity. Flow cytometry and immunocytochemistry do play an important role in evaluating cell heterogeneity. However, these methods are based on separation by antibodies with limited specificity. Cross-reactivity can occur leading to bias in result. Western blot is done to separate the proteins according to molecular weight. Therefore, off-target and on-target signals can be discriminated. Detection of protein expression from a tissue can be done with the help of western blot. However, it is unable to differentiate protein expression of individual cells. For detection of this cell-to-cell variation, a highly advanced technique termed "single-cell western blotting" is carried out. Single-cell western blot has enabled us to detect protein expression at cellular level at a fairly advanced high resolution using a western blot designed to assess cell heterogeneity.

  19. 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.

  20. Flavone Glucoside Uptake into Barley Mesophyll and Arabidopsis Cell Culture Vacuoles. Energization Occurs by H+-Antiport and ATP-Binding Cassette-Type Mechanisms1

    Science.gov (United States)

    Frangne, Nathalie; Eggmann, Thomas; Koblischke, Carsten; Weissenböck, Gottfried; Martinoia, Enrico; Klein, Markus

    2002-01-01

    In many cases, secondary plant products accumulate in the large central vacuole of plant cells. However, the mechanisms involved in the transport of secondary compounds are only poorly understood. Here, we demonstrate that the transport mechanisms for the major barley (Hordeum vulgare) flavonoid saponarin (apigenin 6-C-glucosyl-7-O-glucoside) are different in various plant species: Uptake into barley vacuoles occurs via a proton antiport and is competitively inhibited by isovitexin (apigenin 6-C-glucoside), suggesting that both flavone glucosides are recognized by the same transporter. In contrast, the transport into vacuoles from Arabidopsis, which does not synthesize flavone glucosides, displays typical characteristics of ATP-binding cassette transporters. Transport of saponarin into vacuoles of both the species is saturable with a Km of 50 to 100 μm. Furthermore, the uptake of saponarin into vacuoles from a barley mutant exhibiting a strongly reduced flavone glucoside biosynthesis is drastically decreased when compared with the parent variety. Thus, the barley vacuolar flavone glucoside/H+ antiporter could be modulated by the availability of the substrate. We propose that different vacuolar transporters may be responsible for the sequestration of species-specific/endogenous and nonspecific/xenobiotic secondary compounds in planta. PMID:11842175

  1. Single Molecule Spectroscopy: Single Live Cell

    Indian Academy of Sciences (India)

    kbhattacharjee

    Live Cell Imaging: Seeing inside a cell. • Cell: ~20,000 nm ~ 100 times bigger than focus. • Label different parts of a cell with fluorescent dye. • Cancer Cell: How different from a normal cell? cell. Space & time resolution ...

  2. Isolation of Mesophyll Protoplasts from Leaves of Dalbergia sissoo ...

    African Journals Online (AJOL)

    Isolation of protoplasts was attempted to produce an effective source for the pathogenicity test. This study described a procedure for the rapid isolation, in high yield, of photosynthetically active mesophyll protoplasts from young leaves of D. sissoo. The present study reports the isolation of protoplasts from leaf mesophyll of ...

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

    Science.gov (United States)

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

    2014-10-01

    In conjunction with genomics, transcriptomics, and proteomics, plant metabolomics is providing large data sets that are paving the way towards a comprehensive and holistic understanding of plant growth, development, defense, and productivity. However, dilution effects from organ- and tissue-based sampling of metabolomes have limited our understanding of the intricate regulation of metabolic pathways and networks at the cellular level. Recent advances in metabolomics methodologies, along with the post-genomic expansion of bioinformatics knowledge and functional genomics tools, have allowed the gathering of enriched information on individual cells and single cell types. Here we review progress, current status, opportunities, and challenges presented by single cell-based metabolomics research in plants. Copyright © 2014 Elsevier Ltd. All rights reserved.

  4. Single-cell photoacoustic thermometry.

    Science.gov (United States)

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

    2013-02-01

    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.

  5. Single-Cell Western Blotting.

    Science.gov (United States)

    Sinkala, Elly; Herr, Amy E

    2015-01-01

    Little headway has been made in single cell protein analysis, aside from tools that rely solely on antibody-probe based detection (i.e., flow cytometry, immunocytochemistry), which are limited by low specificity and multiplexing capabilities. To address these protein analysis gaps, we have introduced a single-cell western blot (scWestern). The protein assay is capable of highly specific analysis by coupling antibody-based detection with a polyacrylamide gel electrophoresis (PAGE) protein separation. Cells are settled via gravity into polyacrylamide (PA) microwells, chemically lysed in the wells, and then subjected to PAGE through the walls of the microwells and into the surrounding PA gel. Over a thousand single-cell separations are performed simultaneously, and multiple protein targets of interest are investigated. After PAGE separation, photo-immobilization of all proteins to the gel allows for antibody probing and lends to the archival quality of the scWestern assay where new proteins targets can be investigated months after the initial separations are performed.

  6. Proton extrusion is an essential signalling component in the HR of epidermal single cells in the barley-powdery mildew interaction

    DEFF Research Database (Denmark)

    Zhou, F.S.; Andersen, C.H.; Burhenne, K.

    2000-01-01

    . This will cause an acidification of the apoplast towards the mesophyll cells, thereby activating generation of H2O2 from the mesophyll, which subsequently triggers the epidermal cell to undergo HR. The model is supported by the following data: (1) the earliest HR-related H2O2 is found in the attachment zones...... between the epidermal cell and underlying mesophyll cells; (2) scavenger treatment reduces HR; (3) treatment of leaves with low-pH (3.5) citrate and succinate buffers causes more cells to undergo HR in the compatible interaction, while treatment with the same buffers at pH 5.5 reduces the number of HR...

  7. Single-Cell Genomic Analysis in Plants

    Directory of Open Access Journals (Sweden)

    Yuxuan Yuan

    2018-01-01

    Full Text Available Individual cells in an organism are variable, which strongly impacts cellular processes. Advances in sequencing technologies have enabled single-cell genomic analysis to become widespread, addressing shortcomings of analyses conducted on populations of bulk cells. While the field of single-cell plant genomics is in its infancy, there is great potential to gain insights into cell lineage and functional cell types to help understand complex cellular interactions in plants. In this review, we discuss current approaches for single-cell plant genomic analysis, with a focus on single-cell isolation, DNA amplification, next-generation sequencing, and bioinformatics analysis. We outline the technical challenges of analysing material from a single plant cell, and then examine applications of single-cell genomics and the integration of this approach with genome editing. Finally, we indicate future directions we expect in the rapidly developing field of plant single-cell genomic analysis.

  8. Ability of leaf mesophyll to retain potassium correlates with salinity tolerance in wheat and barley.

    Science.gov (United States)

    Wu, Honghong; Shabala, Lana; Barry, Karen; Zhou, Meixue; Shabala, Sergey

    2013-12-01

    This work investigated the importance of the ability of leaf mesophyll cells to control K(+) flux across the plasma membrane as a trait conferring tissue tolerance mechanism in plants grown under saline conditions. Four wheat (Triticum aestivum and Triticum turgidum) and four barley (Hordeum vulgare) genotypes contrasting in their salinity tolerance were grown under glasshouse conditions. Seven to 10-day-old leaves were excised, and net K(+) and H(+) fluxes were measured from either epidermal or mesophyll cells upon acute 100 mM treatment (mimicking plant failure to restrict Na(+) delivery to the shoot) using non-invasive microelectrode ion flux estimation (the MIFE) system. To enable net ion flux measurements from leaf epidermal cells, removal of epicuticular waxes was trialed with organic solvents. A series of methodological experiments was conducted to test the efficiency of different methods of wax removal, and the impact of experimental procedures on cell viability, in order to optimize the method. A strong positive correlation was found between plants' ability to retain K(+) in salt-treated leaves and their salinity tolerance, in both wheat and especially barley. The observed effects were related to the ionic but not osmotic component of salt stress. Pharmacological experiments have suggested that voltage-gated K(+) -permeable channels mediate K(+) retention in leaf mesophyll upon elevated NaCl levels in the apoplast. It is concluded that MIFE measurements of NaCl-induced K(+) fluxes from leaf mesophyll may be used as an efficient screening tool for breeding in cereals for salinity tissue tolerance. © 2013 Scandinavian Plant Physiology Society.

  9. Relation between Mesophyll Surface Area, Photosynthetic Rate, and Illumination Level during Development for Leaves of Plectranthus parviflorus Henckel 1

    Science.gov (United States)

    Nobel, Park S.; Zaragoza, Lawrence J.; Smith, William K.

    1975-01-01

    The influence of illumination level during leaf development on the mesophyll cell surface area per unit leaf area (Ames/A), CO2 resistances, and the photosynthetic rate was determined for leaves of Plectranthus parviflorus Henckel. The relative importance of Ames/A versus CO2 resistances in accounting for observed changes in photosynthesis was quantitatively evaluated using equations based on analogies to electrical circuits. When the illumination during development was raised from 900 to 42,000 lux, the leaves more than tripled in thickness as the mesophyll cells increased in size and frequency, which caused Ames/A to go from 11 to 50. The net rate of photosynthesis at light saturation concomitantly increased 4-fold, reflecting a corresponding decrease in the total resistance for CO2 movement per unit leaf area. However, the CO2 resistance per unit area of mesophyll cells remained about 580 seconds per centimeter for leaves grown under 900 to 42,000 lux. Thus, for P. parviflorus, the increased photosynthetic rate for leaves developing under higher illuminations resulted from a higher Ames/A, not from changes in the CO2 resistances within individual mesophyll cells, expressed per unit area of cell surface. Results are discussed in terms of previously observed increases in thickness, internal leaf area, and photosynthetic rates for sun versus shade leaves on various plant species. PMID:16659211

  10. Blumeria graminis interactions with barley conditioned by different single R genes demonstrate a temporal and spatial relationship between stomatal dysfunction and cell death.

    Science.gov (United States)

    Prats, Elena; Gay, Alan P; Roberts, Peter C; Thomas, Barry J; Sanderson, Ruth; Paveley, Neil; Lyngkjaer, Michael F; Carver, Tim L W; Mur, Luis A J

    2010-01-01

    Hypersensitive response (HR) against Blumeria graminis f. sp. hordei infection in barley (Hordeum vulgare) was associated with stomata "lock-up" leading to increased leaf water conductance (g(l)). Unique spatio-temporal patterns of HR formation occurred in barley with Mla1, Mla3, or MlLa R genes challenged with B. graminis f. sp. hordei. With Mla1, a rapid HR, limited to epidermal cells, arrested fungal growth before colonies initiated secondary attacks. With Mla3, mesophyll HR preceded that in epidermal cells whose initial survival supported secondary infections. With MlLa, mesophyll survived and not all attacked epidermal cells died immediately, allowing colony growth and secondary infection until arrested. Isolines with Mla1, Mla3, or MlLa genes inoculated with B. graminis f. sp. hordei ranging from 1 to 100 conidia mm(2) showed abnormally high g(l) during dark periods whose timing and extent correlated with those of each HR. Each isoline showed increased dark g(l) with the nonpathogen B. graminis f. sp. avenae which caused a single epidermal cell HR. Guard cell autofluorescence was seen only after drying of epidermal strips and closure of stomata suggesting that locked open stomata were viable. The data link stomatal lock-up to HR associated cell death and has implications for strategies for selecting disease resistant genotypes.

  11. 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 detec...

  12. Purine metabolism in mesophyll protoplasts of tobacco (Nicotiana tabacum) leaves.

    OpenAIRE

    Barankiewicz, J; Paszkowski, J

    1980-01-01

    The overall metabolism of purines was studied in tobacco (Nicotiana tabacum) mesophyll protoplasts. Metabolic pathways were studied by measuring the conversion of radioactive adenine, adenosine, hypoxanthine and guanine into purine ribonucleotides, ribonucleosides, bases and nucleic acid constituents. Adenine was extensively deaminated to hypoxanthine, whereupon it was also converted into AMP and incorporated into nucleic acids. Adenosine was mainly hydrolysed to adenine. Inosinate formed fro...

  13. 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.

  14. 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.

  15. 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.

  16. 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

  17. 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.

  18. 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.

  19. Purine metabolism in mesophyll protoplasts of tobacco (Nicotiana tabacum) leaves.

    Science.gov (United States)

    Barankiewicz, J; Paszkowski, J

    1980-01-15

    The overall metabolism of purines was studied in tobacco (Nicotiana tabacum) mesophyll protoplasts. Metabolic pathways were studied by measuring the conversion of radioactive adenine, adenosine, hypoxanthine and guanine into purine ribonucleotides, ribonucleosides, bases and nucleic acid constituents. Adenine was extensively deaminated to hypoxanthine, whereupon it was also converted into AMP and incorporated into nucleic acids. Adenosine was mainly hydrolysed to adenine. Inosinate formed from hypoxanthine was converted into AMP and GMP, which were then catabolized to adenine and guanosine respectively. Guanine was mainly deaminated to xanthine and also incorporated into nucleic acids via GTP. Increased RNA synthesis in the protoplasts resulted in enhanced incorporation of adenine and guanine, but not of hypoxanthine and adenosine, into the nucleic acid fraction. The overall pattern of purine-nucleotide metabolic pathways in protoplasts of tobacco leaf mesophyll is proposed.

  20. Subcellular western blotting of single cells.

    Science.gov (United States)

    Yamauchi, Kevin A; Herr, Amy E

    2017-01-01

    Although immunoassays are the de facto standard for determining subcellular protein localization in individual cells, antibody probe cross-reactivity and fixation artifacts remain confounding factors. To enhance selectivity while providing single-cell resolution, we introduce a subcellular western blotting technique capable of separately assaying proteins in the 14 pL cytoplasm and 2 pL nucleus of individual cells. To confer precision fluidic control, we describe a passive multilayer microdevice that leverages the rapid transport times afforded by miniaturization. After isolating single cells in microwells, we apply single-cell differential detergent fractionation to lyse and western blot the cytoplasmic lysate, whereas the nucleus remains intact in the microwell. Subsequently, we lyse the intact nucleus and western blot the nuclear lysate. To index each protein analysis to the originating subcellular compartment, we utilize bi-directional electrophoresis, a multidimensional separation that assays the lysate from each compartment in a distinct region of the separation axis. Single-cell bi-directional electrophoresis eliminates the need for semi-subjective image segmentation algorithms required in immunocytochemistry. The subcellular, single-cell western blot is demonstrated for six targets per cell, and successfully localizes spliceosome-associated proteins solubilized from large protein and RNA complexes, even for closely sized proteins (a 7 kDa difference). Measurement of NF-κB translocation dynamics in unfixed cells at 15-min intervals demonstrates reduced technical variance compared with immunofluorescence. This chemical cytometry assay directly measures the nucleocytoplasmic protein distribution in individual unfixed cells, thus providing insight into protein signaling in heterogeneous cell populations.

  1. Single cell-resolution western blotting.

    Science.gov (United States)

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

    2016-08-01

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

  2. 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

  3. Optimization of genetic analysis for single cell

    Directory of Open Access Journals (Sweden)

    hussein mouawia

    2012-03-01

    Full Text Available The molecular genetic analysis of microdissected cells by laser, a method for selecting a starting material of pure DNA or RNA uncontaminated. Our study focuses on technical pre-PCR (polymerase chain reaction for the amplification of DNA from a single cell (leukocyte isolated from human blood after laser microdissection and aims to optimize the yield of DNA extracted of this cell to be amplified without errors and provide reliable genetic analyzes. This study has allowed us to reduce the duration of cell lysis in order to perform the step of expanding genomic PEP (primer extension preamplification directly after lysis the same day and the quality of genomic amplification and eliminate purification step of the product PEP, step with a risk of contamination and risk of loss of genetic material related to manipulation. This approach has shown that the combination of at least 3 STR (short tandem repeat markers for genetic analysis of single cell improves the efficiency and accuracy of PCR and minimizes the loss of allele (allele drop out; ADO. This protocol can be applied to large scale and an effective means suitable for genetic testing for molecular diagnostic from isolated single cell (cancerous - fetal.

  4. Thermoresponsive micropatterned substrates for single cell studies.

    Directory of Open Access Journals (Sweden)

    Kalpana Mandal

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

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

    Directory of Open Access Journals (Sweden)

    Linda E. Kippner

    2014-06-01

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

  6. Single-cell Raman spectroscopy of irradiated tumour cells

    Science.gov (United States)

    Matthews, Quinn

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

  7. Probing bacterial adhesion at the single-cell level

    DEFF Research Database (Denmark)

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

    Bacteria initiate attachment to surfaces with the aid of different extracellular proteins and polymeric adhesins. To quantitatively analyse the cell-cell and cell-surface interactions provided by bacterial adhesins, it is essential to go down to single cell level where cell-to-cell variation can...... 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...

  8. Interaction of E. coli DNA with tobacco mesophyll protoplasts

    International Nuclear Information System (INIS)

    Heyn, R.F.

    1975-01-01

    This chapter is part of a dissertation dealing with the interaction of DNA with protoplasts. Having established the length of time during which tobacco mesophyll protoplasts do not synthesize DNA following their isolation, it is important to know the extent of DNA uptake just before the onset of DNA synthesis (and possible integration) and to find optimal conditions for this uptake. Therefore, the association of E. coli DNA with tobacco protoplasts was studied. Care should be taken with the interpretation of ''uptake'' results: adsorption phenomena play a very important role and may do so at the plasmalemma of naked protoplasts. To solve the problems involved, the use of radiation-damaged DNA was attempted. With E. coli DNA possessing a large number of thymine containing pyrimidine dimers, the loss of dimers from DNA recovered from treated protoplasts was tested in order to obtain an indication of ''real'' uptake. The results are reported

  9. GENOTYPICALY IDENTIFYING WHEAT MESOPHYLL CONDUCTANCE REGULATION UNDER PROGRESSIVE DROUGHT STRESS

    Directory of Open Access Journals (Sweden)

    Katarina Olsovska

    2016-08-01

    Full Text Available AbstractPhotosynthesis limitation by CO2 flow constraints from sub-stomatal cavities to carboxylation sites in chloroplasts under drought stress conditions is, at least in some plant species or crops not fully understood, yet. Leaf mesophyll conductance for CO2 (gm may considerably affect both photosynthesis and water use efficiency in plants under drought conditions. The aim of our study was to detect the responses of gm in leaves of four winter wheat (Triticum aestivum L. genotypes from different origins under long-term progressive drought. Based on the measurement of gas-exchange parameters the variability of genotypic responses was analyzed at stomatal (stomata closure and non-stomatal (diffusional and biochemical limits of net CO2 assimilation rate (AN. In general, progressive drought caused an increasing leaf diffusion resistance against CO2 flow leading to the decrease of AN, gm and stomatal conductance (gs, respectively. Reduction of gm also led to inhibition of carboxylation efficiency (Vcmax. On the basis of achieved results a strong positive relationship between gm and gs was found out indicating a co-regulation and mutual independence of the relationship under the drought conditions. In severely stressed plants, the stomatal limitation of the CO2 assimilation rate was progressively increased, but to a less extent in comparison to gm, while a non-stomatal limitation became more dominant due to the prolonged drought. Mesophyll conductance (gm seems to be a suitable mechanism and parameter for selection of improved diffusional properties and photosynthetic carbon assimilation in C3 plants, thus explaining their better photosynthetic performance at a whole plant level during periods of drought.

  10. 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

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

    Science.gov (United States)

    Efroni, Idan; Birnbaum, Kenneth D

    2016-04-05

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

  12. Differential gene expression and transport functionality in the bundle sheath versus mesophyll - a potential role in leaf mineral homeostasis.

    Science.gov (United States)

    Wigoda, Noa; Pasmanik-Chor, Metsada; Yang, Tianyuan; Yu, Ling; Moshelion, Menachem; Moran, Nava

    2017-06-01

    Under fluctuating ambient conditions, the ability of plants to maintain hydromineral homeostasis requires the tight control of long distance transport. This includes the control of radial transport within leaves, from veins to mesophyll. The bundle sheath is a structure that tightly wraps around leaf vasculature. It has been suggested to act as a selective barrier in the context of radial transport. This suggestion is based on recent physiological transport assays of bundle sheath cells (BSCs), as well as the anatomy of these cells.We hypothesized that the unique transport functionality of BSCs is apparent in their transcriptome. To test this, we compared the transcriptomes of individually hand-picked protoplasts of GFP-labeled BSCs and non-labeled mesophyll cells (MCs) from the leaves of Arabidopsis thaliana. Of the 90 genes differentially expressed between BSCs and MCs, 45% are membrane related and 20% transport related, a prominent example being the proton pump AHA2. Electrophysiological assays showed that the major AKT2-like membrane K+ conductances of BSCs and MCs had different voltage dependency ranges. Taken together, these differences may cause simultaneous but oppositely directed transmembrane K+ fluxes in BSCs and MCs, in otherwise similar conditions. © The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  13. Novel efficient methods for measuring mesophyll anatomical characteristics from fresh thick sections using stereology and confocal microscopy: application on acid rain-treated Norway spruce needles

    Czech Academy of Sciences Publication Activity Database

    Albrechtová, Jana; Janáček, Jiří; Lhotáková, Zuzana; Radochová, Barbora; Kubínová, Lucie

    2007-01-01

    Roč. 58, č. 6 (2007), s. 1451-1461 ISSN 0022-0957 R&D Projects: GA AV ČR IAA5011810; GA AV ČR(CZ) IAA600110507; GA MŠk(CZ) LC06063 Institutional research plan: CEZ:AV0Z50110509; CEZ:AV0Z60050516 Keywords : mesophyll * stereology * confocal microscopy Subject RIV: EA - Cell Biology Impact factor: 3.917, year: 2007

  14. Micro-PIXE for single cell analysis

    International Nuclear Information System (INIS)

    Ortega, Richard

    2012-01-01

    The knowledge of the intracellular distribution of biological relevant metals is important to understand their mechanisms of action in cells, either for physiological, toxicological or pathological processes. However, the direct detection of trace metals in single cells is a challenging task that requires sophisticated analytical developments. The combination of micro-PIXE with RBS and STIM (Scanning Transmission Ion Microscopy) allows the quantitative determination of trace metal content within sub-cellular compartments. The application of STIM analysis provides high spatial resolution imaging (< 200 nm) and excellent mass sensitivity (< 0.1 ng). Application of the STIM-PIXE-RBS methodology is absolutely needed when organic mass loss appears during PIXE-RBS irradiation. This combination of STIM-PIXE-RBS provides fully quantitative determination of trace element content, expressed in μg/g, which is a quite unique capability for micro-PIXE compared to other micro-analytical methods such as the electron and synchrotron x-ray fluorescence. Examples of micro-PIXE studies for sub-cellular imaging of trace elements in various fields of interest will be presented: in patho-physiology of trace elements involved in neurodegenerative diseases such as Parkinson's disease, and in toxicology of metals such as cobalt. (author)

  15. Recent Trends on Micro/Nanofluidic Single Cell Electroporation

    Directory of Open Access Journals (Sweden)

    Tuhin Subhra Santra

    2013-09-01

    Full Text Available The behaviors of cell to cell or cell to environment with their organelles and their intracellular physical or biochemical effects are still not fully understood. Analyzing millions of cells together cannot provide detailed information, such as cell proliferation, differentiation or different responses to external stimuli and intracellular reaction. Thus, single cell level research is becoming a pioneering research area that unveils the interaction details in high temporal and spatial resolution among cells. To analyze the cellular function, single cell electroporation can be conducted by employing a miniaturized device, whose dimension should be similar to that of a single cell. Micro/nanofluidic devices can fulfill this requirement for single cell electroporation. This device is not only useful for cell lysis, cell to cell fusion or separation, insertion of drug, DNA and antibodies inside single cell, but also it can control biochemical, electrical and mechanical parameters using electroporation technique. This device provides better performance such as high transfection efficiency, high cell viability, lower Joule heating effect, less sample contamination, lower toxicity during electroporation experiment when compared to bulk electroporation process. In addition, single organelles within a cell can be analyzed selectively by reducing the electrode size and gap at nanoscale level. This advanced technique can deliver (in/out biomolecules precisely through a small membrane area (micro to nanoscale area of the single cell, known as localized single cell membrane electroporation (LSCMEP. These articles emphasize the recent progress in micro/nanofluidic single cell electroporation, which is potentially beneficial for high-efficient therapeutic and delivery applications or understanding cell to cell interaction.

  16. The Single Cell Proteome Project - Cell-Cycle Dependent Protein Expression in Breast Cancer Cell Lines

    National Research Council Canada - National Science Library

    Dovichi, Norman J

    2005-01-01

    .... Capillary sieving electrophoresis and capillary micellar electrophoresis were used to characterize proteins in single cells in one-dimensional separations, while the two techniques were combined...

  17. Epidermal Micromorphology and Mesophyll Structure of Populus euphratica Heteromorphic Leaves at Different Development Stages.

    Directory of Open Access Journals (Sweden)

    Yubing Liu

    Full Text Available Leaf epidermal micromorphology and mesophyll structure during the development of Populus euphratica heteromorphic leaves, including linear, lanceolate, ovate, dentate ovate, dentate rhombic, dentate broad-ovate and dentate fan-shaped leaves, were studied by using electron and light microscopy. During development of heteromorphic leaves, epidermal appendages (wax crystals and trichomes and special cells (mucilage cells and crystal idioblasts increased in all leaf types while chloroplast ultrastructure and stomatal characters show maximum photosynthetic activity in dentate ovate and rhombic leaves. Also, functional analysis by subordinate function values shows that the maximum adaptability to adverse stress was exhibited in the broad type of mature leaves. The 12 heteromorphic leaf types are classified into three major groups by hierarchical cluster analysis: young, developing and mature leaves. Mature leaves can effectively obtain the highest stress resistance by combining the protection of xerophytic anatomy from drought stress, regulation of water uptake in micro-environment by mucilage and crystal idioblasts, and assistant defense of transpiration reduction through leaf epidermal appendages, which improves photosynthetic activity under arid desert conditions. Our data confirms that the main leaf function is differentiated during the developing process of heteromorphic leaves.

  18. Epidermal Micromorphology and Mesophyll Structure of Populus euphratica Heteromorphic Leaves at Different Development Stages.

    Science.gov (United States)

    Liu, Yubing; Li, Xinrong; Chen, Guoxiong; Li, Mengmeng; Liu, Meiling; Liu, Dan

    2015-01-01

    Leaf epidermal micromorphology and mesophyll structure during the development of Populus euphratica heteromorphic leaves, including linear, lanceolate, ovate, dentate ovate, dentate rhombic, dentate broad-ovate and dentate fan-shaped leaves, were studied by using electron and light microscopy. During development of heteromorphic leaves, epidermal appendages (wax crystals and trichomes) and special cells (mucilage cells and crystal idioblasts) increased in all leaf types while chloroplast ultrastructure and stomatal characters show maximum photosynthetic activity in dentate ovate and rhombic leaves. Also, functional analysis by subordinate function values shows that the maximum adaptability to adverse stress was exhibited in the broad type of mature leaves. The 12 heteromorphic leaf types are classified into three major groups by hierarchical cluster analysis: young, developing and mature leaves. Mature leaves can effectively obtain the highest stress resistance by combining the protection of xerophytic anatomy from drought stress, regulation of water uptake in micro-environment by mucilage and crystal idioblasts, and assistant defense of transpiration reduction through leaf epidermal appendages, which improves photosynthetic activity under arid desert conditions. Our data confirms that the main leaf function is differentiated during the developing process of heteromorphic leaves.

  19. Design and Analysis of Single-Cell Sequencing Experiments

    NARCIS (Netherlands)

    Grün, Dominic; van Oudenaarden, Alexander

    2015-01-01

    Recent advances in single-cell sequencing hold great potential for exploring biological systems with unprecedented resolution. Sequencing the genome of individual cells can reveal somatic mutations and allows the investigation of clonal dynamics. Single-cell transcriptome sequencing can elucidate

  20. Single cell array impedance analysis in a microfluidic device

    Science.gov (United States)

    Altinagac, Emre; Taskin, Selen; Kizil, Huseyin

    2016-10-01

    Impedance analysis of single cells is presented in this paper. Following the separation of a target cell type by dielectrophoresis in our previous work, this paper focuses on capturing the cells as a single array and performing impedance analysis to point out the signature difference between each cell type. Lab-on-a-chip devices having a titanium interdigitated electrode layer on a glass substrate and a PDMS microchannel are fabricated to capture each cell in a single form and perform impedance analysis. HCT116 (homosapiens colon colorectal carcin) and HEK293 (human embryonic kidney) cells are used in our experiments.

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

    Science.gov (United States)

    Okajima, Takaharu

    2012-11-01

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

  2. 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

  3. 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.

  4. Microfluidics-Enabled Enzyme Activity Measurement in Single Cells.

    Science.gov (United States)

    Tesauro, Cinzia; Frøhlich, Rikke; Stougaard, Magnus; Ho, Yi-Ping; Knudsen, Birgitta R

    2015-01-01

    Cellular heterogeneity has presented a significant challenge in the studies of biology. While most of our understanding is based on the analysis of ensemble average, individual cells may process information and respond to perturbations very differently. Presented here is a highly sensitive platform capable of measuring enzymatic activity at the single-cell level. The strategy innovatively combines a rolling circle-enhanced enzyme activity detection (REEAD) assay with droplet microfluidics. The single-molecule sensitivity of REEAD allows highly sensitive detection of enzymatic activities, i.e. at the single catalytic event level, whereas the microfluidics enables isolation of single cells. Further, confined reactions in picoliter-sized droplets significantly improve enzyme extraction from human cells or microorganisms and result in faster reaction kinetics. Taken together, the described protocol is expected to open up new possibilities in the single-cell research, particularly for the elucidation of heterogeneity in a population of cells.

  5. Clinical Outcome Prediction Using Single-Cell Data.

    Science.gov (United States)

    Pouyan, Maziyar Baran; Jindal, Vasu; Nourani, Mehrdad

    2016-10-01

    Single-cell technologies like flow cytometry (FCM) provide valuable biological data for knowledge discovery in complex cellular systems like tissues and organs. FCM data contains multi-dimensional information about the cellular heterogeneity of intricate cellular systems. It is possible to correlate single-cell markers with phenotypic properties of those systems. Cell population identification and clinical outcome prediction from single-cell measurements are challenging problems in the field of single cell analysis. In this paper, we propose a hybrid learning approach to predict clinical outcome using samples' single-cell FCM data. The proposed method is efficient in both i) identification of cellular clusters in each sample's FCM data and ii) predict clinical outcome (healthy versus unhealthy) for each subject. Our method is robust and the experimental results indicate promising performance.

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

    Science.gov (United States)

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

    2015-11-07

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

  7. Relationship between ultrasonic properties and structural changes in the mesophyll during leaf dehydration.

    Science.gov (United States)

    Sancho-Knapik, Domingo; Alvarez-Arenas, Tomás Gómez; Peguero-Pina, José Javier; Fernández, Victoria; Gil-Pelegrín, Eustaquio

    2011-06-01

    The broad-band ultrasonic spectroscopy technique allows the determination of changes in the relative water content (RWC) of leaves with contrasting structural features. Specifically, the standardized frequency associated with the maximum transmittance (f/f(o)) is strongly related to the RWC. This relationship is characterized by the existence of two phases separated by an inflexion point (associated with the turgor loss point). To obtain a better understanding of the strong relationship found between RWC and f/f(o), this work has studied the structural changes experienced by Quercus muehlenbergii leaves during dehydration in terms of ultrasounds measurements, cell wall elasticity, leaf thickness, leaf density, and leaf structure. The results suggest that the decrease found in f/f(o) before the turgor loss point can be attributed to the occurrence of changes in the estimation of the macroscopic effective elastic constant of the leaf (c(33)), mainly associated with changes in the bulk modulus of elasticity of the cell wall (ε). These changes are overriding or compensating for the thickness decreases recorded during this phase. On the other hand, the high degree of cell shrinkage and stretching found in the mesophyll cells during the second phase seem to explain the changes in the acoustic properties of the leaf beyond the turgor loss point. The formation of large intercellular spaces, which increased the irregularity in the acoustic pathway, may explain the increase of the attenuation coefficient of ultrasounds once the turgor loss point threshold is exceeded. The direct measurement of c(33) from ultrasonic measurements would allow a better knowledge of the overall biomechanical properties of the leaf further than those derived from the P-V analysis.

  8. Biology at a single cell level

    CSIR Research Space (South Africa)

    Mthunzi, P

    2012-10-01

    Full Text Available ://www.regenexx.com/wp-content/uploads/2011/05/IPS-cell-problems.jpg Induced pluripotent stem cells differentiated in culture http://www.youtube.com/watch?v=ECllrIzTKbA&feature=related Transfecting neuroblastomas Neuroblastoma ? Brain cells ? 80 ? 120 billion neurons in human... brain ? Non- renewing cell type ? Neurons difficult to transfect with established protocols ? Susceptible to degenerative disorders: - Parkinson?s disease - Multiple sclerosis - Alzheimer's disease http...

  9. Single cell electroporation using microfluidic devices

    NARCIS (Netherlands)

    le Gac, Severine; van den Berg, Albert; Lindstrom, S.; Andersson, Helene

    2012-01-01

    Electroporation is a powerful technique to increase the permeability of cell membranes and subsequently introduce foreign materials into cells. Pores are created in the cell membrane upon application of an electric fi eld (kV/cm). Most applications employ bulk electroporation, at the scale of 1 mL

  10. PRODt;CTION OF SINGLE CELL PROTEIN FROM BREWERY ...

    African Journals Online (AJOL)

    BSN

    The production of single cell protein (SCP) by the propagation of the yeast, Saccharomyces cerevisae ... animal feed but little or no information has been documented as per its explication for the production of single cell .... use of yeasts produced from vatious carbohydrate sources, molasses, sulphite liquors and vegetable.

  11. Single-molecule tracking in living cells using single quantum dot applications.

    Science.gov (United States)

    Baba, Koichi; Nishida, Kohji

    2012-01-01

    Revealing the behavior of single molecules in living cells is very useful for understanding cellular events. Quantum dot probes are particularly promising tools for revealing how biological events occur at the single molecule level both in vitro and in vivo. In this review, we will introduce how single quantum dot applications are used for single molecule tracking. We will discuss how single quantum dot tracking has been used in several examples of complex biological processes, including membrane dynamics, neuronal function, selective transport mechanisms of the nuclear pore complex, and in vivo real-time observation. We also briefly discuss the prospects for single molecule tracking using advanced probes.

  12. 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

  13. 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.

  14. 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.

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

    Directory of Open Access Journals (Sweden)

    Joseph D Steinmeyer

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

  16. Image analysis driven single-cell analytics for systems microbiology.

    Science.gov (United States)

    Balomenos, Athanasios D; Tsakanikas, Panagiotis; Aspridou, Zafiro; Tampakaki, Anastasia P; Koutsoumanis, Konstantinos P; Manolakos, Elias S

    2017-04-04

    Time-lapse microscopy is an essential tool for capturing and correlating bacterial morphology and gene expression dynamics at single-cell resolution. However state-of-the-art computational methods are limited in terms of the complexity of cell movies that they can analyze and lack of automation. The proposed Bacterial image analysis driven Single Cell Analytics (BaSCA) computational pipeline addresses these limitations thus enabling high throughput systems microbiology. BaSCA can segment and track multiple bacterial colonies and single-cells, as they grow and divide over time (cell segmentation and lineage tree construction) to give rise to dense communities with thousands of interacting cells in the field of view. It combines advanced image processing and machine learning methods to deliver very accurate bacterial cell segmentation and tracking (F-measure over 95%) even when processing images of imperfect quality with several overcrowded colonies in the field of view. In addition, BaSCA extracts on the fly a plethora of single-cell properties, which get organized into a database summarizing the analysis of the cell movie. We present alternative ways to analyze and visually explore the spatiotemporal evolution of single-cell properties in order to understand trends and epigenetic effects across cell generations. The robustness of BaSCA is demonstrated across different imaging modalities and microscopy types. BaSCA can be used to analyze accurately and efficiently cell movies both at a high resolution (single-cell level) and at a large scale (communities with many dense colonies) as needed to shed light on e.g. how bacterial community effects and epigenetic information transfer play a role on important phenomena for human health, such as biofilm formation, persisters' emergence etc. Moreover, it enables studying the role of single-cell stochasticity without losing sight of community effects that may drive it.

  17. 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.

  18. New Array Approaches to Explore Single Cells Genomes

    Science.gov (United States)

    Vanneste, Evelyne; Bittman, Lilach; Van der Aa, Niels; Voet, Thierry; Vermeesch, Joris Robert

    2011-01-01

    Microarray analysis enables the genome-wide detection of copy number variations and the investigation of chromosomal instability. Whereas array techniques have been well established for the analysis of unamplified DNA derived from many cells, it has been more challenging to enable the accurate analysis of single cell genomes. In this review, we provide an overview of single cell DNA amplification techniques, the different array approaches, and discuss their potential applications to study human embryos. PMID:22509179

  19. Multimodal sensory integration in single cerebellar granule cells in vivo.

    Science.gov (United States)

    Ishikawa, Taro; Shimuta, Misa; Häusser, Michael

    2015-12-29

    The mammalian cerebellum is a highly multimodal structure, receiving inputs from multiple sensory modalities and integrating them during complex sensorimotor coordination tasks. Previously, using cell-type-specific anatomical projection mapping, it was shown that multimodal pathways converge onto individual cerebellar granule cells (Huang et al., 2013). Here we directly measure synaptic currents using in vivo patch-clamp recordings and confirm that a subset of single granule cells receive convergent functional multimodal (somatosensory, auditory, and visual) inputs via separate mossy fibers. Furthermore, we show that the integration of multimodal signals by granule cells can enhance action potential output. These recordings directly demonstrate functional convergence of multimodal signals onto single granule cells.

  20. Mass spectrometry-based metabolomics of single yeast cells.

    Science.gov (United States)

    Ibáñez, Alfredo J; Fagerer, Stephan R; Schmidt, Anna Mareike; Urban, Pawel L; Jefimovs, Konstantins; Geiger, Philipp; Dechant, Reinhard; Heinemann, Matthias; Zenobi, Renato

    2013-05-28

    Single-cell level measurements are necessary to characterize the intrinsic biological variability in a population of cells. In this study, we demonstrate that, with the microarrays for mass spectrometry platform, we are able to observe this variability. We monitor environmentally (2-deoxy-D-glucose) and genetically (ΔPFK2) perturbed Saccharomyces cerevisiae cells at the single-cell, few-cell, and population levels. Correlation plots between metabolites from the glycolytic pathway, as well as with the observed ATP/ADP ratio as a measure of cellular energy charge, give biological insight that is not accessible from population-level metabolomic data.

  1. 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

  2. The single-cell gel electrophoresis assay to determine apoptosis ...

    African Journals Online (AJOL)

    The aim of the present study was to determine if the pattern of DNA fragmentation determined by the single cell gel electrophoresis assay can be used to determine apoptosis induced by siRNA in Colo 320 cells. When the frequency of appearance of apoptotic cells following was observed over a period of time, there was a ...

  3. 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...

  4. 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.

  5. Reconstructing Cell Lineages from Single-Cell Gene Expression Data: A Pilot Study

    Science.gov (United States)

    2016-08-30

    Reconstructing cell lineages from single -cell gene expression data: a pilot study The goal of this pilot study is to develop novel mathematical...methods, by leveraging tools developed in the bifurcation theory, to infer the underlying cell-state dynamics from single -cell gene expression data. Our...from single -cell gene expression data. The views, opinions and/or findings contained in this report are those of the author(s) and should not contrued

  6. Single-Cell Expression Profiling and Proteomics of Primordial Germ Cells, Spermatogonial Stem Cells, Adult Germ Stem Cells, and Oocytes.

    Science.gov (United States)

    Conrad, Sabine; Azizi, Hossein; Skutella, Thomas

    2018-01-04

    The mammalian germ cells, cell assemblies, tissues, and organs during development and maturation have been extensively studied at the tissue level. However, to investigate and understand the fundamental insights at the molecular basis of germ and stem cells, their cell fate plasticity, and determination, it is of most importance to analyze at the large scale on the single-cell level through different biological windows. Here, modern molecular techniques optimized for single-cell analysis, including single fluorescence-activated cell sorting (FACS) and single-cell RNA sequencing (scRNA-seq) or microfluidic high-throughput quantitative real-time polymerase chain reaction (qRT-PCR) for single-cell gene expression and liquid chromatography coupled to tandem mass spectrometry (LC-MSMS) for protein profiling, have been established and are still getting optimized.This review aims on describing and discussing recent single-cell expression profiling and proteomics of different types of human germ cells, including primordial germ cells (PGCs), spermatogonial stem cells (SSCs), human adult germ stem cells (haGSCs), and oocytes.

  7. 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.

  8. 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.

  9. Single-cell phospho-protein analysis by flow cytometry.

    Science.gov (United States)

    Schulz, Kenneth R; Danna, Erika A; Krutzik, Peter O; Nolan, Garry P

    2012-02-01

    This protocol describes methods for monitoring intracellular phosphorylation-dependent signaling events on a single-cell basis. This approach measures cell signaling by treating cells with exogenous stimuli, fixing cells with formaldehyde, permeabilizing with methanol, and then staining with phospho-specific antibodies. Thus, cell signaling states can be determined as a measure of how cells interact with their environment. This method has applications in clinical research as well as mechanistic studies of basic biology. In clinical research, diagnostic or drug efficacy information can be retrieved by discovering how a disease affects the ability of cells to respond to growth factors. Basic scientists can use this technique to analyze signaling events in cell lines and human or murine primary cells, including rare populations, like B1 cells or stem cells. This technique has broad applications bringing standard biochemical analysis into primary cells in order to garner valuable information about signaling events in physiologic settings. © 2012 by John Wiley & Sons, Inc.

  10. Plasmalemma- and tonoplast-ATPase activity in mesophyll protoplasts, vacuoles and microsomes of the Crassulacean-acid-metabolism plant Kalanchoe daigremontiana.

    Science.gov (United States)

    Balsamo, R A; Uribe, E G

    1988-02-01

    Adenosine-triphosphatase activity on the plasmalemma and tonoplast of isolated mesophyll protoplasts, isolated vacuoles and tonoplast-derived microsomes of the Crassulacean-acid-metabolism plant Kalanchoe daigremontiana Hamet et Perr., was localized by a cytochemical procedure using lead citrate. Enzyme activity was detected on the cytoplasmic surfaces of the plasmalemma and tonoplast. The identity of the enzymes was confirmed by various treatments differentiating the enzymes by their sensitivity to inhibitors of plasmalemma and tonoplast H(+)-ATPase. Isolated vacuoles and microsomes prepared from isolated vacuoles clearly exhibited single-sided deposition on membrane surfaces.

  11. Measurement of gross photosynthesis, respiration in the light, and mesophyll conductance using H218O labeling.

    Science.gov (United States)

    Gauthier, Paul Pg; Battle, Mark O; Griffin, Kevin L; Bender, Michael L

    2018-03-27

    A fundamental challenge in plant physiology is independently determining the rates of gross O2 production by photosynthesis and O2 consumption by respiration, photorespiration, and other processes. Previous studies on isolated chloroplasts or leaves have separately constrained net and gross O2 production (NOP and GOP, respectively) by labeling ambient O2 with 18O while leaf water was unlabeled. Here, we describe a method to accurately measure GOP and NOP of whole detached leaves in a cuvette as a routine gas exchange measurement. The petiole is immersed in water enriched to a δ18O of ~9,000‰, and leaf water is labeled through the transpiration stream. Photosynthesis transfers 18O from H2O to O2. GOP is calculated from the increase in δ18O of O2 as air passes through the cuvette. NOP is determined from the increase in O2/N2. Both terms are measured by isotope ratio mass spectrometry. CO2 assimilation and other standard gas exchange parameters are also measured. Reproducible measurements are made on a single leaf for more than 15 hours. We used this method to measure the light response curve of NOP and GOP in Phaseolus vulgaris at 21% and 2% O2. We then used these data to examine the O2/CO2 ratio of net photosynthesis, the light response curve of mesophyll conductance, and the apparent inhibition of respiration in the light (Kok effect) at both oxygen levels. The results are discussed in the context of evaluating the technique as a tool to study and understand leaf physiological traits. {copyright, serif} 2018 American Society of Plant Biologists. All rights reserved.

  12. 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.

  13. 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.

  14. New insights into human primordial germ cells and early embryonic development from single-cell analysis.

    Science.gov (United States)

    Otte, Jörg; Wruck, Wasco; Adjaye, James

    2017-08-01

    Human preimplantation developmental studies are difficult to accomplish due to associated ethical and moral issues. Preimplantation cells are rare and exist only in transient cell states. From a single cell, it is very challenging to analyse the origination of the heterogeneity and complexity inherent to the human body. However, recent advances in single-cell technology and data analysis have provided new insights into the process of early human development and germ cell specification. In this Review, we examine the latest single-cell datasets of human preimplantation embryos and germ cell development, compare them to bulk cell analyses, and interpret their biological implications. © 2017 Federation of European Biochemical Societies.

  15. Solar cell structure incorporating a novel single crystal silicon material

    Science.gov (United States)

    Pankove, Jacques I.; Wu, Chung P.

    1983-01-01

    A novel hydrogen rich single crystal silicon material having a band gap energy greater than 1.1 eV can be fabricated by forming an amorphous region of graded crystallinity in a body of single crystalline silicon and thereafter contacting the region with atomic hydrogen followed by pulsed laser annealing at a sufficient power and for a sufficient duration to recrystallize the region into single crystal silicon without out-gassing the hydrogen. The new material can be used to fabricate semiconductor devices such as single crystal silicon solar cells with surface window regions having a greater band gap energy than that of single crystal silicon without hydrogen.

  16. Single-cell magnetic imaging using a quantum diamond microscope.

    Science.gov (United States)

    Glenn, D R; Lee, K; Park, H; Weissleder, R; Yacoby, A; Lukin, M D; Lee, H; Walsworth, R L; Connolly, C B

    2015-08-01

    We apply a quantum diamond microscope for detection and imaging of immunomagnetically labeled cells. This instrument uses nitrogen-vacancy (NV) centers in diamond for correlated magnetic and fluorescence imaging. Our device provides single-cell resolution and a field of view (∼1 mm(2)) two orders of magnitude larger than that of previous NV imaging technologies, enabling practical applications. To illustrate, we quantified cancer biomarkers expressed by rare tumor cells in a large population of healthy cells.

  17. Single cell magnetic imaging using a quantum diamond microscope

    Science.gov (United States)

    Park, H.; Weissleder, R.; Yacoby, A.; Lukin, M. D.; Lee, H.; Walsworth, R. L.; Connolly, C. B.

    2015-01-01

    We apply a quantum diamond microscope to detection and imaging of immunomagnetically labeled cells. This instrument uses nitrogen-vacancy (NV) centers in diamond for correlated magnetic and fluorescence imaging. Our device provides single-cell resolution and two orders of magnitude larger field of view (~1 mm2) than previous NV imaging technologies, enabling practical applications. To illustrate, we quantify cancer biomarkers expressed by rare tumor cells in a large population of healthy cells. PMID:26098019

  18. 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

  19. Functional Insights into Sponge Microbiology by Single Cell Genomics

    KAUST Repository

    Hentschel, Ute

    2011-04-09

    Marine Sponges (Porifera) are known to harbor enormous amounts of microorganisms with members belonging to at least 30 different bacterial phyla including several candidate phyla and both archaeal lineages. Here, we applied single cell genomics to the mic

  20. Variation among soybean cultivars in mesophyll conductance and leaf water use efficiency

    Science.gov (United States)

    Improving water use efficiency (WUE) may prove a useful way to adapt crop species to drought. Since the recognition of the importance of mesophyll conductance to CO2 movement from inside stomatal pores to the sites of photosynthetic carboxylation, there has been interest in how much intraspecific v...

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

    Directory of Open Access Journals (Sweden)

    Felix Schmidt

    2016-06-01

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

  2. 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.

  3. 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.

  4. Single Cell Characterization of Prostate Cancer-Circulating Tumor Cells

    Science.gov (United States)

    2013-09-01

    contaminating WBC. Scale bar = 20 microns. (E) MagSweeper versus CellSearch comparison. Samples with 0 CTC were assigned a value of 1. 10...cancer patient blood sample, and contaminating WBC found after MagSweeper isolation. Scale bar = 20 microns. (E) MagSweeper versus CellSearch...Weinberg RA (2011) Hallmarks of cancer: the next generation. Cell 144: 646–674. 4. Ashworth TR (1869) A case of cancer in which cells similar to those in

  5. Single cell transcriptome profiling of developing chick retinal cells.

    Science.gov (United States)

    Laboissonniere, Lauren A; Martin, Gregory M; Goetz, Jillian J; Bi, Ran; Pope, Brock; Weinand, Kallie; Ellson, Laura; Fru, Diane; Lee, Miranda; Wester, Andrea K; Liu, Peng; Trimarchi, Jeffrey M

    2017-08-15

    The vertebrate retina is a specialized photosensitive tissue comprised of six neuronal and one glial cell types, each of which develops in prescribed proportions at overlapping timepoints from a common progenitor pool. While each of these cells has a specific function contributing to proper vision in the mature animal, their differential representation in the retina as well as the presence of distinctive cellular subtypes makes identifying the transcriptomic signatures that lead to each retinal cell's fate determination and development challenging. We have analyzed transcriptomes from individual cells isolated from the chick retina throughout retinogenesis. While we focused our efforts on the retinal ganglion cells, our transcriptomes of developing chick cells also contained representation from multiple retinal cell types, including photoreceptors and interneurons at different stages of development. Most interesting was the identification of transcriptomes from individual mixed lineage progenitor cells in the chick as these cells offer a window into the cell fate decision-making process. Taken together, these data sets will enable us to uncover the most critical genes acting in the steps of cell fate determination and early differentiation of various retinal cell types. © 2017 Wiley Periodicals, Inc.

  6. 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 technology in lab-on-chip systems devoted to accurate individual cell trapping and manipulation....

  7. Single Cell Adhesion Assay Using Computer Controlled Micropipette

    Science.gov (United States)

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

    2014-01-01

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

  8. 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

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

    Science.gov (United States)

    Doud, Devin F R; Woyke, Tanja

    2017-07-01

    Deeper sequencing and improved bioinformatics in conjunction with single-cell and metagenomic approaches continue to illuminate undercharacterized environmental microbial communities. This has propelled the 'who is there, and what might they be doing' paradigm to the uncultivated and has already radically changed the topology of the tree of life and provided key insights into the microbial contribution to biogeochemistry. While characterization of 'who' based on marker genes can describe a large fraction of the community, answering 'what are they doing' remains the elusive pinnacle for microbiology. Function-driven single-cell genomics provides a solution by using a function-based screen to subsample complex microbial communities in a targeted manner for the isolation and genome sequencing of single cells. This enables single-cell sequencing to be focused on cells with specific phenotypic or metabolic characteristics of interest. Recovered genomes are conclusively implicated for both encoding and exhibiting the feature of interest, improving downstream annotation and revealing activity levels within that environment. This emerging approach has already improved our understanding of microbial community functioning and facilitated the experimental analysis of uncharacterized gene product space. Here we provide a comprehensive review of strategies that have been applied for function-driven single-cell genomics and the future directions we envision. © FEMS 2017.

  10. 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...

  11. Single-cell sequencing of the small-RNA transcriptome.

    Science.gov (United States)

    Faridani, Omid R; Abdullayev, Ilgar; Hagemann-Jensen, Michael; Schell, John P; Lanner, Fredrik; Sandberg, Rickard

    2016-12-01

    Little is known about the heterogeneity of small-RNA expression as small-RNA profiling has so far required large numbers of cells. Here we present a single-cell method for small-RNA sequencing and apply it to naive and primed human embryonic stem cells and cancer cells. Analysis of microRNAs and fragments of tRNAs and small nucleolar RNAs (snoRNAs) reveals the potential of microRNAs as markers for different cell types and states.

  12. Review of methods to probe single cell metabolism and bioenergetics.

    Science.gov (United States)

    Vasdekis, Andreas E; Stephanopoulos, Gregory

    2015-01-01

    Single cell investigations have enabled unexpected discoveries, such as the existence of biological noise and phenotypic switching in infection, metabolism and treatment. Herein, we review methods that enable such single cell investigations specific to metabolism and bioenergetics. Firstly, we discuss how to isolate and immobilize individuals from a cell suspension, including both permanent and reversible approaches. We also highlight specific advances in microbiology for its implications in metabolic engineering. Methods for probing single cell physiology and metabolism are subsequently reviewed. The primary focus therein is on dynamic and high-content profiling strategies based on label-free and fluorescence microspectroscopy and microscopy. Non-dynamic approaches, such as mass spectrometry and nuclear magnetic resonance, are also briefly discussed. Published by Elsevier Inc.

  13. 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.

  14. 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.

  15. Counting Legionella cells within single amoeba host cells

    Science.gov (United States)

    Here we present the first attempt to quantify L. pneumophila cell numbers within individual amoebae hosts that may be released into engineered water systems. The maximum numbers of culturable L. pneumophila cells grown within Acanthamoeba polyphaga and Naegleria fowleri were 134...

  16. 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

  17. T cell fate and clonality inference from single-cell transcriptomes.

    Science.gov (United States)

    Stubbington, Michael J T; Lönnberg, Tapio; Proserpio, Valentina; Clare, Simon; Speak, Anneliese O; Dougan, Gordon; Teichmann, Sarah A

    2016-04-01

    We developed TraCeR, a computational method to reconstruct full-length, paired T cell receptor (TCR) sequences from T lymphocyte single-cell RNA sequence data. TraCeR links T cell specificity with functional response by revealing clonal relationships between cells alongside their transcriptional profiles. We found that T cell clonotypes in a mouse Salmonella infection model span early activated CD4(+) T cells as well as mature effector and memory cells.

  18. 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.

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

    Science.gov (United States)

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

    2013-03-01

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

  20. Exploring single electrode reactions in polymer electrolyte fuel cells

    Energy Technology Data Exchange (ETDEWEB)

    Kuhn, H.; Wokaun, A.; Scherer, G.G. [Paul Scherrer Institute, Electrochemistry Laboratory, 5232 Villigen (Switzerland)

    2007-01-20

    Utilising a pseudo-reference electrode in polymer electrolyte fuel cells allows for the separation of anodic and cathodic contributions to the entire cell impedance. Modelling the impedance responses by using equivalent circuits inhibits the investigation of kinetic parameters of the basic electrochemical reactions, which take place at single electrode-electrolyte interfaces. Therefore, we evaluate single electrode impedance measurements by a kinetic model, which is based on specific reaction pathways, either for the oxygen reduction reaction (ORR) or the hydrogen oxidation reaction (HOR). As a consequence, it is possible to obtain kinetic parameters for the specific reaction of interest. Furthermore, the information gained from the single electrode impedance measurements and the kinetic model can give insight into single reactions steps. In particular, the ORR has to include a chemical step in the reaction pathway. (author)

  1. Magnetophoretic circuits for digital control of single particles and cells

    Science.gov (United States)

    Lim, Byeonghwa; Reddy, Venu; Hu, Xinghao; Kim, Kunwoo; Jadhav, Mital; Abedini-Nassab, Roozbeh; Noh, Young-Woock; Lim, Yong Taik; Yellen, Benjamin B.; Kim, Cheolgi

    2014-05-01

    The ability to manipulate small fluid droplets, colloidal particles and single cells with the precision and parallelization of modern-day computer hardware has profound applications for biochemical detection, gene sequencing, chemical synthesis and highly parallel analysis of single cells. Drawing inspiration from general circuit theory and magnetic bubble technology, here we demonstrate a class of integrated circuits for executing sequential and parallel, timed operations on an ensemble of single particles and cells. The integrated circuits are constructed from lithographically defined, overlaid patterns of magnetic film and current lines. The magnetic patterns passively control particles similar to electrical conductors, diodes and capacitors. The current lines actively switch particles between different tracks similar to gated electrical transistors. When combined into arrays and driven by a rotating magnetic field clock, these integrated circuits have general multiplexing properties and enable the precise control of magnetizable objects.

  2. The Use of Evolutionary Approaches to Understand Single Cell Genomes

    Directory of Open Access Journals (Sweden)

    Haiwei eLuo

    2015-03-01

    Full Text Available The vast majority of environmental bacteria and archaea remain uncultivated, yet their genome sequences are rapidly becoming available through single cell sequencing technologies. Reconstructing metabolism is one common way to make use of genome sequences of ecologically important bacteria, but molecular evolutionary analysis is another approach that, while currently underused, can reveal important insights into the function of these uncultivated microbes in nature. Because genome sequences from single cells are often incomplete, metabolic reconstruction based on genome content can be compromised. However, this problem does not necessarily impede the use of phylogenomic and population genomic approaches that are based on patterns of polymorphisms and substitutions at nucleotide and amino acid sites. These approaches explore how various evolutionary forces act to assemble genetic diversity within and between lineages. In this mini-review, I present examples illustrating the benefits of analyzing single cell genomes using evolutionary approaches.

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

    Science.gov (United States)

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

    2014-03-01

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

  4. Virtual microstructural leaf tissue generation based on cell growth modeling

    NARCIS (Netherlands)

    Abera, M.K.; Retta, M.A.; Verboven, P.; Nicolai, B.M.; Berghuijs, H.; Struik, P.

    2016-01-01

    A cell growth algorithm for virtual leaf tissue generation is presented based on the biomechanics of plant cells in tissues. The algorithm can account for typical differences in epidermal layers, palisade mesophyll layer and spongy mesophyll layer which have characteristic differences in the

  5. Translocation of 14C-sucrose from mesophyll to vein and stem bark in mulberry plant

    International Nuclear Information System (INIS)

    Yamashita, Tadaaki

    1982-01-01

    One-year-old seedlings of mulberry, the variety of Shinichinose, were used to fix 14 CO 2 for 10 minutes under light of 18,000 lux at 28 deg C. Photosynthesis was continued after that in the air not containing 14 CO 2 . The specimens were collected immediately after and 10 and 30 minutes after the 14 CO 2 fixation, divided into mesophyll, vein and stem bark, frozen in liquid N 2 , and the contents of sucrose, glucose and fructose and their radioactivity were measured. The ratio of the radioactivity incorporated into sucrose to the total radioactivity incorporated into each tissue was very large, and the incorporation into glucose or fructose was very little. The content of sucrose was also larger than that of the other sugars in all the tissues. These findings showed that the photosynthates were translocated mainly in the form of sucrose. The ratio of specific radioactivity (dpm/mg sugar) in the mesophyll to that in the vein was 0.28, 0.58 and 0.42, respectively, immediately after, 10 minutes after and 30 minutes after the 14 CO 2 fixation for sucrose, 0.16, 0.39 and 0.29, respectively, for glucose, and 0.17, 0.25 and 0.46, respectively, for fructose. The specific radioactivity of sucrose in the vein 10 minutes after the fixation was as high as 58 % of that in the mesophyll, suggesting that the sucrose produced in the mesophyll was rapidly transported into the vein. (Kaihara, S.)

  6. 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

  7. Leaf anatomical properties in relation to differences in mesophyll conductance to CO(2) and photosynthesis in two related Mediterranean Abies species.

    Science.gov (United States)

    Peguero-Pina, José Javier; Flexas, Jaume; Galmés, Jeroni; Niinemets, Ulo; Sancho-Knapik, Domingo; Barredo, Gonzalo; Villarroya, Dido; Gil-Pelegrín, Eustaquio

    2012-12-01

    Abies alba and Abies pinsapo are closely related species with the same ribulose 1·5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit (rbcL) but contrasting hydraulic traits and mesophyll structure occurring in the Iberian Peninsula under contrasting conditions. As photosynthesis and hydraulic capacities often co-scale, we hypothesize that these species differ in mesophyll conductance to CO(2) (g(m) ). g(m) and key anatomical traits were measured in both species. Drought-adapted population of A. pinsapo has higher photosynthesis than the more mesic population of A. alba, in agreement with its higher hydraulic capacity. However, A. alba exhibits the largest stomatal conductance (g(s) ), and so water use efficiency (WUE) is much higher in A. pinsapo. The differences in photosynthesis were explained by differences in g(m) , indicating a correlation between hydraulic capacity and g(m) . We report a case where g(m) is the main factor limiting photosynthesis in one species (A. alba) when compared with the other one (A. pinsapo). The results also highlight the discrepancy between g(m) estimates based on anatomical measurements and those based on gas exchange methods, probably due to the very large resistance exerted by cell walls and the stroma in both species. Thus, the cell wall and chloroplast properties in relation to CO(2) diffusion constitute a near-future research priority. © 2012 Blackwell Publishing Ltd.

  8. 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

  9. The Single Prostate Cell Transcriptome as Biological Assay

    National Research Council Canada - National Science Library

    Nelson, Peter

    1999-01-01

    .... The scope to the research involves the construction of cDNA libraries representing the genes expressed in selected populations of normal and neoplastic prostate cancer cells followed by the construction of microarrays suitable for comprehensive gene expression studies. These arrays are then used to evaluate methods for single-cell transcriptome amplification with the aim of identifying a cohort of cellular transcripts which correlate with, or.

  10. Parallel single-cell sequencing links transcriptional and epigenetic heterogeneity.

    Science.gov (United States)

    Angermueller, Christof; Clark, Stephen J; Lee, Heather J; Macaulay, Iain C; Teng, Mabel J; Hu, Tim Xiaoming; Krueger, Felix; Smallwood, Sebastien; Ponting, Chris P; Voet, Thierry; Kelsey, Gavin; Stegle, Oliver; Reik, Wolf

    2016-03-01

    We report scM&T-seq, a method for parallel single-cell genome-wide methylome and transcriptome sequencing that allows for the discovery of associations between transcriptional and epigenetic variation. Profiling of 61 mouse embryonic stem cells confirmed known links between DNA methylation and transcription. Notably, the method revealed previously unrecognized associations between heterogeneously methylated distal regulatory elements and transcription of key pluripotency genes.

  11. Femtosecond laser fabrication of optofluidic devices for single cell manipulation

    Directory of Open Access Journals (Sweden)

    Bragheri Francesca

    2015-01-01

    Full Text Available In this work we fabricate and validate two optofludic devices for the manipulation and analysis of single cells. The chips are fabricated by femtosecond laser micromachining exploiting the 3D capabilities of the technique and the inherent perfect alignment between microfluidic channels and optical networks. Both devices have been validated by probing the mechanical properties of different cancer cell lines, which are expected to show different elasticity because of their different metastatic potential.

  12. Single-cell mechanics: the parallel plates technique.

    Science.gov (United States)

    Bufi, Nathalie; Durand-Smet, Pauline; Asnacios, Atef

    2015-01-01

    We describe here the parallel plates technique which enables quantifying single-cell mechanics, either passive (cell deformability) or active (whole-cell traction forces). Based on the bending of glass microplates of calibrated stiffness, it is easy to implement on any microscope, and benefits from protocols and equipment already used in biology labs (coating of glass slides, pipette pullers, micromanipulators, etc.). We first present the principle of the technique, the design and calibration of the microplates, and various surface coatings corresponding to different cell-substrate interactions. Then we detail the specific cell preparation for the assays, and the different mechanical assays that can be carried out. Finally, we discuss the possible technical simplifications and the specificities of each mechanical protocol, as well as the possibility of extending the use of the parallel plates to investigate the mechanics of cell aggregates or tissues. Copyright © 2015 Elsevier Inc. All rights reserved.

  13. Bioanalytical tools for single-cell study of exocytosis.

    Science.gov (United States)

    Ge, Shencheng; Koseoglu, Secil; Haynes, Christy L

    2010-08-01

    Regulated exocytosis is a fundamental biological process used to deliver chemical messengers for cell-cell communication via membrane fusion and content secretion. A plethora of cell types employ this chemical-based communication to achieve crucial functions in many biological systems. Neurons in the brain and platelets in the circulatory system are representative examples utilizing exocytosis for neurotransmission and blood clotting. Single-cell studies of regulated exocytosis in the past several decades have greatly expanded our knowledge of this critical process, from vesicle/granule transport and docking at the early stages of exocytosis to membrane fusion and to eventual chemical messenger secretion. Herein, four main approaches that have been widely used to study single-cell exocytosis will be highlighted, including total internal reflection fluorescence microscopy, capillary electrophoresis, single-cell mass spectrometry, and microelectrochemistry. These techniques are arranged in the order following the route of a vesicle/granule destined for secretion. Within each section, the basic principles and experimental strategies are reviewed and representative examples are given revealing critical spatial, temporal, and chemical information of a secretory vesicle/granule at different stages of its lifetime. Lastly, an analytical chemist's perspective on potential future developments in this exciting field is discussed.

  14. Single-cell epigenomics: powerful new methods for understanding gene regulation and cell identity.

    Science.gov (United States)

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

    2016-04-18

    Emerging single-cell epigenomic methods are being developed with the exciting potential to transform our knowledge of gene regulation. Here we review available techniques and future possibilities, arguing that the full potential of single-cell epigenetic studies will be realized through parallel profiling of genomic, transcriptional, and epigenetic information.

  15. 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

  16. 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.

  17. Single-cell analysis of endothelial morphogenesis in vivo

    Science.gov (United States)

    Yu, Jianxin A.; Castranova, Daniel; Pham, Van N.; Weinstein, Brant M.

    2015-01-01

    Vessel formation has been extensively studied at the tissue level, but the difficulty in imaging the endothelium with cellular resolution has hampered study of the morphogenesis and behavior of endothelial cells (ECs) in vivo. We are using endothelial-specific transgenes and high-resolution imaging to examine single ECs in zebrafish. By generating mosaics with transgenes that simultaneously mark endothelial nuclei and membranes we are able to definitively identify and study the morphology and behavior of individual ECs during vessel sprouting and lumen formation. Using these methods, we show that developing trunk vessels are composed of ECs of varying morphology, and that single-cell analysis can be used to quantitate alterations in morphology and dynamics in ECs that are defective in proper guidance and patterning. Finally, we use single-cell analysis of intersegmental vessels undergoing lumen formation to demonstrate the coexistence of seamless transcellular lumens and single or multicellular enclosed lumens with autocellular or intercellular junctions, suggesting that heterogeneous mechanisms contribute to vascular lumen formation in vivo. The tools that we have developed for single EC analysis should facilitate further rigorous qualitative and quantitative analysis of EC morphology and behavior in vivo. PMID:26253401

  18. 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.

  19. Evaluation of yeast single cell protein (SCP) diets on growth ...

    African Journals Online (AJOL)

    An investigation was carried out on the possibility of replacing fishmeal with graded levels of yeast single cell protein (SCP; 10, 20, 30, 40 and 50%) in ... that the 50% yeast SCP fed fish had the highest percentage of body protein (55.35%), but with a lower amount of fat at the end of the feeding trial compared to the control.

  20. PRODt;CTION OF SINGLE CELL PROTEIN FROM BREWERY ...

    African Journals Online (AJOL)

    BSN

    customary food and feed sources of protein (agriculnrre and fishery) to ocher sources like single cell protein (SCP); whose production from hydrocarbons is one ... origin is unicellular or simple multicellular organism such as bacteria, yeasts, fungi, algae. protozoa, mid even bacterinphagcs generally cultivated on substrates ...

  1. Production of single cell proterin from brewery spent grains ...

    African Journals Online (AJOL)

    The production of single cell protein (SCP) by the propagation of the yeast, Saccharomyces cerevisae obtained from the Federal Institute of Industrial Research Oshtxli was studied by using the extract of spent grains obtained from the International Beer and Beverage Industries, Kacltuia, Nigeria as a substrate in a medium ...

  2. Single-cell LEP-type cavity on measurement stand

    CERN Multimedia

    CERN PhotoLab

    1982-01-01

    A single-cell cavity, made of copper, with tapered connectors for impedance measurements. It was used as a model of LEP-type superconducting cavities, to investigate impedance and higher-order modes and operated at around 600 MHz (the LEP acceleration frequency was 352.2 MHz). See 8202500.

  3. Conversion of Food waste to Single Cell Protein using Aspergillus ...

    African Journals Online (AJOL)

    ADOWIE PERE

    2018-03-13

    Mar 13, 2018 ... ABSTRACT: The utilization of food waste into products like single cell protein is an alternative solution to global protein shortage ... as orange, pineapple, banana, watermelon and cucumber waste as growth media for A. niger using standard techniques. ..... Waste to Wealth- Value Recovery from. Agrofood.

  4. Conversion of Food waste to Single Cell Protein using Aspergillus ...

    African Journals Online (AJOL)

    The utilization of food waste into products like single cell protein is an alternative solution to global protein shortage and to alleviate pollution problems. This investigation was carried out with food wastes such as orange, pineapple, banana, watermelon and cucumber waste as growth media for A. niger using standard ...

  5. Single-cell sequencing to quantify genomic integrity in cancer

    NARCIS (Netherlands)

    van den Bos, Hilda; Bakker, Bjorn; Spierings, Diana C J; Lansdorp, Peter M; Foijer, Floris

    The use of single-cell DNA sequencing (sc-seq) techniques for the diagnosis, prognosis and treatment of cancer is a rapidly developing field. Sc-seq research is gaining momentum by decreased sequencing costs and continuous improvements in techniques. In this review, we provide an overview of recent

  6. Evaluation of yeast single cell protein (SCP) diets on growth ...

    African Journals Online (AJOL)

    Jane

    An investigation was carried out on the possibility of replacing fishmeal with graded levels of yeast single cell protein (SCP; 10, 20, 30, 40 and 50%) in isonitrogenous feed formulations (30% protein) in the diet of Oreochromis niloticus fingerlings for a period of 12 weeks. The control diet had fishmeal as the primary protein ...

  7. Correlated receptor transport processes buffer single-cell heterogeneity.

    Science.gov (United States)

    Kallenberger, Stefan M; Unger, Anne L; Legewie, Stefan; Lymperopoulos, Konstantinos; Klingmüller, Ursula; Eils, Roland; Herten, Dirk-Peter

    2017-09-01

    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.

  8. 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.

  9. Mie scatter corrections in single cell infrared microspectroscopy.

    Science.gov (United States)

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

    2016-06-23

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

  10. Photoacoustic imaging of single circulating melanoma cells in vivo

    Science.gov (United States)

    Wang, Lidai; Yao, Junjie; Zhang, Ruiying; Xu, Song; Li, Guo; Zou, Jun; Wang, Lihong V.

    2015-03-01

    Melanoma, one of the most common types of skin cancer, has a high mortality rate, mainly due to a high propensity for tumor metastasis. The presence of circulating tumor cells (CTCs) is a potential predictor for metastasis. Label-free imaging of single circulating melanoma cells in vivo provides rich information on tumor progress. Here we present photoacoustic microscopy of single melanoma cells in living animals. We used a fast-scanning optical-resolution photoacoustic microscope to image the microvasculature in mouse ears. The imaging system has sub-cellular spatial resolution and works in reflection mode. A fast-scanning mirror allows the system to acquire fast volumetric images over a large field of view. A 500-kHz pulsed laser was used to image blood and CTCs. Single circulating melanoma cells were imaged in both capillaries and trunk vessels in living animals. These high-resolution images may be used in early detection of CTCs with potentially high sensitivity. In addition, this technique enables in vivo study of tumor cell extravasation from a primary tumor, which addresses an urgent pre-clinical need.

  11. 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.

  12. Preparation of Single Cells for Imaging Mass Spectrometry

    Energy Technology Data Exchange (ETDEWEB)

    Berman, E S; Fortson, S L; Kulp, K S; Checchi, K D; Wu, L; Felton, J S; Wu, K J

    2007-10-24

    Characterizing chemical changes within single cells is important for determining fundamental mechanisms of biological processes that will lead to new biological insights and improved disease understanding. Imaging biological systems with mass spectrometry (MS) has gained popularity in recent years as a method for creating precise chemical maps of biological samples. In order to obtain high-quality mass spectral images that provide relevant molecular information about individual cells, samples must be prepared so that salts and other cell-culture components are removed from the cell surface and the cell contents are rendered accessible to the desorption beam. We have designed a cellular preparation protocol for imaging MS that preserves the cellular contents for investigation and removes the majority of the interfering species from the extracellular matrix. Using this method, we obtain excellent imaging results and reproducibility in three diverse cell types: MCF7 human breast cancer cells, Madin-Darby canine kidney (MDCK) cells, and NIH/3T3 mouse fibroblasts. This preparation technique allows routine imaging MS analysis of cultured cells, allowing for any number of experiments aimed at furthering scientific understanding of molecular processes within individual cells.

  13. Single-cell Western blotting after whole-cell imaging to assess cancer chemotherapeutic response.

    Science.gov (United States)

    Kang, Chi-Chih; Lin, Jung-Ming G; Xu, Zhuchen; Kumar, Sanjay; Herr, Amy E

    2014-10-21

    Intratumor heterogeneity remains a major obstacle to effective cancer therapy and personalized medicine. Current understanding points to differential therapeutic response among subpopulations of tumor cells as a key challenge to successful treatment. To advance our understanding of how this heterogeneity is reflected in cell-to-cell variations in chemosensitivity and expression of drug-resistance proteins, we optimize and apply a new targeted proteomics modality, single-cell western blotting (scWestern), to a human glioblastoma cell line. To acquire both phenotypic and proteomic data on the same, single glioblastoma cells, we integrate high-content imaging prior to the scWestern assays. The scWestern technique supports thousands of concurrent single-cell western blots, with each assay comprised of chemical lysis of single cells seated in microwells, protein electrophoresis from those microwells into a supporting polyacrylamide (PA) gel layer, and in-gel antibody probing. We systematically optimize chemical lysis and subsequent polyacrylamide gel electrophoresis (PAGE) of the single-cell lysate. The scWestern slides are stored for months then reprobed, thus allowing archiving and later analysis as relevant to sparingly limited, longitudinal cell specimens. Imaging and scWestern analysis of single glioblastoma cells dosed with the chemotherapeutic daunomycin showed both apoptotic (cleaved caspase 8- and annexin V-positive) and living cells. Intriguingly, living glioblastoma subpopulations show up-regulation of a multidrug resistant protein, P-glycoprotein (P-gp), suggesting an active drug efflux pump as a potential mechanism of drug resistance. Accordingly, linking of phenotype with targeted protein analysis with single-cell resolution may advance our understanding of drug response in inherently heterogeneous cell populations, such as those anticipated in tumors.

  14. 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.

  15. 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.

  16. High resolution ultrasound and photoacoustic imaging of single cells

    Directory of Open Access Journals (Sweden)

    Eric M. Strohm

    2016-03-01

    Full Text Available High resolution ultrasound and photoacoustic images of stained neutrophils, lymphocytes and monocytes from a blood smear were acquired using a combined acoustic/photoacoustic microscope. Photoacoustic images were created using a pulsed 532 nm laser that was coupled to a single mode fiber to produce output wavelengths from 532 nm to 620 nm via stimulated Raman scattering. The excitation wavelength was selected using optical filters and focused onto the sample using a 20× objective. A 1000 MHz transducer was co-aligned with the laser spot and used for ultrasound and photoacoustic images, enabling micrometer resolution with both modalities. The different cell types could be easily identified due to variations in contrast within the acoustic and photoacoustic images. This technique provides a new way of probing leukocyte structure with potential applications towards detecting cellular abnormalities and diseased cells at the single cell level.

  17. Single Particle Tracking: Analysis Techniques for Live Cell Nanoscopy

    Science.gov (United States)

    Relich, Peter Kristopher, II

    Single molecule experiments are a set of experiments designed specifically to study the properties of individual molecules. It has only been in the last three decades where single molecule experiments have been applied to the life sciences; where they have been successfully implemented in systems biology for probing the behaviors of sub-cellular mechanisms. The advent and growth of super-resolution techniques in single molecule experiments has made the fundamental behaviors of light and the associated nano-probes a necessary concern amongst life scientists wishing to advance the state of human knowledge in biology. This dissertation disseminates some of the practices learned in experimental live cell microscopy. The topic of single particle tracking is addressed here in a format that is designed for the physicist who embarks upon single molecule studies. Specifically, the focus is on the necessary procedures to generate single particle tracking analysis techniques that can be implemented to answer biological questions. These analysis techniques range from designing and testing a particle tracking algorithm to inferring model parameters once an image has been processed. The intellectual contributions of the author include the techniques in diffusion estimation, localization filtering, and trajectory associations for tracking which will all be discussed in detail in later chapters. The author of this thesis has also contributed to the software development of automated gain calibration, live cell particle simulations, and various single particle tracking packages. Future work includes further evaluation of this laboratory's single particle tracking software, entropy based approaches towards hypothesis validations, and the uncertainty quantification of gain calibration.

  18. 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).

  19. 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 ...

  20. Opto-acoustic microscopy reveals adhesion mechanics of single cells

    Science.gov (United States)

    Abi Ghanem, Maroun; Dehoux, Thomas; Liu, Liwang; Le Saux, Guillaume; Plawinski, Laurent; Durrieu, Marie-Christine; Audoin, Bertrand

    2018-01-01

    Laser-generated GHz-ultrasonic-based technologies have shown the ability to image single cell adhesion and stiffness simultaneously. Using this new modality, we here demonstrate quantitative indicators to investigate contact mechanics and adhesion processes of the cell. We cultured human cells on a rigid substrate, and we used an inverted pulsed opto-acoustic microscope to generate acoustic pulses containing frequencies up to 100 GHz in the substrate. We map the reflection of the acoustic pulses at the cell-substrate interface to obtain images of the acoustic impedance of the cell, Zc, as well as of the stiffness of the interface, K, with 1 μm lateral resolution. Our results show that the standard deviation ΔZc reveals differences between different cell types arising from the multiplicity of local conformations within the nucleus. From the distribution of K-values within the nuclear region, we extract a mean interfacial stiffness, Km, that quantifies the average contact force in areas of the cell displaying weak bonding. By analogy with classical contact mechanics, we also define the ratio of the real to nominal contact areas, Sr/St. We show that Km can be interpreted as a quantitative indicator of passive contact at metal-cell interfaces, while Sr/St is sensitive to active adhesive processes in the nuclear region. The ability to separate the contributions of passive and active adhesion processes should allow gaining insight into cell-substrate interactions, with important applications in tissue engineering.

  1. 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

  2. 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

  3. 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.

  4. 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).

  5. Dissecting the Cell Entry Pathway of Dengue Virus by Single-Particle Tracking in Living Cells

    NARCIS (Netherlands)

    van der Schaar, Hilde M.; Rust, Michael J.; Chen, Chen; van der Ende-Metselaar, Heidi; Wilschut, Jan; Zhuang, Xiaowei; Smit, Jolanda M.

    2008-01-01

    Dengue virus (DENV) is an enveloped RNA virus that causes the most common arthropod-borne infection worldwide. The mechanism by which DENV infects the host cell remains unclear. In this work, we used live-cell imaging and single-virus tracking to investigate the cell entry, endocytic trafficking,

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

    Science.gov (United States)

    Teschendorff, Andrew E.; Enver, Tariq

    2017-06-01

    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.

  7. 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

  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. 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.

  10. Single particle labeling of RNA virus in live cells.

    Science.gov (United States)

    Liu, Xiaohui; Ouyang, Ting; Ouyang, Hongsheng; Ren, Linzhu

    2017-06-02

    Real-time and visual tracking of viral infection is crucial for elucidating the infectious and pathogenesis mechanisms. To track the virus successfully, an efficient labeling method is necessary. In this review, we first discuss the practical labeling techniques for virus tracking in live cells. We then describe the current knowledge of interactions between RNA viruses (especially influenza viruses, immunodeficiency viruses, and Flaviviruses) and host cellular structures, obtained using single particle labeling techniques combined with real-time fluorescence microscopy. Single particle labeling provides an easy system for understanding the RNA virus life cycle. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. Preliminary investigation of single chamber single electrode microbial fuel cell using sewage sludge as a substrate

    Science.gov (United States)

    Sai Chaithanya, M.; Thakur, Somil; Sonu, Kumar; Das, Bhaskar

    2017-11-01

    A microbial fuel cell (MFC) consists of a cathode and anode; micro-organisms transfer electrons acquired from the degradation of organic matter in the substrate to anode; and thereby to cathode; by using an external circuit to generate electricity. In the present study, a single chamber single electrode microbial fuel cell has been fabricated to generate electricity from the sludge of the sewage treatment plant at two different ambient temperature range of 25 ± 4°C and 32 ± 4°C under aerobic condition. No work has been done yet by using the single electrode in any MFC system; it is hypothesized that single electrode submerged partially in substrate and rest to atmosphere can function as both cathode and anode. The maximum voltage obtained was about 2890 mV after 80 (hrs) at temperature range of 25 ± 4°C, with surface power density of 1108.29 mW/m2. When the ambient temperature was 32 ± 4°C, maximum voltage obtained was 1652 mV after 40 (hrs.) surface power density reduced to 865.57 mW/m2. When amount of substrate was decreased for certain area of electrode at 25 ± 4°C range, electricity generation decreased and it also shortened the time to reach peak voltage. On the other hand, when the ambient temperature was increased to 32 ± 4°C, the maximum potential energy generated was less than that of previous experiment at 25 ± 4°C for the same substrate Also the time to reach peak voltage decreased to 40 hrs. When comparing with other single chamber single electrode MFC, the present model is generating more electricity that any MFC using sewage sludge as substrate except platinum electrode, which is much costlier that electrode used in the present study.

  12. Digital cell counting device integrated with a single-cell array.

    Directory of Open Access Journals (Sweden)

    Tatsuya Saeki

    Full Text Available In this paper, we present a novel cell counting method accomplished using a single-cell array fabricated on an image sensor, complementary metal oxide semiconductor sensor. The single-cell array was constructed using a microcavity array, which can trap up to 7,500 single cells on microcavities periodically arranged on a plane metallic substrate via the application of a negative pressure. The proposed method for cell counting is based on shadow imaging, which uses a light diffraction pattern generated by the microcavity array and trapped cells. Under illumination, the cell-occupied microcavities are visualized as shadow patterns in an image recorded by the complementary metal oxide semiconductor sensor due to light attenuation. The cell count is determined by enumerating the uniform shadow patterns created from one-on-one relationships with single cells trapped on the microcavities in digital format. In the experiment, all cell counting processes including entrapment of non-labeled HeLa cells from suspensions on the array and image acquisition of a wide-field-of-view of 30 mm(2 in 1/60 seconds were implemented in a single integrated device. As a result, the results from the digital cell counting had a linear relationship with those obtained from microscopic observation (r(2  = 0.99. This platform could be used at extremely low cell concentrations, i.e., 25-15,000 cells/mL. Our proposed system provides a simple and rapid miniaturized cell counting device for routine laboratory use.

  13. 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.

  14. 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

  15. Current Developments in Prokaryotic Single Cell Whole Genome Amplification

    Energy Technology Data Exchange (ETDEWEB)

    Goudeau, Danielle; Nath, Nandita; Ciobanu, Doina; Cheng, Jan-Fang; Malmstrom, Rex

    2014-03-14

    Our approach to prokaryotic single-cell Whole Genome Amplification at the JGI continues to evolve. To increase both the quality and number of single-cell genomes produced, we explore all aspects of the process from cell sorting to sequencing. For example, we now utilize specialized reagents, acoustic liquid handling, and reduced reaction volumes eliminate non-target DNA contamination in WGA reactions. More specifically, we use a cleaner commercial WGA kit from Qiagen that employs a UV decontamination procedure initially developed at the JGI, and we use the Labcyte Echo for tip-less liquid transfer to set up 2uL reactions. Acoustic liquid handling also dramatically reduces reagent costs. In addition, we are exploring new cell lysis methods including treatment with Proteinase K, lysozyme, and other detergents, in order to complement standard alkaline lysis and allow for more efficient disruption of a wider range of cells. Incomplete lysis represents a major hurdle for WGA on some environmental samples, especially rhizosphere, peatland, and other soils. Finding effective lysis strategies that are also compatible with WGA is challenging, and we are currently assessing the impact of various strategies on genome recovery.

  16. 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.

  17. Gravity research on plants: use of single cell experimental models

    Directory of Open Access Journals (Sweden)

    Youssef eChebli

    2011-09-01

    Full Text Available Future space missions and implementation of permanent bases on Moon and Mars will greatly depend on the availability of ambient air and sustainable food supply. Therefore, understanding the effects of altered gravity conditions on plant metabolism and growth is vital for space missions and extra-terrestrial human existence. In this mini-review we summarize how plant cells are thought to perceive changes in magnitude and orientation of the gravity vector. The particular advantages of several single celled model systems for gravity research are explored and an overview over recent advancements and potential use of these systems is provided.

  18. New library construction method for single-cell genomes.

    Directory of Open Access Journals (Sweden)

    Larry Xi

    Full Text Available A central challenge in sequencing single-cell genomes is the accurate determination of point mutations, phasing of these mutations, and identifying copy number variations with few assumptions. Ideally, this is accomplished under as low sequencing coverage as possible. Here we report our attempt to meet these goals with a novel library construction and library amplification methodology. In our approach, single-cell genomic DNA is first fragmented with saturated transposition to make a primary library that uniformly covers the whole genome by short fragments. The library is then amplified by a carefully optimized PCR protocol in a uniform and synchronized fashion for next-generation sequencing. Each step of the protocol can be quantitatively characterized. Our shallow sequencing data show that the library is tightly distributed and is useful for the determination of copy number variations.

  19. 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.

  20. STABILITY OF AXIALLY COMPRESSED SINGLE-CELL MONO ...

    African Journals Online (AJOL)

    60. N.N. OSADEBE and J.C. EZEH. Figure 1 shows one of the cross sections of a single-cell mono symmetric thin-walled closed column under consideration. Using. Lagrange's principle, Vlasov [6] expressed the displacements in the longitudinal and transverse directions, u(x, s) and v(x, s) of a thin-walled closed structure in ...

  1. Multicolor imaging of cancer cells with fluorophore-tagged aptamers for single cell typing.

    Science.gov (United States)

    Wang, Song; Kong, Hao; Gong, Xiaoyun; Zhang, Sichun; Zhang, Xinrong

    2014-08-19

    The discrimination of the type of cancer cells remains challenging due to the subtle differences in their expression of membrane receptors. In this work, we developed a multicolor cell imaging method for distinguishing the type of cancer cells with fluorophore-tagged aptamers. We found that the interaction between aptamers and cancer cells was affected by both of the sequence of aptamers and the labeled dyes. As the co-ownership of biomarkers for different cancer cell lines, the fluorophore-tagged aptamers interacted with different cancer cell lines in different degree, resulting in a distinct color to discriminate the type of cancer cells at single cell level. Taking advantage of the cross-reactive ability of the fluorophore-tagged aptamers, we could not only distinguish the cancerous cells quickly from large quantities of noncancerous cells, but also identify the type of the cancerous cells. This work has potential application for cancer diagnostic and therapy in the future.

  2. Morphological and anatomical determinants of mesophyll conductance in wild relatives of tomato (Solanum sect. Lycopersicon, sect. Lycopersicoides; Solanaceae).

    Science.gov (United States)

    Muir, Christopher D; Hangarter, Roger P; Moyle, Leonie C; Davis, Phillip A

    2014-06-01

    Natural selection on photosynthetic performance is a primary factor determining leaf phenotypes. The complex CO2 diffusion path from substomatal cavities to the chloroplasts - the mesophyll conductance (g(m)) - limits photosynthetic rate in many species and hence shapes variation in leaf morphology and anatomy. Among sclerophyllous and succulent taxa, structural investment in leaves, measured as the leaf dry mass per area (LMA), has been implicated in decreased gm . However, in herbaceous taxa with high g(m), it is less certain how LMA impacts CO2 diffusion and whether it significantly affects photosynthetic performance. We addressed these questions in the context of understanding the ecophysiological significance of leaf trait variation in wild tomatoes, a closely related group of herbaceous perennials. Although g(m) was high in wild tomatoes, variation in g(m) significantly affected photosynthesis. Even in these tender-leaved herbaceous species, greater LMA led to reduced g(m). This relationship between g(m) and LMA is partially mediated by cell packing and leaf thickness, although amphistomy (equal distribution of stomata on both sides of the leaf) mitigates the effect of leaf thickness. Understanding the costs of increased LMA will inform future work on the adaptive significance of leaf trait variation across ecological gradients in wild tomatoes and other systems. © 2013 John Wiley & Sons Ltd.

  3. [AGGLUTINATION OF MESOPHYLL PLASTIDS AND OBLITERATION OF PHLOEM SIEVE TUBES ARE THE TOTAL RESULT OF SEASONAL PAUSES IN PHOTOSYNTHATE EXPORT].

    Science.gov (United States)

    Gamalei, Yu V

    2015-01-01

    Chloroplast agglutination and sieve tube obliteration are related to the different plant tissues: the agglutination--to the leaf mesophyll, and the obliteration--to the axis phloem. Being equally produced by photosynthate export dynamics, both phenomena are synchronous and can be used for diagnostics of seasonal flashes and pauses of photosynthetic activity with equal success. The nature of the mobility of chloroplast and their shuttle displacements from the nuclear envelope to the cell periphery connected with export dynamics have been established. It is assumed that nuclear envelope is the base structure of the endoplasmic reticulum (ER) inside which the chloroplasts are localized. Activation of photosynthesis and sugar accumulation inside the ER induces its expansion followed by centrifugal diffusion of chloroplasts. Come back effect--ER collapse, its return to the source--can be induced by the blockade of photosynthesis. Centripetal collapse is accompanied by plastid concentration around the nuclear envelope. Displacements of ER and the chloroplasts dislocating inside it are reversible. It depends on seasonal fluctuations of photosynthesis and export intensities. Changes in the volume of sieve tubes, which are due to the same reason, are irreversible. Each seasonal wave of photosynthesis and sugar export forms new series of sieve tubes, replacing obliterated ones.

  4. Large-scale protein-protein interaction analysis in Arabidopsis mesophyll protoplasts by split firefly luciferase complementation.

    Directory of Open Access Journals (Sweden)

    Jian-Feng Li

    Full Text Available Protein-protein interactions (PPIs constitute the regulatory network that coordinates diverse cellular functions. There are growing needs in plant research for creating protein interaction maps behind complex cellular processes and at a systems biology level. However, only a few approaches have been successfully used for large-scale surveys of PPIs in plants, each having advantages and disadvantages. Here we present split firefly luciferase complementation (SFLC as a highly sensitive and noninvasive technique for in planta PPI investigation. In this assay, the separate halves of a firefly luciferase can come into close proximity and transiently restore its catalytic activity only when their fusion partners, namely the two proteins of interest, interact with each other. This assay was conferred with quantitativeness and high throughput potential when the Arabidopsis mesophyll protoplast system and a microplate luminometer were employed for protein expression and luciferase measurement, respectively. Using the SFLC assay, we could monitor the dynamics of rapamycin-induced and ascomycin-disrupted interaction between Arabidopsis FRB and human FKBP proteins in a near real-time manner. As a proof of concept for large-scale PPI survey, we further applied the SFLC assay to testing 132 binary PPIs among 8 auxin response factors (ARFs and 12 Aux/IAA proteins from Arabidopsis. Our results demonstrated that the SFLC assay is ideal for in vivo quantitative PPI analysis in plant cells and is particularly powerful for large-scale binary PPI screens.

  5. Comparative Studies of Polymer Electrolyte Membrane Fuel Cell Stacks and Single Cells

    Science.gov (United States)

    2000-02-01

    in the Catalyst Layer and Effects of Both Perfluorosulfonate Ionomer and PTFE-Loaded Carbon on the Catalyst Layer of Polymer Electrolyte Fuel Cells ...financial support of this project. 12 References 1. T. F. Fuller, "Is a Fuel Cell in Your Future?" 77K Electrochemical Society Interface (Fall...ARMY RESEARCH LABORATORY mm^ n Comparative Studies of Polymer Electrolyte Membrane Fuel Cell Stacks and Single Cells Deryn Chu and Rongzhong

  6. Exploring Arabidopsis thaliana Root Endophytes via Single-Cell Genomics

    Energy Technology Data Exchange (ETDEWEB)

    Lundberg, Derek; Woyke, Tanja; Tringe, Susannah; Dangl, Jeff

    2014-03-19

    Land plants grow in association with microbial communities both on their surfaces and inside the plant (endophytes). The relationships between microbes and their host can vary from pathogenic to mutualistic. Colonization of the endophyte compartment occurs in the presence of a sophisticated plant immune system, implying finely tuned discrimination of pathogens from mutualists and commensals. Despite the importance of the microbiome to the plant, relatively little is known about the specific interactions between plants and microbes, especially in the case of endophytes. The vast majority of microbes have not been grown in the lab, and thus one of the few ways of studying them is by examining their DNA. Although metagenomics is a powerful tool for examining microbial communities, its application to endophyte samples is technically difficult due to the presence of large amounts of host plant DNA in the sample. One method to address these difficulties is single-cell genomics where a single microbial cell is isolated from a sample, lysed, and its genome amplified by multiple displacement amplification (MDA) to produce enough DNA for genome sequencing. This produces a single-cell amplified genome (SAG). We have applied this technology to study the endophytic microbes in Arabidopsis thaliana roots. Extensive 16S gene profiling of the microbial communities in the roots of multiple inbred A. thaliana strains has identified 164 OTUs as being significantly enriched in all the root endophyte samples compared to their presence in bulk soil.

  7. Single Cell Genomics and Transcriptomics for Unicellular Eukaryotes

    Energy Technology Data Exchange (ETDEWEB)

    Ciobanu, Doina; Clum, Alicia; Singh, Vasanth; Salamov, Asaf; Han, James; Copeland, Alex; Grigoriev, Igor; James, Timothy; Singer, Steven; Woyke, Tanja; Malmstrom, Rex; Cheng, Jan-Fang

    2014-03-14

    Despite their small size, unicellular eukaryotes have complex genomes with a high degree of plasticity that allow them to adapt quickly to environmental changes. Unicellular eukaryotes live with prokaryotes and higher eukaryotes, frequently in symbiotic or parasitic niches. To this day their contribution to the dynamics of the environmental communities remains to be understood. Unfortunately, the vast majority of eukaryotic microorganisms are either uncultured or unculturable, making genome sequencing impossible using traditional approaches. We have developed an approach to isolate unicellular eukaryotes of interest from environmental samples, and to sequence and analyze their genomes and transcriptomes. We have tested our methods with six species: an uncharacterized protist from cellulose-enriched compost identified as Platyophrya, a close relative of P. vorax; the fungus Metschnikowia bicuspidate, a parasite of water flea Daphnia; the mycoparasitic fungi Piptocephalis cylindrospora, a parasite of Cokeromyces and Mucor; Caulochytrium protosteloides, a parasite of Sordaria; Rozella allomycis, a parasite of the water mold Allomyces; and the microalgae Chlamydomonas reinhardtii. Here, we present the four components of our approach: pre-sequencing methods, sequence analysis for single cell genome assembly, sequence analysis of single cell transcriptomes, and genome annotation. This technology has the potential to uncover the complexity of single cell eukaryotes and their role in the environmental samples.

  8. 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.

  9. A microfluidic platform for regulating signal transduction in single cells

    Science.gov (United States)

    Wong, Pak Kin; Yu, Fuqu; Sun, Ren; Ho, Chih-Ming

    2004-11-01

    Recent progress in micro cell culture systems has lead to new approaches in cell biology studies. Using micro devices for cell culturing possesses distinctive advantages over traditional methods. Length scale matching facilitates manipulation and detection at the single cell level. Previously, we have demonstrated generation of various stimulations such as spatial chemical gradient, electric field, and shear stress to study the dynamic responses of individual cells. Dynamic stimulations and continuous monitoring in a microfluidic system can be useful in studying different aspects of cellular process. In this work, we present a microfluidic platform for regulating nuclear factor kappa B (NF-kB) signal transduction in human embryonic kidney 293T cells. Time-varying bio-chemical stimulants, such as interleukin 1 and tumor necrosis factor, are introduced into the microchannel to activate the NF-kB signaling pathway. The dynamic responses of individual cells are monitored with the expression of reporter gene, green fluorescent protein. Regulation of the NF-kB activity is successfully demonstrated. This work is supported by CMISE through NASA URETI program.

  10. A membraneless single compartment abiotic glucose fuel cell

    Science.gov (United States)

    Slaughter, Gymama; Sunday, Joshua

    2014-09-01

    A simple energy harvesting strategy has been developed to selectively catalyze glucose in the presence of oxygen in a glucose/O2 fuel cell. The anode consists of an abiotic catalyst Al/Au/ZnO, in which ZnO seed layer was deposited on the surface of Al/Au substrate using hydrothermal method. The cathode is constructed from a single rod of platinum with an outer diameter of 500 μm. The abiotic glucose fuel cell was studied in phosphate buffer solution (pH 7.4) containing 5 mM glucose at a temperature of 22 °C. The cell is characterized according to its open-circuit voltage, polarization profile, and power density plot. Under these conditions, the abiotic glucose fuel cell possesses an open-circuit voltage of 840 mV and delivered a maximum power density of 16.2 μW cm-2 at a cell voltage of 495 mV. These characteristics are comparable to biofuel cell utilizing a much more complex system design. Such low-cost lightweight abiotic catalyzed glucose fuel cells have a great promise to be optimized, miniaturized to power bio-implantable devices.

  11. Single-Walled Carbon Nanotubes in Solar Cells.

    Science.gov (United States)

    Jeon, Il; Matsuo, Yutaka; Maruyama, Shigeo

    2018-01-22

    Photovoltaics, more generally known as solar cells, are made from semiconducting materials that convert light into electricity. Solar cells have received much attention in recent years due to their promise as clean and efficient light-harvesting devices. Single-walled carbon nanotubes (SWNTs) could play a crucial role in these devices and have been the subject of much research, which continues to this day. SWNTs are known to outperform multi-walled carbon nanotubes (MWNTs) at low densities, because of the difference in their optical transmittance for the same current density, which is the most important parameter in comparing SWNTs and MWNTs. SWNT films show semiconducting features, which make SWNTs function as active or charge-transporting materials. This chapter, consisting of two sections, focuses on the use of SWNTs in solar cells. In the first section, we discuss SWNTs as a light harvester and charge transporter in the photoactive layer, which are reviewed chronologically to show the history of the research progress. In the second section, we discuss SWNTs as a transparent conductive layer outside of the photoactive layer, which is relatively more actively researched. This section introduces SWNT applications in silicon solar cells, organic solar cells, and perovskite solar cells each, from their prototypes to recent results. As we go along, the science and prospects of the application of solar cells will be discussed.

  12. Abscisic Acid Induces Rapid Reductions in Mesophyll Conductance to Carbon Dioxide.

    Directory of Open Access Journals (Sweden)

    Giuseppe Sorrentino

    Full Text Available The rate of photosynthesis (A of plants exposed to water deficit is a function of stomatal (gs and mesophyll (gm conductance determining the availability of CO2 at the site of carboxylation within the chloroplast. Mesophyll conductance often represents the greatest impediment to photosynthetic uptake of CO2, and a crucial determinant of the photosynthetic effects of drought. Abscisic acid (ABA plays a fundamental role in signalling and co-ordination of plant responses to drought; however, the effect of ABA on gm is not well-defined. Rose, cherry, olive and poplar were exposed to exogenous ABA and their leaf gas exchange parameters recorded over a four hour period. Application with ABA induced reductions in values of A, gs and gm in all four species. Reduced gm occurred within one hour of ABA treatment in three of the four analysed species; indicating that the effect of ABA on gm occurs on a shorter timescale than previously considered. These declines in gm values associated with ABA were not the result of physical changes in leaf properties due to altered turgor affecting movement of CO2, or caused by a reduction in the sub-stomatal concentration of CO2 (Ci. Increased [ABA] likely induces biochemical changes in the properties of the interface between the sub-stomatal air-space and mesophyll layer through the actions of cooporins to regulate the transport of CO2. The results of this study provide further evidence that gm is highly responsive to fluctuations in the external environment, and stress signals such as ABA induce co-ordinated modifications of both gs and gm in the regulation of photosynthesis.

  13. Single cell cytometry of protein function in RNAi treated cells and in native populations

    Directory of Open Access Journals (Sweden)

    Hill Andrew

    2008-08-01

    Full Text Available Abstract Background High Content Screening has been shown to improve results of RNAi and other perturbations, however significant intra-sample heterogeneity is common and can complicate some analyses. Single cell cytometry can extract important information from subpopulations within these samples. Such approaches are important for immune cells analyzed by flow cytometry, but have not been broadly available for adherent cells that are critical to the study of solid-tumor cancers and other disease models. Results We have directly quantitated the effect of resolving RNAi treatments at the single cell level in experimental systems for both exogenous and endogenous targets. Analyzing the effect of an siRNA that targets GFP at the single cell level permits a stronger measure of the absolute function of the siRNA by gating to eliminate background levels of GFP intensities. Extending these methods to endogenous proteins, we have shown that well-level results of the knockdown of PTEN results in an increase in phospho-S6 levels, but at the single cell level, the correlation reveals the role of other inputs into the pathway. In a third example, reduction of STAT3 levels by siRNA causes an accumulation of cells in the G1 phase of the cell cycle, but does not induce apoptosis or necrosis when compared to control cells that express the same levels of STAT3. In a final example, the effect of reduced p53 levels on increased adriamycin sensitivity for colon carcinoma cells was demonstrated at the whole-well level using siRNA knockdown and in control and untreated cells at the single cell level. Conclusion We find that single cell analysis methods are generally applicable to a wide range of experiments in adherent cells using technology that is becoming increasingly available to most laboratories. It is well-suited to emerging models of signaling dysfunction, such as oncogene addition and oncogenic shock. Single cell cytometry can demonstrate effects on cell

  14. Plant regeneration from mesophyll protoplasts of Matthiola incana (L.) R. Br.

    Science.gov (United States)

    Siemens, J; Sacristán, M D

    1995-04-01

    A protocol for obtaining regenerated fertile plants from mesophyll protoplasts of three lines of Matthiola incana is described. Protoplasts were isolated from leaves of 21-28 days old Matthiola plants grown in controlled environment. Sustained divisions were achieved when protoplasts were embedded in sodium alginate. Up to 2.0 % of the protoplasts developed into colonies which could be transferred to shoot regeneration media. More than 25 % of the obtained calluses regenerated shoots. About 4 % of these shoots could be rooted and after transfer to soil phenotypically normal plants have been obtained.

  15. Genetic biosensors for imaging nitric oxide in single cells.

    Science.gov (United States)

    Eroglu, Emrah; Charoensin, Suphachai; Bischof, Helmut; Ramadani, Jeta; Gottschalk, Benjamin; Depaoli, Maria R; Waldeck-Weiermair, Markus; Graier, Wolfgang F; Malli, Roland

    2018-02-01

    Over the last decades a broad collection of sophisticated fluorescent protein-based probes was engineered with the aim to specifically monitor nitric oxide (NO), one of the most important signaling molecules in biology. Here we report and discuss the characteristics and fields of applications of currently available genetically encoded fluorescent sensors for the detection of NO and its metabolites in different cell types. Because of its radical nature and short half-life, real-time imaging of NO on the level of single cells is challenging. Herein we review state-of-the-art genetically encoded fluorescent sensors for NO and its byproducts such as peroxynitrite, nitrite and nitrate. Such probes enable the real-time visualization of NO signals directly or indirectly on the level of single cells and cellular organelles and, hence, extend our understanding of the spatiotemporal dynamics of NO formation, diffusion and degradation. Here, we discuss the significance of NO detection in individual cells and on subcellular level with genetic biosensors. Currently available genetically encoded fluorescent probes for NO and nitrogen species are critically discussed in order to provide insights in the functionality and applicability of these promising tools. As an outlook we provide ideas for novel approaches for the design and application of improved NO probes and fluorescence imaging protocols. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

  16. 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.

  17. Single material solar cells: the next frontier for organic photovoltaics?

    Energy Technology Data Exchange (ETDEWEB)

    Roncali, Jean [Group Linear Conjugated Systems, CNRS, Moltech-Anjou, UMR 6200, University of Angers, 2 Bd Lavoisier 49045 Angers (France)

    2011-03-18

    An overview of various approaches for the realization of single-material organic solar cells (SMOCs) is presented. Fullerene-conjugated systems dyads, di-block copolymers, and self-organized donor-acceptor molecules all represent different possible approaches towards SMOCs. Although each of them presents specific advantages and poses specific problems of design and synthesis, these different routes have witnessed significant progress in the past few years and SMOCs with efficiencies in the range of 1.50% have been realized. These performances are already higher than those of bi-component bulk heterojunction solar cells some ten years ago, demonstrating that SMOCs can represent a credible approach towards efficient and simple organic solar cells. Possible directions for future research are discussed with the aim of stimulating further research on this exciting topic. (Copyright copyright 2011 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim)

  18. Microfluidic single-cell technology in immunology and antibody screening.

    Science.gov (United States)

    Seah, Yu Fen Samantha; Hu, Hongxing; Merten, Christoph A

    2018-02-01

    Single-cell technology has a major impact on the field of immunology. It enables the kinetics and logic of immune signaling and immune cell migration to be elucidated, facilitates antibody screening and allows massively parallelized analysis of B- and T-cell repertoires. Impressive progress has been made over the last decade, strongly boosted by microfluidic approaches. In this review, we summarize the most powerful microfluidic systems based on continuous flow, nanowells, valves and droplets and we analyze their benefits for phenotypic characterization, drug discovery and next generation sequencing experiments. We describe current limitations and provide an outlook on important future applications. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

  19. 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.

  20. High-throughput full-length single-cell mRNA-seq of rare cells.

    Directory of Open Access Journals (Sweden)

    Chin Chun Ooi

    Full Text Available Single-cell characterization techniques, such as mRNA-seq, have been applied to a diverse range of applications in cancer biology, yielding great insight into mechanisms leading to therapy resistance and tumor clonality. While single-cell techniques can yield a wealth of information, a common bottleneck is the lack of throughput, with many current processing methods being limited to the analysis of small volumes of single cell suspensions with cell densities on the order of 107 per mL. In this work, we present a high-throughput full-length mRNA-seq protocol incorporating a magnetic sifter and magnetic nanoparticle-antibody conjugates for rare cell enrichment, and Smart-seq2 chemistry for sequencing. We evaluate the efficiency and quality of this protocol with a simulated circulating tumor cell system, whereby non-small-cell lung cancer cell lines (NCI-H1650 and NCI-H1975 are spiked into whole blood, before being enriched for single-cell mRNA-seq by EpCAM-functionalized magnetic nanoparticles and the magnetic sifter. We obtain high efficiency (> 90% capture and release of these simulated rare cells via the magnetic sifter, with reproducible transcriptome data. In addition, while mRNA-seq data is typically only used for gene expression analysis of transcriptomic data, we demonstrate the use of full-length mRNA-seq chemistries like Smart-seq2 to facilitate variant analysis of expressed genes. This enables the use of mRNA-seq data for differentiating cells in a heterogeneous population by both their phenotypic and variant profile. In a simulated heterogeneous mixture of circulating tumor cells in whole blood, we utilize this high-throughput protocol to differentiate these heterogeneous cells by both their phenotype (lung cancer versus white blood cells, and mutational profile (H1650 versus H1975 cells, in a single sequencing run. This high-throughput method can help facilitate single-cell analysis of rare cell populations, such as circulating tumor

  1. Rapid acquisition of mean Raman spectra of eukaryotic cells for a robust single cell classification.

    Science.gov (United States)

    Schie, Iwan W; Kiselev, Roman; Krafft, Christoph; Popp, Jürgen

    2016-11-14

    Raman spectroscopy has previously been used to identify eukaryotic and prokaryotic cells. While prokaryotic cells are small in size and can be assessed by a single Raman spectrum, the larger size of eukaryotic cells and their complex organization requires the acquisition of multiple Raman spectra to properly characterize them. A Raman spectrum from a diffraction-limited spot at an arbitrary location within a cell results in spectral variations that affect classification approaches. To probe whole cells with Raman imaging at high spatial resolution is time consuming, because a large number of Raman spectra need to be collected, resulting in low cell throughput and impairing statistical analysis due to low cell numbers. Here we propose a method to overcome the effects of cellular heterogeneity by acquiring integrated Raman spectra covering a large portion of a cell. The acquired spectrum represents the mean macromolecular composition of a cell with an exposure time that is comparable to acquisition of a single Raman spectrum. Data sets were collected from T lymphocyte Jurkat cells, and pancreatic cell lines Capan1 and MiaPaca2. Cell classification by support vector machines was compared for single spectra, spectra of images and integrated Raman spectra of cells. The integrated approach provides better and more stable prediction for individual cells, and in the current implementation, the mean macromolecular information of a cell can be acquired faster than with the acquisition of individual spectra from a comparable region. It is expected that this approach will have a major impact on the implementation of Raman based cell classification.

  2. How much territory can a single E. coli cell control?

    Directory of Open Access Journals (Sweden)

    Ziad W. El-Hajj

    2015-04-01

    Full Text Available Bacteria have been traditionally classified in terms of size and shape and are best known for their very small size. E. coli cells in particular are small rods, each 1-2 microns. However the size varies with the medium, and faster growing cells are larger because they must have more ribosomes to make more protoplasm per unit time, and ribosomes take up space. Indeed, Maaloe's experiments on how E. coli establishes its size began with shifts between rich and poor media.Recently much larger bacteria have been described, including Epulopiscium fishelsoni at 700 μm and Thiomargarita namibiensisis at 750 μm. These are not only much longer than E. coli cells but also much wider, necessitating considerable intracellular organization. Epulopiscium cells for instance, at 80 μm wide, enclose a large enough volume of cytoplasm to present it with major transport problems.This review surveys E. coli cells much longer than those which grow in nature and in usual lab cultures. These include cells mutated in a single gene (metK which are 2-4x longer than their nonmutated parent. This metK mutant stops dividing when slowly starved of S-adenosylmethionine but continues to elongate to 50 μm and more. FtsZ mutants have been routinely isolated as long cells which form during growth at 42°C. The SOS response is a well-characterized regulatory network that is activated in response to DNA damage and also results in cell elongation. Our champion elongated E. coli is a metK strain with a further, as yet unidentified mutation, which reaches 750 μm with no internal divisions and no increase in width.

  3. SmashCell: A software framework for the analysis of single-cell amplified genome sequences

    DEFF Research Database (Denmark)

    Harrington, Eoghan D; Arumugam, Manimozhiyan; Raes, Jeroen

    2010-01-01

    SUMMARY: Recent advances in single-cell manipulation technology, whole genome amplification and high-throughput sequencing have now made it possible to sequence the genome of an individual cell. The bioinformatic analysis of these genomes however is far more complicated than the analysis of those...

  4. Parallel single cell analysis on an integrated microfluidic platform for cell trapping, lysis and analysis

    NARCIS (Netherlands)

    le Gac, Severine; de Boer, Hans L.; Wijnperle, Daniël; Meuleman, W.; Carlen, Edwin; van den Berg, Albert; Kim, Tae Song; Lee, Yoon-Sik; Chung, Taek-Dong; Jeon, Noo Li; Lee, Sang-Hoon; Suh, Kahp-Yang; Choo, Jaebum; Kim, Yong-Kweon

    2009-01-01

    We report here a novel and easily scalable microfluidic platform for the parallel analysis of hundreds of individual cells, with controlled single cell trapping, followed by their lysis and subsequent retrieval of the cellular content for on-chip analysis. The device consists of a main channel and

  5. 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

  6. Sample Preparation Methods Following CellSearch Approach Compatible of Single-Cell Whole-Genome Amplification: An Overview

    NARCIS (Netherlands)

    Swennenhuis, Joost Franciscus; Terstappen, Leonardus Wendelinus Mathias Marie; Kroneis, Thomas

    2015-01-01

    Single cells are increasingly used to determine the heterogeneity of therapy targets in the genome during the course of a disease. The first challenge using single cells is to isolate these cells from the surrounding cells, especially when the targeted cells are rare. A number of techniques have

  7. Molecular Genetic Characterization of Individual Cancer Cells Isolated via Single-Cell Printing.

    Directory of Open Access Journals (Sweden)

    Julian Riba

    Full Text Available Intratumoral genetic heterogeneity may impact disease outcome. Gold standard for dissecting clonal heterogeneity are single-cell analyses. Here, we present an efficient workflow based on an advanced Single-Cell Printer (SCP device for the study of gene variants in single cancer cells. To allow for precise cell deposition into microwells the SCP was equipped with an automatic dispenser offset compensation, and the 384-microwell plates were electrostatically neutralized. The ejection efficiency was 99.7% for fluorescent beads (n = 2304 and 98.7% for human cells (U-2 OS or Kasumi-1 cancer cell line, acute myeloid leukemia [AML] patient; n = 150. Per fluorescence microscopy, 98.8% of beads were correctly delivered into the wells. A subset of single cells (n = 81 was subjected to whole genome amplification (WGA, which was successful in all cells. On empty droplets, a PCR on LINE1 retrotransposons yielded no product after WGA, verifying the absence of free-floating DNA in SCP-generated droplets. Representative gene variants identified in bulk specimens were sequenced in single-cell WGA DNA. In U-2 OS, 22 of 25 cells yielded results for both an SLC34A2 and TET2 mutation site, including cells harboring the SLC34A2 but not the TET2 mutation. In one cell, the TET2 mutation analysis was inconclusive due to allelic dropout, as assessed via polymorphisms located close to the mutation. Of Kasumi-1, 23 of 33 cells with data on both the KIT and TP53 mutation site harbored both mutations. In the AML patient, 21 of 23 cells were informative for a TP53 polymorphism; the identified alleles matched the loss of chromosome arm 17p. The advanced SCP allows efficient, precise and gentle isolation of individual cells for subsequent WGA and routine PCR/sequencing-based analyses of gene variants. This makes single-cell information readily accessible to a wide range of applications and can provide insights into clonal heterogeneity that were indeterminable solely by

  8. Production strategies and applications of microbial single cell oils

    Directory of Open Access Journals (Sweden)

    Katrin Ochsenreither

    2016-10-01

    Full Text Available Polyunsaturated fatty acids (PUFAs of the -3 and -6 class (e.g. -linolenic acid, linoleic acid are essential for maintaining biofunctions in mammalians like humans. Due to the fact that humans cannot synthesize these essential fatty acids, they must be taken up from different food sources. Classical sources for these fatty acids are porcine liver and fish oil. However, microbial lipids or single cell oils, produced by oleaginous microorganisms such as algae, fungi and bacteria, are a promising source as well. These single cell oils can be used for many valuable chemicals with applications not only for nutrition but also for fuels and are therefore an ideal basis for a bio-based economy. A crucial point for the establishment of microbial lipids utilization is the cost-effective production and purification of fuels or products of higher value. The fermentative production can be realized by submerged (SmF or solid state fermentation (SSF. The yield and the composition of the obtained microbial lipids depend on the type of fermentation and the particular conditions (e.g. medium, pH-value, temperature, aeration, nitrogen source. From an economical point of view, waste or by-product streams can be used as cheap and renewable carbon and nitrogen sources. In general, downstream processing costs are one of the major obstacles to be solved for full economic efficiency of microbial lipids. For the extraction of lipids from microbial biomass cell disruption is most important, because efficiency of cell disruption directly influences subsequent downstream operations and overall extraction efficiencies. A multitude of cell disruption and lipid extraction methods are available, conventional as well as newly emerging methods, which will be described and discussed in terms of large scale applicability, their potential in a modern biorefinery and their influence on product quality. Furthermore, an overview is given about applications of microbial lipids

  9. Production Strategies and Applications of Microbial Single Cell Oils.

    Science.gov (United States)

    Ochsenreither, Katrin; Glück, Claudia; Stressler, Timo; Fischer, Lutz; Syldatk, Christoph

    2016-01-01

    Polyunsaturated fatty acids (PUFAs) of the ω-3 and ω-6 class (e.g., α-linolenic acid, linoleic acid) are essential for maintaining biofunctions in mammalians like humans. Due to the fact that humans cannot synthesize these essential fatty acids, they must be taken up from different food sources. Classical sources for these fatty acids are porcine liver and fish oil. However, microbial lipids or single cell oils, produced by oleaginous microorganisms such as algae, fungi and bacteria, are a promising source as well. These single cell oils can be used for many valuable chemicals with applications not only for nutrition but also for fuels and are therefore an ideal basis for a bio-based economy. A crucial point for the establishment of microbial lipids utilization is the cost-effective production and purification of fuels or products of higher value. The fermentative production can be realized by submerged (SmF) or solid state fermentation (SSF). The yield and the composition of the obtained microbial lipids depend on the type of fermentation and the particular conditions (e.g., medium, pH-value, temperature, aeration, nitrogen source). From an economical point of view, waste or by-product streams can be used as cheap and renewable carbon and nitrogen sources. In general, downstream processing costs are one of the major obstacles to be solved for full economic efficiency of microbial lipids. For the extraction of lipids from microbial biomass cell disruption is most important, because efficiency of cell disruption directly influences subsequent downstream operations and overall extraction efficiencies. A multitude of cell disruption and lipid extraction methods are available, conventional as well as newly emerging methods, which will be described and discussed in terms of large scale applicability, their potential in a modern biorefinery and their influence on product quality. Furthermore, an overview is given about applications of microbial lipids or derived fatty

  10. Growth of single T cells and single thymocytes in a high cloning efficiency filler-cell free microculture system.

    Science.gov (United States)

    Chen, W F; Ewing, T; Scollay, R; Shortman, K

    1988-01-01

    A high cloning-efficiency microculture system is described in which single T cells, stimulated to divide by phorbol ester and calcium ionophore, grow rapidly under the influence of purified growth factors in the absence of other cells. The kinetics of clonal growth has been monitored over a five day period by phase-contrast microscopy. Mature peripheral T cells, and mature subpopulations from the thymus, responded with a cloning efficiency over 80%; they required IL-2 as a minimum but several other factors enhanced growth. Ly2+L3T4- thymocytes (mean doubling time 10.4 hr) grew more rapidly than Ly2-L3T4+ thymocytes (mean doubling time 15.2 hr). Early (Ly2-L3T4-) thymocytes responded with a cloning efficiency of 60%; their efficient growth was dependent on both IL-1 and IL-2. The typical Ly2+L3T4+ cortical thymocyte did not grow under these conditions.

  11. Every cell is special: genome-wide studies add a new dimension to single-cell biology.

    Science.gov (United States)

    Junker, Jan Philipp; van Oudenaarden, Alexander

    2014-03-27

    Single-cell analyses have provided invaluable insights into studying heterogenity, signaling, and stochastic gene expression. Recent technological advances now open the door to genome-wide single-cell studies. Copyright © 2014 Elsevier Inc. All rights reserved.

  12. Every cell is special : genome-wide studies add a new dimension to single-cell biology

    NARCIS (Netherlands)

    Junker, Jan Philipp; van Oudenaarden, Alexander

    2014-01-01

    Single-cell analyses have provided invaluable insights into studying heterogenity, signaling, and stochastic gene expression. Recent technological advances now open the door to genome-wide single-cell studies.

  13. Study of DNA uptake locations in single E. coli cells

    Science.gov (United States)

    Xu, C. Shan; Meadow Anderson, L.; Yang, Haw

    2006-03-01

    Artificial gene transfer of bacteria, such as E. coli, has become the main stream technique in genetic engineering and molecular cell biology studies. In spite of the great improvements in transformation efficiency, some fundamental questions remained to be answered. For instance, what are the DNA uptake channels and how do they form and function under external stimuli? Furthermore, where are these channels located on the cell membrane? Here we report a study aimed at DNA uptake locations in the two widely used gene transformation techniques: electroporation and heat shock. A direct visualization of the settling location of single DNA molecules inside individual E. coli cells was obtained by fluorescence imaging and spectroscopy. Electroporation and heat shock exhibit two distinct characteristics of DNA uptake locations. A preferential distribution toward cell poles during electroporation is consistent with earlier experiments and previously proposed models. However, the result from heat shock is unanticipated in which the majority of DNA enters the cell near the center. Such observation suggests that uptake channels form preferentially where newly-synthesized membrane is located under cation and low temperature treatment

  14. Monitoring single-channel water permeability in polarized cells.

    Science.gov (United States)

    Erokhova, Liudmila; Horner, Andreas; Kügler, Philipp; Pohl, Peter

    2011-11-18

    So far the determination of unitary permeability (p(f)) of water channels that are expressed in polarized cells is subject to large errors because the opening of a single water channel does not noticeably increase the water permeability of a membrane patch above the background. That is, in contrast to the patch clamp technique, where the single ion channel conductance may be derived from a single experiment, two experiments separated in time and/or space are required to obtain the single-channel water permeability p(f) as a function of the incremental water permeability (P(f,c)) and the number (n) of water channels that contributed to P(f,c). Although the unitary conductance of ion channels is measured in the native environment of the channel, p(f) is so far derived from reconstituted channels or channels expressed in oocytes. To determine the p(f) of channels from live epithelial monolayers, we exploit the fact that osmotic volume flow alters the concentration of aqueous reporter dyes adjacent to the epithelia. We measure these changes by fluorescence correlation spectroscopy, which allows the calculation of both P(f,c) and osmolyte dilution within the unstirred layer. Shifting the focus of the laser from the aqueous solution to the apical and basolateral membranes allowed the FCS-based determination of n. Here we validate the new technique by determining the p(f) of aquaporin 5 in Madin-Darby canine kidney cell monolayers. Because inhibition and subsequent activity rescue are monitored on the same sample, drug effects on exocytosis or endocytosis can be dissected from those on p(f).

  15. Sulforaphane induces DNA single strand breaks in cultured human cells

    Energy Technology Data Exchange (ETDEWEB)

    Sestili, Piero, E-mail: piero.sestili@uniurb.it [Dipartimento di Scienze Biomolecolari, Via Maggetti, 21, Universita degli Studi di Urbino ' Carlo Bo' , 61029 Urbino, PU (Italy); Paolillo, Marco [Dipartimento di Scienze Biomolecolari, Via Maggetti, 21, Universita degli Studi di Urbino ' Carlo Bo' , 61029 Urbino, PU (Italy); Lenzi, Monia [Dipartimento di Farmacologia, Universita degli Studi di Bologna, Via Irnerio 48, 40126 Bologna (Italy); Colombo, Evelin; Vallorani, Luciana; Casadei, Lucia; Martinelli, Chiara [Dipartimento di Scienze Biomolecolari, Via Maggetti, 21, Universita degli Studi di Urbino ' Carlo Bo' , 61029 Urbino, PU (Italy); Fimognari, Carmela [Dipartimento di Farmacologia, Universita degli Studi di Bologna, Via Irnerio 48, 40126 Bologna (Italy)

    2010-07-07

    Sulforaphane (SFR), an isothiocyanate from cruciferous vegetables, possesses growth-inhibiting and apoptosis-inducing activities in cancer cell lines. Recently, SFR has been shown to promote the mitochondrial formation of reactive oxygen species (ROS) in human cancer cell lines. The present study was undertaken to see whether SFR-derived ROS might cause DNA damage in cultured human cells, namely T limphoblastoid Jurkat and human umbilical vein endothelial cells (HUVEC). 1-3 h treatments with 10-30 {mu}M SFR elicited intracellular ROS formation (as assayed with dihydrorhodamine, DHR, oxidation) as well as DNA breakage (as assessed with fast halo assay, FHA). These effects lacked cell-type specificity, since could be observed in both Jurkat and HUVEC. Differential-pH FHA analysis of damaged DNA showed that SFR causes frank DNA single strand breaks (SSBs); no DNA double strand breaks (DSBs) were found within the considered treatment times (up to 3 h). SFR-derived ROS were formed at the mitochondrial respiratory chain (MRC) level: indeed rotenone or myxothiazol (MRC Complex I and III inhibitors, respectively) abrogated ROS formation. Furthermore ROS were not formed in Jurkat cells pharmacologically depleted of respiring mitochondria (MRC-/Jurkat). Formation of ROS was causally linked to the induction of SSBs: indeed all the experimental conditions capable of preventing ROS formation also prevented the damage of nuclear DNA from SFR-intoxicated cells. As to the toxicological relevance of SSBs, we found that their prevention slightly but significantly attenuated SFR cytotoxicity, suggesting that high-dose SFR toxicity is the result of a complex series of events among which GSH depletion seems to play a pivotal role. In conclusion, the present study identifies a novel mechanism contributing to SFR toxicity which - since DNA damage is a prominent mechanism underlying the cytotoxic activity of established antineoplastic agents - might help to exploit the therapeutic value

  16. Sulforaphane induces DNA single strand breaks in cultured human cells

    International Nuclear Information System (INIS)

    Sestili, Piero; Paolillo, Marco; Lenzi, Monia; Colombo, Evelin; Vallorani, Luciana; Casadei, Lucia; Martinelli, Chiara; Fimognari, Carmela

    2010-01-01

    Sulforaphane (SFR), an isothiocyanate from cruciferous vegetables, possesses growth-inhibiting and apoptosis-inducing activities in cancer cell lines. Recently, SFR has been shown to promote the mitochondrial formation of reactive oxygen species (ROS) in human cancer cell lines. The present study was undertaken to see whether SFR-derived ROS might cause DNA damage in cultured human cells, namely T limphoblastoid Jurkat and human umbilical vein endothelial cells (HUVEC). 1-3 h treatments with 10-30 μM SFR elicited intracellular ROS formation (as assayed with dihydrorhodamine, DHR, oxidation) as well as DNA breakage (as assessed with fast halo assay, FHA). These effects lacked cell-type specificity, since could be observed in both Jurkat and HUVEC. Differential-pH FHA analysis of damaged DNA showed that SFR causes frank DNA single strand breaks (SSBs); no DNA double strand breaks (DSBs) were found within the considered treatment times (up to 3 h). SFR-derived ROS were formed at the mitochondrial respiratory chain (MRC) level: indeed rotenone or myxothiazol (MRC Complex I and III inhibitors, respectively) abrogated ROS formation. Furthermore ROS were not formed in Jurkat cells pharmacologically depleted of respiring mitochondria (MRC-/Jurkat). Formation of ROS was causally linked to the induction of SSBs: indeed all the experimental conditions capable of preventing ROS formation also prevented the damage of nuclear DNA from SFR-intoxicated cells. As to the toxicological relevance of SSBs, we found that their prevention slightly but significantly attenuated SFR cytotoxicity, suggesting that high-dose SFR toxicity is the result of a complex series of events among which GSH depletion seems to play a pivotal role. In conclusion, the present study identifies a novel mechanism contributing to SFR toxicity which - since DNA damage is a prominent mechanism underlying the cytotoxic activity of established antineoplastic agents - might help to exploit the therapeutic value of

  17. 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 preclinical modalities and translation towards clinical application. Conclusions: The development of radiation microbeams has been a valuable tool for the exploration of fundamental radiobiological response mechanisms. The strength of micro-irradiation techniques lies in their ability to deliver precise doses...

  18. 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

  19. Optofluidics for handling and analysis of single living cells

    KAUST Repository

    Perozziello, Gerardo

    2017-12-07

    Optofluidics is a field with important applications in areas such as biotechnology, chemical synthesis and analytical chemistry. Optofluidic devices combine optical elements into microfluidic devices in ways that increase portability and sensitivity of analysis for diagnostic or screening purposes .In fact in these devices fluids give fine adaptability, mobility and accessibility to nanoscale photonic devices which otherwise could not be realized using conventional devices. This review describes several cases inwhich optical or microfluidic approaches are used to trap single cells in proximity of integrated optical sensor for being analysed.

  20. 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.

  1. Laser-guidance-based cell deposition microscope for heterotypic single-cell micropatterning

    Energy Technology Data Exchange (ETDEWEB)

    Ma Zhen; Wan Qin; Yun, Julie X; Gao, Bruce Z [Department of Bioengineering, COMSET, Clemson University, Clemson, SC 29634 (United States); Pirlo, Russell K [Naval Research Laboratory, Washington, DC 20375 (United States); Yuan, Xiaocong [Institute of Modern Optics, Key Laboratory of Optoelectronic Information Science and Technology, Ministry of Education of China, Nankai University, Tianjin (China); Xiang Peng [Institute of Optoelectronics, Shenzhen University, Shenzhen, Guangdong (China); Borg, Thomas K, E-mail: zgao@clemson.edu [Department of Regenerative Medicine and Cell Biology, Medical University of South Carolina, Charleston, SC (United States)

    2011-09-15

    Cell patterning methods enable researchers to control specific homotypic and heterotypic contact-mediated cell-cell and cell-ECM interactions and to impose defined cell and tissue geometries. To micropattern individual cells to specific points on a substrate with high spatial resolution, we have developed a cell deposition microscope based on the laser-guidance technique. We discuss the theory of optical forces for generating laser guidance and the optimization of the optical configuration (NA {approx} 0.1) to manipulate cells with high speed in three dimensions. Our cell deposition microscope is capable of patterning different cell types onto and within standard cell research devices and providing on-stage incubation for long-term cell culturing. Using this cell deposition microscope, rat mesenchymal stem cells from bone marrow were micropatterned with cardiomyocytes into a substrate microfabricated with polydimethylsiloxane on a 22 mm x 22 mm coverglass to form a single-cell coculturing microenvironment, and their electrophysiological property changes were investigated during the coculturing days.

  2. 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

  3. Single-cell analysis of G-protein signal transduction.

    Science.gov (United States)

    Clister, Terri; Mehta, Sohum; Zhang, Jin

    2015-03-13

    The growing use of fluorescent biosensors to directly probe the spatiotemporal dynamics of biochemical processes in living cells has revolutionized the study of intracellular signaling. In this review, we summarize recent developments in the use of biosensors to illuminate the molecular details of G-protein-coupled receptor (GPCR) signaling pathways, which have long served as the model for our understanding of signal transduction, while also offering our perspectives on the future of this exciting field. Specifically, we highlight several ways in which biosensor-based single-cell analyses are being used to unravel many of the enduring mysteries that surround these diverse signaling pathways. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  4. Quantitative single cell analysis of cell population dynamics during submandibular salivary gland development and differentiation

    Science.gov (United States)

    Nelson, Deirdre A.; Manhardt, Charles; Kamath, Vidya; Sui, Yunxia; Santamaria-Pang, Alberto; Can, Ali; Bello, Musodiq; Corwin, Alex; Dinn, Sean R.; Lazare, Michael; Gervais, Elise M.; Sequeira, Sharon J.; Peters, Sarah B.; Ginty, Fiona; Gerdes, Michael J.; Larsen, Melinda

    2013-01-01

    Summary Epithelial organ morphogenesis involves reciprocal interactions between epithelial and mesenchymal cell types to balance progenitor cell retention and expansion with cell differentiation for evolution of tissue architecture. Underlying submandibular salivary gland branching morphogenesis is the regulated proliferation and differentiation of perhaps several progenitor cell populations, which have not been characterized throughout development, and yet are critical for understanding organ development, regeneration, and disease. Here we applied a serial multiplexed fluorescent immunohistochemistry technology to map the progressive refinement of the epithelial and mesenchymal cell populations throughout development from embryonic day 14 through postnatal day 20. Using computational single cell analysis methods, we simultaneously mapped the evolving temporal and spatial location of epithelial cells expressing subsets of differentiation and progenitor markers throughout salivary gland development. We mapped epithelial cell differentiation markers, including aquaporin 5, PSP, SABPA, and mucin 10 (acinar cells); cytokeratin 7 (ductal cells); and smooth muscle α-actin (myoepithelial cells) and epithelial progenitor cell markers, cytokeratin 5 and c-kit. We used pairwise correlation and visual mapping of the cells in multiplexed images to quantify the number of single- and double-positive cells expressing these differentiation and progenitor markers at each developmental stage. We identified smooth muscle α-actin as a putative early myoepithelial progenitor marker that is expressed in cytokeratin 5-negative cells. Additionally, our results reveal dynamic expansion and redistributions of c-kit- and K5-positive progenitor cell populations throughout development and in postnatal glands. The data suggest that there are temporally and spatially discreet progenitor populations that contribute to salivary gland development and homeostasis. PMID:23789091

  5. 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.

  6. In vivo single cell analysis reveals Gata2 dynamics in cells transitioning to hematopoietic fate.

    Science.gov (United States)

    Eich, Christina; Arlt, Jochen; Vink, Chris S; Solaimani Kartalaei, Parham; Kaimakis, Polynikis; Mariani, Samanta A; van der Linden, Reinier; van Cappellen, Wiggert A; Dzierzak, Elaine

    2018-01-02

    Cell fate is established through coordinated gene expression programs in individual cells. Regulatory networks that include the Gata2 transcription factor play central roles in hematopoietic fate establishment. Although Gata2 is essential to the embryonic development and function of hematopoietic stem cells that form the adult hierarchy, little is known about the in vivo expression dynamics of Gata2 in single cells. Here, we examine Gata2 expression in single aortic cells as they establish hematopoietic fate in Gata2Venus mouse embryos. Time-lapse imaging reveals rapid pulsatile level changes in Gata2 reporter expression in cells undergoing endothelial-to-hematopoietic transition. Moreover, Gata2 reporter pulsatile expression is dramatically altered in Gata2 +/- aortic cells, which undergo fewer transitions and are reduced in hematopoietic potential. Our novel finding of dynamic pulsatile expression of Gata2 suggests a highly unstable genetic state in single cells concomitant with their transition to hematopoietic fate. This reinforces the notion that threshold levels of Gata2 influence fate establishment and has implications for transcription factor-related hematologic dysfunctions. © 2018 Eich et al.

  7. Single-cell paired-end genome sequencing reveals structural variation per cell cycle

    Science.gov (United States)

    Voet, Thierry; Kumar, Parveen; Van Loo, Peter; Cooke, Susanna L.; Marshall, John; Lin, Meng-Lay; Zamani Esteki, Masoud; Van der Aa, Niels; Mateiu, Ligia; McBride, David J.; Bignell, Graham R.; McLaren, Stuart; Teague, Jon; Butler, Adam; Raine, Keiran; Stebbings, Lucy A.; Quail, Michael A.; D’Hooghe, Thomas; Moreau, Yves; Futreal, P. Andrew; Stratton, Michael R.; Vermeesch, Joris R.; Campbell, Peter J.

    2013-01-01

    The nature and pace of genome mutation is largely unknown. Because standard methods sequence DNA from populations of cells, the genetic composition of individual cells is lost, de novo mutations in cells are concealed within the bulk signal and per cell cycle mutation rates and mechanisms remain elusive. Although single-cell genome analyses could resolve these problems, such analyses are error-prone because of whole-genome amplification (WGA) artefacts and are limited in the types of DNA mutation that can be discerned. We developed methods for paired-end sequence analysis of single-cell WGA products that enable (i) detecting multiple classes of DNA mutation, (ii) distinguishing DNA copy number changes from allelic WGA-amplification artefacts by the discovery of matching aberrantly mapping read pairs among the surfeit of paired-end WGA and mapping artefacts and (iii) delineating the break points and architecture of structural variants. By applying the methods, we capture DNA copy number changes acquired over one cell cycle in breast cancer cells and in blastomeres derived from a human zygote after in vitro fertilization. Furthermore, we were able to discover and fine-map a heritable inter-chromosomal rearrangement t(1;16)(p36;p12) by sequencing a single blastomere. The methods will expedite applications in basic genome research and provide a stepping stone to novel approaches for clinical genetic diagnosis. PMID:23630320

  8. 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.

  9. 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.

  10. Responses of single germinal-center B cells in T-cell-dependent microculture.

    Science.gov (United States)

    George, A; Cebra, J J

    1991-01-01

    B cells purified from the germinal centers (GCs) of murine Peyer's patches can be stimulated in a clonal microculture containing helper T cells and dendritic cells to divide and secrete immunoglobulin. Intraclonal isotype switching occurs, and a variety of immunoglobulin isotypes, including IgA, is secreted. Memory cells, which generate clones secreting IgA exclusively, are only rarely identified in the GC B-cell subset. Such memory cells can, however, be readily identified among unfractionated Peyer's patch B cells, and in non-GC subsets of B cells. The results suggest that the GC does not contain IgA memory cells that can be restimulated in vitro to secrete only IgA. When division of GC B cells is prevented by irradiation or aphidicholin treatment, a large subset that secretes IgA as the sole immunoglobulin isotype is seen, and the output of presumably single B cells is large enough to be scored by RIA. Both helper T cells and dendritic cells are required for the phenomenon. The data indicate that commitment to IgA secretion occurs in Peyer's patch GCs and suggest that the prolific cell division known to be supported in GCs may forestall terminal differentiation of preplasmablasts to immunoglobulin secretion.

  11. Developmental changes in mesophyll diffusion conductance and photosynthetic capacity under different light and water availabilities in Populus tremula: how structure constrains function.

    Science.gov (United States)

    Tosens, Tiina; Niinemets, Ulo; Vislap, Vivian; Eichelmann, Hillar; Castro Díez, Pilar

    2012-05-01

    Finite mesophyll diffusion conductance (g(m) ) significantly constrains net assimilation rate (A(n) ), but g(m) variations and variation sources in response to environmental stresses during leaf development are imperfectly known. The combined effects of light and water limitations on g(m) and diffusion limitations of photosynthesis were studied in saplings of Populus tremula L. An one-dimensional diffusion model was used to gain insight into the importance of key anatomical traits in determining g(m) . Leaf development was associated with increases in dry mass per unit area, thickness, density, exposed mesophyll (S(mes) /S) and chloroplast (S(c) /S) to leaf area ratio, internal air space (f(ias) ), cell wall thickness and chloroplast dimensions. Development of S(mes) /S and S(c) /S was delayed under low light. Reduction in light availability was associated with lower S(c) /S, but with larger f(ias) and chloroplast thickness. Water stress reduced S(c) /S and increased cell wall thickness under high light. In all treatments, g(m) and A(n) increased and CO(2) drawdown because of g(m) , C(i) -C(c) , decreased with increasing leaf age. Low light and drought resulted in reduced g(m) and A(n) and increased C(i) -C(c) . These results emphasize the importance of g(m) and its components in determining A(n) variations during leaf development and in response to stress. © 2011 Blackwell Publishing Ltd.

  12. Comparison of variations detection between whole-genome amplification methods used in single-cell resequencing

    DEFF Research Database (Denmark)

    Hou, Yong; Wu, Kui; Shi, Xulian

    2015-01-01

    BACKGROUND: Single-cell resequencing (SCRS) provides many biomedical advances in variations detection at the single-cell level, but it currently relies on whole genome amplification (WGA). Three methods are commonly used for WGA: multiple displacement amplification (MDA), degenerate-oligonucleoti......BACKGROUND: Single-cell resequencing (SCRS) provides many biomedical advances in variations detection at the single-cell level, but it currently relies on whole genome amplification (WGA). Three methods are commonly used for WGA: multiple displacement amplification (MDA), degenerate...

  13. In silico lineage tracing through single cell transcriptomics identifies a neural stem cell population in planarians.

    Science.gov (United States)

    Molinaro, Alyssa M; Pearson, Bret J

    2016-04-27

    The planarian Schmidtea mediterranea is a master regenerator with a large adult stem cell compartment. The lack of transgenic labeling techniques in this animal has hindered the study of lineage progression and has made understanding the mechanisms of tissue regeneration a challenge. However, recent advances in single-cell transcriptomics and analysis methods allow for the discovery of novel cell lineages as differentiation progresses from stem cell to terminally differentiated cell. Here we apply pseudotime analysis and single-cell transcriptomics to identify adult stem cells belonging to specific cellular lineages and identify novel candidate genes for future in vivo lineage studies. We purify 168 single stem and progeny cells from the planarian head, which were subjected to single-cell RNA sequencing (scRNAseq). Pseudotime analysis with Waterfall and gene set enrichment analysis predicts a molecularly distinct neoblast sub-population with neural character (νNeoblasts) as well as a novel alternative lineage. Using the predicted νNeoblast markers, we demonstrate that a novel proliferative stem cell population exists adjacent to the brain. scRNAseq coupled with in silico lineage analysis offers a new approach for studying lineage progression in planarians. The lineages identified here are extracted from a highly heterogeneous dataset with minimal prior knowledge of planarian lineages, demonstrating that lineage purification by transgenic labeling is not a prerequisite for this approach. The identification of the νNeoblast lineage demonstrates the usefulness of the planarian system for computationally predicting cellular lineages in an adult context coupled with in vivo verification.

  14. Protoplast isolation, transient transformation of leaf mesophyll protoplasts and improved Agrobacterium-mediated leaf disc infiltration of Phaseolus vulgaris: tools for rapid gene expression analysis.

    Science.gov (United States)

    Nanjareddy, Kalpana; Arthikala, Manoj-Kumar; Blanco, Lourdes; Arellano, Elizabeth S; Lara, Miguel

    2016-06-24

    Phaseolus vulgaris is one of the most extensively studied model legumes in the world. The P. vulgaris genome sequence is available; therefore, the need for an efficient and rapid transformation system is more imperative than ever. The functional characterization of P. vulgaris genes is impeded chiefly due to the non-amenable nature of Phaseolus sp. to stable genetic transformation. Transient transformation systems are convenient and versatile alternatives for rapid gene functional characterization studies. Hence, the present work focuses on standardizing methodologies for protoplast isolation from multiple tissues and transient transformation protocols for rapid gene expression analysis in the recalcitrant grain legume P. vulgaris. Herein, we provide methodologies for the high-throughput isolation of leaf mesophyll-, flower petal-, hypocotyl-, root- and nodule-derived protoplasts from P. vulgaris. The highly efficient polyethylene glycol-mannitol magnesium (PEG-MMG)-mediated transformation of leaf mesophyll protoplasts was optimized using a GUS reporter gene. We used the P. vulgaris SNF1-related protein kinase 1 (PvSnRK1) gene as proof of concept to demonstrate rapid gene functional analysis. An RT-qPCR analysis of protoplasts that had been transformed with PvSnRK1-RNAi and PvSnRK1-OE vectors showed the significant downregulation and ectopic constitutive expression (overexpression), respectively, of the PvSnRK1 transcript. We also demonstrated an improved transient transformation approach, sonication-assisted Agrobacterium-mediated transformation (SAAT), for the leaf disc infiltration of P. vulgaris. Interestingly, this method resulted in a 90 % transformation efficiency and transformed 60-85 % of the cells in a given area of the leaf surface. The constitutive expression of YFP further confirmed the amenability of the system to gene functional characterization studies. We present simple and efficient methodologies for protoplast isolation from multiple P

  15. 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

  16. Cell type discovery and representation in the era of high-content single cell phenotyping.

    Science.gov (United States)

    Bakken, Trygve; Cowell, Lindsay; Aevermann, Brian D; Novotny, Mark; Hodge, Rebecca; Miller, Jeremy A; Lee, Alexandra; Chang, Ivan; McCorrison, Jamison; Pulendran, Bali; Qian, Yu; Schork, Nicholas J; Lasken, Roger S; Lein, Ed S; Scheuermann, Richard H

    2017-12-21

    A fundamental characteristic of multicellular organisms is the specialization of functional cell types through the process of differentiation. These specialized cell types not only characterize the normal functioning of different organs and tissues, they can also be used as cellular biomarkers of a variety of different disease states and therapeutic/vaccine responses. In order to serve as a reference for cell type representation, the Cell Ontology has been developed to provide a standard nomenclature of defined cell types for comparative analysis and biomarker discovery. Historically, these cell types have been defined based on unique cellular shapes and structures, anatomic locations, and marker protein expression. However, we are now experiencing a revolution in cellular characterization resulting from the application of new high-throughput, high-content cytometry and sequencing technologies. The resulting explosion in the number of distinct cell types being identified is challenging the current paradigm for cell type definition in the Cell Ontology. In this paper, we provide examples of state-of-the-art cellular biomarker characterization using high-content cytometry and single cell RNA sequencing, and present strategies for standardized cell type representations based on the data outputs from these cutting-edge technologies, including "context annotations" in the form of standardized experiment metadata about the specimen source analyzed and marker genes that serve as the most useful features in machine learning-based cell type classification models. We also propose a statistical strategy for comparing new experiment data to these standardized cell type representations. The advent of high-throughput/high-content single cell technologies is leading to an explosion in the number of distinct cell types being identified. It will be critical for the bioinformatics community to develop and adopt data standard conventions that will be compatible with these new

  17. Systems biology. Conditional density-based analysis of T cell signaling in single-cell data.

    Science.gov (United States)

    Krishnaswamy, Smita; Spitzer, Matthew H; Mingueneau, Michael; Bendall, Sean C; Litvin, Oren; Stone, Erica; Pe'er, Dana; Nolan, Garry P

    2014-11-28

    Cellular circuits sense the environment, process signals, and compute decisions using networks of interacting proteins. To model such a system, the abundance of each activated protein species can be described as a stochastic function of the abundance of other proteins. High-dimensional single-cell technologies, such as mass cytometry, offer an opportunity to characterize signaling circuit-wide. However, the challenge of developing and applying computational approaches to interpret such complex data remains. Here, we developed computational methods, based on established statistical concepts, to characterize signaling network relationships by quantifying the strengths of network edges and deriving signaling response functions. In comparing signaling between naïve and antigen-exposed CD4(+) T lymphocytes, we find that although these two cell subtypes had similarly wired networks, naïve cells transmitted more information along a key signaling cascade than did antigen-exposed cells. We validated our characterization on mice lacking the extracellular-regulated mitogen-activated protein kinase (MAPK) ERK2, which showed stronger influence of pERK on pS6 (phosphorylated-ribosomal protein S6), in naïve cells as compared with antigen-exposed cells, as predicted. We demonstrate that by using cell-to-cell variation inherent in single-cell data, we can derive response functions underlying molecular circuits and drive the understanding of how cells process signals. Copyright © 2014, American Association for the Advancement of Science.

  18. Single-Cell Mass Cytometry Analysis of Human Tonsil T Cell Remodeling by Varicella Zoster Virus

    Directory of Open Access Journals (Sweden)

    Nandini Sen

    2014-07-01

    Full Text Available Although pathogens must infect differentiated host cells that exhibit substantial diversity, documenting the consequences of infection against this heterogeneity is challenging. Single-cell mass cytometry permits deep profiling based on combinatorial expression of surface and intracellular proteins. We used this method to investigate varicella-zoster virus (VZV infection of tonsil T cells, which mediate viral transport to skin. Our results indicate that VZV induces a continuum of changes regardless of basal phenotypic and functional T cell characteristics. Contrary to the premise that VZV selectively infects T cells with skin trafficking profiles, VZV infection altered T cell surface proteins to enhance or induce these properties. Zap70 and Akt signaling pathways that trigger such surface changes were activated in VZV-infected naive and memory cells by a T cell receptor (TCR-independent process. Single-cell mass cytometry is likely to be broadly relevant for demonstrating how intracellular pathogens modulate differentiated cells to support pathogenesis in the natural host.

  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. Isolation and functional interrogation of adult human prostate epithelial stem cells at single cell resolution

    Directory of Open Access Journals (Sweden)

    Wen-Yang Hu

    2017-08-01

    Full Text Available Using primary cultures of normal human prostate epithelial cells, we developed a novel prostasphere-based, label-retention assay that permits identification and isolation of stem cells at a single cell level. Their bona fide stem cell nature was corroborated using in vitro and in vivo regenerative assays and documentation of symmetric/asymmetric division. Robust WNT10B and KRT13 levels without E-cadherin or KRT14 staining distinguished individual stem cells from daughter progenitors in spheroids. Following FACS to isolate label-retaining stem cells from label-free progenitors, RNA-seq identified unique gene signatures for the separate populations which may serve as useful biomarkers. Knockdown of KRT13 or PRAC1 reduced sphere formation and symmetric self-renewal highlighting their role in stem cell maintenance. Pathways analysis identified ribosome biogenesis and membrane estrogen-receptor signaling enriched in stem cells with NF-ĸB signaling enriched in progenitors; activities that were biologically confirmed. Further, bioassays identified heightened autophagy flux and reduced metabolism in stem cells relative to progenitors. These approaches similarly identified stem-like cells from prostate cancer specimens and prostate, breast and colon cancer cell lines suggesting wide applicability. Together, the present studies isolate and identify unique characteristics of normal human prostate stem cells and uncover processes that maintain stem cell homeostasis in the prostate gland.

  1. Live-Cell Imaging Tool Optimization To Study Gene Expression Levels and Dynamics in Single Cells of Bacillus cereus

    NARCIS (Netherlands)

    Eijlander, Robyn T.; Kuipers, Oscar P.

    Single-cell methods are a powerful application in microbial research to study the molecular mechanism underlying phenotypic heterogeneity and cell-to-cell variability. Here, we describe the optimization and application of single-cell time-lapse fluorescence microscopy for the food spoilage bacterium

  2. 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.

  3. Using single cell sequencing data to model the evolutionary history of a tumor

    OpenAIRE

    Kim, Kyung In; Simon, Richard

    2014-01-01

    Background The introduction of next-generation sequencing (NGS) technology has made it possible to detect genomic alterations within tumor cells on a large scale. However, most applications of NGS show the genetic content of mixtures of cells. Recently developed single cell sequencing technology can identify variation within a single cell. Characterization of multiple samples from a tumor using single cell sequencing can potentially provide information on the evolutionary history of that tumo...

  4. Bidirectional Promoter Engineering for Single Cell MicroRNA Sensors in Embryonic Stem Cells.

    Directory of Open Access Journals (Sweden)

    Hanna L Sladitschek

    Full Text Available MicroRNAs have emerged as important markers and regulators of cell identity. Precise measurements of cellular miRNA levels rely traditionally on RNA extraction and thus do not allow to follow miRNA expression dynamics at the level of single cells. Non-invasive miRNA sensors present an ideal solution but they critically depend on the performance of suitable ubiquitous promoters that reliably drive expression both in pluripotent and differentiated cell types. Here we describe the engineering of bidirectional promoters that drive the expression of precise ratiometric fluorescent miRNA sensors in single mouse embryonic stem cells (mESCs and their differentiated derivatives. These promoters are based on combinations of the widely used CAG, EF1α and PGK promoters as well as the CMV and PGK enhancers. miR-142-3p, which is known to be bimodally expressed in mESCs, served as a model miRNA to gauge the precision of the sensors. The performance of the resulting miRNA sensors was assessed by flow cytometry in single stable transgenic mESCs undergoing self-renewal or differentiation. EF1α promoters arranged back-to-back failed to drive the robustly correlated expression of two transgenes. Back-to-back PGK promoters were shut down during mESC differentiation. However, we found that a back-to-back arrangement of CAG promoters with four CMV enhancers provided both robust expression in mESCs undergoing differentiation and the best signal-to-noise for measurement of miRNA activity in single cells among all the sensors we tested. Such a bidirectional promoter is therefore particularly well suited to study the dynamics of miRNA expression during cell fate transitions at the single cell level.

  5. Decoding speech perception from single cell activity in humans.

    Science.gov (United States)

    Ossmy, Ori; Fried, Itzhak; Mukamel, Roy

    2015-08-15

    Deciphering the content of continuous speech is a challenging task performed daily by the human brain. Here, we tested whether activity of single cells in auditory cortex could be used to support such a task. We recorded neural activity from auditory cortex of two neurosurgical patients while presented with a short video segment containing speech. Population spiking activity (~20 cells per patient) allowed detection of word onset and decoding the identity of perceived words with significantly high accuracy levels. Oscillation phase of local field potentials (8-12Hz) also allowed decoding word identity although with lower accuracy levels. Our results provide evidence that the spiking activity of a relatively small population of cells in human primary auditory cortex contains significant information for classification of words in ongoing speech. Given previous evidence for overlapping neural representation during speech perception and production, this may have implications for developing brain-machine interfaces for patients with deficits in speech production. Copyright © 2015 Elsevier Inc. All rights reserved.

  6. 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.

  7. Microencapsulation of single-cell protein from various microalgae species

    Directory of Open Access Journals (Sweden)

    Purnama Sukardi

    2015-10-01

    Full Text Available ABSTRACT The objective of the research was to evaluate nutritional values of microencapsulated diet made from single cell protein of microalgae. Complete randomized design was applied using three different types of microalgae for inclusion trials i.e. (A Nannochloropsis sp., (B Chlorella sp., and (C Spirulina sp. with five replications respectively. Microencapsulated diet was produced by a modification method based on thermal cross-linking with stable temperature. Phytoplankton was cultured in sea water for which fertilized by a modification of Walne and Guillard fertilizer. The results showed that the highest value of nutrition content was Spirulina sp. and the average composition of protein, crude lipid, carbohydrate, ash, nitrogen free extract, and water content was 34.80%, 0.30%, 18.53%, 20.09%, 26.29%, and 13.32%, respectively. Organoleptically, microcapsule showed that the color of capsule was dark green and smell fresh phytoplankton. Keywords: microcapsule, single-cell protein, thermal cross-linking, microalgae, phytoplankton  ABSTRAK Tujuan penelitian adalah mengevaluasi kandungan nutrisi pakan mikrokapsul protein sel tunggal (single cell protein yang berasal dari berbagai jenis mikroalga (fitoplankton. Rancangan percobaan yang digunakan adalah rancangan acak lengkap, dengan perlakuan inklusi mikrokapsul dari jenis fitoplankton (A Nannochloropsis sp., (B Chlorella sp., dan (C Spirulina sp., masing-masing diulang lima kali. Pembuatan mikrokapsul dilakukan dengan menggunakan modifikasi metode dasar thermal cross-linking, serta menerapkan teknik pengeringan suhu konstan. Proses pembuatan mikrokapsul protein diawali dengan kultur fitoplankton jenis Nannochloropsis sp., Chlorella sp., dan Spirulina sp. Kultur dilakukan di dalam laboratorium menggunakan media air laut dan modifikasi pupuk Walne dan Guillard. Hasil penelitian menunjukkan bahwa kandungan nutrisi tertinggi terdapat pada jenis mikrokapsul protein sel tunggal yang berasal dari

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

    Directory of Open Access Journals (Sweden)

    Ariza de Schellenberger A

    2016-04-01

    Full Text Available Angela Ariza de Schellenberger,1 Harald Kratz,1 Tracy D Farr,2,3 Norbert Löwa,4 Ralf Hauptmann,1 Susanne Wagner,1 Matthias Taupitz,1 Jörg Schnorr,1 Eyk A Schellenberger1 1Department of Radiology, 2Department of Experimental Neurology, Center for Stroke Research Berlin, Charité – Universitätsmedizin Berlin, Berlin, Germany; 3School of Life Sciences, University of Nottingham, Medical School, Nottingham, UK; 4Department of Biomagnetic Signals, Physikalisch-Technische Bundesanstalt Berlin, Berlin, Germany Abstract: Sensitive cell detection by magnetic resonance imaging (MRI is an important tool for the development of cell therapies. However, clinically approved contrast agents that allow single-cell detection are currently not available. Therefore, we compared very small iron oxide nanoparticles (VSOP and new multicore carboxymethyl dextran-coated iron oxide nanoparticles (multicore particles, MCP designed by our department for magnetic particle imaging (MPI with discontinued Resovist® regarding their suitability for detection of single mesenchymal stem cells (MSC by MRI. We achieved an average intracellular nanoparticle (NP load of >10 pg Fe per cell without the use of transfection agents. NP loading did not lead to significantly different results in proliferation, colony formation, and multilineage in vitro differentiation assays in comparison to controls. MRI allowed single-cell detection using VSOP, MCP, and Resovist® in conjunction with high-resolution T2*-weighted imaging at 7 T with postprocessing of phase images in agarose cell phantoms and in vivo after delivery of 2,000 NP-labeled MSC into mouse brains via the left carotid artery. With optimized labeling conditions, a detection rate of ~45% was achieved; however, the experiments were limited by nonhomogeneous NP loading of the MSC population. Attempts should be made to achieve better cell separation for homogeneous NP loading and to thus improve NP

  9. Mesophyll conductance to CO2 transport estimated by two independent methods: effect of variable CO2 concentration and abscisic acid

    Czech Academy of Sciences Publication Activity Database

    Vrábl, D.; Vašková, M.; Hronková, Marie; Flexas, J.; Šantrůček, Jiří

    2009-01-01

    Roč. 60, č. 8 (2009), s. 2315-2323 ISSN 0022-0957 R&D Projects: GA AV ČR(CZ) IAA601410505 Institutional research plan: CEZ:AV0Z50510513 Keywords : Carbon dioxide * mesophyll conductance * Helianthus annuus Subject RIV: ED - Physiology Impact factor: 4.271, year: 2009

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

    Directory of Open Access Journals (Sweden)

    Fieguth Paul

    2010-10-01

    of localizing single cells in microwells and can be adapted for the other cell types that may not have circular shape. This method can be potentially used for single cell analysis to study the temporal dynamics of cells.

  11. An automated system for high-throughput single cell-based breeding

    Science.gov (United States)

    Yoshimoto, Nobuo; Kida, Akiko; Jie, Xu; Kurokawa, Masaya; Iijima, Masumi; Niimi, Tomoaki; Maturana, Andrés D.; Nikaido, Itoshi; Ueda, Hiroki R.; Tatematsu, Kenji; Tanizawa, Katsuyuki; Kondo, Akihiko; Fujii, Ikuo; Kuroda, Shun'ichi

    2013-01-01

    When establishing the most appropriate cells from the huge numbers of a cell library for practical use of cells in regenerative medicine and production of various biopharmaceuticals, cell heterogeneity often found in an isogenic cell population limits the refinement of clonal cell culture. Here, we demonstrated high-throughput screening of the most suitable cells in a cell library by an automated undisruptive single-cell analysis and isolation system, followed by expansion of isolated single cells. This system enabled establishment of the most suitable cells, such as embryonic stem cells with the highest expression of the pluripotency marker Rex1 and hybridomas with the highest antibody secretion, which could not be achieved by conventional high-throughput cell screening systems (e.g., a fluorescence-activated cell sorter). This single cell-based breeding system may be a powerful tool to analyze stochastic fluctuations and delineate their molecular mechanisms. PMID:23378922

  12. Cotton fiber: a powerful single-cell model for cell wall and celluloseresearch

    Directory of Open Access Journals (Sweden)

    Candace Hope Haigler

    2012-05-01

    Full Text Available Cotton fibers are single-celled extensions of the seed epidermis. They can be isolated in pureform as they undergo staged differentiation including primary cell wall synthesis duringelongation and nearly pure cellulose synthesis during secondary wall thickening. Thiscombination of features supports clear interpretation of data about cell walls and cellulosesynthesis in the context of high throughput modern experimental technologies. Priorcontributions of cotton fiber to building fundamental knowledge about cell walls will besummarized and the dynamic changes in cell wall polymers throughout cotton fiberdifferentiation will be described. Recent successes in using stable cotton transformation to altercotton fiber cell wall properties as well as cotton fiber quality will be discussed. Future prospectsto perform experiments more rapidly through altering cotton fiber wall properties via virusinduced gene silencing will be evaluated.

  13. Single-Cell Phosphospecific Flow Cytometric Analysis of Canine and Murine Adipose-Derived Stem Cells

    Directory of Open Access Journals (Sweden)

    Harumichi Itoh

    2017-01-01

    Full Text Available This study aimed to demonstrate single-cell phosphospecific flow cytometric analysis of canine and murine adipose-derived stem/stromal cells (ADSCs. ADSCs were obtained from clinically healthy laboratory beagles and C57BL/6 mice. Cell differentiation into adipocytes, osteocytes, and chondrocytes was observed for the cultured canine ADSCs (cADSCs and murine ADSCs (mADSCs to determine their multipotency. We also performed single-cell phosphospecific flow cytometric analysis related to cell differentiation and stemness. Cultured cADSCs and mADSCs exhibited the potential to differentiate into adipocytes, osteocytes, and chondrocytes. In addition, single-cell phosphospecific flow cytometric analysis revealed similar β-catenin and Akt phosphorylation between mADSCs and cADSCs. On the other hand, it showed the phosphorylation of different Stat proteins. It was determined that cADSCs and mADSCs show the potential to differentiate into adipocytes, osteocytes, and chondrocytes. Furthermore, a difference in protein phosphorylation between undifferentiated cADSCs and mADSCs was identified.

  14. Modeling bacterial population growth from stochastic single-cell dynamics.

    Science.gov (United States)

    Alonso, Antonio A; Molina, Ignacio; Theodoropoulos, Constantinos

    2014-09-01

    A few bacterial cells may be sufficient to produce a food-borne illness outbreak, provided that they are capable of adapting and proliferating on a food matrix. This is why any quantitative health risk assessment policy must incorporate methods to accurately predict the growth of bacterial populations from a small number of pathogens. In this aim, mathematical models have become a powerful tool. Unfortunately, at low cell concentrations, standard deterministic models fail to predict the fate of the population, essentially because the heterogeneity between individuals becomes relevant. In this work, a stochastic differential equation (SDE) model is proposed to describe variability within single-cell growth and division and to simulate population growth from a given initial number of individuals. We provide evidence of the model ability to explain the observed distributions of times to division, including the lag time produced by the adaptation to the environment, by comparing model predictions with experiments from the literature for Escherichia coli, Listeria innocua, and Salmonella enterica. The model is shown to accurately predict experimental growth population dynamics for both small and large microbial populations. The use of stochastic models for the estimation of parameters to successfully fit experimental data is a particularly challenging problem. For instance, if Monte Carlo methods are employed to model the required distributions of times to division, the parameter estimation problem can become numerically intractable. We overcame this limitation by converting the stochastic description to a partial differential equation (backward Kolmogorov) instead, which relates to the distribution of division times. Contrary to previous stochastic formulations based on random parameters, the present model is capable of explaining the variability observed in populations that result from the growth of a small number of initial cells as well as the lack of it compared to

  15. 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.

  16. 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.

  17. 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.

  18. A highly-occupied, single-cell trapping microarray for determination of cell membrane permeability.

    Science.gov (United States)

    Weng, Lindong; Ellett, Felix; Edd, Jon; Wong, Keith H K; Uygun, Korkut; Irimia, Daniel; Stott, Shannon L; Toner, Mehmet

    2017-11-21

    Semi- and selective permeability is a fundamentally important characteristic of the cell membrane. Membrane permeability can be determined by monitoring the volumetric change of cells following exposure to a non-isotonic environment. For this purpose, several microfluidic perfusion chambers have been developed recently. However, these devices only allow the observation of one single cell or a group of cells that may interact with one another in an uncontrolled way. Some of these devices have integrated on-chip temperature control to investigate the temperature-dependence of membrane permeability, but they inevitably require sophisticated fabrication and assembly, and delicate temperature and pressure calibration. Therefore, it is highly desirable to design a simple single-cell trapping device that allows parallel monitoring of multiple separate, individual cells subjected to non-isotonic exposure at various temperatures. In this study, we developed a pumpless, single-layer microarray with high trap occupancy of single cells. The benchmark performance of the device was conducted by targeting spherical particles of 18.8 μm in diameter as a model, yielding trap occupancy of up to 86.8% with a row-to-row shift of 10-30 μm. It was also revealed that in each array the particles larger than a corresponding critical size would be excluded by the traps in a deterministic lateral displacement mode. Demonstrating the utility of this approach, we used the single-cell trapping device to determine the membrane permeability of rat hepatocytes and patient-derived circulating tumor cells (Brx-142) at 4, 22 and 37 °C. The membrane of rat hepatocytes was found to be highly permeable to water and small molecules such as DMSO and glycerol, via both lipid- and aquaporin-mediated pathways. Brx-142 cells, however, displayed lower membrane permeability than rat hepatocytes, which was associated with strong coupling of water and DMSO transport but less interaction between water and

  19. Single-cell analysis of the fate of c-kit-positive bone marrow cells

    Science.gov (United States)

    Czarna, Anna; Sanada, Fumihiro; Matsuda, Alex; Kim, Junghyun; Signore, Sergio; Pereira, João D.; Sorrentino, Andrea; Kannappan, Ramaswamy; Cannatà, Antonio; Hosoda, Toru; Rota, Marcello; Crea, Filippo; Anversa, Piero; Leri, Annarosa

    2017-10-01

    The plasticity of c-kit-positive bone marrow cells (c-kit-BMCs) in tissues different from their organ of origin remains unclear. We tested the hypothesis that c-kit-BMCs are functionally heterogeneous and only a subgroup of these cells possesses cardiomyogenic potential. Population-based assays fall short of identifying the properties of individual stem cells, imposing on us the introduction of single cell-based approaches to track the fate of c-kit-BMCs in the injured heart; they included viral gene-tagging, multicolor clonal-marking and transcriptional profiling. Based on these strategies, we report that single mouse c-kit-BMCs expand clonally within the infarcted myocardium and differentiate into specialized cardiac cells. Newly-formed cardiomyocytes, endothelial cells, fibroblasts and c-kit-BMCs showed in their genome common sites of viral integration, providing strong evidence in favor of the plasticity of a subset of BMCs expressing the c-kit receptor. Similarly, individual c-kit-BMCs, which were infected with multicolor reporters and injected in infarcted hearts, formed cardiomyocytes and vascular cells organized in clusters of similarly colored cells. The uniform distribution of fluorescent proteins in groups of specialized cells documented the polyclonal nature of myocardial regeneration. The transcriptional profile of myogenic c-kit-BMCs and whole c-kit-BMCs was defined by RNA sequencing. Genes relevant for engraftment, survival, migration, and differentiation were enriched in myogenic c-kit-BMCs, a cell subtype which could not be assigned to a specific hematopoietic lineage. Collectively, our findings demonstrate that the bone marrow comprises a category of cardiomyogenic, vasculogenic and/or fibrogenic c-kit-positive cells and a category of c-kit-positive cells that retains an undifferentiated state within the damaged heart.

  20. 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

  1. Pseudotemporal Ordering of Single Cells Reveals Metabolic Control of Postnatal β Cell Proliferation.

    Science.gov (United States)

    Zeng, Chun; Mulas, Francesca; Sui, Yinghui; Guan, Tiffany; Miller, Nathanael; Tan, Yuliang; Liu, Fenfen; Jin, Wen; Carrano, Andrea C; Huising, Mark O; Shirihai, Orian S; Yeo, Gene W; Sander, Maike

    2017-05-02

    Pancreatic β cell mass for appropriate blood glucose control is established during early postnatal life. β cell proliferative capacity declines postnatally, but the extrinsic cues and intracellular signals that cause this decline remain unknown. To obtain a high-resolution map of β cell transcriptome dynamics after birth, we generated single-cell RNA-seq data of β cells from multiple postnatal time points and ordered cells based on transcriptional similarity using a new analytical tool. This analysis captured signatures of immature, proliferative β cells and established high expression of amino acid metabolic, mitochondrial, and Srf/Jun/Fos transcription factor genes as their hallmark feature. Experimental validation revealed high metabolic activity in immature β cells and a role for reactive oxygen species and Srf/Jun/Fos transcription factors in driving postnatal β cell proliferation and mass expansion. Our work provides the first high-resolution molecular characterization of state changes in postnatal β cells and paves the way for the identification of novel therapeutic targets to stimulate β cell regeneration. Copyright © 2017 Elsevier Inc. All rights reserved.

  2. 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

  3. 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...... with a commercial cell adhesive CellTakTM. The method was applied to four different bacterial strains, and single-cell adhesion was measured on three surfaces (fresh glass, hydrophilic glass, mica). Attachment to the cantilever was stable during the 2 h of AFM force measurements, and viability was confirmed by Live....../Dead fluorescence staining at the end of each experiment. The adhesion force and final rupture length were dependent on bacterial strains, surfaces properties, and time of contact. The single-cell probe offers control of the cell immobilization, thus holds advantages over the commonly used multi-cell probes where...

  4. 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.

  5. 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.

  6. Single-Cell Phenotype Classification Using Deep Convolutional Neural Networks.

    Science.gov (United States)

    Dürr, Oliver; Sick, Beate

    2016-10-01

    Deep learning methods are currently outperforming traditional state-of-the-art computer vision algorithms in diverse applications and recently even surpassed human performance in object recognition. Here we demonstrate the potential of deep learning methods to high-content screening-based phenotype classification. We trained a deep learning classifier in the form of convolutional neural networks with approximately 40,000 publicly available single-cell images from samples treated with compounds from four classes known to lead to different phenotypes. The input data consisted of multichannel images. The construction of appropriate feature definitions was part of the training and carried out by the convolutional network, without the need for expert knowledge or handcrafted features. We compare our results against the recent state-of-the-art pipeline in which predefined features are extracted from each cell using specialized software and then fed into various machine learning algorithms (support vector machine, Fisher linear discriminant, random forest) for classification. The performance of all classification approaches is evaluated on an untouched test image set with known phenotype classes. Compared to the best reference machine learning algorithm, the misclassification rate is reduced from 8.9% to 6.6%. © 2016 Society for Laboratory Automation and Screening.

  7. PCR amplification of microsatellites from single cells of Karenia brevis preserved in Lugol's iodine solution.

    Science.gov (United States)

    Henrichs, D W; Renshaw, M A; Santamaria, C A; Richardson, B; Gold, J R; Campbell, L

    2008-01-01

    A simple and effective protocol is described for multiplex polymerase chain reaction (PCR) amplification of single cells of Karenia brevis. The protocol requires minimum processing, avoids additions that might dilute target DNA template, and can be used on cells preserved in Lugol's iodine preservative. Destaining of Lugol's-preserved cells with sodium thiosulfate allowed successful amplification of single-copy, nuclear-encoded microsatellites in single cells of K. brevis that have been preserved for up to 6 years.

  8. Functional mapping of cell surface proteins with localized stimulation of single cells

    Science.gov (United States)

    Sun, Bingyun; Chiu, Daniel T.

    2003-11-01

    This paper describes the development of using individual micro and nano meter-sized vesicles as delivery vessels to functionally map the distribution of cell surface proteins at the level of single cells. The formation of different sizes of vesicles from tens of nanometers to a few micrometers in diameter that contain the desired molecules is addressed. An optical trap is used to manipulate the loaded vesicle to specific cell morphology of interest, and a pulsed UV laser is used to photo-release the stimuli onto the cell membrane. Carbachol activated cellular calcium flux, upon binding to muscarinic acetylcholine receptors, is studied by this method, and the potential of using this method for the functional mapping of localized proteins on the cell surface membrane is discussed.

  9. 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.

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

    Science.gov (United States)

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

    2016-03-01

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

  11. 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.

  12. Single cell lineage analysis of mouse embryonic stem cells at the exit from pluripotency

    Directory of Open Access Journals (Sweden)

    Jamie Trott

    2013-08-01

    Understanding how interactions between extracellular signalling pathways and transcription factor networks influence cellular decision making will be crucial for understanding mammalian embryogenesis and for generating specialised cell types in vitro. To this end, pluripotent mouse Embryonic Stem (mES cells have proven to be a useful model system. However, understanding how transcription factors and signalling pathways affect decisions made by individual cells is confounded by the fact that measurements are generally made on groups of cells, whilst individual mES cells differentiate at different rates and towards different lineages, even in conditions that favour a particular lineage. Here we have used single-cell measurements of transcription factor expression and Wnt/β-catenin signalling activity to investigate their effects on lineage commitment decisions made by individual cells. We find that pluripotent mES cells exhibit differing degrees of heterogeneity in their expression of important regulators from pluripotency, depending on the signalling environment to which they are exposed. As mES cells differentiate, downregulation of Nanog and Oct4 primes cells for neural commitment, whilst loss of Sox2 expression primes cells for primitive streak commitment. Furthermore, we find that Wnt signalling acts through Nanog to direct cells towards a primitive streak fate, but that transcriptionally active β-catenin is associated with both neural and primitive streak commitment. These observations confirm and extend previous suggestions that pluripotency genes influence lineage commitment and demonstrate how their dynamic expression affects the direction of lineage commitment, whilst illustrating two ways in which the Wnt signalling pathway acts on this network during cell fate assignment.

  13. 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.

  14. First steps towards fuel cells testing harmonisation: Procedures and parameters for single cell performance evaluation

    Energy Technology Data Exchange (ETDEWEB)

    Lunghi, P. [Department of Industrial Engineering, University of Perugia, Via Duranti 93, Perugia (Italy); Ubertini, S. [Department of Mechanical Engineering, University of Rome ' ' Tor Vergata' ' , Via di Torvergata, 110, Rome (Italy)

    2004-01-01

    The great interest in Fuel Cell Systems, combined with the innovation of the device itself, has led to a huge developmental effort to make the steps necessary for future FC plant commissioning. The clearest and most effective way to evaluate the performance of a fuel cell is to measure it directly and, since few fuel cell test rigs are available at the moment, standard experimental procedures have not been realized so far. Our research group is currently performing single cell testing at the University of Perugia fuel cell laboratory and particular emphasis has been put on the definition of procedures and the testing of parameterisation. The work team strongly believes that this is the key to effective system testing and reliable performance evaluation. In this work, the test parameterisation developed by the team, and the resulting advanced control procedure used for a single MCFC experimental characterization are presented. Efforts have been dedicated to obtain some relevant non-dimensional parameters to allow an easy understanding of the results and quick comparisons with other tests under different operating conditions, or with results obtained on different cells eventually tested in different laboratories. The authors strongly emphasise this topic to avoid the data that developers and research institutions collect being of no practical use due to a lack of shared rules. (Abstract Copyright [2003], Wiley Periodicals, Inc.)

  15. Low aspect ratio micropores for single-particle and single-cell analysis.

    Science.gov (United States)

    Goyal, Gaurav; Mulero, Rafael; Ali, Jamel; Darvish, Armin; Kim, Min Jun

    2015-05-01

    This paper describes microparticle and bacterial translocation studies using low aspect ratio solid-state micropores. Micropores, 5 μm in diameter, were fabricated in 200 nm thick free-standing silicon nitride membranes, resulting in pores with an extremely low aspect ratio, nominally 0.04. For microparticle translocation experiments, sulfonated polystyrene microparticles and magnetic microbeads in size range of 1-4 μm were used. Using the microparticle translocation characteristics, we find that particle translocations result in a change only in the pore's geometrical resistance while the access resistance remains constant. Furthermore, we demonstrate the ability of our micropore to probe high-resolution shape information of translocating analytes using concatenated magnetic microspheres. Distinct current drop peaks were observed for each microsphere of the multibead architecture. For bacterial translocation experiments, nonflagellated Escherichia coli (strain HCB 5) and wild type flagellated Salmonella typhimurium (strain SJW1103) were used. Distinct current signatures for the two bacteria were obtained and this difference in translocation behavior was attributed to different surface protein distributions on the bacteria. Our findings may help in developing low aspect ratio pores for high-resolution microparticle characterization and single-cell analysis. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Integrated Raman and angular scattering of single biological cells

    Science.gov (United States)

    Smith, Zachary J.

    2009-12-01

    epi- and trans-illumination modalities are also discussed. In addition, transilluminated Raman and elastic-scattering spectra were obtained from several biological test-cases, including Streptococcus pneumoniae, baker's yeast, and single human immune cells. Both the Raman and elastic-scattering channels extract information from these samples that are well in line with their known characteristics from the literature. Finally, we report on an experiment in which CD8+ T lymphocytes were stimulated by exposure to the antigens staphylococcal enterotoxin B and phorbol myristate acetate. Clear chemical and morphological differences were observed between the activated and unactivated cells, with the results correlating well to analysis performed on parallel samples using fluorescent stains and a flow cytometer.

  17. Development of single chamber solid oxide fuel cells (SCFC)

    Energy Technology Data Exchange (ETDEWEB)

    Viricelle, J.-P.; Udroiu, S.; Gadacz, G.; Pijolat, M.; Pijolat, C. [Ecole Nationale Superieure des Mines de Saint-Etienne, Centre SPIN, LPMG-UMR CNRS 5148, 158 cours Fauriel, 42023 Saint-Etienne Cedex 02 (France)

    2010-08-15

    Single Chamber Solid Oxide Fuel Cells (SCFC) have been prepared using an electrolyte as support (Ce{sub 0.9}Gd{sub 0.1}O{sub 1.95} named GDC). Anode (Ni-GDC) and different cathodes (Sm{sub 0.5}Sr{sub 0.5}CoO{sub 3} (SSC), Ba{sub 0.5}Sr{sub 0.5}Co{sub 0.2}Fe{sub 0.8}O{sub 3} (BSCF) and La{sub 0.8}Sr{sub 0.2}MnO{sub 3} (LSM)) were placed on the same side of the electrolyte. All the electrodes were deposited using screen-printing technology. A gold collector was also deposited on the cathode to decrease the over-potential. The different materials and fuel cell devices were tested under propane/air mixture, after a preliminary treatment under hydrogen to reduce the as-deposited nickel oxide anode. The results show that SSC and BSCF cathodes are not stable in these conditions, leading to a very low open circuit voltage (OCV) of 150 mV. Although LSM material is not the more adequate cathode regarding its high catalytic activity towards hydrocarbon conversion, it has a better chemical stability than SSC and BSCF. Ni-GDC-LSM SCFC devices were elaborated and tested; an OCV of nearly 750 mV could be obtained with maximum power densities around 20 mW cm{sup -2} at 620 C, under air-propane mixture with C{sub 3}H{sub 8}/O{sub 2} ratio equal to 0.53. (Abstract Copyright [2010], Wiley Periodicals, Inc.)

  18. Repopulation dynamics of single haematopoietic stem cells in mouse transplantation experiments: Importance of stem cell composition in competitor cells.

    Science.gov (United States)

    Ema, Hideo; Uchinomiya, Kouki; Morita, Yohei; Suda, Toshio; Iwasa, Yoh

    2016-04-07

    The transplantation of blood tissues from bone marrow into a lethally irradiated animal is an experimental procedure that is used to study how the blood system is reconstituted by haematopoietic stem cells (HSC). In a competitive repopulation experiment, a lethally irradiated mouse was transplanted with a single HSC as a test cell together with a number of bone marrow cells as competitor cells, and the fraction of the test cell progeny (percentage of chimerism) was traced over time. In this paper, we studied the stem cell kinetics in this experimental procedure. The balance between symmetric self-renewal and differentiation divisions in HSC determined the number of cells which HSC produce and the length of time for which HSC live after transplantation. The percentage of chimerism depended on the type of test cell (long-, intermediate-, or short-term HSC), as well as the type and number of HSC included in competitor cells. We next examined two alternative HSC differentiation models, one-step and multi-step differentiation models. Although these models differed in blood cell production, the percentage of chimerism appeared very similar. We also estimated the numbers of different types of HSC in competitor cells. Based on these results, we concluded that the experimental results inevitably include stochasticity with regard to the number and the type of HSC in competitor cells, and that, in order to detect different types of HSC, an appropriate number of competitor cells needs to be used in transplantation experiments. Copyright © 2016. Published by Elsevier Ltd.

  19. 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

  20. Micromagnetic Cancer Cell Immobilization and Release for Real-Time Single Cell Analysis

    Science.gov (United States)

    Jaiswal, Devina; Rad, Armin Tahmasbi; Nieh, Mu-Ping; Claffey, Kevin P.; Hoshino, Kazunori

    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.

  1. 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.

  2. Evaluation of single cell protein for nutrition of farm animals

    Energy Technology Data Exchange (ETDEWEB)

    Oslage, H.J.; Schulz, E.

    1981-08-01

    For the production of microorganisms with high content of protein technologies on the basis of carbon rich substrates have been developed during the past years. Thus, signification of Single Cell Protein (SCP) for nutrition of farm animals has changed. While, in former times, yeasts were added only in small portions (1-2%) as vitamin supplementation today it is the aim to use microbial biomass as a protein component. The use of SCP as a feedstuff requires a careful physiological and toxicological evaluation as well as extensive investigations of possible use and frontiers of those products for farm animals. Topic of this work were bacteria, bred on methanol as well as yeasts, grown on alcanes and on whey/lactic acid respectively. SCP is preferently used as a feedstuff for poultry, pigs, calves and fishes. Digestibility and utilisation of protein is good till very good, for the a.m. animals, digestibility being between 75-93% and net protein utilisation (NPU) being between 60-76%. In rations of young animals (chicken, piglets and calves) contents of 5-10% SCP have been proved to be without any negative effect on acceptance, body gain, feed utilisation and mortality. For older animals SCP can be used as the only protein source beside the basic feedstuffs.

  3. An RF input coupler for a superconducting single cell cavity

    International Nuclear Information System (INIS)

    Fechner, B.; Ouchi, Nobuo; Kusano, Joichi; Mizumoto, Motoharu; Mukugi, Ken; Krawczyk, F.

    1999-03-01

    Japan Atomic Energy Research Institute proposes a high intensity proton accelerator for the Neutron Science Project. A superconducting linac is a main option for the high energy part of the accelerator. Design and development work for the superconducting accelerating cavities (resonant frequency of 600 MHz) is in progress. Superconducting cavities have an advantage of very high accelerating efficiency because RF wall loss is very small and much of the RF power fed to the cavity is consumed for the beam acceleration. On the other hand, an RF input coupler for the superconducting cavity has to be matched to the beam loading. Therefore, estimation of coupling coefficient or external quality factor (Qext) of the RF input coupler is important for the design of the couplers. In this work, Qext's were calculated by the electromagnetic analysis code (MAFIA) and were compared with those by the measurements. A β (ratio of the particle velocity to the light velocity) = 0.5 single-cell cavity with either axial coupler or side coupler was used in this work. In the experiments, a model cavity made by copper is applied. Both 2- and 3-dimensional calculations were performed in the axial coupler geometry and the results were compared. The agreements between calculated and measured values are good and this method for calculation of Qext is confirmed to be proper for the design of the RF input couplers. (author)

  4. 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.

  5. Polydimethylsiloxane (PDMS Sub-Micron Traps for Single-Cell Analysis of Bacteria

    Directory of Open Access Journals (Sweden)

    Dietrich Kohlheyer

    2013-10-01

    Full Text Available Microfluidics has become an essential tool in single-cell analysis assays for gaining more accurate insights into cell behavior. Various microfluidics methods have been introduced facilitating single-cell analysis of a broad range of cell types. However, the study of prokaryotic cells such as Escherichia coli and others still faces the challenge of achieving proper single-cell immobilization simply due to their small size and often fast growth rates. Recently, new approaches were presented to investigate bacteria growing in monolayers and single-cell tracks under environmental control. This allows for high-resolution time-lapse observation of cell proliferation, cell morphology and fluorescence-coupled bioreporters. Inside microcolonies, interactions between nearby cells are likely and may cause interference during perturbation studies. In this paper, we present a microfluidic device containing hundred sub-micron sized trapping barrier structures for single E. coli cells. Descendant cells are rapidly washed away as well as components secreted by growing cells. Experiments show excellent growth rates, indicating high cell viability. Analyses of elongation and growth rates as well as morphology were successfully performed. This device will find application in prokaryotic single-cell studies under constant environment where by-product interference is undesired.

  6. 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.

  7. Single?Cell Mass Spectrometry for Discovery Proteomics: Quantifying Translational Cell Heterogeneity in the 16?Cell Frog (Xenopus) Embryo

    OpenAIRE

    Lombard?Banek, Camille; Moody, Sally A.; Nemes, Peter

    2016-01-01

    Abstract We advance mass spectrometry from a cell population?averaging tool to one capable of quantifying the expression of diverse proteins in single embryonic cells. Our instrument combines capillary electrophoresis (CE), electrospray ionization, and a tribrid ultrahigh?resolution mass spectrometer (HRMS) to enable untargeted (discovery) proteomics with ca. 25?amol lower limit of detection. CE??ESI?HRMS enabled the identification of 500?800 nonredundant protein groups by measuring 20?ng, or

  8. High-Efficient Transfection of Human Embryonic Stem Cells by Single-Cell Plating and Starvation.

    Science.gov (United States)

    Liu, Hui; Ren, Caiping; Zhu, Bin; Wang, Lei; Liu, Weidong; Shi, Jia; Lin, Jianxing; Xia, Xiaomeng; Zeng, Fei; Chen, Jiawen; Jiang, Xingjun

    2016-03-15

    Nowadays, the low efficiency of small interfering RNA (siRNA) or plasmid DNA (pDNA) transfection is a critical issue in genetic manipulation of human embryonic stem (hES) cells. Development of an efficient transfection method for delivery of siRNAs and plasmids into hES cells becomes more and more imperative. In this study, we tried to modify the traditional transfection protocol by introducing two crucial processes, single-cell plating and starvation, to increase the transfection efficiency in hES cells. Furthermore, we comparatively examined the transfection efficiency of some commercially available siRNA or pDNA transfection reagents in hES cells. Our results showed that the new developed method markedly enhanced the transfection efficiency without influencing the proliferation and pluripotency of hES cells. Lipofectamine RNAiMAX exhibited much higher siRNA transfection efficiency than the other reagents, and FuGENE HD was identified as the best suitable reagent for efficient pDNA transfection of hES cells among the tested reagents.

  9. 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.

  10. Novel exons and splice variants in the human antibody heavy chain identified by single cell and single molecule sequencing.

    Directory of Open Access Journals (Sweden)

    Christopher Vollmers

    Full Text Available Antibody heavy chains contain a variable and a constant region. The constant region of the antibody heavy chain is encoded by multiple groups of exons which define the isotype and therefore many functional characteristics of the antibody. We performed both single B cell RNAseq and long read single molecule sequencing of antibody heavy chain transcripts and were able to identify novel exons for IGHA1 and IGHA2 as well as novel isoforms for IGHM antibody heavy chain.

  11. Separation and Analysis of Adherent and Non-Adherent Cancer Cells Using a Single-Cell Microarray Chip.

    Science.gov (United States)

    Yamamura, Shohei; Yamada, Eriko; Kimura, Fukiko; Miyajima, Kumiko; Shigeto, Hajime

    2017-10-21

    A new single-cell microarray chip was designed and developed to separate and analyze single adherent and non-adherent cancer cells. The single-cell microarray chip is made of polystyrene with over 60,000 microchambers of 10 different size patterns (31-40 µm upper diameter, 11-20 µm lower diameter). A drop of suspension of adherent carcinoma (NCI-H1650) and non-adherent leukocyte (CCRF-CEM) cells was placed onto the chip, and single-cell occupancy of NCI-H1650 and CCRF-CEM was determined to be 79% and 84%, respectively. This was achieved by controlling the chip design and surface treatment. Analysis of protein expression in single NCI-H1650 and CCRF-CEM cells was performed on the single-cell microarray chip by multi-antibody staining. Additionally, with this system, we retrieved positive single cells from the microchambers by a micromanipulator. Thus, this system demonstrates the potential for easy and accurate separation and analysis of various types of single cells.

  12. An assessment of the cultural capabilities of Trifolium repens L. (white clover) and Onobrychis viciifolia Scop. (sainfoin) mesophyll protoplasts.

    Science.gov (United States)

    Ahuja, P S; Lu, D Y; Cocking, E C; Davey, M R

    1983-10-01

    Mesophyll protoplasts isolated from white clover and sainfoin divided to form callus under similar cultural conditions. White clover protoplasts showed varietal differences in their plating efficiency. Sainfoin tissues regenerated readily by forming shoots, but induction of morphogenesis in white clover was only achieved after testing several media and culture sequences. Many of the white clover shoots were abnormal in being fused together to form green plate-like structures, but the latter still developed into plantlets while attached to the parent callus. The ability to isolate, culture, and regenerate mesophyll protoplasts of these two forage legumes is discussed in relation to future attempts to produce somatic hybrids between high tannin containing bloat-safe sainfoin and other major forage legumes such as alfalfa, white clover, and red clover.

  13. 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.

  14. Localization and abundance analysis of human lncRNAs at single-cell and single-molecule resolution.

    Science.gov (United States)

    Cabili, Moran N; Dunagin, Margaret C; McClanahan, Patrick D; Biaesch, Andrew; Padovan-Merhar, Olivia; Regev, Aviv; Rinn, John L; Raj, Arjun

    2015-01-29

    Long non-coding RNAs (lncRNAs) have been implicated in diverse biological processes. In contrast to extensive genomic annotation of lncRNA transcripts, far fewer have been characterized for subcellular localization and cell-to-cell variability. Addressing this requires systematic, direct visualization of lncRNAs in single cells at single-molecule resolution. We use single-molecule RNA-FISH to systematically quantify and categorize the subcellular localization patterns of a representative set of 61 lncRNAs in three different cell types. Our survey yields high-resolution quantification and stringent validation of the number and spatial positions of these lncRNA, with an mRNA set for comparison. Using this highly quantitative image-based dataset, we observe a variety of subcellular localization patterns, ranging from bright sub-nuclear foci to almost exclusively cytoplasmic localization. We also find that the low abundance of lncRNAs observed from cell population measurements cannot be explained by high expression in a small subset of 'jackpot' cells. Additionally, nuclear lncRNA foci dissolve during mitosis and become widely dispersed, suggesting these lncRNAs are not mitotic bookmarking factors. Moreover, we see that divergently transcribed lncRNAs do not always correlate with their cognate mRNA, nor do they have a characteristic localization pattern. Our systematic, high-resolution survey of lncRNA localization reveals aspects of lncRNAs that are similar to mRNAs, such as cell-to-cell variability, but also several distinct properties. These characteristics may correspond to particular functional roles. Our study also provides a quantitative description of lncRNAs at the single-cell level and a universally applicable framework for future study and validation of lncRNAs.

  15. Centering Single Cells in Microgels via Delayed Crosslinking Supports Long-Term 3D Culture by Preventing Cell Escape

    NARCIS (Netherlands)

    Kamperman, Tom; Henke, Sieger; Visser, Claas Willem; Karperien, Marcel; Leijten, Jeroen

    2017-01-01

    Single-cell-laden microgels support physiological 3D culture conditions while enabling straightforward handling and high-resolution readouts of individual cells. However, their widespread adoption for long-term cultures is limited by cell escape. In this work, it is demonstrated that cell escape is

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

    Science.gov (United States)

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

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

  17. Computation and measurement of cell decision making errors using single cell data.

    Science.gov (United States)

    Habibi, Iman; Cheong, Raymond; Lipniacki, Tomasz; Levchenko, Andre; Emamian, Effat S; Abdi, Ali

    2017-04-01

    In this study a new computational method is developed to quantify decision making errors in cells, caused by noise and signaling failures. Analysis of tumor necrosis factor (TNF) signaling pathway which regulates the transcription factor Nuclear Factor κB (NF-κB) using this method identifies two types of incorrect cell decisions called false alarm and miss. These two events represent, respectively, declaring a signal which is not present and missing a signal that does exist. Using single cell experimental data and the developed method, we compute false alarm and miss error probabilities in wild-type cells and provide a formulation which shows how these metrics depend on the signal transduction noise level. We also show that in the presence of abnormalities in a cell, decision making processes can be significantly affected, compared to a wild-type cell, and the method is able to model and measure such effects. In the TNF-NF-κB pathway, the method computes and reveals changes in false alarm and miss probabilities in A20-deficient cells, caused by cell's inability to inhibit TNF-induced NF-κB response. In biological terms, a higher false alarm metric in this abnormal TNF signaling system indicates perceiving more cytokine signals which in fact do not exist at the system input, whereas a higher miss metric indicates that it is highly likely to miss signals that actually exist. Overall, this study demonstrates the ability of the developed method for modeling cell decision making errors under normal and abnormal conditions, and in the presence of transduction noise uncertainty. Compared to the previously reported pathway capacity metric, our results suggest that the introduced decision error metrics characterize signaling failures more accurately. This is mainly because while capacity is a useful metric to study information transmission in signaling pathways, it does not capture the overlap between TNF-induced noisy response curves.

  18. 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

    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......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...... advantage of studying different liver cell types that have been isolated from the same organism....

  19. Isolation and characterization of protoplasts and vacuoles from sugar beet leaf mesophyll

    International Nuclear Information System (INIS)

    Dubinina, I.M.; Kudryavtseva, L.F.; Burakhanova, E.A.

    1989-01-01

    The present paper describes methods for isolation of protoplasts and vacuoles from sugar beet (Beta vulgaris L.) leaf mesophyll. Protoplasts were isolated by the enzymatic method in two stages. The yield of protoplasts in the crude suspension attained 3-10 units from 1g of fresh tissue mass. Two methods of purifying the crude protoplast suspension are compared in the paper, the indicated methods employing gradients of Percoll (method 1) and Ficoll (method 2). The final yield comprised 4.5-9.0-10.5 protoplasts from 1g of fresh tissue mass after purification method 1 and 6.0-10.5-1.2-10 protoplasts after method 2. The photosynthesis rate in such protoplasts under optimal conditions comprised 75-100 μmoles of CO 2 h per mg of chlorophyll as compared with 100-130 μmoles in leaf blade disks. The two methods were used to obtain vacuoles, method 1 involving osmotic lysis of protoplasts (the yield constituting 6-15% of vacuoles of the protoplasts taken) and method 2 consisting of ultracentrifugation in a Ficoll gradient (giving a yield of 25-45%). As was monitored microscopically and from the absence of activity of extravacuolar enzymes (NADH-cytochrome-c reductase and cytochrome-c oxidase), vacuoles free of foreign impurities were obtained in both cases. The time needed to obtain protoplasts from leaf tissue comprised 2-3 h, whereas 1.5-2 h was needed to obtain vacuoles from protoplasts

  20. Robust synchronization of coupled circadian and cell cycle oscillators in single mammalian cells

    Science.gov (United States)

    Bieler, Jonathan; Cannavo, Rosamaria; Gustafson, Kyle; Gobet, Cedric; Gatfield, David; Naef, Felix

    2014-01-01

    Circadian cycles and cell cycles are two fundamental periodic processes with a period in the range of 1 day. Consequently, coupling between such cycles can lead to synchronization. Here, we estimated the mutual interactions between the two oscillators by time-lapse imaging of single mammalian NIH3T3 fibroblasts during several days. The analysis of thousands of circadian cycles in dividing cells clearly indicated that both oscillators tick in a 1:1 mode-locked state, with cell divisions occurring tightly 5 h before the peak in circadian Rev-Erbα-YFP reporter expression. In principle, such synchrony may be caused by either unidirectional or bidirectional coupling. While gating of cell division by the circadian cycle has been most studied, our data combined with stochastic modeling unambiguously show that the reverse coupling is predominant in NIH3T3 cells. Moreover, temperature, genetic, and pharmacological perturbations showed that the two interacting cellular oscillators adopt a synchronized state that is highly robust over a wide range of parameters. These findings have implications for circadian function in proliferative tissues, including epidermis, immune cells, and cancer. PMID:25028488

  1. Single-Cell RNA Sequencing Identifies Extracellular Matrix Gene Expression by Pancreatic Circulating Tumor Cells

    Directory of Open Access Journals (Sweden)

    David T. Ting

    2014-09-01

    Full Text Available Circulating tumor cells (CTCs are shed from primary tumors into the bloodstream, mediating the hematogenous spread of cancer to distant organs. To define their composition, we compared genome-wide expression profiles of CTCs with matched primary tumors in a mouse model of pancreatic cancer, isolating individual CTCs using epitope-independent microfluidic capture, followed by single-cell RNA sequencing. CTCs clustered separately from primary tumors and tumor-derived cell lines, showing low-proliferative signatures, enrichment for the stem-cell-associated gene Aldh1a2, biphenotypic expression of epithelial and mesenchymal markers, and expression of Igfbp5, a gene transcript enriched at the epithelial-stromal interface. Mouse as well as human pancreatic CTCs exhibit a very high expression of stromal-derived extracellular matrix (ECM proteins, including SPARC, whose knockdown in cancer cells suppresses cell migration and invasiveness. The aberrant expression by CTCs of stromal ECM genes points to their contribution of microenvironmental signals for the spread of cancer to distant organs.

  2. Single-cell RNA sequencing identifies extracellular matrix gene expression by pancreatic circulating tumor cells.

    Science.gov (United States)

    Ting, David T; Wittner, Ben S; Ligorio, Matteo; Vincent Jordan, Nicole; Shah, Ajay M; Miyamoto, David T; Aceto, Nicola; Bersani, Francesca; Brannigan, Brian W; Xega, Kristina; Ciciliano, Jordan C; Zhu, Huili; MacKenzie, Olivia C; Trautwein, Julie; Arora, Kshitij S; Shahid, Mohammad; Ellis, Haley L; Qu, Na; Bardeesy, Nabeel; Rivera, Miguel N; Deshpande, Vikram; Ferrone, Cristina R; Kapur, Ravi; Ramaswamy, Sridhar; Shioda, Toshi; Toner, Mehmet; Maheswaran, Shyamala; Haber, Daniel A

    2014-09-25

    Circulating tumor cells (CTCs) are shed from primary tumors into the bloodstream, mediating the hematogenous spread of cancer to distant organs. To define their composition, we compared genome-wide expression profiles of CTCs with matched primary tumors in a mouse model of pancreatic cancer, isolating individual CTCs using epitope-independent microfluidic capture, followed by single-cell RNA sequencing. CTCs clustered separately from primary tumors and tumor-derived cell lines, showing low-proliferative signatures, enrichment for the stem-cell-associated gene Aldh1a2, biphenotypic expression of epithelial and mesenchymal markers, and expression of Igfbp5, a gene transcript enriched at the epithelial-stromal interface. Mouse as well as human pancreatic CTCs exhibit a very high expression of stromal-derived extracellular matrix (ECM) proteins, including SPARC, whose knockdown in cancer cells suppresses cell migration and invasiveness. The aberrant expression by CTCs of stromal ECM genes points to their contribution of microenvironmental signals for the spread of cancer to distant organs. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

  3. 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.

  4. 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.

  5. SC3: consensus clustering of single-cell RNA-seq data.

    Science.gov (United States)

    Kiselev, Vladimir Yu; Kirschner, Kristina; Schaub, Michael T; Andrews, Tallulah; Yiu, Andrew; Chandra, Tamir; Natarajan, Kedar N; Reik, Wolf; Barahona, Mauricio; Green, Anthony R; Hemberg, Martin

    2017-05-01

    Single-cell RNA-seq enables the quantitative characterization of cell types based on global transcriptome profiles. We present single-cell consensus clustering (SC3), a user-friendly tool for unsupervised clustering, which achieves high accuracy and robustness by combining multiple clustering solutions through a consensus approach (http://bioconductor.org/packages/SC3). We demonstrate that SC3 is capable of identifying subclones from the transcriptomes of neoplastic cells collected from patients.

  6. Understanding gene expression variability in its biological context using theoretical and experimental analyses of single cells

    NARCIS (Netherlands)

    Kempe, H.

    2017-01-01

    Traditional gene expression studies have largely ignored cell-to-cell variability in transcription. Current methods allow for single cell analyses and have shown considerable variability in gene expression, even in populations of isogenic cells exposed to the same growth environment. In this thesis,

  7. Up-scaling single cell-inoculated suspension culture of human embryonic stem cells.

    Science.gov (United States)

    Singh, Harmeet; Mok, Pamela; Balakrishnan, Thavamalar; Rahmat, Siti Norfiza Binte; Zweigerdt, Robert

    2010-05-01

    We have systematically developed single cell-inoculated suspension cultures of human embryonic stem cells (hESC) in defined media. Cell survival was dependent on hESC re-aggregation. In the presence of the Rho kinase inhibitor Y-27632 (Ri) only approximately 44% of the seeded cells were rescued, but an optimized heat shock treatment combined with Ri significantly increased cell survival to approximately 60%. Mechanistically, our data suggest that E-cadherin plays a role in hESC aggregation and that dissociation and re-aggregation upon passaging functions as a purification step towards a pluripotency markers-enriched population. Mass expansion of hESC was readily achieved by up-scaling 2 ml cultures to serial passaging in 50 ml spinner flasks. A media comparison revealed that mTeSR was superior to KnockOut-SR in supporting cell proliferation and pluripotency. Persistent expression of pluripotency markers was achieved for two lines (hES2, hES3) that were used at higher passages (>86). In contrast, rapid down regulation of Oct4, Tra-1-60, and SSEA4 was observed for ESI049, a clinically compliant line, used at passages 20-36. The up-scaling strategy has significant potential to provide pluripotent cells on a clinical scale. Nevertheless, our data also highlights a significant line-to-line variability and the need for a critical assessment of novel methods with numerous relevant cell lines. Copyright 2010 Elsevier B.V. All rights reserved.

  8. 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.

  9. Optimizing cryopreservation of human spermatogonial stem cells: comparing the effectiveness of testicular tissue and single cell suspension cryopreservation.

    Science.gov (United States)

    Yango, Pamela; Altman, Eran; Smith, James F; Klatsky, Peter C; Tran, Nam D

    2014-11-01

    To determine whether optimal human spermatogonial stem cell (SSC) cryopreservation is best achieved with testicular tissue or single cell suspension cryopreservation. This study compares the effectiveness between these two approaches by using testicular SSEA-4+ cells, a known population containing SSCs. In vitro human testicular tissues. Academic research unit. Adult testicular tissues (n=4) collected from subjects with normal spermatogenesis and normal fetal testicular tissues (n=3). Testicular tissue versus single cell suspension cryopreservation. Cell viability, total cell recovery per milligram of tissue, as well as viable and SSEA-4+ cell recovery. Single cell suspension cryopreservation yielded higher recovery of SSEA-4+ cells enriched in adult SSCs, whereas fetal SSEA-4+ cell recovery was similar between testicular tissue and single cell suspension cryopreservation. Adult and fetal human SSEA-4+ populations exhibited differential sensitivity to cryopreservation based on whether they were cryopreserved in situ as testicular tissues or as single cells. Thus, optimal preservation of human SSCs depends on the patient's age, type of samples cryopreserved, and end points of therapeutic applications. Copyright © 2014 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

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

    Science.gov (United States)

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

    2015-04-01

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

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

    Science.gov (United States)

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

    2017-09-14

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

  12. Quantum cascade laser infrared spectroscopy of single cancer cells

    KAUST Repository

    Patel, Imran

    2017-03-27

    Quantum cascade laser infrared spectroscopy is a next generation novel imaging technique allowing high resolution spectral imaging of cells. We show after spectral pre-processing, identification of different cancer cell populations within minutes.

  13. A simple and effective method to encapsulate tobacco mesophyll protoplasts to maintain cell viability

    Directory of Open Access Journals (Sweden)

    Rong Lei

    2015-01-01

    • It is very convenient to change or collect the solution without mechanically disturbing the protoplasts. This simple and effective silica sol–gel/alginate two-step immobilization of protoplasts in Transwell has great potential for applications in genetic transformation, metabolite production, and migration assays.

  14. Quantifying biomass changes of single CD8+ T cells during antigen specific cytotoxicity.

    Directory of Open Access Journals (Sweden)

    Thomas A Zangle

    Full Text Available Existing approaches that quantify cytotoxic T cell responses rely on bulk or surrogate measurements which impede the direct identification of single activated T cells of interest. Single cell microscopy or flow cytometry methodologies typically rely on fluorescent labeling, which limits applicability to primary cells such as human derived T lymphocytes. Here, we introduce a quantitative method to track single T lymphocyte mediated cytotoxic events within a mixed population of cells using live cell interferometry (LCI, a label-free microscopy technique that maintains cell viability. LCI quantifies the mass distribution within individual cells by measuring the phase shift caused by the interaction of light with intracellular biomass. Using LCI, we imaged cytotoxic T cells killing cognate target cells. In addition to a characteristic target cell mass decrease of 20-60% over 1-4 h following attack by a T cell, there was a significant 4-fold increase in T cell mass accumulation rate at the start of the cytotoxic event and a 2-3 fold increase in T cell mass relative to the mass of unresponsive T cells. Direct, label-free measurement of CD8+ T and target cell mass changes provides a kinetic, quantitative assessment of T cell activation and a relatively rapid approach to identify specific, activated patient-derived T cells for applications in cancer immunotherapy.

  15. 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.

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

    Science.gov (United States)

    Mohammed, Hisham; Hernando-Herraez, Irene; Savino, Aurora; Scialdone, Antonio; Macaulay, Iain; Mulas, Carla; Chandra, Tamir; Voet, Thierry; Dean, Wendy; Nichols, Jennifer; Marioni, John C; Reik, Wolf

    2017-08-01

    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. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  17. 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)

  18. DNA template strand sequencing of single-cells maps genomic rearrangements at high resolution

    NARCIS (Netherlands)

    Falconer, Ester; Hills, Mark; Naumann, Ulrike; Poon, Steven S. S.; Chavez, Elizabeth A.; Sanders, Ashley D.; Zhao, Yongjun; Hirst, Martin; Lansdorp, Peter M.

    2012-01-01

    DNA rearrangements such as sister chromatid exchanges (SCEs) are sensitive indicators of genomic stress and instability, but they are typically masked by single-cell sequencing techniques. We developed Strand-seq to independently sequence parental DNA template strands from single cells, making it

  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. Genome wide single cell analysis of chemotherapy resistant metastatic cells in a case of gastroesophageal adenocarcinoma

    International Nuclear Information System (INIS)

    Hjortland, Geir Olav; Fodstad, Oystein; Smeland, Sigbjorn; Hovig, Eivind; Meza-Zepeda, Leonardo A; Beiske, Klaus; Ree, Anne H; Tveito, Siri; Hoifodt, Hanne; Bohler, Per J; Hole, Knut H; Myklebost, Ola

    2011-01-01

    Metastatic progression due to development or enrichment of therapy-resistant tumor cells is eventually lethal. Molecular characterization of such chemotherapy resistant tumor cell clones may identify markers responsible for malignant progression and potential targets for new treatment. Here, in a case of stage IV adenocarcinoma of the gastroesophageal junction, we report the successful genome wide analysis using array comparative genomic hybridization (CGH) of DNA from only fourteen tumor cells using a bead-based single cell selection method from a bone metastasis progressing during chemotherapy. In a case of metastatic adenocarcinoma of the gastroesophageal junction, the progression of bone metastasis was observed during a chemotherapy regimen of epirubicin, oxaliplatin and capecitabine, whereas lung-, liver and lymph node metastases as well as the primary tumor were regressing. A bone marrow aspirate sampled at the site of progressing metastasis in the right iliac bone was performed, and single cell molecular analysis using array-CGH of Epithelial Specific Antigen (ESA)-positive metastatic cells, and revealed two distinct regions of amplification, 12p12.1 and 17q12-q21.2 amplicons, containing the KRAS (12p) and ERBB2 (HER2/NEU) (17q) oncogenes. Further intrapatient tumor heterogeneity of these highlighted gene copy number changes was analyzed by fluorescence in situ hybridization (FISH) in all available primary and metastatic tumor biopsies, and ErbB2 protein expression was investigated by immunohistochemistry. ERBB2 was heterogeneously amplified by FISH analysis in the primary tumor, as well as liver and bone metastasis, but homogenously amplified in biopsy specimens from a progressing bone metastasis after three initial cycles of chemotherapy, indicating a possible enrichment of erbB2 positive tumor cells in the progressing bone marrow metastasis during chemotherapy. A similar amplification profile was detected for wild-type KRAS, although more heterogeneously

  1. Probing living bacterial adhesion by single cell force spectroscopy using atomic force microscopy

    DEFF Research Database (Denmark)

    Zeng, Guanghong; Ogaki, Ryosuke; Regina, Viduthalai R.

    Bacteria initiate attachment to the surfaces with the aid of different extracellular polymers. To quantitatively study how these polymers mediate bacterial adhesion and possibly their interactions, it is essential to go down to single cell level, with in mind that cell-to-cell variation should...... with a commercial cell adhesive CellTakTM. The method was applied to four different bacterial strains, and single-cell adhesion was measured on three surfaces (fresh glass, hydrophilic glass, mica). Attachment to the cantilever was stable during the 2 h of AFM force measurements, and viability was confirmed by Live...

  2. Microselection - Affinity Selecting Antibodies against a Single Rare Cell in a Heterogeneous Population

    DEFF Research Database (Denmark)

    Sørensen, Morten Dræby; Agerholm, Inge Errebo; Christensen, Britta

    2009-01-01

    antibodies. Here we have generated a microselection method allowing antibody selection, by phage display, targeting a single cell in a heterogeneous population. One K562 cell (female origin) was positioned on glass-slide among millions of lymphocytes from male donor, identifying the K562 cell by FISH (XX......). Several single cell selections were performed on such individual slides. The phage particles bound to the target cell is protected by a minute disc, while inactivating all remaining phage by UV-irradiation; leaving only the phage bound to the target cell viable. We hereby retrieved up to eight antibodies...

  3. Ablation of a single cell from eight-cell embryos of the amphipod crustacean Parhyale hawaiensis.

    Science.gov (United States)

    Nast, Anastasia R; Extavour, Cassandra G

    2014-03-16

    The amphipod Parhyale hawaiensis is a small crustacean found in intertidal marine habitats worldwide. Over the past decade, Parhyale has emerged as a promising model organism for laboratory studies of development, providing a useful outgroup comparison to the well studied arthropod model organism Drosophila melanogaster. In contrast to the syncytial cleavages of Drosophila, the early cleavages of Parhyale are holoblastic. Fate mapping using tracer dyes injected into early blastomeres have shown that all three germ layers and the germ line are established by the eight-cell stage. At this stage, three blastomeres are fated to give rise to the ectoderm, three are fated to give rise to the mesoderm, and the remaining two blastomeres are the precursors of the endoderm and germ line respectively. However, blastomere ablation experiments have shown that Parhyale embryos also possess significant regulatory capabilities, such that the fates of blastomeres ablated at the eight-cell stage can be taken over by the descendants of some of the remaining blastomeres. Blastomere ablation has previously been described by one of two methods: injection and subsequent activation of phototoxic dyes or manual ablation. However, photoablation kills blastomeres but does not remove the dead cell body from the embryo. Complete physical removal of specific blastomeres may therefore be a preferred method of ablation for some applications. Here we present a protocol for manual removal of single blastomeres from the eight-cell stage of Parhyale embryos, illustrating the instruments and manual procedures necessary for complete removal of the cell body while keeping the remaining blastomeres alive and intact. This protocol can be applied to any Parhyale cell at the eight-cell stage, or to blastomeres of other early cleavage stages. In addition, in principle this protocol could be applicable to early cleavage stage embryos of other holoblastically cleaving marine invertebrates.

  4. 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.

  5. 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.

  6. Single-Cell Analysis of the Impact of Host Cell Heterogeneity on Infection with Foot-and-Mouth Disease Virus.

    Science.gov (United States)

    Xin, Xiu; Wang, Hailong; Han, Lingling; Wang, Mingzhen; Fang, Hui; Hao, Yao; Li, Jiadai; Zhang, Hu; Zheng, Congyi; Shen, Chao

    2018-05-01

    Viral infection and replication are affected by host cell heterogeneity, but the mechanisms underlying the effects remain unclear. Using single-cell analysis, we investigated the effects of host cell heterogeneity, including cell size, inclusion, and cell cycle, on foot-and-mouth disease virus (FMDV) infection (acute and persistent infections) and replication. We detected various viral genome replication levels in FMDV-infected cells. Large cells and cells with a high number of inclusions generated more viral RNA copies and viral protein and a higher proportion of infectious cells than other cells. Additionally, we found that the viral titer was 10- to 100-fold higher in cells in G 2 /M than those in other cell cycle phases and identified a strong correlation between cell size, inclusion, and cell cycle heterogeneity, which all affected the infection and replication of FMDV. Furthermore, we demonstrated that host cell heterogeneity influenced the adsorption of FMDV due to differences in the levels of FMDV integrin receptors expression. Collectively, these results further our understanding of the evolution of a virus in a single host cell. IMPORTANCE It is important to understand how host cell heterogeneity affects viral infection and replication. Using single-cell analysis, we found that viral genome replication levels exhibited dramatic variability in foot-and-mouth disease virus (FMDV)-infected cells. We also found a strong correlation between heterogeneity in cell size, inclusion number, and cell cycle status and that all of these characteristics affect the infection and replication of FMDV. Moreover, we found that host cell heterogeneity influenced the viral adsorption as differences in the levels of FMDV integrin receptors' expression. This study provided new ideas for the studies of correlation between FMDV infection mechanisms and host cells. Copyright © 2018 American Society for Microbiology.

  7. 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.

  8. 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.

  9. High-throughput microfluidic single-cell digital polymerase chain reaction.

    Science.gov (United States)

    White, A K; Heyries, K A; Doolin, C; Vaninsberghe, M; Hansen, C L

    2013-08-06

    Here we present an integrated microfluidic device for the high-throughput digital polymerase chain reaction (dPCR) analysis of single cells. This device allows for the parallel processing of single cells and executes all steps of analysis, including cell capture, washing, lysis, reverse transcription, and dPCR analysis. The cDNA from each single cell is distributed into a dedicated dPCR array consisting of 1020 chambers, each having a volume of 25 pL, using surface-tension-based sample partitioning. The high density of this dPCR format (118,900 chambers/cm(2)) allows the analysis of 200 single cells per run, for a total of 204,000 PCR reactions using a device footprint of 10 cm(2). Experiments using RNA dilutions show this device achieves shot-noise-limited performance in quantifying single molecules, with a dynamic range of 10(4). We performed over 1200 single-cell measurements, demonstrating the use of this platform in the absolute quantification of both high- and low-abundance mRNA transcripts, as well as micro-RNAs that are not easily measured using alternative hybridization methods. We further apply the specificity and sensitivity of single-cell dPCR to performing measurements of RNA editing events in single cells. High-throughput dPCR provides a new tool in the arsenal of single-cell analysis methods, with a unique combination of speed, precision, sensitivity, and specificity. We anticipate this approach will enable new studies where high-performance single-cell measurements are essential, including the analysis of transcriptional noise, allelic imbalance, and RNA processing.

  10. Single-cell RNA-seq reveals changes in cell cycle and differentiation programs upon aging of hematopoietic stem cells

    Science.gov (United States)

    Kowalczyk, Monika S.; Tirosh, Itay; Heckl, Dirk; Rao, Tata Nageswara; Dixit, Atray; Haas, Brian J.; Schneider, Rebekka K.; Wagers, Amy J.; Ebert, Benjamin L.; Regev, Aviv

    2015-01-01

    Both intrinsic cell state changes and variations in the composition of stem cell populations have been implicated as contributors to aging. We used single-cell RNA-seq to dissect variability in hematopoietic stem cell (HSC) and hematopoietic progenitor cell populations from young and old mice from two strains. We found that cell cycle dominates the variability within each population and that there is a lower frequency of cells in the G1 phase among old compared with young long-term HSCs, suggesting that they traverse through G1 faster. Moreover, transcriptional changes in HSCs during aging are inversely related to those upon HSC differentiation, such that old short-term (ST) HSCs resemble young long-term (LT-HSCs), suggesting that they exist in a less differentiated state. Our results indicate both compositional changes and intrinsic, population-wide changes with age and are consistent with a model where a relationship between cell cycle progression and self-renewal versus differentiation of HSCs is affected by aging and may contribute to the functional decline of old HSCs. PMID:26430063

  11. Decoding Signal Processing at the Single-Cell Level

    Energy Technology Data Exchange (ETDEWEB)

    Wiley, H. Steven

    2017-12-01

    The ability of cells to detect and decode information about their extracellular environment is critical to generating an appropriate response. In multicellular organisms, cells must decode dozens of signals from their neighbors and extracellular matrix to maintain tissue homeostasis while still responding to environmental stressors. How cells detect and process information from their surroundings through a surprisingly limited number of signal transduction pathways is one of the most important question in biology. Despite many decades of research, many of the fundamental principles that underlie cell signal processing remain obscure. However, in this issue of Cell Systems, Gillies et al present compelling evidence that the early response gene circuit can act as a linear signal integrator, thus providing significant insight into how cells handle fluctuating signals and noise in their environment.

  12. Nuclear size regulation: from single cells to development and disease.

    Science.gov (United States)

    Edens, Lisa J; White, Karen H; Jevtic, Predrag; Li, Xiaoyang; Levy, Daniel L

    2013-04-01

    Cell size varies greatly among different cell types and organisms, especially during early development when cell division is rapid with little overall growth. A fundamental question is how organelle size is regulated relative to cell size. The nucleus exhibits exquisite size scaling during development and between species, and nuclear size is often altered in cancer cells. Recent studies have elucidated mechanisms of nuclear size regulation in a variety of experimental systems, opening the door to future research on how nuclear size impacts upon cell and nuclear function and subnuclear organization. In this review we discuss studies that have clarified nuclear size control mechanisms and how these results have or will contribute to our understanding of the functional significance of nuclear size. Copyright © 2012 Elsevier Ltd. All rights reserved.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2009-05-01

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

  14. An Immunofluorescence-Assisted Microfluidic Single Cell Quantitative Reverse Transcription Polymerase Chain Reaction Analysis of Tumour Cells Separated from Blood

    Directory of Open Access Journals (Sweden)

    Kazunori Hoshino

    2015-11-01

    Full Text Available Circulating tumour cells (CTCs are important indicators of metastatic cancer and may provide critical information for individualized treatment. As CTCs are usually very rare, the techniques to obtain information from very small numbers of cells are crucial. Here, we propose a method to perform a single cell quantitative reverse transcription polymerase chain reaction (qPCR analysis of rare tumour cells. We utilized a microfluidic immunomagnetic assay to separate cancer cells from blood. A combination of detailed immunofluorescence and laser microdissection enabled the precise selection of individual cells. Cancer cells that were spiked into blood were successfully separated and picked up for a single cell PCR analysis. The breast cancer cell lines MCF7, SKBR3 and MDAMB231 were tested with 10 different genes. The result of the single cell analysis matched the results from a few thousand cells. Some markers (e.g., ER, HER2 that are commonly used for cancer identification showed relatively large deviations in expression levels. However, others (e.g., GRB7 showed deviations that are small enough to supplement single cell disease profiling.

  15. 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.

  16. Raman spectral dynamics of single cells in the early stages of growth factor stimulation.

    Science.gov (United States)

    Takanezawa, Sota; Morita, Shin-ichi; Ozaki, Yukihiro; Sako, Yasushi

    2015-05-05

    Cell fates change dynamically in response to various extracellular signals, including growth factors that stimulate differentiation and proliferation. The processes underlying cell-fate decisions are complex and often include large cell-to-cell variations, even within a clonal population in the same environment. To understand the origins of these cell-to-cell variations, we must detect the internal dynamics of single cells that reflect their changing chemical milieu. In this study, we used the Raman spectra of single cells to trace their internal dynamics during the early stages of growth factor stimulation. This method allows nondestructive and inclusive time-series analyses of chemical compositions of the same single cells. Applying a Gaussian mixture model to the major principal components of the single-cell Raman spectra, we detected the dynamics of the chemical states in MCF-7 cancer-derived cells in the absence and presence of differentiation and proliferation factors. The dynamics displayed characteristic variations according to the functions of the growth factors. In the differentiation pathway, the chemical composition changed directionally between multiple states, including both reversible and irreversible state transitions. In contrast, in the proliferation pathway, the chemical composition was homogenized into a single state. The differentiation factor also stimulated fluctuations in the chemical composition, whereas the proliferation factor did not. Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  17. Single-centre experience of allogeneic haemopoietic stem cell ...

    African Journals Online (AJOL)

    There were 31 sibling matched peripheral-blood stem cell (PBSC) transplants and 1 maternal haploidentical PBSC transplant. Stem cells were mobilised from bone marrow into peripheral blood by administering granulocyte-colony stimulating factor to donors. PBSCs were harvested by apheresis. Eight patients received ...

  18. Optical and hydrodynamic stretching of single cells from blood

    DEFF Research Database (Denmark)

    Thirstrup, Henrik; Rungling, Tony B.; Khalil Al-Hamdani, Mustafa Zyad

    2017-01-01

    as an optical stretcher, in a microfluidic chip in which optical fibers have been placed during a post-processing step. Another strategy is to exert hydrodynamic shear forces on the cells by forcing the cells through a narrow constriction. The latter method has the advantage of a considerably higher throughput...

  19. Physical chemistry in a single live cell: confocal microscopy.

    Science.gov (United States)

    Amin, Md Asif; Nandi, Somen; Mondal, Prasenjit; Mahata, Tanushree; Ghosh, Surajit; Bhattacharyya, Kankan

    2017-05-24

    A live cell is a complex, yet extremely important container. Understanding the dynamics in a selected intracellular component is a challenging task. We have recently made significant progress in this direction using a confocal microscope as a tool. The smallest size of the focused spot in a confocal microscope is ∼0.2 μm (200 nm). This is nearly one hundred times smaller than the size of a live cell. Thus, one can selectively study different intracellular components/organelles in a live cell. In this paper, we discuss how one can image different intracellular components/organelles, record fluorescence spectra and decay at different locations, ascertain local polarity and viscosity, and monitor the dynamics of solvation, proton transfer, red-ox and other phenomena at specified locations/organelles inside a cell. We will highlight how this knowledge enriched us in differentiating between cancer and non-cancer cells, 3D tumor spheroids and towards drug delivery.

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

    Science.gov (United States)

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

    2011-02-01

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

  1. A modified single-cell electroporation method for molecule delivery into a motile protist, Euglena gracilis.

    Science.gov (United States)

    Ohmachi, Masashi; Fujiwara, Yoshie; Muramatsu, Shuki; Yamada, Koji; Iwata, Osamu; Suzuki, Kengo; Wang, Dan Ohtan

    2016-11-01

    Single-cell transfection is a powerful technique for delivering chemicals, drugs, or probes into arbitrary, specific single cells. This technique is especially important when the analysis of molecular function and cellular behavior in individual microscopic organisms such as protists requires the precise identification of the target cell, as fluorescence labeling of bulk populations makes tracking of individual motile protists virtually impossible. Herein, we have modified current single-cell electroporation techniques for delivering fluorescent markers into single Euglena gracilis, a motile photosynthetic microalga. Single-cell electroporation introduced molecules into individual living E. gracilis cells after a negative pressure was applied through a syringe connected to the micropipette to the target cell. The new method achieves high transfection efficiency and viability after electroporation. With the new technique, we successfully introduced a variety of molecules such as GFP, Alexa Fluor 488, and exciton-controlled hybridization-sensitive fluorescent oligonucleotide (ECHO) RNA probes into individual motile E. gracilis cells. We demonstrate imaging of endogenous mRNA in living E. gracilis without interfering with their physiological functions, such as swimming or division, over an extended period of time. Thus the modified single-cell electroporation technique is suitable for delivering versatile functional molecules into individual motile protists. Copyright © 2016 Elsevier B.V. All rights reserved.

  2. Using single cell sequencing data to model the evolutionary history of a tumor.

    Science.gov (United States)

    Kim, Kyung In; Simon, Richard

    2014-01-24

    The introduction of next-generation sequencing (NGS) technology has made it possible to detect genomic alterations within tumor cells on a large scale. However, most applications of NGS show the genetic content of mixtures of cells. Recently developed single cell sequencing technology can identify variation within a single cell. Characterization of multiple samples from a tumor using single cell sequencing can potentially provide information on the evolutionary history of that tumor. This may facilitate understanding how key mutations accumulate and evolve in lineages to form a heterogeneous tumor. We provide a computational method to infer an evolutionary mutation tree based on single cell sequencing data. Our approach differs from traditional phylogenetic tree approaches in that our mutation tree directly describes temporal order relationships among mutation sites. Our method also accommodates sequencing errors. Furthermore, we provide a method for estimating the proportion of time from the earliest mutation event of the sample to the most recent common ancestor of the sample of cells. Finally, we discuss current limitations on modeling with single cell sequencing data and possible improvements under those limitations. Inferring the temporal ordering of mutational sites using current single cell sequencing data is a challenge. Our proposed method may help elucidate relationships among key mutations and their role in tumor progression.

  3. Transfection efficiency of normal and cancer cell lines and monitoring of promoter activity by single-cell bioluminescence imaging.

    Science.gov (United States)

    Horibe, Tomohisa; Torisawa, Aya; Akiyoshi, Ryutaro; Hatta-Ohashi, Yoko; Suzuki, Hirobumi; Kawakami, Koji

    2014-02-01

    The bioluminescence system (luciferase reporter assay system) is widely used to study gene expression, signal transduction and other cellular activities. Although transfection of reporter plasmid DNA to mammalian cell lines is an indispensable experimental step, the transfection efficiency of DNA varies among cell lines, and several cell lines are not suitable for this type of assay because of the low transfection efficiency. In this study, we confirm the transfection efficiency of reporter DNA to several cancer and normal cell lines after transient transfection by single-cell imaging. Luminescence images could be obtained from living single cells after transient transfection, and the calculated transfection efficiency of this method was similar to that of the conventional reporter assay using a luminometer. We attempted to measure the activity of the Bip promoter under endoplasmic reticulum stress conditions using both high and low transfection efficiency cells for plasmid DNA at the single-cell level, and observed activation of this promoter even in cells with the lowest transfection efficiency. These results show that bioluminescence imaging of single cells is a powerful tool for the analysis of gene expression based on a reporter assay using limited samples such as clinical specimens or cells from primary culture, and could provide additional information compared with the conventional assay. Copyright © 2013 John Wiley & Sons, Ltd.

  4. Estimation of turgor pressure through comparison between single plant cell and pressurized shell mechanics

    Science.gov (United States)

    Durand-Smet, P.; Gauquelin, E.; Chastrette, N.; Boudaoud, A.; Asnacios, A.

    2017-10-01

    While plant growth is well known to rely on turgor pressure, it is challenging to quantify the contribution of turgor pressure to plant cell rheology. Here we used a custom-made micro-rheometer to quantify the viscoelastic behavior of isolated plant cells while varying their internal turgor pressure. To get insight into how plant cells adapt their internal pressure to the osmolarity of their medium, we compared the mechanical behavior of single plant cells to that of a simple, passive, pressurized shell: a soccer ball. While both systems exhibited the same qualitative behavior, a simple mechanical model allowed us to quantify turgor pressure regulation at the single cell scale.

  5. Heterogeneity of Metazoan Cells and Beyond: To Integrative Analysis of Cellular Populations at Single-Cell Level.

    Science.gov (United States)

    Barteneva, Natasha S; Vorobjev, Ivan A

    2018-01-01

    In this paper, we review some of the recent advances in cellular heterogeneity and single-cell analysis methods. In modern research of cellular heterogeneity, there are four major approaches: analysis of pooled samples, single-cell analysis, high-throughput single-cell analysis, and lately integrated analysis of cellular population at a single-cell level. Recently developed high-throughput single-cell genetic analysis methods such as RNA-Seq require purification step and destruction of an analyzed cell often are providing a snapshot of the investigated cell without spatiotemporal context. Correlative analysis of multiparameter morphological, functional, and molecular information is important for differentiation of more uniform groups in the spectrum of different cell types. Simplified distributions (histograms and 2D plots) can underrepresent biologically significant subpopulations. Future directions may include the development of nondestructive methods for dissecting molecular events in intact cells, simultaneous correlative cellular analysis of phenotypic and molecular features by hybrid technologies such as imaging flow cytometry, and further progress in supervised and non-supervised statistical analysis algorithms.

  6. Single crystalline silicon solar cells with rib structure

    Directory of Open Access Journals (Sweden)

    Shuhei Yoshiba

    2017-02-01

    Full Text Available To improve the conversion efficiency of Si solar cells, we have developed a thin Si wafer-based solar cell that uses a rib structure. The open-circuit voltage of a solar cell is known to increase with deceasing wafer thickness if the cell is adequately passivated. However, it is not easy to handle very thin wafers because they are brittle and are subject to warpage. We fabricated a lattice-shaped rib structure on the rear side of a thin Si wafer to improve the wafer’s strength. A silicon nitride film was deposited on the Si wafer surface and patterned to form a mask to fabricate the lattice-shaped rib, and the wafer was then etched using KOH to reduce the thickness of the active area, except for the rib region. Using this structure in a Si heterojunction cell, we demonstrated that a high open-circuit voltage (VOC could be obtained by thinning the wafer without sacrificing its strength. A wafer with thickness of 30 μm was prepared easily using this structure. We then fabricated Si heterojunction solar cells using these rib wafers, and measured their implied VOC as a function of wafer thickness. The measured values were compared with device simulation results, and we found that the measured VOC agrees well with the simulated results. To optimize the rib and cell design, we also performed device simulations using various wafer thicknesses and rib dimensions.

  7. Concurrent Isolation of 3 Distinct Cardiac Stem Cell Populations From a Single Human Heart Biopsy.

    Science.gov (United States)

    Monsanto, Megan M; White, Kevin S; Kim, Taeyong; Wang, Bingyan J; Fisher, Kristina; Ilves, Kelli; Khalafalla, Farid G; Casillas, Alexandria; Broughton, Kathleen; Mohsin, Sadia; Dembitsky, Walter P; Sussman, Mark A

    2017-07-07

    The relative actions and synergism between distinct myocardial-derived stem cell populations remain obscure. Ongoing debates on optimal cell population(s) for treatment of heart failure prompted implementation of a protocol for isolation of multiple stem cell populations from a single myocardial tissue sample to develop new insights for achieving myocardial regeneration. Establish a robust cardiac stem cell isolation and culture protocol to consistently generate 3 distinct stem cell populations from a single human heart biopsy. Isolation of 3 endogenous cardiac stem cell populations was performed from human heart samples routinely discarded during implantation of a left ventricular assist device. Tissue explants were mechanically minced into 1 mm 3 pieces to minimize time exposure to collagenase digestion and preserve cell viability. Centrifugation removes large cardiomyocytes and tissue debris producing a single cell suspension that is sorted using magnetic-activated cell sorting technology. Initial sorting is based on tyrosine-protein kinase Kit (c-Kit) expression that enriches for 2 c-Kit + cell populations yielding a mixture of cardiac progenitor cells and endothelial progenitor cells. Flowthrough c-Kit - mesenchymal stem cells are positively selected by surface expression of markers CD90 and CD105. After 1 week of culture, the c-Kit + population is further enriched by selection for a CD133 + endothelial progenitor cell population. Persistence of respective cell surface markers in vitro is confirmed both by flow cytometry and immunocytochemistry. Three distinct cardiac cell populations with individualized phenotypic properties consistent with cardiac progenitor cells, endothelial progenitor cells, and mesenchymal stem cells can be successfully concurrently isolated and expanded from a single tissue sample derived from human heart failure patients. © 2017 American Heart Association, Inc.

  8. 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

  9. On-slide detection of enzymatic activities in selected single cells

    DEFF Research Database (Denmark)

    Keller, Josephine Geertsen; Tesauro, Cinzia; Coletta, Andrea

    2017-01-01

    With increasing recognition of the importance in addressing cell-to-cell heterogeneity for the understanding of complex biological systems, there is a growing need for assays capable of single cell analyses. In the current study, we describe the measurement of human topoisomerase I activity in si...

  10. The volumes and transcript counts of single cells reveal concentration homeostasis and capture biological noise.

    NARCIS (Netherlands)

    Kempe, H.; Schwabe, A.; Crémazy, F.; Verschure, P.J.; Bruggeman, F.J.

    2015-01-01

    Transcriptional stochasticity can be measured by counting the number of mRNA molecules per cell. Cell-to-cell variability is best captured in terms of concentration rather than molecule counts, because reaction rates depend on concentrations. We combined single-molecule mRNA counting with

  11. 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

  12. Sonoporation of suspension cells with a single cavitation bubble in a microfluidic confinement

    NARCIS (Netherlands)

    le Gac, Severine; Zwaan, Ed; van den Berg, Albert; Ohl, C.D.

    2007-01-01

    We report here the sonoporation of HL60 (human promyelocytic leukemia) suspension cells in a microfluidic confinement using a single laser-induced cavitation bubble. Cavitation bubbles can induce membrane poration of cells located in their close vicinity. Membrane integrity of suspension cells

  13. Single cell-type comparative metabolomics of epidermal bladder cells from the halophyte Mesembryanthemum crystallinum.

    Directory of Open Access Journals (Sweden)

    Bronwyn Jane Barkla

    2015-06-01

    Full Text Available 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 transcriptomics studies of the EBC establishing that these cells are metabolically active and have roles other than sodium sequestration. To continue our investigation into the function of these unusual cells we carried out a comprehensive global analysis of the metabolites present in the EBC extract by gas chromatography Time-of-Flight mass spectrometry (GC-TOF and identified 194 known and 722 total molecular features. Statistical analysis of the metabolic changes between control and salt-treated samples was used to identify 352 significantly differing metabolites (268 after correction for FDR. Principal components analysis provided an unbiased evaluation of the data variance structure. Biochemical pathway enrichment analysis suggested significant perturbations in 13 biochemical pathways as defined in KEGG. More than 50% of the metabolites that show significant changes in the EBC, can be classified as compatible solutes and include sugars, sugar alcohols, protein and non-protein amino acids, and organic acids, highlighting the need to maintain osmotic homeostasis to balance the accumulation of Na and Cl ions. Overall, the comparison of metabolic changes in salt treated relative to control samples suggest large alterations in Mesembryanthemum crystallinum epidermal bladder cells.

  14. Single HeLa and MCF-7 cell measurement using minimized impedance spectroscopy and microfluidic device

    Science.gov (United States)

    Wang, Min-Haw; Kao, Min-Feng; Jang, Ling-Sheng

    2011-06-01

    This study presents an impedance measurement system for single-cell capture and measurement. The microwell structure which utilizes nDEP force is used to single-cell capture and a minimized impedance spectroscopy which includes a power supply chip, an impedance measurement chip and a USB microcontroller chip is used to single-cell impedance measurement. To improve the measurement accuracy of the proposed system, Biquadratic fitting is used in this study. The measurement accuracy and reliability of the proposed system are compared to those of a conventional precision impedance analyzer. Moreover, a stable material, latex beads, is used to study the impedance measurement using the minimized impedance spectroscopy with cell-trapping device. Finally, the proposed system is used to measure the impedance of HeLa cells and MCF-7 cells. The impedance of single HeLa cells decreased from 9.55 × 103 to 3.36 × 103 Ω and the impedance of single MCF-7 cells decreased from 3.48 × 103 to 1.45 × 103 Ω at an operate voltage of 0.5 V when the excitation frequency was increased from 11 to 101 kHz. The results demonstrate that the proposed impedance measurement system successfully distinguishes HeLa cells and MCF-7 cells.

  15. Geometry-induced injection dispersion in single-cell protein electrophoresis.

    Science.gov (United States)

    Pan, Qiong; Herr, Amy E

    2018-02-13

    Arrays of microwells are widely used to isolate individual cells, facilitate high throughput cytometry assays, and ensure compatibility of those assays with whole-cell imaging. Microwell geometries have recently been utilized for handling and preparation of single-cell lysate, prior to single-cell protein electrophoresis. It is in the context of single-cell electrophoresis that we investigate the interplay of microwell geometry (circular, rectangular, triangular) and transport (diffusion, electromigration) on the subsequent performance of single-cell polyacrylamide gel electrophoresis (PAGE) for protein targets. We define and measure injector-induced dispersion during PAGE, and develop a numerical model of band broadening sources, experimentally validate the numerical model, and then identify operating conditions (characterized through the Peclet number, Pe) that lead to microwell-geometry induced losses in separation performance. With analysis of mammalian cells as a case study, we sought to understand at what Pe is the PAGE separation performance adversely sensitized to the microwell geometry. In developing design rules, we find that for the microwell geometries that are the most suitable for isolation of mammalian cells and moderate mass protein targets, the Pe is usually small enough (Pe geometry on protein PAGE of single-cell lysate. In extreme cases where the largest mammalian cells are analyzed (Pe > ∼20), consideration of Pe suggests using a rectangular - and not the widely used circular - microwell geometry to maximize protein PAGE separation performance. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Raman spectroscopy of single human tumour cells exposed to ionizing radiation in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Matthews, Q; Jirasek, A [Department of Physics and Astronomy, University of Victoria, Victoria BC V8W 3P6 (Canada); Brolo, AG [Department of Chemistry, University of Victoria, Victoria BC V8W 3V6 (Canada); Lum, J; Duan, X, E-mail: qmatthew@uvic.c, E-mail: jirasek@uvic.c [Deeley Research Centre, BC Cancer Agency-Vancouver Island Centre, Victoria BC V8R 6V5 (Canada)

    2011-01-07

    This work investigates the capability of Raman spectroscopy (RS) to study the effects of ionizing radiation on single human tumour cells. Prostate tumour cells (cell line DU145) are cultured in vitro and irradiated to doses between 15 and 50 Gy with single fractions of 6 MV photons. Single-cell Raman spectra are acquired from irradiated and unirradiated cultures up to 5 days post-irradiation. Principal component analysis is used to distinguish the uniquely radiation-induced spectral changes from inherent sources of spectral variability arising from cell cycle differences and other known factors. We observe uniquely radiation-induced spectral changes which are correlated with both the irradiated dose and the incubation time post-irradiation. The spectral changes induced by radiation arise from biochemical differences in lipids, nucleic acids, amino acids and conformational protein structures between irradiated and unirradiated cells. To our knowledge, this study is the first use of RS to observe radiation-induced biochemical differences in single cells, and is the first use of vibrational spectroscopy to observe uniquely radiation-induced biochemical differences in single cells independent of concurrent cell-cycle- or cell-death-related processes.

  17. 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.

  18. Single cell analysis facilitates staging of Blimp1-dependent primordial germ cells derived from mouse embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    John J Vincent

    Full Text Available The cell intrinsic programming that regulates mammalian primordial germ cell (PGC development in the pre-gonadal stage is challenging to investigate. To overcome this we created a transgene-free method for generating PGCs in vitro (iPGCs from mouse embryonic stem cells (ESCs. Using labeling for SSEA1 and cKit, two cell surface molecules used previously to isolate presumptive iPGCs, we show that not all SSEA1+/cKit+ double positive cells exhibit a PGC identity. Instead, we determined that selecting for cKit(bright cells within the SSEA1+ fraction significantly enriches for the putative iPGC population. Single cell analysis comparing SSEA1+/cKit(bright iPGCs to ESCs and embryonic PGCs demonstrates that 97% of single iPGCs co-express PGC signature genes Blimp1, Stella, Dnd1, Prdm14 and Dazl at similar levels to e9.5-10.5 PGCs, whereas 90% of single mouse ESC do not co-express PGC signature genes. For the 10% of ESCs that co-express PGC signature genes, the levels are significantly lower than iPGCs. Microarray analysis shows that iPGCs are transcriptionally distinct from ESCs and repress gene ontology groups associated with mesoderm and heart development. At the level of chromatin, iPGCs contain 5-methyl cytosine bases in their DNA at imprinted and non-imprinted loci, and are enriched in histone H3 lysine 27 trimethylation, yet do not have detectable levels of Mvh protein, consistent with a Blimp1-positive pre-gonadal PGC identity. In order to determine whether iPGC formation is dependent upon Blimp1, we generated Blimp1 null ESCs and found that loss of Blimp1 significantly depletes SSEA1/cKit(bright iPGCs. Taken together, the generation of Blimp1-positive iPGCs from ESCs constitutes a robust model for examining cell-intrinsic regulation of PGCs during the Blimp1-positive stage of development.

  19. Volatility of Mutator Phenotypes at Single Cell Resolution.

    Directory of Open Access Journals (Sweden)

    Scott R Kennedy

    2015-04-01

    Full Text Available Mutator phenotypes accelerate the evolutionary process of neoplastic transformation. Historically, the measurement of mutation rates has relied on scoring the occurrence of rare mutations in target genes in large populations of cells. Averaging mutation rates over large cell populations assumes that new mutations arise at a constant rate during each cell division. If the mutation rate is not constant, an expanding mutator population may contain subclones with widely divergent rates of evolution. Here, we report mutation rate measurements of individual cell divisions of mutator yeast deficient in DNA polymerase ε proofreading and base-base mismatch repair. Our data are best fit by a model in which cells can assume one of two distinct mutator states, with mutation rates that differ by an order of magnitude. In error-prone cell divisions, mutations occurred on the same chromosome more frequently than expected by chance, often in DNA with similar predicted replication timing, consistent with a spatiotemporal dimension to the hypermutator state. Mapping of mutations onto predicted replicons revealed that mutations were enriched in the first half of the replicon as well as near termination zones. Taken together, our findings show that individual genome replication events exhibit an unexpected volatility that may deepen our understanding of the evolution of mutator-driven malignancies.

  20. Light acclimation of photosynthesis in two closely related firs (Abies pinsapo Boiss. and Abies alba Mill.): the role of leaf anatomy and mesophyll conductance to CO2.

    Science.gov (United States)

    Peguero-Pina, José Javier; Sancho-Knapik, Domingo; Flexas, Jaume; Galmés, Jeroni; Niinemets, Ülo; Gil-Pelegrín, Eustaquio

    2016-03-01

    Leaves growing in the forest understory usually present a decreased mesophyll conductance (gm) and photosynthetic capacity. The role of leaf anatomy in determining the variability in gm among species is known, but there is a lack of information on how the acclimation of gm to shade conditions is driven by changes in leaf anatomy. Within this context, we demonstrated that Abies pinsapo Boiss. experienced profound modifications in needle anatomy to drastic changes in light availability that ultimately led to differential photosynthetic performance between trees grown in the open field and in the forest understory. In contrast to A. pinsapo, its congeneric Abies alba Mill. did not show differences either in needle anatomy or in photosynthetic parameters between trees grown in the open field and in the forest understory. The increased gm values found in trees of A. pinsapo grown in the open field can be explained by occurrence of stomata at both needle sides (amphistomatous needles), increased chloroplast surface area exposed to intercellular airspace, decreased cell wall thickness and, especially, decreased chloroplast thickness. To the best of our knowledge, the role of such drastic changes in ultrastructural needle anatomy in explaining the response of gm to the light environment has not been demonstrated in field conditions. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  1. Single-Cell Mass Spectrometry for Discovery Proteomics: Quantifying Translational Cell Heterogeneity in the 16-Cell Frog (Xenopus) Embryo.

    Science.gov (United States)

    Lombard-Banek, Camille; Moody, Sally A; Nemes, Peter

    2016-02-12

    We advance mass spectrometry from a cell population-averaging tool to one capable of quantifying the expression of diverse proteins in single embryonic cells. Our instrument combines capillary electrophoresis (CE), electrospray ionization, and a tribrid ultrahigh-resolution mass spectrometer (HRMS) to enable untargeted (discovery) proteomics with ca. 25 amol lower limit of detection. CE-μESI-HRMS enabled the identification of 500-800 nonredundant protein groups by measuring 20 ng, or embryo, amounting to a total of 1709 protein groups identified between n=3 biological replicates. By quantifying ≈150 nonredundant protein groups between all blastomeres and replicate measurements, we found significant translational cell heterogeneity along multiple axes of the embryo at this very early stage of development when the transcriptional program of the embryo has yet to begin. © 2016 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.

  2. 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

  3. 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.

  4. All-in-polymer injection molded device for single cell capture using multilevel silicon master fabrication

    DEFF Research Database (Denmark)

    Tanzi, S.; Larsen, S.T.; Matteucci, M.

    2012-01-01

    This work demonstrates a novel all-in-polymer device for single cell capture applicable for biological recordings. The chip is injection molded and comprises a "cornered" (non planar) aperture. It has been demonstrated how cornered apertures are straightforward to mold in PDMS [1,2]. In this stud...... defects during demolding. Capturing of single PC12 cells has been demonstrated.......This work demonstrates a novel all-in-polymer device for single cell capture applicable for biological recordings. The chip is injection molded and comprises a "cornered" (non planar) aperture. It has been demonstrated how cornered apertures are straightforward to mold in PDMS [1,2]. In this study...

  5. ZIFA: Dimensionality reduction for zero-inflated single-cell gene expression analysis.

    Science.gov (United States)

    Pierson, Emma; Yau, Christopher

    2015-11-02

    Single-cell RNA-seq data allows insight into normal cellular function and various disease states through molecular characterization of gene expression on the single cell level. Dimensionality reduction of such high-dimensional data sets is essential for visualization and analysis, but single-cell RNA-seq data are challenging for classical dimensionality-reduction methods because of the prevalence of dropout events, which lead to zero-inflated data. Here, we develop a dimensionality-reduction method, (Z)ero (I)nflated (F)actor (A)nalysis (ZIFA), which explicitly models the dropout characteristics, and show that it improves modeling accuracy on simulated and biological data sets.

  6. Identity and Diversity of Human Peripheral Th and T Regulatory Cells Defined by Single-Cell Mass Cytometry.

    Science.gov (United States)

    Kunicki, Matthew A; Amaya Hernandez, Laura C; Davis, Kara L; Bacchetta, Rosa; Roncarolo, Maria-Grazia

    2018-01-01

    Human CD3 + CD4 + Th cells, FOXP3 + T regulatory (Treg) cells, and T regulatory type 1 (Tr1) cells are essential for ensuring peripheral immune response and tolerance, but the diversity of Th, Treg, and Tr1 cell subsets has not been fully characterized. Independent functional characterization of human Th1, Th2, Th17, T follicular helper (Tfh), Treg, and Tr1 cells has helped to define unique surface molecules, transcription factors, and signaling profiles for each subset. However, the adequacy of these markers to recapitulate the whole CD3 + CD4 + T cell compartment remains questionable. In this study, we examined CD3 + CD4 + T cell populations by single-cell mass cytometry. We characterize the CD3 + CD4 + Th, Treg, and Tr1 cell populations simultaneously across 23 memory T cell-associated surface and intracellular molecules. High-dimensional analysis identified several new subsets, in addition to the already defined CD3 + CD4 + Th, Treg, and Tr1 cell populations, for a total of 11 Th cell, 4 Treg, and 1 Tr1 cell subsets. Some of these subsets share markers previously thought to be selective for Treg, Th1, Th2, Th17, and Tfh cells, including CD194 (CCR4) + FOXP3 + Treg and CD183 (CXCR3) + T-bet + Th17 cell subsets. Unsupervised clustering displayed a phenotypic organization of CD3 + CD4 + T cells that confirmed their diversity but showed interrelation between the different subsets, including similarity between Th1-Th2-Tfh cell populations and Th17 cells, as well as similarity of Th2 cells with Treg cells. In conclusion, the use of single-cell mass cytometry provides a systems-level characterization of CD3 + CD4 + T cells in healthy human blood, which represents an important baseline reference to investigate abnormalities of different subsets in immune-mediated pathologies. Copyright © 2017 by The American Association of Immunologists, Inc.

  7. 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.

  8. Targeted genetics in Drosophila cell lines: Inserting single transgenes in vitro.

    Science.gov (United States)

    Manivannan, Sathiya N; Simcox, Amanda

    2016-07-02

    A long-standing problem with analyzing transgene expression in tissue-culture cells is the variation caused by random integration of different copy numbers of transfected transgenes. In mammalian cells, single transgenes can be inserted by homologous recombination but this process is inefficient in Drosophila cells. To tackle this problem, our group, and the Cherbas group, used recombination-mediated cassette exchange (RMCE) to introduce single-copy transgenes into specific locations in the Drosophila genome. In both cases, ϕC31 was used to catalyze recombination between its target sequences attP in the genome, and attB flanking the donor sequence. We generated cell lines de novo with a single attP-flanked cassette for recombination, whereas, Cherbas et al. introduced a single attP-flanked cassette into existing cell lines. In both approaches, a 2-drug selection scheme was used to select for cells with a single copy of the donor sequence inserted by RMCE and against cells with random integration of multiple copies. Here we describe the general advantages of using RMCE to introduce genes into fly cells, the different attributes of the 2 methods, and how future work could make use of other recombinases and CRISPR/Cas9 genome editing to further enable genetic manipulation of Drosophila cells in vitro.

  9. Functionalized nanopipettes: toward label-free, single cell biosensors

    OpenAIRE

    Actis, Paolo; Mak, Andy C.; Pourmand, Nader

    2010-01-01

    Nanopipette technology has been proven to be a label-free biosensor capable of identifying DNA and proteins. The nanopipette can include specific recognition elements for analyte discrimination based on size, shape, and charge density. The fully electrical read-out and the ease and low-cost fabrication are unique features that give this technology an enormous potential. Unlike other biosensing platforms, nanopipettes can be precisely manipulated with submicron accuracy and used to study singl...

  10. 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.

  11. Single fibre and multifibre unit cell analysis of strength and cracking of unidirectional composites

    DEFF Research Database (Denmark)

    Wang, H.W.; Zhou, H.W.; Mishnaevsky, Leon

    2009-01-01

    Numerical simulations of damage evolution in composites reinforced with single and multifibre are presented. Several types of unit cell models are considered: single fibre unit cell, multiple fibre unit cell with one and several damageable sections per fibres, unit cells with homogeneous...... and inhomogeneous interfaces, etc. Two numerical damage models, cohesive elements, and damageable layers are employed for the simulation of the damage evolution in single fibre and multifibre unit cells. The two modelling approaches were compared and lead to the very close results. Competition among the different...... damageable parts in composites (matrix cracks, fibre/matrix interface damage and fibre fracture) was observed in the simulations. The strength of interface begins to influence the deformation behaviour of the cell only after the fibre is broken. In this case, the higher interface layer strength leads...

  12. Single nucleotide variations in cultured cancer cells: Effect of mismatch repair.

    Science.gov (United States)

    Panyutin, Igor G; Panyutin, Irina V; Powell-Castilla, Ian; Felix, Laura; Neumann, Ronald D

    2017-10-01

    We assessed single nucleotide variations (SNVs) between individual cells in two cancer cell lines; DU145, from brain metastasis of prostate tumor with deficient mismatch repair; and HT1080, a fibrosarcoma cell line. Clones of individual cells were isolated, and sequenced using Ion Ampliseq comprehensive cancer panel that covered the exomes of 409 oncogenes and tumor suppressor genes. Five clones of DU145 and four clones of HT1080 cells were analyzed. We found from 7 to 12 unique SNVs between DU145 clones, while HT1080 clones showed no more than one unique SNV. We then sub-cloned individual cells from some of these isolated clones of DU145 and HT1080 cells. The sub-clones were expanded from a single cell to approximately one million cells after about 20 cell divisions. The sub-clones of DU145 cells had from one to four new unique SNVs within the sequenced regions. No unique SNVs were found between sub-clones of HT1080 cells. Our data demonstrate that the extent of genetic variation at the single nucleotide level in cultured cancer cells is significantly affected by the status of the DNA mismatch repair system. Published by Elsevier B.V.

  13. From single cells to tissues: interactions between the matrix and human breast cells in real time.

    Directory of Open Access Journals (Sweden)

    Clifford Barnes

    Full Text Available Mammary gland morphogenesis involves ductal elongation, branching, and budding. All of these processes are mediated by stroma--epithelium interactions. Biomechanical factors, such as matrix stiffness, have been established as important factors in these interactions. For example, epithelial cells fail to form normal acinar structures in vitro in 3D gels that exceed the stiffness of a normal mammary gland. Additionally, heterogeneity in the spatial distribution of acini and ducts within individual collagen gels suggests that local organization of the matrix may guide morphogenesis. Here, we quantified the effects of both bulk material stiffness and local collagen fiber arrangement on epithelial morphogenesis.The formation of ducts and acini from single cells and the reorganization of the collagen fiber network were quantified using time-lapse confocal microscopy. MCF10A cells organized the surrounding collagen fibers during the first twelve hours after seeding. Collagen fiber density and alignment relative to the epithelial surface significantly increased within the first twelve hours and were a major influence in the shaping of the mammary epithelium. The addition of Matrigel to the collagen fiber network impaired cell-mediated reorganization of the matrix and increased the probability of spheroidal acini rather than branching ducts. The mechanical anisotropy created by regions of highly aligned collagen fibers facilitated elongation and branching, which was significantly correlated with fiber organization. In contrast, changes in bulk stiffness were not a strong predictor of this epithelial morphology.Localized regions of collagen fiber alignment are required for ductal elongation and branching suggesting the importance of local mechanical anisotropy in mammary epithelial morphogenesis. Similar principles may govern the morphology of branching and budding in other tissues and organs.

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

    Science.gov (United States)

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

    2017-03-01

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

  15. 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

    Characterization of directed differentiation of pluripotent stem cells towards therapeutically relevant cell types, including pancreatic beta-cells and hepatocytes, depends on molecular markers and assays that resolve the signature of individual cells. Pancreas and liver both have a common origin...... 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...

  16. pH and expansin action on single suspension-cultured tomato (Lycopersicon esculentum) cells.

    Science.gov (United States)

    Wang, C X; Wang, L; McQueen-Mason, S J; Pritchard, J; Thomas, C R

    2008-09-01

    The aim of this study was to measure key material properties of the cell walls of single suspension-cultured plant cells and relate these to cell-wall biochemistry. To this end, micromanipulation was used to compress single tomato cells between two flat surfaces until they ruptured, and force-deformation data were obtained. In addition to measuring the bursting force, we also determined the elastic (Young's) modulus of the cell walls by matching low strain (composites and proposed mechanisms of such failure. Through the measurement of cell-wall material properties using micromanipulation, it may be possible to understand more fully how cell-wall composition, structure and biochemistry lead to cell mechanical behaviour.

  17. DNA-electrophoresis of single cells - a method to screen for irradiated foodstuffs

    International Nuclear Information System (INIS)

    Leffke, A.; Helle, N.; Boegl, K.W.; Schreiber, G.A.

    1993-01-01

    Microelectrophoresis of single cells can be used to detect γ-irradiation over a wide dose range and for a variety of products. It is a simple and rapid test for DNA damages and can be used for screening. The method was tested on cell suspensions of bone marrow and muscle cells from frozen chicken legs, chicken heart, turkey liver, beef and pork irradiated with doses up to 3 kGy. Cell suspensions were prepared by incubation of tissues in EDTA-SDS-buffer at pH 8. Single cell electrophoresis was performed in 0.75% agarose gel. DNA was visualised by silver staining. In unirradiated samples no or only a small amount of DNA penetrated the cell membranes. Cells of irradiated samples appeared like a ''comet'' due to to migration of DNA-fragments out of cell. (orig.)

  18. Bioconductor workflow for single-cell RNA sequencing: Normalization, dimensionality reduction, clustering, and lineage inference.

    Science.gov (United States)

    Perraudeau, Fanny; Risso, Davide; Street, Kelly; Purdom, Elizabeth; Dudoit, Sandrine

    2017-01-01

    Novel single-cell transcriptome sequencing assays allow researchers to measure gene expression levels at the resolution of single cells and offer the unprecendented opportunity to investigate at the molecular level fundamental biological questions, such as stem cell differentiation or the discovery and characterization of rare cell types. However, such assays raise challenging statistical and computational questions and require the development of novel methodology and software. Using stem cell differentiation in the mouse olfactory epithelium as a case study, this integrated workflow provides a step-by-step tutorial to the methodology and associated software for the following four main tasks: (1) dimensionality reduction accounting for zero inflation and over dispersion and adjusting for gene and cell-level covariates; (2) cell clustering using resampling-based sequential ensemble clustering; (3) inference of cell lineages and pseudotimes; and (4) differential expression analysis along lineages.

  19. Magnetic tweezers for manipulation of magnetic particles in single cells

    Science.gov (United States)

    Ebrahimian, H.; Giesguth, M.; Dietz, K.-J.; Reiss, G.; Herth, S.

    2014-02-01

    Magnetic tweezers gain increasing interest for applications in biology. Here, a setup of magnetic tweezers is introduced using micropatterned conducting lines on transparent glass slides. Magnetic particles of 1 μm diameter were injected in barley cell vacuoles using a microinject system under microscopic control. Time dependent tracking of the particles after application of a magnetic field was used to determine the viscosity of vacuolar sap in vivo relative to water and isolated vacuolar fluid. The viscosity of vacuolar sap in cells was about 2-fold higher than that of extracted vacuolar fluid and 5 times higher than that of water.

  20. Analysis of the paired TCR α- and β-chains of single human T cells.

    Directory of Open Access Journals (Sweden)

    Song-Min Kim

    Full Text Available Analysis of the paired i.e. matching TCR α- and β-chain rearrangements of single human T cells is required for a precise investigation of clonal diversity, tissue distribution and specificity of protective and pathologic T-cell mediated immune responses. Here we describe a multiplex RT-PCR based technology, which for the first time allows for an unbiased analysis of the complete sequences of both α- and β-chains of TCR from single T cells. We validated our technology by the analysis of the pathologic T-cell infiltrates from tissue lesions of two T-cell mediated autoimmune diseases, psoriasis vulgaris (PV and multiple sclerosis (MS. In both disorders we could detect various T cell clones as defined by multiple T cells with identical α- and β-chain rearrangements distributed across the tissue lesions. In PV, single cell TCR analysis of lesional T cells identified clonal CD8(+ T cell expansions that predominated in the epidermis of psoriatic plaques. An MS brain lesion contained two dominant CD8(+ T-cell clones that extended over the white and grey matter and meninges. In both diseases several clonally expanded T cells carried dual TCRs composed of one Vβ and two different Vα-chain rearrangements. These results show that our technology is an efficient instrument to analyse αβ-T cell responses with single cell resolution in man. It should facilitate essential new insights into the mechanisms of protective and pathologic immunity in many human T-cell mediated conditions and allow for resurrecting functional TCRs from any αβ-T cell of choice that can be used for investigating their specificity.

  1. Simultaneous detection of mRNA and protein in single cells using immunofluorescence-combined single-molecule RNA FISH.

    Science.gov (United States)

    Kochan, Jakub; Wawro, Mateusz; Kasza, Aneta

    2015-10-01

    Although the concept of combining immunofluorescence (IF) with single-molecule RNA fluorescence in situ hybridization (smRNA FISH) seems obvious, the specific materials used during IF and smRNA FISH make it difficult to perform these procedures simultaneously on the same specimen. Even though there are reports where IF and smRNA FISH were combined with success, these were insufficient in terms of signal intensities, staining patterns, and GFP-compatibility, and a detailed exploration of the various factors that influence IF and smRNA FISH outcome has not been published yet. Here, we report a detailed study of conditions and reagents used in classic IF and smRNA FISH that allowed us to establish an easy, robust, and GFP-compatible procedure. Our protocol enables simultaneous detection of mRNA and protein quantity as well as the subcellular distribution of these molecules in single cells by combining an RNase-free modification of the IF technique and the more recent smRNA FISH method. Using this procedure, we have shown the direct interaction of RNase MCPIP1 with IL-6 mRNA. We also demonstrate the use of our protocol in heterogeneous cell population analysis, revealing cell-to-cell differences in mRNA and protein content.

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

    Directory of Open Access Journals (Sweden)

    Takashi Nozoe

    2017-03-01

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

  3. 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.

  4. Human organomics: a fresh approach to understanding human development using single-cell transcriptomics.

    Science.gov (United States)

    Camp, J Gray; Treutlein, Barbara

    2017-05-01

    Innovative methods designed to recapitulate human organogenesis from pluripotent stem cells provide a means to explore human developmental biology. New technologies to sequence and analyze single-cell transcriptomes can deconstruct these 'organoids' into constituent parts, and reconstruct lineage trajectories during cell differentiation. In this Spotlight article we summarize the different approaches to performing single-cell transcriptomics on organoids, and discuss the opportunities and challenges of applying these techniques to generate organ-level, mechanistic models of human development and disease. Together, these technologies will move past characterization to the prediction of human developmental and disease-related phenomena. © 2017. Published by The Company of Biologists Ltd.

  5. 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...

  6. Real-time visualization of intracellular hydrodynamics in single living cells

    NARCIS (Netherlands)

    Potma, Eric O.; Boeij, Wim P. de; Haastert, Peter J.M. van; Wiersma, Douwe A.

    2001-01-01

    Intracellular water concentrations in single living cells were visualized by nonlinear coherent anti-Stokes Raman scattering (CARS) microscopy. In combination with isotopic exchange measurements, CARS microscopy allowed the real-time observation of transient intracellular hydrodynamics at a high

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

    DEFF Research Database (Denmark)

    Yang, Tie; Bragheri, Francesca; Nava, Giovanni

    2016-01-01

    We realized an integrated microfluidic chip that allows measuring both optical deformability and acoustic compressibility on single cells, by optical stretching and acoustophoresis experiments respectively. Additionally, we propose a measurement protocol that allows evaluating the experimental ap...

  8. 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.

  9. 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.

  10. How to design a single-cell RNA-sequencing experiment: pitfalls, challenges and perspectives.

    Science.gov (United States)

    Dal Molin, Alessandra; Di Camillo, Barbara

    2018-01-31

    The sequencing of the transcriptome of single cells, or single-cell RNA-sequencing, has now become the dominant technology for the identification of novel cell types in heterogeneous cell populations or for the study of stochastic gene expression. In recent years, various experimental methods and computational tools for analysing single-cell RNA-sequencing data have been proposed. However, most of them are tailored to different experimental designs or biological questions, and in many cases, their performance has not been benchmarked yet, thus increasing the difficulty for a researcher to choose the optimal single-cell transcriptome sequencing (scRNA-seq) experiment and analysis workflow. In this review, we aim to provide an overview of the current available experimental and computational methods developed to handle single-cell RNA-sequencing data and, based on their peculiarities, we suggest possible analysis frameworks depending on specific experimental designs. Together, we propose an evaluation of challenges and open questions and future perspectives in the field. In particular, we go through the different steps of scRNA-seq experimental protocols such as cell isolation, messenger RNA capture, reverse transcription, amplification and use of quantitative standards such as spike-ins and Unique Molecular Identifiers (UMIs). We then analyse the current methodological challenges related to preprocessing, alignment, quantification, normalization, batch effect correction and methods to control for confounding effects. © The Author(s) 2018. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  11. Single Cell Transfection through Precise Microinjection with Quantitatively Controlled Injection Volumes

    Science.gov (United States)

    Chow, Yu Ting; Chen, Shuxun; Wang, Ran; Liu, Chichi; Kong, Chi-Wing; Li, Ronald A.; Cheng, Shuk Han; Sun, Dong

    2016-04-01

    Cell transfection is a technique wherein foreign genetic molecules are delivered into cells. To elucidate distinct responses during cell genetic modification, methods to achieve transfection at the single-cell level are of great value. Herein, we developed an automated micropipette-based quantitative microinjection technology that can deliver precise amounts of materials into cells. The developed microinjection system achieved precise single-cell microinjection by pre-patterning cells in an array and controlling the amount of substance delivered based on injection pressure and time. The precision of the proposed injection technique was examined by comparing the fluorescence intensities of fluorescent dye droplets with a standard concentration and water droplets with a known injection amount of the dye in oil. Injection of synthetic modified mRNA (modRNA) encoding green fluorescence proteins or a cocktail of plasmids encoding green and red fluorescence proteins into human foreskin fibroblast cells demonstrated that the resulting green fluorescence intensity or green/red fluorescence intensity ratio were well correlated with the amount of genetic material injected into the cells. Single-cell transfection via the developed microinjection technique will be of particular use in cases where cell transfection is challenging and genetically modified of selected cells are desired.

  12. Accounting for technical noise in differential expression analysis of single-cell RNA sequencing data.

    Science.gov (United States)

    Jia, Cheng; Hu, Yu; Kelly, Derek; Kim, Junhyong; Li, Mingyao; Zhang, Nancy R

    2017-11-02

    Recent technological breakthroughs have made it possible to measure RNA expression at the single-cell level, thus paving the way for exploring expression heterogeneity among individual cells. Current single-cell RNA sequencing (scRNA-seq) protocols are complex and introduce technical biases that vary across cells, which can bias downstream analysis without proper adjustment. To account for cell-to-cell technical differences, we propose a statistical framework, TASC (Toolkit for Analysis of Single Cell RNA-seq), an empirical Bayes approach to reliably model the cell-specific dropout rates and amplification bias by use of external RNA spike-ins. TASC incorporates the technical parameters, which reflect cell-to-cell batch effects, into a hierarchical mixture model to estimate the biological variance of a gene and detect differentially expressed genes. More importantly, TASC is able to adjust for covariates to further eliminate confounding that may originate from cell size and cell cycle differences. In simulation and real scRNA-seq data, TASC achieves accurate Type I error control and displays competitive sensitivity and improved robustness to batch effects in differential expression analysis, compared to existing methods. TASC is programmed to be computationally efficient, taking advantage of multi-threaded parallelization. We believe that TASC will provide a robust platform for researchers to leverage the power of scRNA-seq. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  13. Application of the comet assay in studies of programmed cell death (PCD in plants

    Directory of Open Access Journals (Sweden)

    Maria Charzyńska

    2014-01-01

    Full Text Available Programmed cell death (PCD in plants is an intensively investigated process. One of the main characteristics of PCD in both animal and plant organisms is the non-random, internucleosomal fragmentation of nuclear DNA, usually analysed using total DNA gel electrophoresis or TUNEL method. In this paper we present application of the "comet assay" (Single Cell Gel Electrophoresis for detection of nDNA degradation in studies of PCD during plant life cycle. We analyzed three types of tissue: anther tapetum, endosperm and mesophyll which were prepared in different ways to obtain a suspension of viable cells (without cell walls. The comet assay gives a possibility of examination of the nDNA degradation in individual cell. This method is significant for studies of the plant tissue differentiation and senescence especially in the cases when it is not possible to isolate large number of cells at the same developmental stage.

  14. Lung Injury; Relates to Real-Time Endoscopic Monitoring of Single Cells Respiratory Health in Lung

    Science.gov (United States)

    2017-09-01

    AWARD NUMBER: W81XWH-16-1-0253 TITLE: Lung Injury; Relates to Real- Time Endoscopic Monitoring of Single Cells Respiratory Health in Lung...response, including the time for reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and completing and...Sep 2016 - 31 Aug 2017 5a. CONTRACT NUMBER 4. TITLE AND SUBTITLE Lung Injury; Relates to Real- Time Endoscopic Monitoring of Single Cells Respiratory

  15. 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

  16. Rapid Evaluation of Power Degradation in Series Connection of Single Feeding Microsized Microbial Fuel Cells

    KAUST Repository

    Rojas, Jhonathan Prieto

    2014-07-08

    We have developed a sustainable, single feeding, microsized, air-cathode and membrane-free microbial fuel cells with a volume of 40 mu L each, which we have used for rapid evaluation of power generation and viability of a series array of three cells seeking higher voltage levels. Contrary to expectations, the achieved power density was modest (45 mWm(-3)), limited due to non-uniformities in assembly and the single-channel feeding system.

  17. Generation of functionally competent single bovine adrenal chromaffin cells from cell aggregates using the neutral protease dispase.

    Science.gov (United States)

    Craviso, Gale L

    2004-08-30

    A simple and efficient procedure has been developed to enzymatically dissociate aggregates of bovine adrenal chromaffin cells in suspension culture into viable, responsive single cells. For dissociation, the neutral protease dispase is added directly to the culture medium for a minimum of 3 h, followed by incubation of the cells in Hank's calcium-magnesium-free balanced salt solution at 37 degrees C with intermittent trituration to facilitate dispersion. This procedure generates a population of phase-bright single cells that are round in morphology, take up the dye neutral red, exclude the dye trypan blue and readily attach to tissue culture dishes coated with collagen, fibronectin or polylysine, thereby permitting applications that require plated-down conditions. When transferred to culture medium, the cells begin to reaggregate. By altering the length of time the cells are incubated in culture medium prior to attachment, the degree of reaggregation can be controlled to obtain plate-down profiles that consist of both isolated cells and cells in aggregates of varying sizes. Returning dissociated cells to suspension culture results in the reformation of large cell aggregates. Several measures of chromaffin cell function were indistinguishable for dissociated cells placed either in monolayer culture or suspension culture versus non-dissociated cells, implying that the dissociation procedure does not alter cellular responses or cause cellular damage.

  18. 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.

  19. 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

  20. 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

  1. Diaphragm pico-liter pump for single-cell manipulation.

    Science.gov (United States)

    Anis, Yasser; Houkal, Jeffrey; Holl, Mark; Johnson, Roger; Meldrum, Deirdre

    2011-08-01

    A pico-liter pump is developed and integrated into a robotic manipulation system that automatically selects and transfers individual living cells of interest to analysis locations. The pump is a displacement type pump comprising one cylindrical chamber connected to a capillary micropipette. The top of the chamber is a thin diaphragm which, when deflected, causes the volume of the fluid-filled cylindrical chamber to change thereby causing fluid in the chamber to flow in and out of the micropipette. This enables aspirating and dispensing individual living cells. The diaphragm is deflected by a piezoelectric actuator that pushes against its center. The pump aspirates and dispenses volumes of fluid between 500 pL and 250 nL at flow rates up to 250 nL/s. The piezo-driven diaphragm arrangement provides exquisite control of the flow rate in and out of the capillary orifice. This feature, in turn, allows reduced perturbation of live cells by controlling and minimizing the applied shear stresses.

  2. An Immunofluorescence-assisted Microfluidic Single Cell Quantitative Reverse Transcription Polymerase Chain Reaction Analysis of Tumour Cells Separated from Blood

    Directory of Open Access Journals (Sweden)

    Kazunori Hoshino

    2015-11-01

    matched the results from a few thousand cells. Some markers (e.g., ER, HER2 that are commonly used for cancer identification showed relatively large deviations in expres‐ sion levels. However, others (e.g., GRB7 showed devia‐ tions that are small enough to supplement single cell disease profiling.

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

    Science.gov (United States)

    Kussell, Edo

    2017-01-01

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

  4. 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.

  5. Monitoring single-cell gene regulation under dynamically controllable conditions with integrated microfluidics and software.

    Science.gov (United States)

    Kaiser, Matthias; Jug, Florian; Julou, Thomas; Deshpande, Siddharth; Pfohl, Thomas; Silander, Olin K; Myers, Gene; van Nimwegen, Erik

    2018-01-15

    Much is still not understood about how gene regulatory interactions control cell fate decisions in single cells, in part due to the difficulty of directly observing gene regulatory processes in vivo. We introduce here a novel integrated setup consisting of a microfluidic chip and accompanying analysis software that enable long-term quantitative tracking of growth and gene expression in single cells. The dual-input Mother Machine (DIMM) chip enables controlled and continuous variation of external conditions, allowing direct observation of gene regulatory responses to changing conditions in single cells. The Mother Machine Analyzer (MoMA) software achieves unprecedented accuracy in segmenting and tracking cells, and streamlines high-throughput curation with a novel leveraged editing procedure. We demonstrate the power of the method by uncovering several novel features of an iconic gene regulatory program: the induction of Escherichia coli's lac operon in response to a switch from glucose to lactose.

  6. Experimental and Numerical Study on the Cold Start Performance of a Single PEM Fuel Cell

    Directory of Open Access Journals (Sweden)

    Calvin H. Li

    2010-01-01

    Full Text Available A combined experimental and analytical investigation of single proton exchange membrane (PEM fuel cells, during cold start, has been conducted. The temperature influence on the performance of a single PEM fuel cell and the cold start failure of the PEM fuel cell was evaluated experimentally to determine the failure mechanisms and performance. The voltage, current, and power characteristics were investigated as a function of the load, the hydrogen fuel flow rate, and the cell temperature. The characteristics of cold start for a single PEM fuel cell were analyzed, and the various failure mechanisms were explored and characterized. In an effort to better understand the operational behavior and failure modes, a numerical simulation was also developed. The results of this analysis were then compared with the previously obtained experimental results and confirmed the accuracy of the failure mechanisms identified.

  7. Single-Cell Chemical Lysis on Microfluidic Chips with Arrays of Microwells

    Directory of Open Access Journals (Sweden)

    Nikolay A. Maslov

    2011-12-01

    Full Text Available Many conventional biochemical assays are performed using populations of cells to determine their quantitative biomolecular profiles. However, population averages do not reflect actual physiological processes in individual cells, which occur either on short time scales or nonsynchronously. Therefore, accurate analysis at the single-cell level has become a highly attractive tool for investigating cellular content. Microfluidic chips with arrays of microwells were developed for single-cell chemical lysis in the present study. The cellular occupancy in 30-mm-diameter microwells (91.45% was higher than that in 20-mm-diameter microwells (83.19% at an injection flow rate of 2.8 mL/min. However, most of the occupied 20-mm-diameter microwells contained individual cells. The results of chemical lysis experiments at the single-cell level indicate that cell membranes were gradually lysed as the lysis buffer was injected; they were fully lysed after 12 s. Single-cell chemical lysis was demonstrated in the proposed microfluidic chip, which is suitable for high-throughput cell lysis.

  8. Single exosome study reveals subpopulations distributed among cell lines with variability related to membrane content

    Directory of Open Access Journals (Sweden)

    Zachary J. Smith

    2015-12-01

    Full Text Available Current analysis of exosomes focuses primarily on bulk analysis, where exosome-to-exosome variability cannot be assessed. In this study, we used Raman spectroscopy to study the chemical composition of single exosomes. We measured spectra of individual exosomes from 8 cell lines. Cell-line-averaged spectra varied considerably, reflecting the variation in total exosomal protein, lipid, genetic, and cytosolic content. Unexpectedly, single exosomes isolated from the same cell type also exhibited high spectral variability. Subsequent spectral analysis revealed clustering of single exosomes into 4 distinct groups that were not cell-line specific. Each group contained exosomes from multiple cell lines, and most cell lines had exosomes in multiple groups. The differences between these groups are related to chemical differences primarily due to differing membrane composition. Through a principal components analysis, we identified that the major sources of spectral variation among the exosomes were in cholesterol content, relative expression of phospholipids to cholesterol, and surface protein expression. For example, exosomes derived from cancerous versus non-cancerous cell lines can be largely separated based on their relative expression of cholesterol and phospholipids. We are the first to indicate that exosome subpopulations are shared among cell types, suggesting distributed exosome functionality. The origins of these differences are likely related to the specific role of extracellular vesicle subpopulations in both normal cell function and carcinogenesis, and they may provide diagnostic potential at the single exosome level.

  9. How Single-Cell Genomics Is Changing Evolutionary and Developmental Biology.

    Science.gov (United States)

    Marioni, John C; Arendt, Detlev

    2017-10-06

    The recent flood of single-cell data not only boosts our knowledge of cells and cell types, but also provides new insight into development and evolution from a cellular perspective. For example, assaying the genomes of multiple cells during development reveals developmental lineage trees-the kinship lineage-whereas cellular transcriptomes inform us about the regulatory state of cells and their gradual restriction in potency-the Waddington lineage. Beyond that, the comparison of single-cell data across species allows evolutionary changes to be tracked at all stages of development from the zygote, via different kinds of stem cells, to the differentiating cells. We discuss recent insights into the evolution of stem cells and initial attempts to reconstruct the evolutionary cell type tree of the mammalian forebrain, for example, by the comparative analysis of neuron types in the mesencephalic floor. These studies illustrate the immense potential of single-cell genomics to open up a new era in developmental and evolutionary research.

  10. Spatio-temporally controlled transfection by quantitative injection into a single cell.

    Science.gov (United States)

    Kwon, Hyosung; Park, Hang-soo; Yu, Jewon; Hong, Sunghoi; Choi, Yeonho

    2015-10-01

    Transfection-based cellular control has been widely used in biology; however, conventional transfection methods cannot control spatio-temporal differences in gene expression or the quantity of delivered materials such as external DNA or RNA. Here, we present a non-viral and spatio-temporally controlled transfection technique of a quantitative injection into a single cell. DNA was quantitatively injected into a single cell at a desired location and time, and the optimal gene delivery and expression conditions were determined based on the amount of the delivered DNA and the transfection efficacy. Interestingly, an injection of 1500 DNAs produced an about average 30% gene expression efficiency, which was the optimal condition, and gene expression was sustained for more than 14 days. In a single cell, fluorescent intensity and polymerase chain reaction (PCR) results were compared for the quantity of gene expression. The high coincidence of both results suggests that the fluorescence intensity can reveal gene expression level which was investigated by PCR. In addition, 3 multiple DNA genes were successfully expressed in a single cell with different ratio. Overall, these results demonstrate that spatio-temporally controlled transfection by quantitative transfection is a useful technique for regulating gene expression in a single cell, which suggests that this technique may be used for stem cell research, including the creation of induced pluripotent stem (iPS) cells. Copyright © 2015 Elsevier Ltd. All rights reserved.

  11. Changes in photosynthesis, mesophyll conductance to CO2, and isoprenoid emissions in Populus nigra plants exposed to excess nickel

    International Nuclear Information System (INIS)

    Velikova, Violeta; Tsonev, Tsonko; Loreto, Francesco; Centritto, Mauro

    2011-01-01

    Poplar (Populus nigra) plants were grown hydroponically with 30 and 200 μM Ni (Ni 30 and Ni 200 ). Photosynthesis limitations and isoprenoid emissions were investigated in two leaf types (mature and developing). Ni stress significantly decreased photosynthesis, and this effect depended on the leaf Ni content, which was lower in mature than in developing leaves. The main limitations to photosynthesis were attributed to mesophyll conductance and metabolism impairment. In Ni-stressed developing leaves, isoprene emission was significantly stimulated. We attribute such stimulation to the lower chloroplastic [CO 2 ] than in control leaves. However chloroplastic [CO 2 ] did not control isoprene emission in mature leaves. Ni stress induced the emission of cis-β-ocimene in mature leaves, and of linalool in both leaf types. Induced biosynthesis and emission of isoprenoids reveal the onset of antioxidant processes that may also contribute to reduce Ni stress, especially in mature poplar leaves. - Graphical abstract: Visible damage caused by Ni treatment. 1 - Ni 0 (control plants); 2 - Ni 200 ; M = mature and D = developing Populus nigra leaves. Display Omitted Highlights: → We study the effect of Ni pollution on photosynthesis and isoprenoid emissions. → Ni stress significantly decreases photosynthesis. The main limitations are attributed to mesophyll conductance and metabolism impairment. → Constitutive isoprene emission was significantly stimulated in Ni-stressed leaves. Exposure to enhanced Ni concentration induces cis-beta-ocimene and linalool emissions. - The study reveals consequences of Ni stress on plant physiology, namely increasing diffusional limitation to photosynthesis and isoprenoid emissions.

  12. Mesophyll conductance in Zea mays responds transiently to CO2availability: implications for transpiration efficiency in C4crops.

    Science.gov (United States)

    Kolbe, Allison R; Cousins, Asaph B

    2018-03-01

    Mesophyll conductance (g m ) describes the movement of CO 2 from the intercellular air spaces below the stomata to the site of initial carboxylation in the mesophyll. In contrast with C 3 -g m , little is currently known about the intraspecific variation in C 4 -g m or its responsiveness to environmental stimuli. To address these questions, g m was measured on five maize (Zea mays) lines in response to CO 2 , employing three different estimates of g m . Each of the methods indicated a significant response of g m to CO 2 . Estimates of g m were similar between methods at ambient and higher CO 2 , but diverged significantly at low partial pressures of CO 2 . These differences are probably driven by incomplete chemical and isotopic equilibrium between CO 2 and bicarbonate under these conditions. Carbonic anhydrase and phosphoenolpyruvate carboxylase in vitro activity varied significantly despite similar values of g m and leaf anatomical traits. These results provide strong support for a CO 2 response of g m in Z. mays, and indicate that g m in maize is probably driven by anatomical constraints rather than by biochemical limitations. The CO 2 response of g m indicates a potential role for facilitated diffusion in C 4 -g m . These results also suggest that water-use efficiency could be enhanced in C 4 species by targeting g m . © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

  13. Axitinib in metastatic renal cell carcinoma: single center experience

    Directory of Open Access Journals (Sweden)

    Agnieszka Buraczewska

    2017-01-01

    Full Text Available Aim of the study : Due to the emergence of new therapeutic opportunities in the second-line treatment of metastatic renal cell carcinoma, the choice of the appropriate medication requires consideration. Making the selection one should take into account the likelihood of response, the probability of toxicity, properties of the drug and the clinical characteristics of the patient. Aim of the work was to confirm antitumor efficacy of axitinib in patients with metastatic clear-cell renal-cell carcinoma in the second line treatment remaining under the care of our institution. The primary objective was to determine antitumor activity, secondary – to evaluate progression free survival, safety of the treatment and to analyse clinical characteristics of treated population. Results: Treatment records of 27 patients (9 females, 18 males treated from October 2014 to the present (July 2016 were reviewed. The median duration of treatment which corresponds to the time to disease progression in observed population was 6 months (range: under 1 month – 16 months. 1 patient (3.7% had got objective response (PR, partial remission. Clinical benefit rate (PR + SD (stable disease was 66%. 9 patients (33.33% experienced treatment toxicity only in the first degree of CTCAE (common toxicity criteria for adverse events, 11 patients (40.74% presented the second degree toxicity and 5 patients (18.5% – third degree. The most commonly reported treatment related adverse events were diarrhea (47%, fatigue (26%, hand-foot syndrome (26%, deterioration of blood pressure control (22.2%, abnormal liver function tests (18.5%, mucositis (11.1%.We observed 3 cases of unacceptable toxicity. Conclusions : Axitinib confirms its effectiveness also in situ ation outside clinical trials, however, it is characterized by significant toxicity. Therefore, qualification for treatment should take into account the clinical patient characteristics. Effective diagnosis and treatment of side

  14. Development of Visible-Wavelength MALDI Cell Mass Spectrometry for High-Efficiency Single-Cell Analysis.

    Science.gov (United States)

    Xiong, Caiqiao; Zhou, Xiaoyu; He, Qing; Huang, Xi; Wang, Jiyun; Peng, Wen-Ping; Chang, Huan-Cheng; Nie, Zongxiu

    2016-12-06

    Mass is a fundamental physical property of an individual cell, from which is revealed the cell growth, cycle, and activity. Taking advantage of cell mass spectrometry (CMS), accurate mass measurement of a charged single cell has been achieved. However, with the increasing demand for high-efficiency single-cell analysis in biology, the limited throughput and inefficient cell desorption/ionization of the CMS inevitably become important issues. To address the challenge, a state of the art visible-wavelength matrix assisted laser desorption/ionization (MALDI) CMS was developed. The employed transmission mode laser ablation and fast evaporation sample preparation enabled the visible-wavelength MALDI to be soft enough and to generate intact charged cells for mass measurement. By using resorufin as matrix, ten sorts of cells, viz., red blood cells (RBCs), Jurkat (JK), CCRF-CEM, SNU-5, BGC-803, MCF-7, L-O2, 293T, Hep G2, and A549 cells, have been successfully analyzed. It was found that the desorption/ionization efficiency of visible-wavelength MALDI was at least 3-fold higher than that of conventional laser-induced acoustic desorption (LIAD) and relevant to the suspension/adherent property of analyzed cells. Based on the measured mass, different cell types in either the individual or mixed state can be differentiated successfully.

  15. Single-cell sequencing analysis characterizes common and cell-lineage-specific mutations in a muscle-invasive bladder cancer

    DEFF Research Database (Denmark)

    Li, Yingrui; Xu, Xun; Song, Luting

    2012-01-01

    sequencing of 66 individual tumor cells from a muscle-invasive bladder transitional cell carcinoma (TCC). Analyses of the somatic mutant allele frequency spectrum and clonal structure revealed that the tumor cells were derived from a single ancestral cell, but that subsequent evolution occurred, leading...... to two distinct tumor cell subpopulations. By analyzing recurrently mutant genes in an additional cohort of 99 TCC tumors, we identified genes that might play roles in the maintenance of the ancestral clone and in the muscle-invasive capability of subclones of this bladder cancer, respectively...

  16. Photoconversion of organic materials into single-cell protein

    Energy Technology Data Exchange (ETDEWEB)

    Weaver, P.F.

    1991-12-31

    A process is described for converting organic materials (such as biomass wastes) into sterile, high-grade bacterial protein suitable for use an animal feed or human food supplements. In a preferred embodiment the process involves thermally gasifying the organic material into primarily carbon monoxide, hydrogen and nitrogen products, followed by photosynthetic bacterial assimilation of the gases into cell material, which can be high as 65% protein. The process is ideally suited for waste recycling and for food production under zero-gravity or extra-terrestrial conditions.

  17. Transcriptional Networks in Single Perivascular Cells Sorted from Human Adipose Tissue Reveal a Hierarchy of Mesenchymal Stem Cells.

    Science.gov (United States)

    Hardy, W Reef; Moldovan, Nicanor I; Moldovan, Leni; Livak, Kenneth J; Datta, Krishna; Goswami, Chirayu; Corselli, Mirko; Traktuev, Dmitry O; Murray, Iain R; Péault, Bruno; March, Keith

    2017-05-01

    Adipose tissue is a rich source of multipotent mesenchymal stem-like cells, located in the perivascular niche. Based on their surface markers, these have been assigned to two main categories: CD31 - /CD45 - /CD34 + /CD146 - cells (adventitial stromal/stem cells [ASCs]) and CD31 - /CD45 - /CD34 - /CD146 + cells (pericytes [PCs]). These populations display heterogeneity of unknown significance. We hypothesized that aldehyde dehydrogenase (ALDH) activity, a functional marker of primitivity, could help to better define ASC and PC subclasses. To this end, the stromal vascular fraction from a human lipoaspirate was simultaneously stained with fluorescent antibodies to CD31, CD45, CD34, and CD146 antigens and the ALDH substrate Aldefluor, then sorted by fluorescence-activated cell sorting. Individual ASCs (n = 67) and PCs (n = 73) selected from the extremities of the ALDH-staining spectrum were transcriptionally profiled by Fluidigm single-cell quantitative polymerase chain reaction for a predefined set (n = 429) of marker genes. To these single-cell data, we applied differential expression and principal component and clustering analysis, as well as an original gene coexpression network reconstruction algorithm. Despite the stochasticity at the single-cell level, covariation of gene expression analysis yielded multiple network connectivity parameters suggesting that these perivascular progenitor cell subclasses possess the following order of maturity: (a) ALDH br ASC (most primitive); (b) ALDH dim ASC; (c) ALDH br PC; (d) ALDH dim PC (least primitive). This order was independently supported by specific combinations of class-specific expressed genes and further confirmed by the analysis of associated signaling pathways. In conclusion, single-cell transcriptional analysis of four populations isolated from fat by surface markers and enzyme activity suggests a developmental hierarchy among perivascular mesenchymal stem cells supported by markers and coexpression

  18. Direct electrochemiluminescence imaging of a single cell on a chitosan film modified electrode.

    Science.gov (United States)

    Liu, Gen; Ma, Cheng; Jin, Baokang; Chen, Zixuan; Zhu, Jun-Jie

    2018-03-06

    Single-cell imaging is essential for elucidating the biological mechanism of cell function because it accurately reveals the heterogeneity among cells. The electrochemiluminescence (ECL) microscopy technique has been considered a powerful tool to study cells because of its high throughput and zero cellular background light. However, since cells are immobilized on the electrode surface, the steric hindrance and the insulation from the cells make it difficult to obtain a luminous cell ECL image. To solve this problem, direct ECL imaging of a single cell was investigated and achieved on chitosan and nano-TiO2 modified fluoride-doped tin oxide conductive glass (FTO/TiO2/CS). The permeable chitosan film is not only favorable for cell immobilization but also increases the space between the bottom of cells and the electrode, thus, more ECL reagent can exist below the cells compared with the cells on a bare electrode, which guarantees the high sensitivity of quantitative analysis. The modification of nano-TiO2 strengthens the ECL visual signal in luminol solution and effectively improves the signal-to-noise ratio. The light intensity is correlated with the H2O2 concentration on FTO/TiO2/CS, which can be applied to analyze the H2O2 released from cells at the single-cell level. As far as we know, this is the first work to achieve cell ECL imaging without the steric hindrance effect of the cell, and it expands the applications of a modified electrode in visualization study.

  19. Parameter Optimization of Single-Diode Model of Photovoltaic Cell Using Memetic Algorithm

    Directory of Open Access Journals (Sweden)

    Yourim Yoon

    2015-01-01

    Full Text Available This study proposes a memetic approach for optimally determining the parameter values of single-diode-equivalent solar cell model. The memetic algorithm, which combines metaheuristic and gradient-based techniques, has the merit of good performance in both global and local searches. First, 10 single algorithms were considered including genetic algorithm, simulated annealing, particle swarm optimization, harmony search, differential evolution, cuckoo search, least squares method, and pattern search; then their final solutions were used as initial vectors for generalized reduced gradient technique. From this memetic approach, we could further improve the accuracy of the estimated solar cell parameters when compared with single algorithm approaches.

  20. Time-lapse electrical impedance spectroscopy for monitoring the cell cycle of single immobilized S. pombe cells

    Science.gov (United States)

    Zhu, Zhen; Frey, Olivier; Haandbaek, Niels; Franke, Felix; Rudolf, Fabian; Hierlemann, Andreas

    2015-11-01

    As a complement and alternative to optical methods, wide-band electrical impedance spectroscopy (EIS) enables multi-parameter, label-free and real-time detection of cellular and subcellular features. We report on a microfluidics-based system designed to reliably capture single rod-shaped Schizosaccharomyces pombe cells by applying suction through orifices in a channel wall. The system enables subsequent culturing of immobilized cells in an upright position, while dynamic changes in cell-cycle state and morphology were continuously monitored through EIS over a broad frequency range. Besides measuring cell growth, clear impedance signals for nuclear division have been obtained. The EIS system has been characterized with respect to sensitivity and detection limits. The spatial resolution in measuring cell length was 0.25 μm, which corresponds to approximately a 5-min interval of cell growth under standard conditions. The comprehensive impedance data sets were also used to determine the occurrence of nuclear division and cytokinesis. The obtained results have been validated through concurrent confocal imaging and plausibilized through comparison with finite-element modeling data. The possibility to monitor cellular and intracellular features of single S. pombe cells during the cell cycle at high spatiotemporal resolution renders the presented microfluidics-based EIS system a suitable tool for dynamic single-cell investigations.

  1. Detection of colony-stimulating factor messenger RNA in single T cells by in situ hybridization

    DEFF Research Database (Denmark)

    Williamson, D J; Owens, T; Pearse, M

    1989-01-01

    In situ hybridization has been used to study the accumulation of colony-stimulating factor (CSF) mRNA in single cells of a T lymphocyte clone (E9.D4) following antibody-mediated (F23.1) activation via the Ti-T3 complex in filler-independent bulk cultures. The specificity of hybridization for cell...

  2. Noninvasive imaging of protein metabolic labeling in single human cells using stable isotopes and Raman microscopy

    NARCIS (Netherlands)

    van Manen, H.J.; Lenferink, Aufrid T.M.; Otto, Cornelis

    2008-01-01

    We have combined nonresonant Raman microspectroscopy and spectral imaging with stable isotope labeling by amino acids in cell culture (SILAC) to selectively detect the incorporation of deuterium-labeled phenylalanine, tyrosine, and methionine into proteins in intact, single HeLa cells. The C−D

  3. DeActs : genetically encoded tools for perturbing the actin cytoskeleton in single cells

    NARCIS (Netherlands)

    Harterink, Martin; Santos Esteves da Silva, Marta; Will, Lena; Turan, Julia; Ibrahim, Adiljan; Lang, Alexander E; Van Battum, Eljo Y; Pasterkamp, R Jeroen; Kapitein, Lukas C; Kudryashov, Dmitri; Barres, Ben A; Hoogenraad, Casper C; Zuchero, J Bradley

    2017-01-01

    The actin cytoskeleton is essential for many fundamental biological processes, but tools for directly manipulating actin dynamics are limited to cell-permeable drugs that preclude single-cell perturbations. Here we describe DeActs, genetically encoded actin-modifying polypeptides, which effectively

  4. Parallel probing of drug uptake of single cancer cells on a microfluidic device

    NARCIS (Netherlands)

    Yang, Yoon Sun; le Gac, Severine; Terstappen, Leon; Rho, Hoon Suk

    2017-01-01

    Drug resistance is frequently developing during treatment of cancer patients. Intracellular drug uptake is one of the important characteristics to understand mechanism of drug resistance. However, the heterogeneity of cancer cells requires the investigation of drug uptake at the single cell level.

  5. 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.

  6. 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.

  7. Formation of monofluorocarbon compounds by single cell cultures of Glycine max growing on inorganic fluoride

    Energy Technology Data Exchange (ETDEWEB)

    Peters, R.A.; Shorthouse, M.

    1972-01-01

    Single cell cultures of Glycine max were exposed to sodium fluoride to determine the capacity for synthesis of fluoroacetate and fluorocitrate. This capacity had previously been observed in soybean plants and alfalfa. The results show that cell cultures of Glycine max can also synthesize these fluoroacids.

  8. Preparing Viable Single Cells from Human Tissue and Tumors for Cytomic Analysis.

    Science.gov (United States)

    Leelatian, Nalin; Doxie, Deon B; Greenplate, Allison R; Sinnaeve, Justine; Ihrie, Rebecca A; Irish, Jonathan M

    2017-04-03

    Mass cytometry is a single-cell biology technique that samples >500 cells per second, measures >35 features per cell, and is sensitive across a dynamic range of >10 4 relative intensity units per feature. This combination of technical assets has powered a series of recent cytomic studies where investigators used mass cytometry to measure protein and phospho-protein expression in millions of cells, characterize rare cell types in healthy and diseased tissues, and reveal novel, unexpected cells. However, these advances largely occurred in studies of blood, lymphoid tissues, and bone marrow, since the cells in these tissues are readily obtained in single-cell suspensions. This unit establishes a primer for single-cell analysis of solid tumors and tissues, and has been tested with mass cytometry. The cells obtained from these protocols can be fixed for study, cryopreserved for long-term storage, or perturbed ex vivo to dissect responses to stimuli and inhibitors. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

  9. The logic of single-cell projections from visual cortex.

    Science.gov (United States)

    Han, Yunyun; Kebschull, Justus M; Campbell, Robert A A; Cowan, Devon; Imhof, Fabia; Zador, Anthony M; Mrsic-Flogel, Thomas D

    2018-04-05

    Neocortical areas communicate through extensive axonal projections, but the logic of information transfer remains poorly understood, because the projections of individual neurons have not been systematically characterized. It is not known whether individual neurons send projections only to single cortical areas or distribute signals across multiple targets. Here we determine the projection patterns of 591 individual neurons in the mouse primary visual cortex using whole-brain fluorescence-based axonal tracing and high-throughput DNA sequencing of genetically barcoded neurons (MAPseq). Projections were highly diverse and divergent, collectively targeting at least 18 cortical and subcortical areas. Most neurons targeted multiple cortical areas, often in non-random combinations, suggesting that sub-classes of intracortical projection neurons exist. Our results indicate that the dominant mode of intracortical information transfer is not based on 'one neuron-one target area' mapping. Instead, signals carried by individual cortical neurons are shared across subsets of target areas, and thus concurrently contribute to multiple functional pathways.

  10. Inertial-ordering-assisted droplet microfluidics for high-throughput single-cell RNA-sequencing.

    Science.gov (United States)

    Moon, Hui-Sung; Je, Kwanghwi; Min, Jae-Woong; Park, Donghyun; Han, Kyung-Yeon; Shin, Seung-Ho; Park, Woong-Yang; Yoo, Chang Eun; Kim, Shin-Hyun

    2018-02-27

    Single-cell RNA-seq reveals the cellular heterogeneity inherent in the population of cells, which is very important in many clinical and research applications. Recent advances in droplet microfluidics have achieved the automatic isolation, lysis, and labeling of single cells in droplet compartments without complex instrumentation. However, barcoding errors occurring in the cell encapsulation process because of the multiple-beads-in-droplet and insufficient throughput because of the low concentration of beads for avoiding multiple-beads-in-a-droplet remain important challenges for precise and efficient expression profiling of single cells. In this study, we developed a new droplet-based microfluidic platform that significantly improved the throughput while reducing barcoding errors through deterministic encapsulation of inertially ordered beads. Highly concentrated beads containing oligonucleotide barcodes were spontaneously ordered in a spiral channel by an inertial effect, which were in turn encapsulated in droplets one-by-one, while cells were simultaneously encapsulated in the droplets. The deterministic encapsulation of beads resulted in a high fraction of single-bead-in-a-droplet and rare multiple-beads-in-a-droplet although the bead concentration increased to 1000 μl -1 , which diminished barcoding errors and enabled accurate high-throughput barcoding. We successfully validated our device with single-cell RNA-seq. In addition, we found that multiple-beads-in-a-droplet, generated using a normal Drop-Seq device with a high concentration of beads, underestimated transcript numbers and overestimated cell numbers. This accurate high-throughput platform can expand the capability and practicality of Drop-Seq in single-cell analysis.

  11. Mechanics governs single-cell signaling and multi-cell robustness in biofilm infections

    Science.gov (United States)

    Gordon, Vernita

    In biofilms, bacteria and other microbes are embedded in extracellular polymers (EPS). Multiple types of EPS can be produced by a single bacterial strain - the reasons for this redundancy are not well-understood. Our work suggests that different polymers may confer distinct mechanical benefits. Our model organism is Pseudomonas aeruginosa, an opportunistic human pathogen that forms chronic biofilm infections associated with increased antibiotic resistance and evasion of the immune defense. Biofilms initiate when bacteria attach to a surface, sense the surface, and change their gene expression. Changes in gene expression are regulated by a chemical signal, cyclic-di-GMP. We find that one EPS material, called ``PEL,'' enhances surface sensing by increasing mechanical coupling of single bacteria to the surface. Measurements of bacterial motility suggest that PEL may increase frictional interactions between the surface and the bacteria. Consistent with this, we show that bacteria increase cyclic-di-GMP signaling in response to mechanical shear stress. Mechanosensing has long been known to be important to the function of cells in higher eukaryotes, but this is one of only a handful of studies showing that bacteria can sense and respond to mechanical forces. For the mature biofilm, the embedding polymer matrix can protect bacteria both chemically and mechanically. P. aeruginosa infections in the cystic fibrosis (CF) lung often last for decades, ample time for the infecting strain(s) to evolve. Production of another EPS material, alginate, is well-known to tend to increase over time in CF infections. Alginate chemically protects biofilms, but also makes them softer and weaker. Recently, it is being increasingly recognized that bacteria in chronic CF infections also evolve to increase PSL production. We use oscillatory bulk rheology to determine the unique contributions of EPS materials to biofilm mechanics. Unlike alginate, increased PSL stiffens biofilms. Increasing both

  12. Single-Cell Analysis Identifies Distinct Stages of Human Endothelial-to-Hematopoietic Transition

    Directory of Open Access Journals (Sweden)

    Carolina Guibentif

    2017-04-01

    Full Text Available During development, hematopoietic cells originate from endothelium in a process known as endothelial-to-hematopoietic transition (EHT. To study human EHT, we coupled flow cytometry and single-cell transcriptional analyses of human pluripotent stem cell-derived CD34+ cells. The resulting transcriptional hierarchy showed a continuum of endothelial and hematopoietic signatures. At the interface of these two signatures, a unique group of cells displayed both an endothelial signature and high levels of key hematopoietic stem cell-associated genes. This interphase group was validated via sort and subculture as an immediate precursor to hematopoietic cells. Differential expression analyses further divided this population into subgroups, which, upon subculture, showed distinct hematopoietic lineage differentiation potentials. We therefore propose that immediate precursors to hematopoietic cells already have their hematopoietic lineage restrictions defined prior to complete downregulation of the endothelial signature. These findings increase our understanding of the processes of de novo hematopoietic cell generation in the human developmental context.

  13. Investigation of adhesion and mechanical properties of human glioma cells by single cell force spectroscopy and atomic force microscopy.

    Science.gov (United States)

    Andolfi, Laura; Bourkoula, Eugenia; Migliorini, Elisa; Palma, Anita; Pucer, Anja; Skrap, Miran; Scoles, Giacinto; Beltrami, Antonio Paolo; Cesselli, Daniela; Lazzarino, Marco

    2014-01-01

    Active cell migration and invasion is a peculiar feature of glioma that makes this tumor able to rapidly infiltrate into the surrounding brain tissue. In our recent work, we identified a novel class of glioma-associated-stem cells (defined as GASC for high-grade glioma--HG--and Gasc for low-grade glioma--LG) that, although not tumorigenic, act supporting the biological aggressiveness of glioma-initiating stem cells (defined as GSC for HG and Gsc for LG) favoring also their motility. Migrating cancer cells undergo considerable molecular and cellular changes by remodeling their cytoskeleton and cell interactions with surrounding environment. To get a better understanding about the role of the glioma-associated-stem cells in tumor progression, cell deformability and interactions between glioma-initiating stem cells and glioma-associated-stem cells were investigated. Adhesion of HG/LG-cancer cells on HG/LG-glioma-associated stem cells was studied by time-lapse microscopy, while cell deformability and cell-cell adhesion strengths were quantified by indentation measurements by atomic force microscopy and single cell force spectroscopy. Our results demonstrate that for both HG and LG glioma, cancer-initiating-stem cells are softer than glioma-associated-stem cells, in agreement with their neoplastic features. The adhesion strength of GSC on GASC appears to be significantly lower than that observed for Gsc on Gasc. Whereas, GSC spread and firmly adhere on Gasc with an adhesion strength increased as compared to that obtained on GASC. These findings highlight that the grade of glioma-associated-stem cells plays an important role in modulating cancer cell adhesion, which could affect glioma cell migration, invasion and thus cancer aggressiveness. Moreover this work provides evidence about the importance of investigating cell adhesion and elasticity for new developments in disease diagnostics and therapeutics.

  14. Temporal Variation in Single-Cell Power-Law Rheology Spans the Ensemble Variation of Cell Population.

    Science.gov (United States)

    Cai, PingGen; Takahashi, Ryosuke; Kuribayashi-Shigetomi, Kaori; Subagyo, Agus; Sueoka, Kazuhisa; Maloney, John M; Van Vliet, Krystyn J; Okajima, Takaharu

    2017-08-08

    Changes in the cytoskeletal organization within cells can be characterized by large spatial and temporal variations in rheological properties of the cell (e.g., the complex shear modulus G ∗ ). Although the ensemble variation in G ∗ of single cells has been elucidated, the detailed temporal variation of G ∗ remains unknown. In this study, we investigated how the rheological properties of individual fibroblast cells change under a spatially confined environment in which the cell translational motion is highly restricted and the whole cell shape remains unchanged. The temporal evolution of single-cell rheology was probed at the same measurement location within the cell, using atomic force microscopy-based oscillatory deformation. The measurements reveal that the temporal variation in the power-law rheology of cells is quantitatively consistent with the ensemble variation, indicating that the cell system satisfies an ergodic hypothesis in which the temporal statistics are identical to the ensemble statistics. The autocorrelation of G ∗ implies that the cell mechanical state evolves in the ensemble of possible states with a characteristic timescale. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  15. 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.

  16. Lab-on-a-Chip Platforms for Biophysical Studies of Cancer with Single-Cell Resolution.

    Science.gov (United States)

    Shukla, Vasudha C; Kuang, Tai-Rong; Senthilvelan, Abirami; Higuita-Castro, Natalia; Duarte-Sanmiguel, Silvia; Ghadiali, Samir N; Gallego-Perez, Daniel

    2018-03-17

    Recent cancer research has more strongly emphasized the biophysical aspects of tumor development, progression, and microenvironment. In addition to genetic modifications and mutations in cancer cells, it is now well accepted that the physical properties of cancer cells such as stiffness, electrical impedance, and refractive index vary with tumor progression and can identify a malignant phenotype. Moreover, cancer heterogeneity renders population-based characterization techniques inadequate, as individual cellular features are lost in the average. Hence, platforms for fast and accurate characterization of biophysical properties of cancer cells at the single-cell level are required. Here, we highlight some of the recent advances in the field of cancer biophysics and the development of lab-on-a-chip platforms for single-cell biophysical analyses of cancer cells. Copyright © 2018 Elsevier Ltd. All rights reserved.

  17. 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\\%.

  18. Label-free isolation and deposition of single bacterial cells from heterogeneous samples for clonal culturing

    OpenAIRE

    J. Riba; T. Gleichmann; S. Zimmermann; R. Zengerle; P. Koltay

    2016-01-01

    The isolation and analysis of single prokaryotic cells down to 1??m and less in size poses a special challenge and requires micro-engineered devices to handle volumes in the picoliter to nanoliter range. Here, an advanced Single-Cell Printer (SCP) was applied for automated and label-free isolation and deposition of bacterial cells encapsulated in 35?pl droplets by inkjet-like printing. To achieve this, dispenser chips to generate micro droplets have been fabricated with nozzles 20??m in size....

  19. Hydrodynamic lift for single cell manipulation in a femtosecond laser fabricated optofluidic chip

    Directory of Open Access Journals (Sweden)

    Bragheri Francesca

    2017-08-01

    Full Text Available Single cell sorting based either on fluorescence or on mechanical properties has been exploited in the last years in microfluidic devices. Hydrodynamic focusing allows increasing the efficiency of theses devices by improving the matching between the region of optical analysis and that of cell flow. Here we present a very simple solution fabricated by femtosecond laser micromachining that exploits flow laminarity in microfluidic channels to easily lift the sample flowing position to the channel portion illuminated by the optical waveguides used for single cell trapping and analysis.

  20. 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...

  1. Miniaturization Technologies for Efficient Single-Cell Library Preparation for Next-Generation Sequencing.

    Science.gov (United States)

    Mora-Castilla, Sergio; To, Cuong; Vaezeslami, Soheila; Morey, Robert; Srinivasan, Srimeenakshi; Dumdie, Jennifer N; Cook-Andersen, Heidi; Jenkins, Joby; Laurent, Louise C

    2016-08-01

    As the cost of next-generation sequencing has decreased, library preparation costs have become a more significant proportion of the total cost, especially for high-throughput applications such as single-cell RNA profiling. Here, we have applied novel technologies to scale down reaction volumes for library preparation. Our system consisted of in vitro differentiated human embryonic stem cells representing two stages of pancreatic differentiation, for which we prepared multiple biological and technical replicates. We used the Fluidigm (San Francisco, CA) C1 single-cell Autoprep System for single-cell complementary DNA (cDNA) generation and an enzyme-based tagmentation system (Nextera XT; Illumina, San Diego, CA) with a nanoliter liquid handler (mosquito HTS; TTP Labtech, Royston, UK) for library preparation, reducing the reaction volume down to 2 µL and using as little as 20 pg of input cDNA. The resulting sequencing data were bioinformatically analyzed and correlated among the different library reaction volumes. Our results showed that decreasing the reaction volume did not interfere with the quality or the reproducibility of the sequencing data, and the transcriptional data from the scaled-down libraries allowed us to distinguish between single cells. Thus, we have developed a process to enable efficient and cost-effective high-throughput single-cell transcriptome sequencing. © 2016 Society for Laboratory Automation and Screening.

  2. Simultaneous live cell imaging using dual FRET sensors with a single excitation light.

    Directory of Open Access Journals (Sweden)

    Yusuke Niino

    Full Text Available Fluorescence resonance energy transfer (FRET between fluorescent proteins is a powerful tool for visualization of signal transduction in living cells, and recently, some strategies for imaging of dual FRET pairs in a single cell have been reported. However, these necessitate alteration of excitation light between two different wavelengths to avoid the spectral overlap, resulting in sequential detection with a lag time. Thus, to follow fast signal dynamics or signal changes in highly motile cells, a single-excitation dual-FRET method should be required. Here we reported this by using four-color imaging with a single excitation light and subsequent linear unmixing to distinguish fluorescent proteins. We constructed new FRET sensors with Sapphire/RFP to combine with CFP/YFP, and accomplished simultaneous imaging of cAMP and cGMP in single cells. We confirmed that signal amplitude of our dual FRET measurement is comparable to of conventional single FRET measurement. Finally, we demonstrated to monitor both intracellular Ca(2+ and cAMP in highly motile cardiac myocytes. To cancel out artifacts caused by the movement of the cell, this method expands the applicability of the combined use of dual FRET sensors for cell samples with high motility.

  3. High-sensitivity Mass Spectrometry for Probing Gene Translation in Single Embryonic Cells in the Early Frog (Xenopus Embryo

    Directory of Open Access Journals (Sweden)

    Camille Lombard-Banek

    2016-10-01

    Full Text Available Direct measurement of protein expression with single-cell resolution promises to deepen the understanding of basic molecular processes during normal and impaired development. High-resolution mass spectrometry provides detailed coverage of the proteomic composition of large numbers of cells. Here we discuss recent mass spectrometry developments based on single-cell capillary electrophoresis that extend discovery proteomics to sufficient sensitivity to enable the measurement of proteins in single cells. The single-cell mass spectrometry system is used to detect a large number of proteins in single embryonic cells in blastomeres in the 16-cell embryo of the South African clawed frog (Xenopus laevis that give rise to distinct tissue types. Single-cell measurements of protein expression provide complementary information on gene transcription during early development of the vertebrate embryo, raising a potential to understand how differential gene expression coordinates normal cell heterogeneity during development.

  4. Quantifying cellular mechanics and adhesion in renal tubular injury using single cell force spectroscopy.

    Science.gov (United States)

    Siamantouras, Eleftherios; Hills, Claire E; Squires, Paul E; Liu, Kuo-Kang

    2016-05-01

    Tubulointerstitial fibrosis represents the major underlying pathology of diabetic nephropathy where loss of cell-to-cell adhesion is a critical step. To date, research has predominantly focussed on the loss of cell surface molecular binding events that include altered protein ligation. In the current study, atomic force microscopy single cell force spectroscopy (AFM-SCFS) was used to quantify changes in cellular stiffness and cell adhesion in TGF-β1 treated kidney cells of the human proximal tubule (HK2). AFM indentation of TGF-β1 treated HK2 cells showed a significant increase (42%) in the elastic modulus (stiffness) compared to control. Fluorescence microscopy confirmed that increased cell stiffness is accompanied by reorganization of the cytoskeleton. The corresponding changes in stiffness, due to F-actin rearrangement, affected the work of detachment by changing the separation distance between two adherent cells. Overall, our novel data quantitatively demonstrate a correlation between cellular elasticity, adhesion and early morphologic/phenotypic changes associated with tubular injury. Diabetes affects many patients worldwide. One of the long term problems is diabetic nephropathy. Here, the authors utilized atomic force microscopy single cell force spectroscopy (AFM- SCFS) to study cellular stiffness and cell adhesion after TGF1 treatment in human proximal tubule kidney cells. The findings would help further understand the overall disease mechanism in diabetic patients. Copyright © 2015 Elsevier Inc. All rights reserved.

  5. 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.

  6. Measurement of adenosine triphosphate (ATP) content in single red blood cells using the firefly bioluminescent reaction

    Energy Technology Data Exchange (ETDEWEB)

    Kostuk, R.K.; Muhs, A.G.; Kirkpatrick, F.H.; Gabel, C.W.

    1977-01-01

    A unique optical instrument is described which uses the firefly bioluminscent reaction to measure adenosine triphosphate (ATP) levels in single red blood cells. The method allows chemical content level to be associated with individual cell features. The optical instrument consists of a phase contrast microscope to view cells, a pulsed argon-ion laser to rupture the cell membrane, and a photon counting system to measure the bioluminescent yield. The technique has been calibrated against a standard ATP measurement using bulk analysis methods. The ATP loss mechanism for blood cells in a controlled depletion experiment was also investigated.

  7. Fluorescence/luminescence circadian imaging of complex tissues at single-cell resolution.

    Science.gov (United States)

    Sellix, Michael T; Currie, Jake; Menaker, Michael; Wijnen, Herman

    2010-06-01

    The use of luciferase reporter genes together with luminescence detection has enabled high frequency monitoring of molecular circadian clock function in living tissues. With the help of an intensified CCD camera combined with an inverted epifluorescence microscope, the authors have established a new imaging strategy that makes use of transgenic cell type-specific expression of fluorescent proteins to identify cells of interest for subsequent circadian luminescence recording at single-cell resolution.

  8. Organic and inorganic solar cells parameters evaluation from single I-V plot

    International Nuclear Information System (INIS)

    Chegaar, M.; Nehaoua, N.; Bouhemadou, A.

    2008-01-01

    This paper presents a new method to determine the five solar cell parameters of the single diode lumped circuit model. These parameters are usually the saturation current, the series resistance, the ideality factor, the shunt conductance and the photocurrent. This method is based on the measured current-voltage data. The method has been successfully applied to a commercial silicon solar cell, a module and an organic solar cell

  9. Single-Cell Enumeration of an Uncultivated TM7 Subgroup in the Human Subgingival Crevice

    OpenAIRE

    Ouverney, Cleber C.; Armitage, Gary C.; Relman, David A.

    2003-01-01

    Specific oligonucleotide hybridization conditions were established for single-cell enumeration of uncultivated TM7 and IO25 bacteria by using clones expressing heterologous 16S rRNA. In situ analysis of human subgingival crevice specimens revealed that a greater proportion of samples from sites of chronic periodontitis than from healthy sites contained TM7 subgroup IO25. In addition, IO25 bacterial cells from periodontitis site samples were more abundant and fourfold longer than IO25 cells fr...

  10. Single-Cell Transcriptomic Analysis Defines Heterogeneity and Transcriptional Dynamics in the Adult Neural Stem Cell Lineage

    Directory of Open Access Journals (Sweden)

    Ben W. Dulken

    2017-01-01

    Full Text Available Neural stem cells (NSCs in the adult mammalian brain serve as a reservoir for the generation of new neurons, oligodendrocytes, and astrocytes. Here, we use single-cell RNA sequencing to characterize adult NSC populations and examine the molecular identities and heterogeneity of in vivo NSC populations. We find that cells in the NSC lineage exist on a continuum through the processes of activation and differentiation. Interestingly, rare intermediate states with distinct molecular profiles can be identified and experimentally validated, and our analysis identifies putative surface markers and key intracellular regulators for these subpopulations of NSCs. Finally, using the power of single-cell profiling, we conduct a meta-analysis to compare in vivo NSCs and in vitro cultures, distinct fluorescence-activated cell sorting strategies, and different neurogenic niches. These data provide a resource for the field and contribute to an integrative understanding of the adult NSC lineage.

  11. Single-cell RNA-sequencing: The future of genome biology is now.

    Science.gov (United States)

    Picelli, Simone

    2017-05-04

    Genome-wide single-cell analysis represents the ultimate frontier of genomics research. In particular, single-cell RNA-sequencing (scRNA-seq) studies have been boosted in the last few years by an explosion of new technologies enabling the study of the transcriptomic landscape of thousands of single cells in complex multicellular organisms. More sensitive and automated methods are being continuously developed and promise to deliver better data quality and higher throughput with less hands-on time. The outstanding amount of knowledge that is going to be gained from present and future studies will have a profound impact in many aspects of our society, from the introduction of truly tailored cancer treatments, to a better understanding of antibiotic resistance and host-pathogen interactions; from the discovery of the mechanisms regulating stem cell differentiation to the characterization of the early event of human embryogenesis.

  12. DeepCpG: accurate prediction of single-cell DNA methylation states using deep learning.

    Science.gov (United States)

    Angermueller, Christof; Lee, Heather J; Reik, Wolf; Stegle, Oliver

    2017-04-11

    Recent technological advances have enabled DNA methylation to be assayed at single-cell resolution. However, current protocols are limited by incomplete CpG coverage and hence methods to predict missing methylation states are critical to enable genome-wide analyses. We report DeepCpG, a computational approach based on deep neural networks to predict methylation states in single cells. We evaluate DeepCpG on single-cell methylation data from five cell types generated using alternative sequencing protocols. DeepCpG yields substantially more accurate predictions than previous methods. Additionally, we show that the model parameters can be interpreted, thereby providing insights into how sequence composition affects methylation variability.

  13. 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.

  14. Comparative proteomics of chloroplasts envelopes from bundle sheath and mesophyll chloroplasts reveals novel membrane proteins with a possible role in C4-related metabolite fluxes and development.

    Directory of Open Access Journals (Sweden)

    Kalpana eManandhar-Shrestha

    2013-03-01

    Full Text Available As the world population grows, our need for food increases drastically. Limited amounts of arable land lead to a competition between food and fuel crops, while changes in the global climate may impact future crop yields. Thus, a second green revolution will need a better understanding of the processes essential for plant growth and development. One approach toward the solution of this problem is to better understand regulatory and transport processes in C4 plants. C4 plants display an up to 10-fold higher apparent CO2 assimilation and higher yields while maintaining high water use efficiency. This requires differential regulation of mesophyll (M and bundle sheath (BS chloroplast development as well as higher metabolic fluxes of photosynthetic intermediates between cells and across chloroplast envelopes. While previous analyses of overall chloroplast membranes have yielded significant insight, our comparative proteomics approach using enriched BS and M chloroplast envelopes of Zea mays allowed us to identify 37 proteins of unknown function that have not been seen in these earlier studies. We identified 280 proteins, 84% of which are known/predicted to be present in chloroplasts (cp. 74% have a known or predicted membrane association. 21 membrane proteins were 2-15 times more abundant in BS cells, while 36 proteins were more abundant in M cp envelopes. These proteins could represent additional candidates of proteins essential for development or metabolite transport processes in C4 plants. RT-PCR confirmed differential expression of thirteen candidate genes. Cp association was confirmed using GFP labeling. Genes for a PIC-like protein and an ER-AP-like protein show an early transient increase in gene expression during the transition to light. In addition, PIC gene expression is increased in the immature part of the leaf and was lower in the fully developed parts of the leaf, suggesting a need for/incorporation of the protein during chloroplast

  15. Management of Islet Cell Tumours: A Single Hospital Experience.

    Science.gov (United States)

    Fu, Wenguang; Li, Jing; Wen, Jian; Lin, Gao; Wei, Zeng; Deng, Jiaqi; Li, Qiu; Lei, Zhengming

    2015-05-01

    Islet cell tumours (ICTs) are uncommon tumours in clinical practice. Surgical resection is the treatment of choice for ICTs, but localisation of these lesions can be challenging. The aim of this study was to analyse the clinical diagnosis and treatment for ICTs. Thirty-one patients with ICTs who were diagnosed and who underwent surgical treatment in the affiliate hospital of Luzhou Medical College from 1 January 2000 to 31 July 2013 were enrolled. The clinical data of these patients were retrospectively reviewed. Among 31 patients (6 males, 25 females), 15 cases (48.39%) had non-functional ICTs and 16 (51.61%) cases were insulinoma. The mean age of patients with non-functional ICTs was 42.73 ± 12.34 years and of those with insulinoma was 48.88 ± 13 years. Non-functional ICTs had a non-specific presentation. Insulinoma makes different clinical presentations mostly with symptoms of hypoglycaemia. Preoperative and/or intra-operative localisation is needed for ICTs; CT scan or MRI is used routinely as the first choice. If the lesion is very small, DSA is also good for localisation before operation. IOUS is a reliable technique in exactly localising insulinoma. ICTs are considered to be cured with successful surgical removal.

  16. Preparation, characterization and single cell testing of new ionic conducting polymers for fuel cell applications

    Science.gov (United States)

    Escribano, P. G.; del Río y, C.; Acosta, J. L.

    In this work, heterogeneous sulfonation and both, structural and electrical characterization of a mixture composed on block copolymer ionomers (HSBS and EPDM) and commercial silica, are studied. The incorporation of sulfonic groups was checked by infrared spectroscopy (FTIR-ATR). Microstructure was studied by means of dynamic mechanical analysis (DMA). Also, water uptake and methanol crossover were determined, and the results were compared with those of Nafion ® 117. Electrical behavior was recorded by means of electrochemical impedance spectroscopy (EIS) at different hydration times. Results show that sulfonation of the styrene rings has effectively occurred. Conductivity values are similar to Nafion and they improve with hydration time. Methanol crossover is lower than in Nafion. Finally, a single complete proton exchange membrane fuel cell (PEMFC) as a whole was tested obtaining the polarization and power curves at different temperatures and pressures, and modeling it by an electrical equivalent circuit (EC) in the symmetrical mode (SM) configuration using the EIS technique. This study offers a physical interpretation relating physical parameters to several processes occurring in the system. Power density values are higher than in Nafion.

  17. Single-Cell Microfluidics to Study the Effects of Genome Deletion on Bacterial Growth Behavior.

    Science.gov (United States)

    Yuan, Xiaofei; Couto, Jillian M; Glidle, Andrew; Song, Yanqing; Sloan, William; Yin, Huabing

    2017-12-15

    By directly monitoring single cell growth in a microfluidic platform, we interrogated genome-deletion effects in Escherichia coli strains. We compared the growth dynamics of a wild type strain with a clean genome strain, and their derived mutants at the single-cell level. A decreased average growth rate and extended average lag time were found for the clean genome strain, compared to those of the wild type strain. Direct correlation between the growth rate and lag time of individual cells showed that the clean genome population was more heterogeneous. Cell culturability (the ratio of growing cells to the sum of growing and nongrowing cells) of the clean genome population was also lower. Interestingly, after the random mutations induced by a glucose starvation treatment, for the clean genome population mutants that had survived the competition of chemostat culture, each parameter markedly improved (i.e., the average growth rate and cell culturability increased, and the lag time and heterogeneity decreased). However, this effect was not seen in the wild type strain; the wild type mutants cultured in a chemostat retained a high diversity of growth phenotypes. These results suggest that quasi-essential genes that were deleted in the clean genome might be required to retain a diversity of growth characteristics at the individual cell level under environmental stress. These observations highlight that single-cell microfluidics can reveal subtle individual cellular responses, enabling in-depth understanding of the population.

  18. Timing the start of division in E. coli: a single-cell study

    Science.gov (United States)

    Reshes, G.; Vanounou, S.; Fishov, I.; Feingold, M.

    2008-12-01

    We monitor the shape dynamics of individual E. coli cells using time-lapse microscopy together with accurate image analysis. This allows measuring the dynamics of single-cell parameters throughout the cell cycle. In previous work, we have used this approach to characterize the main features of single-cell morphogenesis between successive divisions. Here, we focus on the behavior of the parameters that are related to cell division and study their variation over a population of 30 cells. In particular, we show that the single-cell data for the constriction width dynamics collapse onto a unique curve following appropriate rescaling of the corresponding variables. This suggests the presence of an underlying time scale that determines the rate at which the cell cycle advances in each individual cell. For the case of cell length dynamics a similar rescaling of variables emphasizes the presence of a breakpoint in the growth rate at the time when division starts, τc. We also find that the τc of individual cells is correlated with their generation time, τg, and inversely correlated with the corresponding length at birth, L0. Moreover, the extent of the T-period, τg - τc, is apparently independent of τg. The relations between τc, τg and L0 indicate possible compensation mechanisms that maintain cell length variability at about 10%. Similar behavior was observed for both fast-growing cells in a rich medium (LB) and for slower growth in a minimal medium (M9-glucose). To reveal the molecular mechanisms that lead to the observed organization of the cell cycle, we should further extend our approach to monitor the formation of the divisome.

  19. On-slide detection of enzymatic activities in selected single cells.

    Science.gov (United States)

    Keller, Josephine Geertsen; Tesauro, Cinzia; Coletta, Andrea; Graversen, Astrid Damgaard; Ho, Yi-Ping; Kristensen, Peter; Stougaard, Magnus; Knudsen, Birgitta Ruth

    2017-09-21

    With increasing recognition of the importance in addressing cell-to-cell heterogeneity for the understanding of complex biological systems, there is a growing need for assays capable of single cell analyses. In the current study, we describe the measurement of human topoisomerase I activity in single CD44 positive Caco2 cells specifically captured from a mixed population on glass slides, which were dual functionalized with anti-CD44-antibodies and specific DNA primers. On-slide lysis of captured CD44 positive cells, resulted in the release of human topoisomerase I, allowing the enzyme to circularize a specific linear DNA substrate added to the slides. The generated circles hybridized to the anchored DNA primers and acted as templates for a solid support rolling circle amplification reaction leading to the generation of long tandem repeat products that were detected at the single molecule level in a fluorescent microscope upon hybridization of fluorescent labelled probes. The on-slide detection system was demonstrated to be directly quantitative and specific towards CD44 positive cells. Moreover, it allowed reproducible detection of human topoisomerase I activity in single cells.

  20. Vibrio cholerae biofilm growth program and architecture revealed by single-cell live imaging.

    Science.gov (United States)

    Yan, Jing; Sharo, Andrew G; Stone, Howard A; Wingreen, Ned S; Bassler, Bonnie L

    2016-09-06

    Biofilms are surface-associated bacterial communities that are crucial in nature and during infection. Despite extensive work to identify biofilm components and to discover how they are regulated, little is known about biofilm structure at the level of individual cells. Here, we use state-of-the-art microscopy techniques to enable live single-cell resolution imaging of a Vibrio cholerae biofilm as it develops from one single founder cell to a mature biofilm of 10,000 cells, and to discover the forces underpinning the architectural evolution. Mutagenesis, matrix labeling, and simulations demonstrate that surface adhesion-mediated compression causes V. cholerae biofilms to transition from a 2D branched morphology to a dense, ordered 3D cluster. We discover that directional proliferation of rod-shaped bacteria plays a dominant role in shaping the biofilm architecture in V. cholerae biofilms, and this growth pattern is controlled by a single gene, rbmA Competition analyses reveal that the dense growth mode has the advantage of providing the biofilm with superior mechanical properties. Our single-cell technology can broadly link genes to biofilm fine structure and provides a route to assessing cell-to-cell heterogeneity in response to external stimuli.

  1. 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.

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

    Science.gov (United States)

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

    2015-05-12

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

  3. Three-dimensional anatomy of the Ciona intestinalis tailbud embryo at single-cell resolution.

    Science.gov (United States)

    Nakamura, Mitsuru J; Terai, Jun; Okubo, Reiko; Hotta, Kohji; Oka, Kotaro

    2012-12-15

    During embryogenesis, chordates pass through a tailbud stage in which the larval tail is formed. Since acquisition of a tadpole-like tail during tailbud stage is one of the key events in the evolution of chordates, understanding the anatomy of the tailbud stage chordate embryo is of special interest. In this study, to understand comprehensively the anatomy of the tailbud embryo at single-cell-level, real microscopic image stacks of the tailbud embryo in Ciona intestinalis were reconstructed into a 3D computer model. This comprehensive 3D model of the ascidian tailbud embryo was based on real images of confocal laser scanning microscope (CLSM) and therefore, cell shape, location and cell arrangement reflect real geometries of the tailbud embryo. We found that the tailbud embryo consists of 1579 cells, including 836 epidermal cells, 228 cells in the central nervous system, 218 mesenchymal cells, four trunk ventral cells, two B/B(⁎)8.11 cells, 36 muscle cells, 40 notochord cells, four primordial germ cells, and 199 endodermal cells. Moreover, we identified for the first time two populations of previously undefined cells (a total of 12 cells) in Ciona: one located in the lateral trunk and the other located under the tail dorsal epidermis. This information provides a first step for understanding how the body plan of the chordate tailbud embryo formed and evolved. Copyright © 2012 Elsevier Inc. All rights reserved.

  4. A finite element study of micropipette aspiration of single cells: effect of compressibility.

    Science.gov (United States)

    Bidhendi, Amirhossein Jafari; Korhonen, Rami K

    2012-01-01

    Micropipette aspiration (MA) technique has been widely used to measure the viscoelastic properties of different cell types. Cells experience nonlinear large deformations during the aspiration procedure. Neo-Hookean viscohyperelastic (NHVH) incompressible and compressible models were used to simulate the creep behavior of cells in MA, particularly accounting for the effect of compressibility, bulk relaxation, and hardening phenomena under large strain. In order to find optimal material parameters, the models were fitted to the experimental data available for mesenchymal stem cells. Finally, through Neo-Hookean porohyperelastic (NHPH) material model for the cell, the influence of fluid flow on the aspiration length of the cell was studied. Based on the results, we suggest that the compressibility and bulk relaxation/fluid flow play a significant role in the deformation behavior of single cells and should be taken into account in the analysis of the mechanics of cells.

  5. Cellular Taxonomy of the Mouse Striatum as Revealed by Single-Cell RNA-Seq

    Directory of Open Access Journals (Sweden)

    Ozgun Gokce

    2016-07-01

    Full Text Available The striatum contributes to many cognitive processes and disorders, but its cell types are incompletely characterized. We show that microfluidic and FACS-based single-cell RNA sequencing of mouse striatum provides a well-resolved classification of striatal cell type diversity. Transcriptome analysis revealed ten differentiated, distinct cell types, including neurons, astrocytes, oligodendrocytes, ependymal, immune, and vascular cells, and enabled the discovery of numerous marker genes. Furthermore, we identified two discrete subtypes of medium spiny neurons (MSNs that have specific markers and that overexpress genes linked to cognitive disorders and addiction. We also describe continuous cellular identities, which increase heterogeneity within discrete cell types. Finally, we identified cell type-specific transcription and splicing factors that shape cellular identities by regulating splicing and expression patterns. Our findings suggest that functional diversity within a complex tissue arises from a small number of discrete