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Sample records for living cells challenges

  1. Fluorescence Live Cell Imaging

    PubMed Central

    Ettinger, Andreas

    2014-01-01

    Fluorescence microscopy of live cells has become an integral part of modern cell biology. Fluorescent protein tags, live cell dyes, and other methods to fluorescently label proteins of interest provide a range of tools to investigate virtually any cellular process under the microscope. The two main experimental challenges in collecting meaningful live cell microscopy data are to minimize photodamage while retaining a useful signal-to-noise ratio, and to provide a suitable environment for cells or tissues to replicate physiological cell dynamics. This chapter aims to give a general overview on microscope design choices critical for fluorescence live cell imaging that apply to most fluorescence microscopy modalities, and on environmental control with a focus on mammalian tissue culture cells. In addition, we provide guidance on how to design and evaluate fluorescent protein constructs by spinning disk confocal microscopy. PMID:24974023

  2. Living-Cell Microarrays

    PubMed Central

    Yarmush, Martin L.; King, Kevin R.

    2011-01-01

    Living cells are remarkably complex. To unravel this complexity, living-cell assays have been developed that allow delivery of experimental stimuli and measurement of the resulting cellular responses. High-throughput adaptations of these assays, known as living-cell microarrays, which are based on microtiter plates, high-density spotting, microfabrication, and microfluidics technologies, are being developed for two general applications: (a) to screen large-scale chemical and genomic libraries and (b) to systematically investigate the local cellular microenvironment. These emerging experimental platforms offer exciting opportunities to rapidly identify genetic determinants of disease, to discover modulators of cellular function, and to probe the complex and dynamic relationships between cells and their local environment. PMID:19413510

  3. Live-cell imaging

    PubMed Central

    Cole, Richard

    2014-01-01

    It would be hard to argue that live-cell imaging has not changed our view of biology. The past 10 years have seen an explosion of interest in imaging cellular processes, down to the molecular level. There are now many advanced techniques being applied to live cell imaging. However, cellular health is often under appreciated. For many researchers, if the cell at the end of the experiment has not gone into apoptosis or is blebbed beyond recognition, than all is well. This is simply incorrect. There are many factors that need to be considered when performing live-cell imaging in order to maintain cellular health such as: imaging modality, media, temperature, humidity, PH, osmolality, and photon dose. The wavelength of illuminating light, and the total photon dose that the cells are exposed to, comprise two of the most important and controllable parameters of live-cell imaging. The lowest photon dose that achieves a measureable metric for the experimental question should be used, not the dose that produces cover photo quality images. This is paramount to ensure that the cellular processes being investigated are in their in vitro state and not shifted to an alternate pathway due to environmental stress. The timing of the mitosis is an ideal canary in the gold mine, in that any stress induced from the imaging will result in the increased length of mitosis, thus providing a control model for the current imagining conditions. PMID:25482523

  4. Microencapsulation Of Living Cells

    NASA Technical Reports Server (NTRS)

    Chang, Manchium; Kendall, James M.; Wang, Taylor G.

    1989-01-01

    In experimental technique, living cells and other biological materials encapsulated within submillimeter-diameter liquid-filled spheres. Sphere material biocompatible, tough, and compliant. Semipermeable, permitting relatively small molecules to move into and out of sphere core but preventing passage of large molecules. New technique promises to make such spherical capsules at high rates and in uniform, controllable sizes. Capsules injected into patient through ordinary hypodermic needle. Promising application for technique in treatment of diabetes. Also used to encapsulate pituitary cells and thyroid hormone adrenocortical cells for treatment of other hormonal disorders, to encapsulate other secreting cells for transplantation, and to package variety of pharmaceutical products and agricultural chemicals for controlled release.

  5. Freezing of living cells

    SciTech Connect

    Mazur, P.

    1985-01-01

    It can be calculated that a living cell will survive more than 5000 years at -196/sup 0/C. This ability to essentially stop biological time has important implications in medicine and agriculture, and in biological research. In medicine the chief implications are in the banking of transplantable tissues and organs and in in vitro fertilization. In agriculture the applications stem in part from the role of frozen embryos in amplifying the number of calves produced by high quanlity cows. The problem is how can cells survive both the cooling to such very low temperatures and the return to normal temperatures. The answers involve fundamental characteristics of cells such as the permeability of their surface membranes to water and solutes. These characteristics determine whether or not cells undergo lethal internal ice formation and other response during freezing and thawing. 27 refs., 12 figs.

  6. Nucleic Acid Aptamers for Living Cell Analysis

    NASA Astrophysics Data System (ADS)

    Xiong, Xiangling; Lv, Yifan; Chen, Tao; Zhang, Xiaobing; Wang, Kemin; Tan, Weihong

    2014-06-01

    Cells as the building blocks of life determine the basic functions and properties of a living organism. Understanding the structure and components of a cell aids in the elucidation of its biological functions. Moreover, knowledge of the similarities and differences between diseased and healthy cells is essential to understanding pathological mechanisms, identifying diagnostic markers, and designing therapeutic molecules. However, monitoring the structures and activities of a living cell remains a challenging task in bioanalytical and life science research. To meet the requirements of this task, aptamers, as “chemical antibodies,” have become increasingly powerful tools for cellular analysis. This article reviews recent advances in the development of nucleic acid aptamers in the areas of cell membrane analysis, cell detection and isolation, real-time monitoring of cell secretion, and intracellular delivery and analysis with living cell models. Limitations of aptamers and possible solutions are also discussed.

  7. Very small embryonic-like stem cells (VSELs) represent a real challenge in stem cell biology: recent pros and cons in the midst of a lively debate

    PubMed Central

    Ratajczak, M Z; Zuba-Surma, E; Wojakowski, W; Suszynska, M; Mierzejewska, K; Liu, R; Ratajczak, J; Shin, D M; Kucia, M

    2014-01-01

    The concept that adult tissue, including bone marrow (BM), contains early-development cells with broader differentiation potential has again been recently challenged. In response, we would like to review the accumulated evidence from several independent laboratories that adult tissues, including BM, harbor a population of very rare stem cells that may cross germ layers in their differentiation potential. Thus, the BM stem cell compartment hierarchy needs to be revisited. These dormant, early-development cells that our group described as very small embryonic-like stem cells (VSELs) most likely overlap with similar populations of stem cells that have been identified in adult tissues by other investigators as the result of various experimental strategies and have been given various names. As reported, murine VSELs have some pluripotent stem cell characteristics. Moreover, they display several epiblast/germline markers that suggest their embryonic origin and developmental deposition in adult BM. Moreover, at the molecular level, changes in expression of parentally imprinted genes (for example, Igf2–H19) and resistance to insulin/insulin-like growth factor signaling (IIS) regulates their quiescent state in adult tissues. In several emergency situations related to organ damage, VSELs can be activated and mobilized into peripheral blood, and in appropriate animal models they contribute to tissue organ/regeneration. Interestingly, their number correlates with lifespan in mice, and they may also be involved in some malignancies. VSELs have been successfully isolated in several laboratories; however, some investigators experience problems with their isolation. PMID:24018851

  8. Very small embryonic-like stem cells (VSELs) represent a real challenge in stem cell biology: recent pros and cons in the midst of a lively debate.

    PubMed

    Ratajczak, M Z; Zuba-Surma, E; Wojakowski, W; Suszynska, M; Mierzejewska, K; Liu, R; Ratajczak, J; Shin, D M; Kucia, M

    2014-03-01

    The concept that adult tissue, including bone marrow (BM), contains early-development cells with broader differentiation potential has again been recently challenged. In response, we would like to review the accumulated evidence from several independent laboratories that adult tissues, including BM, harbor a population of very rare stem cells that may cross germ layers in their differentiation potential. Thus, the BM stem cell compartment hierarchy needs to be revisited. These dormant, early-development cells that our group described as very small embryonic-like stem cells (VSELs) most likely overlap with similar populations of stem cells that have been identified in adult tissues by other investigators as the result of various experimental strategies and have been given various names. As reported, murine VSELs have some pluripotent stem cell characteristics. Moreover, they display several epiblast/germline markers that suggest their embryonic origin and developmental deposition in adult BM. Moreover, at the molecular level, changes in expression of parentally imprinted genes (for example, Igf2-H19) and resistance to insulin/insulin-like growth factor signaling (IIS) regulates their quiescent state in adult tissues. In several emergency situations related to organ damage, VSELs can be activated and mobilized into peripheral blood, and in appropriate animal models they contribute to tissue organ/regeneration. Interestingly, their number correlates with lifespan in mice, and they may also be involved in some malignancies. VSELs have been successfully isolated in several laboratories; however, some investigators experience problems with their isolation. PMID:24018851

  9. Live-cell imaging of cyanobacteria.

    PubMed

    Yokoo, Rayka; Hood, Rachel D; Savage, David F

    2015-10-01

    Cyanobacteria are a diverse bacterial phylum whose members possess a high degree of ultrastructural organization and unique gene regulatory mechanisms. Unraveling this complexity will require the use of live-cell fluorescence microscopy, but is impeded by the inherent fluorescent background associated with light-harvesting pigments and the need to feed photosynthetic cells light. Here, we outline a roadmap for overcoming these challenges. Specifically, we show that although basic cyanobacterial biology creates challenging experimental constraints, these restrictions can be mitigated by the careful choice of fluorophores and microscope instrumentation. Many of these choices are motivated by recent successful live-cell studies. We therefore also highlight how live-cell imaging has advanced our understanding of bacterial microcompartments, circadian rhythm, and the organization and segregation of the bacterial nucleoid. PMID:25366827

  10. Optical nanoscopy of a living cell

    NASA Astrophysics Data System (ADS)

    Ahluwalia, Balpreet S.; Wolfson, Deanna L.; Chuang, Frank Y. S.; Huser, Thomas

    2014-08-01

    Optical nanoscopy allows to study biological and functional processes of sub-cellular organelles. In structured illumination microscopy (SIM) the sample is illuminated with a grid-like interference pattern to encode higher spatial frequency information into observable Moiré patterns. By acquiring multiple images and a computation trick a superresolved image is obtained. SIM provides resolution enhancement of 2X in each axis as compared to conventional microscopes. For a visible light, SIM provides an optical resolution of 100 nm. The challenges associated with optical nanoscopy of a living cell are photo-toxicity, special dye requirements and artifacts due to cell movement. SIM works with conventional dyes and is a wide-field technique making it suitable for imaging living cells. In this work, we will discuss the opportunities and challenges of imaging living cells using SIM. Two applications of optical nanoscopy of living cells will be discussed; a) imaging of mitochondria in a keratinocyte cell and Optical microscopy based on fluorescence has emerged as a vital tool in modern bio-medical imaging and diagnosis. Super-resolution bio-imaging allows gathering information from sub-cellular organelles. In structured illumination microscopy (SIM) the sample is illuminated with a grid-like interference patterns to encode higher spatial frequencies information into observable images (Moiré fringes). A super-resolved image is then decoded using computational trick. In this work, we used SIM to acquired super-resolved optical images of mitochondria from a live keratinocyte cell (see Fig 1). SIM provides resolution enhancement of 2X in each axis and contrast enhancement of 8X on a projected image. Time-lapsed imaging was used to study the dynamics of mitochondria in a live cell.

  11. Lives of the cell.

    PubMed

    Mendelsohn, J Andrew

    2003-01-01

    What is the relation between things and theories, the material world and its scientific representations? This is a staple philosophical problem that rarely counts as historically legitimate or fruitful. In the following dialogue, the interlocutors do not argue for or against realism. Instead, they explore changing relations between theories and things, between contested objects of knowledge (like the cell) and less contested, more everyday things (like frog eggs scooped from a pond). Widely seen as the life sciences' first general theory, the cell theory underwent dramatic changes during the nineteenth century. The dialogue established that each successive version of the cell theory was formulated - each identity of the object cell was formed - around a different material: cork, cartilage, eggs in cleavage, muscle. Such things thus serve as exemplary materials, in ways not described by standard concepts like induction, theory-testing, theory-laden observation, and construction. Still, how can theories and perspective possibly be honed on things if these are apprehended differently by different observers according to their interests, training, culture, or indeed theories? The second part of the dialogue addresses this problem, partly through the verbal and visual schemata that were used by nineteenth-century microscopists and that are comparable to schemata in the visual arts. The third part of the dialogue considers the exemplary materials as a historical sequence, itself needing explanation. Theoretical change devolved partly from wider histories and geographies of the prevalence, availability, or scientific and cultural status of materials such as plants, animals, and muscle. PMID:12778897

  12. Acoustic microscopy of living cells.

    PubMed Central

    Hildebrand, J A; Rugar, D; Johnston, R N; Quate, C F

    1981-01-01

    This paper reports preliminary results of the observation by acoustic microscopy of living cells in vitro. The scanning acoustic microscope uses high-frequency sound waves to produce images with submicrometer resolution. The contrast observed in acoustic micrographs of living cells depends on the acoustic properties (i.e., density, stiffness, and attenuation) and on the topographic contour of the cell. Variation in distance separating the acoustic lens and the viewed cell also has a profound effect on the image. When the substratum is located at the focal plane, thick regions of the cell show a darkening that can be related to cellular acoustic attenuation (a function of cytoplasmic viscosity). When the top of the cell is placed near the focal plane, concentric bright and dark rings appear in the image. The location of the rings can be related to cell topography, and the ring contrast can be correlated to the stiffness and density of the cell. In addition, the character of the images of single cells varies dramatically when the substratum upon which they are grown is changed to a different material. By careful selection of the substratum, the information content of the acoustic images can be increased. Our analysis of acoustic images of actively motile cells indicates that leading lamella are less dense or stiff than the quiescent trailing processes of the cells. Images PMID:6940179

  13. Imaging Transcription in Living Cells

    PubMed Central

    Darzacq, Xavier; Yao, Jie; Larson, Daniel R.; Causse, Sebastien Z.; Bosanac, Lana; de Turris, Valeria; Ruda, Vera M.; Lionnet, Timothee; Zenklusen, Daniel; Guglielmi, Benjamin; Tjian, Robert; Singer, Robert H.

    2011-01-01

    The advent of new technologies for the imaging of living cells has made it possible to determine the properties of transcription, the kinetics of polymerase movement, the association of transcription factors, and the progression of the polymerase on the gene. We report here the current state of the field and the progress necessary to achieve a more complete understanding of the various steps in transcription. Our Consortium is dedicated to developing and implementing the technology to further this understanding. PMID:19416065

  14. Electronic cages for living cells.

    PubMed

    Al Saeed, Sarah; Bakewell, David J

    2015-08-01

    Design and construction of an electronic cage is described which enables real-time manipulation of live and dead eukaryotic cells. Non-uniform, radio frequency (RF) AC electric fields are used to enable translational and rotational movement of cells, known as dielectrophoresis (DEP) and electro-rotation (EROT), and distinguish their state as viable and non-viable. A concentric multilayered mathematical model, applicable for eukaryotic cells, is also developed, coded and implemented. The simulations predict three dielectric dispersions in the DEP and EROT spectra, though in practice the third is very small so that two are observed. The cage is part of a multi-staged project incorporating controller and DEP/EROT digital signal generator and image processing. PMID:26736401

  15. ``Backpack'' Functionalized Living Immune Cells

    NASA Astrophysics Data System (ADS)

    Swiston, Albert; Um, Soong Ho; Irvine, Darrell; Cohen, Robert; Rubner, Michael

    2009-03-01

    We demonstrate that functional polymeric ``backpacks'' built from polyelectrolyte multilayers (PEMs) can be attached to a fraction of the surface area of living, individual lymphocytes. Backpacks containing fluorescent polymers, superparamagnetic nanoparticles, and commercially available quantum dots have been attached to B and T-cells, which may be spatially manipulated using a magnetic field. Since the backpack does not occlude the entire cellular surface from the environment, this technique allows functional synthetic payloads to be attached to a cell that is free to perform its native functions, thereby synergistically utilizing both biological and synthetic functionalities. For instance, we have shown that backpack-modified T-cells are able to migrate on surfaces for several hours following backpack attachment. Possible payloads within the PEM backpack include drugs, vaccine antigens, thermally responsive polymers, nanoparticles, and imaging agents. We will discuss how this approach has broad potential for applications in bioimaging, single-cell functionalization, immune system and tissue engineering, and cell-based therapeutics where cell-environment interactions are critical.

  16. Information theory in living systems, methods, applications, and challenges.

    PubMed

    Gatenby, Robert A; Frieden, B Roy

    2007-02-01

    Living systems are distinguished in nature by their ability to maintain stable, ordered states far from equilibrium. This is despite constant buffeting by thermodynamic forces that, if unopposed, will inevitably increase disorder. Cells maintain a steep transmembrane entropy gradient by continuous application of information that permits cellular components to carry out highly specific tasks that import energy and export entropy. Thus, the study of information storage, flow and utilization is critical for understanding first principles that govern the dynamics of life. Initial biological applications of information theory (IT) used Shannon's methods to measure the information content in strings of monomers such as genes, RNA, and proteins. Recent work has used bioinformatic and dynamical systems to provide remarkable insights into the topology and dynamics of intracellular information networks. Novel applications of Fisher-, Shannon-, and Kullback-Leibler informations are promoting increased understanding of the mechanisms by which genetic information is converted to work and order. Insights into evolution may be gained by analysis of the the fitness contributions from specific segments of genetic information as well as the optimization process in which the fitness are constrained by the substrate cost for its storage and utilization. Recent IT applications have recognized the possible role of nontraditional information storage structures including lipids and ion gradients as well as information transmission by molecular flux across cell membranes. Many fascinating challenges remain, including defining the intercellular information dynamics of multicellular organisms and the role of disordered information storage and flow in disease. PMID:17083004

  17. Living the Questions: Rilke's Challenge to Our Quest for Certainty

    ERIC Educational Resources Information Center

    Gordon, Mordechai

    2007-01-01

    In this essay, Mordechai Gordon explores the significance of Rilke's challenge to "live the questions" and embrace uncertainty with respect to the quest for certainty in education. The quest for certainty in education refers to our desire to gain a sense of psychological security and more control over a field that is fundamentally indeterminate.…

  18. Thermodynamics of protein destabilization in live cells

    PubMed Central

    Danielsson, Jens; Mu, Xin; Lang, Lisa; Wang, Huabing; Binolfi, Andres; Theillet, François-Xavier; Bekei, Beata; Logan, Derek T.; Selenko, Philipp; Wennerström, Håkan; Oliveberg, Mikael

    2015-01-01

    Although protein folding and stability have been well explored under simplified conditions in vitro, it is yet unclear how these basic self-organization events are modulated by the crowded interior of live cells. To find out, we use here in-cell NMR to follow at atomic resolution the thermal unfolding of a β-barrel protein inside mammalian and bacterial cells. Challenging the view from in vitro crowding effects, we find that the cells destabilize the protein at 37 °C but with a conspicuous twist: While the melting temperature goes down the cold unfolding moves into the physiological regime, coupled to an augmented heat-capacity change. The effect seems induced by transient, sequence-specific, interactions with the cellular components, acting preferentially on the unfolded ensemble. This points to a model where the in vivo influence on protein behavior is case specific, determined by the individual protein’s interplay with the functionally optimized “interaction landscape” of the cellular interior. PMID:26392565

  19. Atomic Force Microscopy of Living Cells

    NASA Astrophysics Data System (ADS)

    Ushiki, Tatsuo; Yamamoto, Susumu; Hitomi, Jiro; Ogura, Shigeaki; Umemoto, Takeshi; Shigeno, Masatsugu

    2000-06-01

    This paper is a review of our results of the application of atomic force microscopy (AFM) to the three-dimensional observation of living cells. First, we showed AFM images of living cultured cells in fluid. Contact mode AFM of living cells provided precise information on the shape of cellular processes (such as spike-like processes or lamellipodia) at the cellular margin. The contour of cytoskeletal elements just beneath the cell membrane was also clearly observable on the upper surface of the cells. Secondly, we showed the data on the discrepancy between the AFM images of living cells and fixed cells. These findings were useful for evaluating AFM images of living cells. Finally, we described the time-lapse AFM of living cells. A fluid chamber system enabled us to obtain AFM images of living cells for over 1 h at time intervals of 2-4 min. A series of these AFM images were useful for examining the movements of cellular processes in relation to subcellular cytoskeletal elements. Time-lapse movies produced by sequential AFM images also gave a realistic view of the cellular dynamics.

  20. Nanobiomechanics of living cells: a review

    PubMed Central

    Chen, Jinju

    2014-01-01

    Nanobiomechanics of living cells is very important to understand cell–materials interactions. This would potentially help to optimize the surface design of the implanted materials and scaffold materials for tissue engineering. The nanoindentation techniques enable quantifying nanobiomechanics of living cells, with flexibility of using indenters of different geometries. However, the data interpretation for nanoindentation of living cells is often difficult. Despite abundant experimental data reported on nanobiomechanics of living cells, there is a lack of comprehensive discussion on testing with different tip geometries, and the associated mechanical models that enable extracting the mechanical properties of living cells. Therefore, this paper discusses the strategy of selecting the right type of indenter tips and the corresponding mechanical models at given test conditions. PMID:24748952

  1. A Live Specimen Cell for the Microscope.

    ERIC Educational Resources Information Center

    McNeil, D. W.

    1991-01-01

    Provides background and instructions for the assembly of a microaquarium, or specimen cell, in which the dynamic world of living microorganisms can be viewed through a microscope overextended periods of time utilizing the simplest of materials in the process. (JJK)

  2. Live cell immunogold labelling of RNA polymerase II

    PubMed Central

    Orlov, Igor; Schertel, Andreas; Zuber, Guy; Klaholz, Bruno; Drillien, Robert; Weiss, Etienne; Schultz, Patrick; Spehner, Danièle

    2015-01-01

    Labeling nuclear proteins with electron dense probes in living cells has been a major challenge due to their inability to penetrate into nuclei. We developed a lipid-based approach for delivering antibodies coupled to 0.8 nm ultrasmall gold particles into the nucleus to label RNA polymerase II. Focussed Ion Beam slicing coupled to Scanning Electron Microscopy (FIB/SEM) enabled visualization of entire cells with probe localization accuracy in the 10 nm range. PMID:25662860

  3. A family living with Alzheimer's disease: The communicative challenges.

    PubMed

    Jones, Danielle

    2015-09-01

    Alzheimer's disease irrevocably challenges a person's capacity to communicate with others. Earlier research on these challenges focused on the language disorders associated with the condition and situated language deficit solely in the limitations of a person's cognitive and semantic impairments. This research falls short of gaining insight into the actual interactional experiences of a person with Alzheimer's and their family. Drawing on a UK data set of 70 telephone calls recorded over a two-and-a-half year period (2006-2008) between one elderly woman with Alzheimer's disease, and her daughter and son-in-law, this paper explores the role which communication (and its degeneration) plays in family relationships. Investigating these interactions, using a conversation analytic approach, reveals that there are clearly communicative difficulties, but closer inspection suggests that they arise due to the contingencies that are generated by the other's contributions in the interaction. That being so, this paper marks a departure from the traditional focus on language level analysis and the assumption that deficits are intrinsic to the individual with Alzheimer's, and instead focuses on the collaborative communicative challenges that arise in the interaction itself and which have a profound impact on people's lives and relationships. PMID:24339113

  4. Live cell imaging in Drosophila melanogaster.

    PubMed

    Parton, Richard M; Vallés, Ana Maria; Dobbie, Ian M; Davis, Ilan

    2010-04-01

    Although many of the techniques of live cell imaging in Drosophila melanogaster are also used by the greater community of cell biologists working on other model systems, studying living fly tissues presents unique difficulties with regard to keeping the cells alive, introducing fluorescent probes, and imaging through thick, hazy cytoplasm. This article outlines the major tissue types amenable to study by time-lapse cinematography and different methods for keeping the cells alive. It describes various imaging and associated techniques best suited to following changes in the distribution of fluorescently labeled molecules in real time in these tissues. Imaging, in general, is a rapidly developing discipline, and recent advances in imaging technology are able to greatly extend what can be achieved with live cell imaging of Drosophila tissues. As far as possible, this article includes the latest technical developments and discusses likely future developments in imaging methods that could have an impact on research using Drosophila. PMID:20360379

  5. Live Cell Imaging during Mechanical Stretch

    PubMed Central

    Rápalo, Gabriel; Herwig, Josh D.; Hewitt, Robert; Wilhelm, Kristina R.; Waters, Christopher M.; Roan, Esra

    2015-01-01

    There is currently a significant interest in understanding how cells and tissues respond to mechanical stimuli, but current approaches are limited in their capability for measuring responses in real time in live cells or viable tissue. A protocol was developed with the use of a cell actuator to distend live cells grown on or tissues attached to an elastic substrate while imaging with confocal and atomic force microscopy (AFM). Preliminary studies show that tonic stretching of human bronchial epithelial cells caused a significant increase in the production of mitochondrial superoxide. Moreover, using this protocol, alveolar epithelial cells were stretched and imaged, which showed direct damage to the epithelial cells by overdistention simulating one form of lung injury in vitro. A protocol to conduct AFM nano-indentation on stretched cells is also provided. PMID:26325607

  6. Imaging gene expression in single living cells

    PubMed Central

    Shav-Tal, Yaron; Singer, Robert H.; Darzacq, Xavier

    2016-01-01

    Technical advances in the field of live-cell imaging have introduced the cell biologist to a new, dynamic, subcellular world. The static world of molecules in fixed cells has now been extended to the time dimension. This allows the visualization and quantification of gene expression and intracellular trafficking events of the studied molecules and the associated enzymatic processes in individual cells, in real time. PMID:15459666

  7. Nanometer scale thermometry in a living cell

    PubMed Central

    Kucsko, G.; Maurer, P. C.; Yao, N. Y.; Kubo, M.; Noh, H. J.; Lo, P. K.; Park, H.; Lukin, M. D.

    2014-01-01

    Sensitive probing of temperature variations on nanometer scales represents an outstanding challenge in many areas of modern science and technology1. In particular, a thermometer capable of sub-degree temperature resolution over a large range of temperatures as well as integration within a living system could provide a powerful new tool for many areas of biological, physical and chemical research; possibilities range from the temperature-induced control of gene expression2–5 and tumor metabolism6 to the cell-selective treatment of disease7,8 and the study of heat dissipation in integrated circuits1. By combining local light-induced heat sources with sensitive nanoscale thermometry, it may also be possible to engineer biological processes at the sub-cellular level2–5. Here, we demonstrate a new approach to nanoscale thermometry that utilizes coherent manipulation of the electronic spin associated with nitrogen-vacancy (NV) color centers in diamond. We show the ability to detect temperature variations down to 1.8 mK (sensitivity of 9mK/Hz) in an ultra-pure bulk diamond sample. Using NV centers in diamond nanocrystals (nanodiamonds, NDs), we directly measure the local thermal environment at length scales down to 200 nm. Finally, by introducing both nanodiamonds and gold nanoparticles into a single human embryonic fibroblast, we demonstrate temperature-gradient control and mapping at the sub-cellular level, enabling unique potential applications in life sciences. PMID:23903748

  8. Colon-targeted delivery of live bacterial cell biotherapeutics including microencapsulated live bacterial cells

    PubMed Central

    Prakash, Satya; Malgorzata Urbanska, Aleksandra

    2008-01-01

    There has been an ample interest in delivery of therapeutic molecules using live cells. Oral delivery has been stipulated as best way to deliver live cells to humans for therapy. Colon, in particular, is a part of gastrointestinal (GI) tract that has been proposed to be an oral targeted site. The main objective of these oral therapy procedures is to deliver live cells not only to treat diseases like colorectal cancer, inflammatory bowel disease, and other GI tract diseases like intestinal obstruction and gastritis, but also to deliver therapeutic molecules for overall therapy in various diseases such as renal failure, coronary heart disease, hypertension, and others. This review provides a comprehensive summary of recent advancement in colon targeted live bacterial cell biotherapeutics. Current status of bacterial cell therapy, principles of artificial cells and its potentials in oral delivery of live bacterial cell biotherapeutics for clinical applications as well as biotherapeutic future perspectives are also discussed in our review. PMID:19707368

  9. Shedding Light on Living Cells.

    PubMed

    Antognazza, Maria Rosa; Martino, Nicola; Ghezzi, Diego; Feyen, Paul; Colombo, Elisabetta; Endeman, Duco; Benfenati, Fabio; Lanzani, Guglielmo

    2015-12-01

    An overview of the optical methods available to modulate the cellular activity in cell cultures and biological tissues is presented, with a focus on the use of exogenous functional materials that absorb electromagnetic radiation and transduce it into a secondary stimulus for cell excitation, with high temporal and spatial resolution. Both organic and inorganic materials are critically evaluated, for in vitro and in vivo applications. Finally, as a direct practical application of optical-stimulation techniques, the most recent results in the realization of artificial visual implants are discussed. PMID:25469452

  10. Minimally invasive surgery for live kidney donors: techniques and challenges.

    PubMed

    Brook, Nicholas R; Nicholson, Michael L

    2005-09-01

    Live kidney donation is assuming an increasingly prominent role in kidney transplantation programs. The traditional operative approach has been through an incision in the upper quadrant of the abdomen or in the loin, with the attendant potential postoperative complications associated with a large surgical wound. These problems may act as disincentives to prospective donors. The introduction of laparoscopic donor surgery in 1995 heralded a new era offering reduced post-operative pain and improved cosmetic result. It is hoped that these benefits may counter some disincentives and thereby increase donation rates. Three minimal-access approaches and their advantages and disadvantages are described: classical laparoscopic, hand-assisted laparoscopic, and retroperitoneoscopic surgery. Published reports indicate extensive experience with the first 2 of these approaches and less experience with the latter. All 3 approaches present technical, physiological, and anatomical challenges in the context of retrieving an organ that is fit for transplantation. For minimal-access surgery to be accepted as the procedure of choice for live kidney donors, it must be demonstrated that morbidity is not transferred from donor to recipient when these techniques are used. Some concerns about these procedures are addressed. High-level evidence in the form of randomized controlled trials is generally lacking, but experiences of surgeons and patients suggest that, with appropriate modifications, these techniques are safe for both donors and allografts and also benefit donors' recovery. PMID:16252632

  11. Healthy Living with Persuasive Technologies: Framework, Issues, and Challenges

    PubMed Central

    Chatterjee, Samir; Price, Alan

    2009-01-01

    While our Y2K worries about old computers “retiring” at midnight captured the television and news media attention, a more significant “old age” phenomenon snuck onto the scene with hardly a headline: the dawn of the age of the aged. 1 The over burdened health care system will face a worldwide wave of retirees who will live longer, cost more to treat, and demand new goods and services to help them stay healthy, active, and independent. Research in persuasive technologies and the associated usage of a computing system, device, or application intentionally designed to change a person's attitude or behavior in a predetermined way is showing the potential to assist in improving healthy living, reduce the costs on the health care system, and allow the aged to maintain a more independent life. This article gives a deeper insight into the evolution of persuasive technologies and presents a framework that can guide a researcher or practitioner in comprehending more effectively the work being done in this novel research field. It also provides categories of domains within health care in which these technologies are used and surveys exemplars from published literature. The article's goal is to provide greater understanding by addressing the challenges that lie ahead for all key stakeholders that design and/or use persuasive technologies in health care. PMID:19074300

  12. Live attenuated vaccines: Historical successes and current challenges

    SciTech Connect

    Minor, Philip D.

    2015-05-15

    Live attenuated vaccines against human viral diseases have been amongst the most successful cost effective interventions in medical history. Smallpox was declared eradicated in 1980; poliomyelitis is nearing global eradication and measles has been controlled in most parts of the world. Vaccines function well for acute diseases such as these but chronic infections such as HIV are more challenging for reasons of both likely safety and probable efficacy. The derivation of the vaccines used has in general not been purely rational except in the sense that it has involved careful clinical trials of candidates and subsequent careful follow up in clinical use; the identification of the candidates is reviewed. - Highlights: • Live vaccines against human diseases caused by viruses have been very successful. • They have been developed by empirical clinical studies and problems identified in later use. • It can be difficult to balance ability to cause disease and ability to immunise for a strain. • There is currently no reliable basis for predicting success from pure virological studies. • Vaccinia, which eradicated smallpox, is the paradigm for all successes and issues.

  13. Living Cell Microarrays: An Overview of Concepts.

    PubMed

    Jonczyk, Rebecca; Kurth, Tracy; Lavrentieva, Antonina; Walter, Johanna-Gabriela; Scheper, Thomas; Stahl, Frank

    2016-01-01

    Living cell microarrays are a highly efficient cellular screening system. Due to the low number of cells required per spot, cell microarrays enable the use of primary and stem cells and provide resolution close to the single-cell level. Apart from a variety of conventional static designs, microfluidic microarray systems have also been established. An alternative format is a microarray consisting of three-dimensional cell constructs ranging from cell spheroids to cells encapsulated in hydrogel. These systems provide an in vivo-like microenvironment and are preferably used for the investigation of cellular physiology, cytotoxicity, and drug screening. Thus, many different high-tech microarray platforms are currently available. Disadvantages of many systems include their high cost, the requirement of specialized equipment for their manufacture, and the poor comparability of results between different platforms. In this article, we provide an overview of static, microfluidic, and 3D cell microarrays. In addition, we describe a simple method for the printing of living cell microarrays on modified microscope glass slides using standard DNA microarray equipment available in most laboratories. Applications in research and diagnostics are discussed, e.g., the selective and sensitive detection of biomarkers. Finally, we highlight current limitations and the future prospects of living cell microarrays. PMID:27600077

  14. Living Cell Microarrays: An Overview of Concepts

    PubMed Central

    Jonczyk, Rebecca; Kurth, Tracy; Lavrentieva, Antonina; Walter, Johanna-Gabriela; Scheper, Thomas; Stahl, Frank

    2016-01-01

    Living cell microarrays are a highly efficient cellular screening system. Due to the low number of cells required per spot, cell microarrays enable the use of primary and stem cells and provide resolution close to the single-cell level. Apart from a variety of conventional static designs, microfluidic microarray systems have also been established. An alternative format is a microarray consisting of three-dimensional cell constructs ranging from cell spheroids to cells encapsulated in hydrogel. These systems provide an in vivo-like microenvironment and are preferably used for the investigation of cellular physiology, cytotoxicity, and drug screening. Thus, many different high-tech microarray platforms are currently available. Disadvantages of many systems include their high cost, the requirement of specialized equipment for their manufacture, and the poor comparability of results between different platforms. In this article, we provide an overview of static, microfluidic, and 3D cell microarrays. In addition, we describe a simple method for the printing of living cell microarrays on modified microscope glass slides using standard DNA microarray equipment available in most laboratories. Applications in research and diagnostics are discussed, e.g., the selective and sensitive detection of biomarkers. Finally, we highlight current limitations and the future prospects of living cell microarrays. PMID:27600077

  15. Live attenuated and inactivated viral vaccine formulation and nasal delivery: potential and challenges.

    PubMed

    Tlaxca, José L; Ellis, Scott; Remmele, Richard L

    2015-10-01

    Vaccines are cost-effective for the prevention of infectious diseases and have significantly reduced mortality and morbidity. Novel approaches are needed to develop safe and effective vaccines against disease. Major challenges in vaccine development include stability in a suitable dosage form and effective modes of delivery. Many live attenuated vaccines are capable of eliciting both humoral and cell mediated immune responses if physicochemically stable in an appropriate delivery vehicle. Knowing primary stresses that impart instability provides a general rationale for formulation development and mode of delivery. Since most pathogens enter the body through the mucosal route, live-attenuated vaccines have the advantage of mimicking natural immunization via non-invasive delivery. This presentation will examine aspects of formulation design, types of robust dosage forms to consider, effective routes of delivery (invasive and noninvasive), and distinctions between live attenuated or inactivated vaccines. PMID:25312673

  16. Imaging neurotransmitter release kinetics in living cells

    SciTech Connect

    Tan, Weihong; Yeung, E.S.; Haydon, P.G.

    1996-12-31

    A new UV-laser based optical microscope and CCD detection system has been developed to image neurotransmitter in living biological cells. We demonstrate the detection of serotonin that has been taken up into and released from individual living glial cells (astrocytes) based on its native fluorescence. The detection methodology has high sensitivity, low limit of detection and does not require coupling to fluorescence dyes. We have studied serotonin uptake kinetics and its release dynamics in single glial cells. Different regions of a glial cell have taken up different amounts of serotonin with a variety of kinetics. Similarly, different serotonin release mechanisms have been observed in different astrocyte cell regions. The temporal resolution of this detection system is as fast as 50 ms, and the spatial resolution is diffraction limited. We will also report on single enzyme molecule reaction studies and single metal ion detection based on CCD imaging of pL reaction vials formed by micromachining on fused silica.

  17. Viscoelastic Mapping of Living Cell Interiors

    NASA Astrophysics Data System (ADS)

    Heinrich, Doris; Sackmann, Erich; Koehler, Jana; Gerisch, Guenther

    2004-03-01

    We performed spatially resolved mapping of the viscoelastic properties of the cytoplasm of living cell interiors. A magnetic tweezer was applied as a local probe for the investigation of active and passive transport inside the slime mold cells Dictyostelium discoideum. Fluorescence labeled components, i.e. the microtubulins, the endoplasmatic reticulum or the core, allow for the determination of the interaction of the magnetic probes with the cytoplasm. By comparing the trajectories of the magnetic beads in the presence of an external magnetic force and in the absence of an external force, we can measure the viscosity at any given position within the cell. These experiments show that the cytoplasm consists of soft pathways (yield stress less or equal 10 Pa) and hard pathways (yield stress less or equal 500 Pa). Selective actin, myosin II or microtubulin network removal in the living cells allows for the determination of the influence of these cell parts on the viscoelastic properties.

  18. Apparatus and method for transforming living cells

    DOEpatents

    Okandan, Murat; Galambos, Paul C.

    2003-11-11

    An apparatus and method are disclosed for in vitro transformation of living cells. The apparatus, which is formed as a microelectromechanical device by surface micromachining, can be used to temporarily disrupt the cell walls or membrane of host cells one at a time so that a particular substance (e.g. a molecular tag, nucleic acid, bacteria, virus etc.) can be introduced into the cell. Disruption of the integrity of the host cells (i.e. poration) can be performed mechanically or electrically, or by both while the host cells are contained within a flow channel. Mechanical poration is possible using a moveable member which has a pointed or serrated edge and which is driven by an electrostatic actuator to abrade, impact or penetrate the host cell. Electroporation is produced by generating a relatively high electric field across the host cell when the host cell is located in the flow channel between a pair of electrodes having a voltage applied therebetween.

  19. Labeling Cytosolic Targets in Live Cells with Blinking Probes

    PubMed Central

    Xu, Jianmin; Chang, Jason; Yan, Qi; Dertinger, Thomas; Bruchez, Marcel; Weiss, Shimon

    2013-01-01

    With the advent of superresolution imaging methods, fast dynamic imaging of biological processes in live cells remains a challenge. A subset of these methods requires the cellular targets to be labeled with spontaneously blinking probes. The delivery and specific targeting of cytosolic targets and the control of the probes’ blinking properties are reviewed for three types of blinking probes: quantum dots, synthetic dyes, and fluorescent proteins. PMID:23930154

  20. Transition metal catalysis in the mitochondria of living cells.

    PubMed

    Tomás-Gamasa, María; Martínez-Calvo, Miguel; Couceiro, José R; Mascareñas, José L

    2016-01-01

    The development of transition metal catalysts capable of promoting non-natural transformations within living cells can open significant new avenues in chemical and cell biology. Unfortunately, the complexity of the cell makes it extremely difficult to translate standard organometallic chemistry to living environments. Therefore, progress in this field has been very slow, and many challenges, including the possibility of localizing active metal catalysts into specific subcellular sites or organelles, remain to be addressed. Herein, we report a designed ruthenium complex that accumulates preferentially inside the mitochondria of mammalian cells, while keeping its ability to react with exogenous substrates in a bioorthogonal way. Importantly, we show that the subcellular catalytic activity can be used for the confined release of fluorophores, and even allows selective functional alterations in the mitochondria by the localized transformation of inert precursors into uncouplers of the membrane potential. PMID:27600651

  1. Bioluminescence imaging in live cells and animals.

    PubMed

    Tung, Jack K; Berglund, Ken; Gutekunst, Claire-Anne; Hochgeschwender, Ute; Gross, Robert E

    2016-04-01

    The use of bioluminescent reporters in neuroscience research continues to grow at a rapid pace as their applications and unique advantages over conventional fluorescent reporters become more appreciated. Here, we describe practical methods and principles for detecting and imaging bioluminescence from live cells and animals. We systematically tested various components of our conventional fluorescence microscope to optimize it for long-term bioluminescence imaging. High-resolution bioluminescence images from live neurons were obtained with our microscope setup, which could be continuously captured for several hours with no signs of phototoxicity. Bioluminescence from the mouse brain was also imaged noninvasively through the intact skull with a conventional luminescence imager. These methods demonstrate how bioluminescence can be routinely detected and measured from live cells and animals in a cost-effective way with common reagents and equipment. PMID:27226972

  2. Detection of living cells in stratospheric samples

    NASA Astrophysics Data System (ADS)

    Harris, Melanie J.; Wickramasinghe, N. C.; Lloyd, David; Narlikar, J. V.; Rajaratnam, P.; Turner, Michael P.; Al-Mufti, Shirwan; Wallis, Max K.; Ramadurai, S.; Hoyle, Fred

    2002-02-01

    Air samples collected aseptically over tropical India at various stratospheric altitudes ranging from 20 to 41 km using cryosampler assemblies carried on balloons flown from Hyderabad have shown evidence of living microbial cells. Unambiguous evidence of living cells came from examining micropore filters on which the samples were recovered with the use of voltage sensitive lipophilic dyes that could detect the presents of active cells. Clumps of viable cells were found at all altitudes using this technique, and this conclusion was found to be consistent with images obtained from electron microscopy. Since the 41 km sample was collected well above the local tropopause, a prima facie case for a space incidence of these microorganisms is established. Further work on culturing, PCR analysis and isotopic analysis is in progress.

  3. Gas Plasma Effects on Living Cells

    NASA Astrophysics Data System (ADS)

    Stoffels, E.; Sladek, R. E. J.; Kieft, I. E.

    This paper surveys the research activities at the Eindhoven University of Technology (The Netherlands) in the area of biomedical applications of gas discharge plasmas. A non-thermal atmospheric plasma source (the plasma needle) has been developed, and its interactions with living mammalian cells and bacteria are studied. It is concluded that plasma can efficiently kill bacteria without harming the cells, and also influence the cells without causing cell death (necrosis). In future it will lead to applications like skin (wound) and caries treatment.

  4. 3D structure determination of a protein in living cells using paramagnetic NMR spectroscopy.

    PubMed

    Pan, Bin-Bin; Yang, Feng; Ye, Yansheng; Wu, Qiong; Li, Conggang; Huber, Thomas; Su, Xun-Cheng

    2016-08-11

    Determining the three-dimensional structure of a protein in living cells remains particularly challenging. We demonstrated that the integration of site-specific tagging proteins and GPS-Rosetta calculations provides a fast and effective way of determining the structures of proteins in living cells, and in principle the interactions and dynamics of protein-ligand complexes. PMID:27470136

  5. PeakForce Tapping resolves individual microvilli on living cells.

    PubMed

    Schillers, Hermann; Medalsy, Izhar; Hu, Shuiqing; Slade, Andrea L; Shaw, James E

    2016-02-01

    Microvilli are a common structure found on epithelial cells that increase the apical surface thus enhancing the transmembrane transport capacity and also serve as one of the cell's mechanosensors. These structures are composed of microfilaments and cytoplasm, covered by plasma membrane. Epithelial cell function is usually coupled to the density of microvilli and its individual size illustrated by diseases, in which microvilli degradation causes malabsorption and diarrhea. Atomic force microscopy (AFM) has been widely used to study the topography and morphology of living cells. Visualizing soft and flexible structures such as microvilli on the apical surface of a live cell has been very challenging because the native microvilli structures are displaced and deformed by the interaction with the probe. PeakForce Tapping® is an AFM imaging mode, which allows reducing tip-sample interactions in time (microseconds) and controlling force in the low pico-Newton range. Data acquisition of this mode was optimized by using a newly developed PeakForce QNM-Live Cell probe, having a short cantilever with a 17-µm-long tip that minimizes hydrodynamic effects between the cantilever and the sample surface. In this paper, we have demonstrated for the first time the visualization of the microvilli on living kidney cells with AFM using PeakForce Tapping. The structures observed display a force dependence representing either the whole microvilli or just the tips of the microvilli layer. Together, PeakForce Tapping allows force control in the low pico-Newton range and enables the visualization of very soft and flexible structures on living cells under physiological conditions. PMID:26414320

  6. Indicator displacement assays inside live cells.

    PubMed

    Norouzy, Amir; Azizi, Zahra; Nau, Werner M

    2015-01-12

    The macrocycle p-sulfonatocalix[4]arene (CX4) and the fluorescent dye lucigenin (LCG) form a stable host-guest complex, in which the dye fluorescence is quenched. Incubation of live V79 and CHO cells with the CX4/LCG chemosensing ensemble resulted in its spontaneous uptake. Subsequent addition of choline, acetylcholine, or protamine, which have a high affinity for CX4 and are capable of entering cells, resulted in a fluorescence switch-on response. This can be traced to the displacement of LCG from CX4 by the analytes. The results establish the principal functionality of indicator displacement assays with synthetic receptors for the detection of the uptake of bioorganic analytes by live cells. PMID:25430503

  7. Activity-driven fluctuations in living cells

    NASA Astrophysics Data System (ADS)

    Fodor, É.; Guo, M.; Gov, N. S.; Visco, P.; Weitz, D. A.; van Wijland, F.

    2015-05-01

    We propose a model for the dynamics of a probe embedded in a living cell, where both thermal fluctuations and nonequilibrium activity coexist. The model is based on a confining harmonic potential describing the elastic cytoskeletal matrix, which undergoes random active hops as a result of the nonequilibrium rearrangements within the cell. We describe the probe's statistics and we bring forth quantities affected by the nonequilibrium activity. We find an excellent agreement between the predictions of our model and experimental results for tracers inside living cells. Finally, we exploit our model to arrive at quantitative predictions for the parameters characterizing nonequilibrium activity, such as the typical time scale of the activity and the amplitude of the active fluctuations.

  8. Scanning ion conductance microscopy of living cells.

    PubMed Central

    Korchev, Y E; Bashford, C L; Milovanovic, M; Vodyanoy, I; Lab, M J

    1997-01-01

    Currently there is a great interest in using scanning probe microscopy to study living cells. However, in most cases the contact the probe makes with the soft surface of the cell deforms or damages it. Here we report a scanning ion conductance microscope specially developed for imaging living cells. A key feature of the instrument is its scanning algorithm, which maintains the working distance between the probe and the sample such that they do not make direct physical contact with each other. Numerical simulation of the probe/sample interaction, which closely matches the experimental observations, provides the optimum working distance. The microscope scans highly convoluted surface structures without damaging them and reveals the true topography of cell surfaces. The images resemble those produced by scanning electron microscopy, with the significant difference that the cells remain viable and active. The instrument can monitor small-scale dynamics of cell surfaces as well as whole-cell movement. Images FIGURE 1 FIGURE 2 FIGURE 3 FIGURE 4 PMID:9251784

  9. Efficient delivery of quantum dots in live cells by gold nanoparticle mediated photoporation

    NASA Astrophysics Data System (ADS)

    Xiong, Ranhua; Joris, Freya; De Cock, Ine; Demeester, Jo; De Smedt, Stefaan C.; Skirtach, Andre G.; Braeckmans, Kevin

    2015-03-01

    There is considerable interest in using Quantum Dots (QDs) as fluorescent probes such for cellular imaging due to unique advantages in comparison with conventional molecular dyes. However, cytosolic delivery of QDs into live cells remains a major challenge. Here we demonstrate highly efficient delivery of PEG-coated QDs into live cells by means of laser-induced vapour nanobubbles. Using this procedure we succeeded in high-throughput loading of ~80% of cells while maintaining a cell viability of ~85%.

  10. Biomimetic silica encapsultation of living cells

    NASA Astrophysics Data System (ADS)

    Jaroch, David Benjamin

    Living cells perform complex chemical processes on size and time scales that artificial systems cannot match. Cells respond dynamically to their environment, acting as biological sensors, factories, and drug delivery devices. To facilitate the use of living systems in engineered constructs, we have developed several new approaches to create stable protective microenvironments by forming bioinspired cell-membrane-specific silica-based encapsulants. These include vapor phase deposition of silica gels, use of endogenous membrane proteins and polysaccharides as a site for silica nucleation and polycondensation in a saturated environment, and protein templated ordered silica shell formation. We demonstrate silica layer formation at the surface of pluripotent stem-like cells, bacterial biofilms, and primary murine and human pancreatic islets. Materials are characterized by AFM, SEM and EDS. Viability assays confirm cell survival, and metabolite flux measurements demonstrate normal function and no major diffusion limitations. Real time PCR mRNA analysis indicates encapsulated islets express normal levels of genetic markers for β-cells and insulin production. The silica glass encapsulant produces a secondary bone like calcium phosphate mineral layer upon exposure to media. Such bioactive materials can improve device integration with surrounding tissue upon implantation. Given the favorable insulin response, bioactivity, and long-term viability observed in silica-coated islets, we are currently testing the encapsulant's ability to prevent immune system recognition of foreign transplants for the treatment of diabetes. Such hybrid silica-cellular constructs have a wide range of industrial, environmental, and medical applications.

  11. Single-Molecule Studies in Live Cells

    NASA Astrophysics Data System (ADS)

    Yu, Ji

    2016-05-01

    Live-cell single-molecule experiments are now widely used to study complex biological processes such as signal transduction, self-assembly, active trafficking, and gene regulation. These experiments' increased popularity results in part from rapid methodological developments that have significantly lowered the technical barriers to performing them. Another important advance is the development of novel statistical algorithms, which, by modeling the stochastic behaviors of single molecules, can be used to extract systemic parameters describing the in vivo biochemistry or super-resolution localization of biological molecules within their physiological environment. This review discusses recent advances in experimental and computational strategies for live-cell single-molecule studies, as well as a selected subset of biological studies that have utilized these new technologies.

  12. IMAGING ENDOCYTIC CLATHRIN STRUCTURES IN LIVING CELLS

    PubMed Central

    Kirchhausen, Tom

    2009-01-01

    Our understanding of the clathrin-dependent endocytic pathway owes much to new visualization techniques. Budding coated pits and clathrin-coated structures are transient molecular machines with distinctive morphological characteristics, and fluorescently labeled versions of a variety of marker proteins have given us a tantalizing glimpse of the dynamics of the system in living cells. Recent live-cell imaging studies reveal unexpected modes of coat assembly, with distinct kinetics, recruitment of associated proteins, requirements for the participation of actin and its accessory proteins, and apparently, distinct mechanisms of membrane deformation. A crucial issue is to connect the events detected by light microscopy with the structures and properties of the molecular constituents. Here, I outline descriptions of coat assembly in different circumstances that are consistent with what is known from x-ray crystallography and electron microscopy. PMID:19836955

  13. Single-Molecule Studies in Live Cells.

    PubMed

    Yu, Ji

    2016-05-27

    Live-cell single-molecule experiments are now widely used to study complex biological processes such as signal transduction, self-assembly, active trafficking, and gene regulation. These experiments' increased popularity results in part from rapid methodological developments that have significantly lowered the technical barriers to performing them. Another important advance is the development of novel statistical algorithms, which, by modeling the stochastic behaviors of single molecules, can be used to extract systemic parameters describing the in vivo biochemistry or super-resolution localization of biological molecules within their physiological environment. This review discusses recent advances in experimental and computational strategies for live-cell single-molecule studies, as well as a selected subset of biological studies that have utilized these new technologies. PMID:27070321

  14. Imaging Single Cells in the Living Retina

    PubMed Central

    Williams, David R.

    2011-01-01

    A quarter century ago, we were limited to a macroscopic view of the retina inside the living eye. Since then, new imaging technologies, including confocal scanning laser ophthalmoscopy, optical coherence tomography, and adaptive optics fundus imaging, transformed the eye into a microscope in which individual cells can now be resolved noninvasively. These technologies have enabled a wide range of studies of the retina that were previously impossible. PMID:21596053

  15. Surface Charge Visualization at Viable Living Cells.

    PubMed

    Perry, David; Paulose Nadappuram, Binoy; Momotenko, Dmitry; Voyias, Philip D; Page, Ashley; Tripathi, Gyanendra; Frenguelli, Bruno G; Unwin, Patrick R

    2016-03-01

    Scanning ion conductance microscopy (SICM) is demonstrated to be a powerful technique for quantitative nanoscale surface charge mapping of living cells. Utilizing a bias modulated (BM) scheme, in which the potential between a quasi-reference counter electrode (QRCE) in an electrolyte-filled nanopipette and a QRCE in bulk solution is modulated, it is shown that both the cell topography and the surface charge present at cellular interfaces can be measured simultaneously at high spatial resolution with dynamic potential measurements. Surface charge is elucidated by probing the properties of the diffuse double layer (DDL) at the cellular interface, and the technique is sensitive at both low-ionic strength and under typical physiological (high-ionic strength) conditions. The combination of experiments that incorporate pixel-level self-referencing (calibration) with a robust theoretical model allows for the analysis of local surface charge variations across cellular interfaces, as demonstrated on two important living systems. First, charge mapping at Zea mays root hairs shows that there is a high negative surface charge at the tip of the cell. Second, it is shown that there are distinct surface charge distributions across the surface of human adipocyte cells, whose role is the storage and regulation of lipids in mammalian systems. These are new features, not previously recognized, and their implications for the functioning of these cells are highlighted. PMID:26871001

  16. Electromicroinjection of particles into living cells

    DOEpatents

    Ray, F. Andrew; Cram, L. Scott; Galey, William R.

    1988-01-01

    Method and apparatus for introducing particles into living cells. Fluorescently-stained human chromosomes are introduced into cultured, mitotic Chinese hamster cells using electromicroinjection. The recipient cells frequently survived the physiological perturbation imposed by a successful chromosome injection. Successfully injected recipient cells maintained viability as evidenced by their ability to be expanded. The technique relies on the surface charge of fluorescently stained chromosomes and their ability to be attracted and repelled to and from the tip of a micropipette. The apparatus includes a micropipette having a tip suitable for piercing the membrane of a target cell and an electrode inserted into the lumen thereof. The target cells and suspended particles are located in an electrically conducted solution, and the lumen of the micropipette is filled with an electrically conducting solution which contacts the electrode located therein. A second electrode is also located in the conducting solution containing the target cells and particles. Voltages applied to the electrode within the micropipette attract the particles to the region of the tip thereof. The particles adhere to the surface of the micropipette with sufficient force that insertion of the micropipette tip and attached particle through the membrane of a target cell will not dislodge the particle. By applying a voltage having the opposite polarity of the attraction voltage, the particles are expelled from the micropipette to which is then withdrawn from the cell body.

  17. Circumventing photodamage in live-cell microscopy

    PubMed Central

    Magidson, Valentin; Khodjakov, Alexey

    2013-01-01

    Fluorescence microscopy has become an essential tool in cell biology. This technique allows researchers to visualize the dynamics of tissue, cells, individual organelles and macromolecular assemblies inside the cell. Unfortunately, fluorescence microscopy is not completely ‘non-invasive’ as the high-intensity excitation light required for excitation of fluorophores is inherently toxic for live cells. Physiological changes induced by excessive illumination can lead to artifacts and abnormal responses. In this chapter we review major factors that contribute to phototoxicity and discuss practical solutions for circumventing photodamage. These solutions include the proper choice of image acquisition parameters, optimization of filter sets, hardware synchronization, and the use of intelligent illumination to avoid unnecessary light exposure. PMID:23931522

  18. Optical magnetic imaging of living cells

    PubMed Central

    Le Sage, D.; Arai, K.; Glenn, D. R.; DeVience, S. J.; Pham, L. M.; Rahn-Lee, L.; Lukin, M. D.; Yacoby, A.; Komeili, A.; Walsworth, R. L.

    2013-01-01

    Magnetic imaging is a powerful tool for probing biological and physical systems. However, existing techniques either have poor spatial resolution compared to optical microscopy and are hence not generally applicable to imaging of sub-cellular structure (e.g., magnetic resonance imaging [MRI]1), or entail operating conditions that preclude application to living biological samples while providing sub-micron resolution (e.g., scanning superconducting quantum interference device [SQUID] microscopy2, electron holography3, and magnetic resonance force microscopy [MRFM]4). Here we demonstrate magnetic imaging of living cells (magnetotactic bacteria) under ambient laboratory conditions and with sub-cellular spatial resolution (400 nm), using an optically-detected magnetic field imaging array consisting of a nanoscale layer of nitrogen-vacancy (NV) colour centres implanted at the surface of a diamond chip. With the bacteria placed on the diamond surface, we optically probe the NV quantum spin states and rapidly reconstruct images of the vector components of the magnetic field created by chains of magnetic nanoparticles (magnetosomes) produced in the bacteria, and spatially correlate these magnetic field maps with optical images acquired in the same apparatus. Wide-field sCMOS acquisition allows parallel optical and magnetic imaging of multiple cells in a population with sub-micron resolution and >100 micron field-of-view. Scanning electron microscope (SEM) images of the bacteria confirm that the correlated optical and magnetic images can be used to locate and characterize the magnetosomes in each bacterium. The results provide a new capability for imaging bio-magnetic structures in living cells under ambient conditions with high spatial resolution, and will enable the mapping of a wide range of magnetic signals within cells and cellular networks5, 6. PMID:23619694

  19. Optical magnetic imaging of living cells.

    PubMed

    Le Sage, D; Arai, K; Glenn, D R; DeVience, S J; Pham, L M; Rahn-Lee, L; Lukin, M D; Yacoby, A; Komeili, A; Walsworth, R L

    2013-04-25

    Magnetic imaging is a powerful tool for probing biological and physical systems. However, existing techniques either have poor spatial resolution compared to optical microscopy and are hence not generally applicable to imaging of sub-cellular structure (for example, magnetic resonance imaging), or entail operating conditions that preclude application to living biological samples while providing submicrometre resolution (for example, scanning superconducting quantum interference device microscopy, electron holography and magnetic resonance force microscopy). Here we demonstrate magnetic imaging of living cells (magnetotactic bacteria) under ambient laboratory conditions and with sub-cellular spatial resolution (400 nanometres), using an optically detected magnetic field imaging array consisting of a nanometre-scale layer of nitrogen-vacancy colour centres implanted at the surface of a diamond chip. With the bacteria placed on the diamond surface, we optically probe the nitrogen-vacancy quantum spin states and rapidly reconstruct images of the vector components of the magnetic field created by chains of magnetic nanoparticles (magnetosomes) produced in the bacteria. We also spatially correlate these magnetic field maps with optical images acquired in the same apparatus. Wide-field microscopy allows parallel optical and magnetic imaging of multiple cells in a population with submicrometre resolution and a field of view in excess of 100 micrometres. Scanning electron microscope images of the bacteria confirm that the correlated optical and magnetic images can be used to locate and characterize the magnetosomes in each bacterium. Our results provide a new capability for imaging bio-magnetic structures in living cells under ambient conditions with high spatial resolution, and will enable the mapping of a wide range of magnetic signals within cells and cellular networks. PMID:23619694

  20. Creep Function of a Single Living Cell

    PubMed Central

    Desprat, Nicolas; Richert, Alain; Simeon, Jacqueline; Asnacios, Atef

    2005-01-01

    We used a novel uniaxial stretching rheometer to measure the creep function J(t) of an isolated living cell. We show, for the first time at the scale of the whole cell, that J(t) behaves as a power-law J(t) = Atα. For N = 43 mice myoblasts (C2-7), we find α = 0.24 ± 0.01 and A = (2.4 ± 0.3) 10−3 Pa−1 s−α. Using Laplace Transforms, we compare A and α to the parameters G0 and β of the complex modulus G*(ω) = G0ωβ measured by other authors using magnetic twisting cytometry and atomic force microscopy. Excellent agreement between A and G0 on the one hand, and between α and β on the other hand, indicated that the power-law is an intrinsic feature of cell mechanics and not the signature of a particular technique. Moreover, the agreement between measurements at very different size scales, going from a few tens of nanometers to the scale of the whole cell, suggests that self-similarity could be a central feature of cell mechanical structure. Finally, we show that the power-law behavior could explain previous results first interpreted as instantaneous elasticity. Thus, we think that the living cell must definitely be thought of as a material with a large and continuous distribution of relaxation time constants which cannot be described by models with a finite number of springs and dash-pots. PMID:15596508

  1. On strain and stress in living cells

    NASA Astrophysics Data System (ADS)

    Cox, Brian N.; Smith, David W.

    2014-11-01

    Recent theoretical simulations of amelogenesis and network formation and new, simple analyses of the basic multicellular unit (BMU) allow estimation of the order of magnitude of the strain energy density in populations of living cells in their natural environment. A similar simple calculation translates recent measurements of the force-displacement relation for contacting cells (cell-cell adhesion energy) into equivalent volume energy densities, which are formed by averaging the changes in contact energy caused by a cell's migration over the cell's volume. The rates of change of these mechanical energy densities (energy density rates) are then compared to the order of magnitude of the metabolic activity of a cell, expressed as a rate of production of metabolic energy per unit volume. The mechanical energy density rates are 4-5 orders of magnitude smaller than the metabolic energy density rate in amelogenesis or bone remodeling in the BMU, which involve modest cell migration velocities, and 2-3 orders of magnitude smaller for innervation of the gut or angiogenesis, where migration rates are among the highest for all cell types. For representative cell-cell adhesion gradients, the mechanical energy density rate is 6 orders of magnitude smaller than the metabolic energy density rate. The results call into question the validity of using simple constitutive laws to represent living cells. They also imply that cells need not migrate as inanimate objects of gradients in an energy field, but are better regarded as self-powered automata that may elect to be guided by such gradients or move otherwise. Thus Ġel=d/dt 1/2 >[(C11+C12)ɛ02+2μγ02]=(C11+C12)ɛ0ɛ˙0+2μγ0γ˙0 or Ġel=ηEɛ0ɛ˙0+η‧Eγ0γ˙0 with 1.4≤η≤3.4 and 0.7≤η‧≤0.8 for Poisson's ratio in the range 0.2≤ν≤0.4 and η=1.95 and η‧=0.75 for ν=0.3. The spatial distribution of shear strains arising within an individual cell as cells slide past one another during amelogenesis is not known

  2. Direct Visualization of De novo Lipogenesis in Single Living Cells

    NASA Astrophysics Data System (ADS)

    Li, Junjie; Cheng, Ji-Xin

    2014-10-01

    Increased de novo lipogenesis is being increasingly recognized as a hallmark of cancer. Despite recent advances in fluorescence microscopy, autoradiography and mass spectrometry, direct observation of de novo lipogenesis in living systems remains to be challenging. Here, by coupling stimulated Raman scattering (SRS) microscopy with isotope labeled glucose, we were able to trace the dynamic metabolism of glucose in single living cells with high spatial-temporal resolution. As the first direct visualization, we observed that glucose was largely utilized for lipid synthesis in pancreatic cancer cells, which occurs at a much lower rate in immortalized normal pancreatic epithelial cells. By inhibition of glycolysis and fatty acid synthase (FAS), the key enzyme for fatty acid synthesis, we confirmed the deuterium labeled lipids in cancer cells were from de novo lipid synthesis. Interestingly, we also found that prostate cancer cells exhibit relatively lower level of de novo lipogenesis, but higher fatty acid uptake compared to pancreatic cancer cells. Together, our results demonstrate a valuable tool to study dynamic lipid metabolism in cancer and other disorders.

  3. [Living donors for kidney transplantation: ethical and legal challenges].

    PubMed

    Mamzer-Bruneel, Marie-France; Fournier, Catherine; Legendre, Christophe

    2010-05-01

    Living donor kidney transplantation has developed very heterogeneously worldwide despite excellent results and without taking into account the context of global organ shortage. Such a heterogeneity highlights persistent ethical issues, whereas organ trafficking is emerging as an organized transplant tourism reinforcing the need for strong national legal frameworks. Despite its powerful regulation system, which ensures standardization, transparency and accountability of support for donation, France remains reluctant to enlarge the circle of legal donors, whereas it would be the first step to give a greater role to living organ donation. PMID:20510152

  4. Microfluidic tissue model for live cell screening.

    PubMed

    Lee, Philip J; Gaige, Terry A; Ghorashian, Navid; Hung, Paul J

    2007-01-01

    We have developed a microfluidic platform modeled after the physiologic microcirculation for multiplexed tissue-like culture and high-throughput analysis. Each microfabricated culture unit consisted of three functional components: a 50 microm wide cell culture pocket, an artificial endothelial barrier with 2 microm pores, and a nutrient transport channel. This configuration enabled a high density of cancer cells to be maintained for over 1 week in a solid tumor-like morphology when fed with continuous flow. The microfluidic chip contained 16 parallel units for "flow cell" based experiments where live cells were exposed to a soluble factor and analyzed via fluorescence microscopy or flow-through biochemistry. Each fluidically independent tissue unit contained approximately 500 cells fed with a continuous flow of 10 nL/min. As a demonstration, the toxicity profile of the anti-cancer drug paclitaxel was collected on HeLa cells cultured in the microfluidic format and compared with a 384-well dish for up to 5 days of continuous drug exposure. PMID:17585775

  5. Recent advances in live cell imaging of hepatoma cells

    PubMed Central

    2014-01-01

    Live cell imaging enables the study of dynamic processes of living cells in real time by use of suitable reporter proteins and the staining of specific cellular structures and/or organelles. With the availability of advanced optical devices and improved cell culture protocols it has become a rapidly growing research methodology. The success of this technique relies mainly on the selection of suitable reporter proteins, construction of recombinant plasmids possessing cell type specific promoters as well as reliable methods of gene transfer. This review aims to provide an overview of the recent developments in the field of marker proteins (bioluminescence and fluorescent) and methodologies (fluorescent resonance energy transfer, fluorescent recovery after photobleaching and proximity ligation assay) employed as to achieve an improved imaging of biological processes in hepatoma cells. Moreover, different expression systems of marker proteins and the modes of gene transfer are discussed with emphasis on the study of lipid droplet formation in hepatocytes as an example. PMID:25005127

  6. Magnetic Manipulation of Nanorods in the Nucleus of Living Cells

    PubMed Central

    Celedon, Alfredo; Hale, Christopher M.; Wirtz, Denis

    2011-01-01

    The organization of chromatin in the cell nucleus is crucial for gene expression regulation. However, physically probing the nuclear interior is challenging because high forces have to be applied using minimally invasive techniques. Here, magnetic nanorods embedded in the nucleus of living cells are subjected to controlled rotational forces, producing micron-sized displacements in the nuclear interior. The resulting time-dependent rotation of the nanorods is analyzed in terms of viscoelastic parameters of the nucleus, in wild-type and Lamin A/C deficient cells. This method and analysis reveal that Lamin A/C knockout, together perhaps with other changes that result from the knockout, induce significant decreases in the nuclear viscosity and elasticity. PMID:22004741

  7. Automated live cell imaging systems reveal dynamic cell behavior.

    PubMed

    Chirieleison, Steven M; Bissell, Taylor A; Scelfo, Christopher C; Anderson, Jordan E; Li, Yong; Koebler, Doug J; Deasy, Bridget M

    2011-07-01

    Automated time-lapsed microscopy provides unique research opportunities to visualize cells and subcellular components in experiments with time-dependent parameters. As accessibility to these systems is increasing, we review here their use in cell science with a focus on stem cell research. Although the use of time-lapsed imaging to answer biological questions dates back nearly 150 years, only recently have the use of an environmentally controlled chamber and robotic stage controllers allowed for high-throughput continuous imaging over long periods at the cell and subcellular levels. Numerous automated imaging systems are now available from both companies that specialize in live cell imaging and from major microscope manufacturers. We discuss the key components of robots used for time-lapsed live microscopic imaging, and the unique data that can be obtained from image analysis. We show how automated features enhance experimentation by providing examples of uniquely quantified proliferation and migration live cell imaging data. In addition to providing an efficient system that drastically reduces man-hours and consumes fewer laboratory resources, this technology greatly enhances cell science by providing a unique dataset of temporal changes in cell activity. PMID:21692197

  8. Raster image correlation spectroscopy in live cells.

    PubMed

    Rossow, Molly J; Sasaki, Jennifer M; Digman, Michelle A; Gratton, Enrico

    2010-11-01

    Raster image correlation spectroscopy (RICS) is a noninvasive technique to detect and quantify events in a live cell, including concentration of molecules and diffusion coefficients of molecules; in addition, by measuring changes in diffusion coefficients, RICS can indirectly detect binding. Any specimen containing fluorophores that can be imaged with a laser scanning microscope can be analyzed using RICS. There are other techniques to measure diffusion coefficients and binding; however, RICS fills a unique niche. It provides spatial information and can be performed in live cells using a conventional confocal microscope. It can measure a range of diffusion coefficients that is not accessible with any other single optical correlation-based technique. In this article we describe a protocol to obtain raster scanned images with an Olympus FluoView FV1000 confocal laser scanning microscope using Olympus FluoView software to acquire data and SimFCS software to perform RICS analysis. Each RICS measurement takes several minutes. The entire procedure can be completed in ∼2 h. This procedure includes focal volume calibration using a solution of fluorophores with a known diffusion coefficient and measurement of the diffusion coefficients of cytosolic enhanced green fluorescent protein (EGFP) and EGFP-paxillin. PMID:21030952

  9. Freezing of living cells: mechanisms and implications

    SciTech Connect

    Mazur, P.

    1984-01-01

    Cells can endure storage at low temperatures such as -196/sup 0/C for centuries. The challenge is to determine how they can survive both the cooling to such temperatures and the subsequent return to physiological conditions. A major factor is whether they freeze intracellularly. They do so if cooling is too rapid, because with rapid cooling insufficient cell water is removed osmotically to eliminate supercooling. Equations have been developed that describe the kinetics of this water loss and permit one to predict the likelihood of intracellular freezing as a function of cooling rate. Such predictions agree well with observations. Although the avoidance of intracellular freezing is usually necessary for survival, it is not sufficient. Slow freezing itself can be injurious. As ice forms outside the cell, the residual unfrozen medium forms channels of decreasing size and increasing solute concentration. The cells lie in the channels and shrink in osmotic response to the rising solute concentration. Prior theories have ascribed slow freezing injury to the concentration of solutes or the cell shrinkage. Recent experiments, however, indicate that the damage is due more to the decrease in the size of the unfrozen channels. This new view of the mechanism of slow freezing injury ought to facilitate the development of procedures for the preservation of complex assemblages of cells of biological, medical, and agricultural significance. 126 references, 18 figures, 2 tables.

  10. Lives of Quality in the Face of Challenge in Israel

    ERIC Educational Resources Information Center

    Neikrug, S.; Roth, D.; Judes, J.

    2011-01-01

    Purpose: The purpose of this study is to describe and analyse the quality of life of Israeli families raising a child with a disability while challenged with all the usual demands of family life. Methods: Respondents were main caregivers of 103 children with disability receiving services at Beit Issie Shapiro, a service agency in Israel. The…

  11. Photoacoustics of individual live cells and particles

    NASA Astrophysics Data System (ADS)

    Kudryashov, Sergey I.; Allen, Susan D.; Galanzha, Ekaterina I.; Galitovskaya, E.; Zharov, Vladimir P.

    2006-02-01

    The photoacoustic (PA) technique has been employed to a number of new biomedical applications based of highly sensitive detection of laser-induced acoustic waves from individual live cells and single absorbing micro-particles or clusters of nanoparticles. These applications involve both linear and non-linear thermoacoustic phenomena initiated by focused nanosecond single laser pulse and detected with a fast PZT-ceramic acoustic transducer. Particularly, we present the following experimental results: 1) monitoring of linear and non-linear PA responses from red blood cells in suspensions in vitro; 2) detection of PA responses from breast cancer cell targeted with gold nanoparticles; 3) PA study of linear and non-linear interaction of laser with colored polystyrene micro-particles as model single absorbers; 4) monitoring of PA responses from moving absorbers in flow in vitro (PA flow cytometry in vitro); 5) recording of PA responses from blood flow in vivo on rat mesentery as animal model (PA flow cytometery in vivo); and 6) monitoring of sedimentation kinetics of particles and cells. The obtained results demonstrate the high sensitivity, low background, simple detection principle, easy data acquisition, and straightforward interpretation of the PA data.

  12. DEMENTIA CARE IN ASSISTED LIVING: NEEDS AND CHALLENGES

    PubMed Central

    Smith, Marianne; Buckwalter, Kathleen C.; Kang, Hyunwook; Ellingrod, Vicki; Schultz, Susan K.

    2011-01-01

    Assisted living (AL) is an increasingly popular long-term care option for older adults with dementia. Recent reports suggest that as many as 68% of AL residents have dementia, and that frequency of both behavioral symptoms and psychotropic medications are high. This pilot project explored the feasibility of research methods for use in AL facilities. Findings suggest that most AL residents with dementia have moderate to severe dementia, and the majority are taking one or more psychotropic medication. Descriptive and qualitative findings related to health records, caregiver perceptions of behavioral symptoms, and practicality of assessment methods undertaken are described and implications for psychiatric nursing practice and research are reviewed. PMID:18649209

  13. The lived experiences of adolescents with sickle cell disease in Kingston, Jamaica

    PubMed Central

    Forrester, Andrea Brown; Barton-Gooden, Antoinette; Pitter, Cynthia; Lindo, Jascinth L. M.

    2015-01-01

    Aim To explore the lived experiences of adolescents with sickle cell disease, in Kingston, Jamaica. Method A descriptive qualitative design was used for this research. In-depth interviews were conducted with six adolescents with sickle cell disease at a Sickle Cell Unit operated by the University of the West Indies. Interviews were audiotaped, transcribed, and thematically analyzed. Results The majority of the adolescents demonstrated a positive self-concept. They reported strong family, school, and peer support which made them feel accepted. All were actively engaged in social activities such as parties, but had challenges participating in sporting activities. Various coping strategies were utilized to address challenges of the disease including praying, watching television, and surfing the Internet. Conclusion Sickle cell disease can be very challenging for the adolescent, but with positive self-concept and increased social support, especially from family and peers, these adolescents were able to effectively cope with their condition and live productive lives. PMID:26341889

  14. Microscale microbial fuel cells: Advances and challenges.

    PubMed

    Choi, Seokheun

    2015-07-15

    The next generation of sustainable energy could come from microorganisms; evidence that it can be seen with the given rise of Electromicrobiology, the study of microorganisms' electrical properties. Many recent advances in electromicrobiology stem from studying microbial fuel cells (MFCs), which are gaining acceptance as a future alternative "green" energy technology and energy-efficient wastewater treatment method. MFCs are powered by living microorganisms with clean and sustainable features; they efficiently catalyse the degradation of a broad range of organic substrates under natural conditions. There is also increasing interest in photosynthetic MFCs designed to harness Earth's most abundant and promising energy source (solar irradiation). Despite their vast potential and promise, however, MFCs and photosynthetic MFCs have not yet successfully translated into commercial applications because they demonstrate persistent performance limitations and bottlenecks associated with scaling up. Instead, microscale MFCs have received increasing attention as a unique platform for various applications such as powering small portable electronic elements in remote locations, performing fundamental studies of microorganisms, screening bacterial strains, and toxicity detection in water. Furthermore, the stacking of miniaturized MFCs has been demonstrated to offer larger power densities than a single macroscale MFC in terms of scaling up. In this overview, we discuss recent achievements in microscale MFCs as well as their potential applications. Further scientific and technological challenges are also reviewed. PMID:25703724

  15. Engineered T cells: the promise and challenges of cancer immunotherapy.

    PubMed

    Fesnak, Andrew D; June, Carl H; Levine, Bruce L

    2016-08-23

    The immune system evolved to distinguish non-self from self to protect the organism. As cancer is derived from our own cells, immune responses to dysregulated cell growth present a unique challenge. This is compounded by mechanisms of immune evasion and immunosuppression that develop in the tumour microenvironment. The modern genetic toolbox enables the adoptive transfer of engineered T cells to create enhanced anticancer immune functions where natural cancer-specific immune responses have failed. Genetically engineered T cells, so-called 'living drugs', represent a new paradigm in anticancer therapy. Recent clinical trials using T cells engineered to express chimeric antigen receptors (CARs) or engineered T cell receptors (TCRs) have produced stunning results in patients with relapsed or refractory haematological malignancies. In this Review we describe some of the most recent and promising advances in engineered T cell therapy with a particular emphasis on what the next generation of T cell therapy is likely to entail. PMID:27550819

  16. Imaging CREB Activation in Living Cells*

    PubMed Central

    Friedrich, Michael W.; Aramuni, Gayane; Mank, Marco; Mackinnon, Jonathan A. G.; Griesbeck, Oliver

    2010-01-01

    The Ca2+- and cAMP-responsive element-binding protein (CREB) and the related ATF-1 and CREM are stimulus-inducible transcription factors that link certain forms of cellular activity to changes in gene expression. They are attributed to complex integrative activation characteristics, but current biochemical technology does not allow dynamic imaging of CREB activation in single cells. Using fluorescence resonance energy transfer between mutants of green fluorescent protein we here develop a signal-optimized genetically encoded indicator that enables imaging activation of CREB due to phosphorylation of the critical serine 133. The indicator of CREB activation due to phosphorylation (ICAP) was used to investigate the role of the scaffold and anchoring protein AKAP79/150 in regulating signal pathways converging on CREB. We show that disruption of AKAP79/150-mediated protein kinase A anchoring or knock-down of AKAP150 dramatically reduces the ability of protein kinase A to activate CREB. In contrast, AKAP79/150 regulation of CREB via L-type channels may only have minor importance. ICAP allows dynamic and reversible imaging in living cells and may become useful in studying molecular components and cell-type specificity of activity-dependent gene expression. PMID:20484048

  17. Imaging Specific Genomic DNA in Living Cells.

    PubMed

    Chen, Baohui; Guan, Juan; Huang, Bo

    2016-07-01

    The three-dimensional organization of the genome plays important roles in regulating the functional output of the genome and even in the maintenance of epigenetic inheritance and genome stability. Here, we review and compare a number of newly developed methods-especially those that utilize the CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 (CRISPR-associated protein 9) system-that enable the direct visualization of specific, endogenous DNA sequences in living cells. We also discuss the practical considerations in implementing the CRISPR imaging technique to achieve sufficient signal-to-background levels, high specificity, and high labeling efficiency. These DNA labeling methods enable tracking of the copy number, localization, and movement of genomic elements, and we discuss the potential applications of these methods in understanding the searching and targeting mechanism of the Cas9-sgRNA complex, investigating chromosome organization, and visualizing genome instability and rearrangement. PMID:27145877

  18. Challenges and Limitations of Intelligent Ambient Assisted Living Environments

    NASA Astrophysics Data System (ADS)

    Wichert, Reiner

    As a result of changing demographics, residing and being cared for in one's own familiar environment versus in an institutionalised inpatient setting is becoming the more attractive alternative for an ever increasing portion of the population. Despite its tremendous market potential, the AAL (Ambient Assisted Living) branch is still on the cusp of a mainstream breakthrough. A lack of viable business models is considered almost unanimously to be the greatest market obstacle to a broad implementation of innovative AAL systems. This paper highlights possible explanations for this deficit and shows why the AAL community has yet to arrive at joint solutions based on a unified AAL reference platform. Furthermore, this paper describes the enormous potential of AmI and AAL, as the first real opportunity for their success is provided through universAAL and AALOA.

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

    PubMed Central

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

    2014-01-01

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

  20. Mechanical force characterization in manipulating live cells with optical tweezers.

    PubMed

    Wu, Yanhua; Sun, Dong; Huang, Wenhao

    2011-02-24

    Laser trapping with optical tweezers is a noninvasive manipulation technique and has received increasing attentions in biological applications. Understanding forces exerted on live cells is essential to cell biomechanical characterizations. Traditional numerical or experimental force measurement assumes live cells as ideal objects, ignoring their complicated inner structures and rough membranes. In this paper, we propose a new experimental method to calibrate the trapping and drag forces acted on live cells. Binding a micro polystyrene sphere to a live cell and moving the mixture with optical tweezers, we can obtain the drag force on the cell by subtracting the drag force on the sphere from the total drag force on the mixture, under the condition of extremely low Reynolds number. The trapping force on the cell is then obtained from the drag force when the cell is in force equilibrium state. Experiments on numerous live cells demonstrate the effectiveness of the proposed force calibration approach. PMID:21087769

  1. Living donor liver transplantation in Taiwan-challenges beyond surgery.

    PubMed

    Pillai, Vinod G; Chen, Chao-Long

    2016-04-01

    Taiwan has a high prevalence of hepatitis B and C viral infections, and consequently a high burden of chronic liver diseases. Liver transplantation (LT) began in Taiwan in 1984, and living donor liver transplantation (LDLT) in 1994. Education and collaboration between physicians on a national and international scale were important factors in the development of transplantation in East Asia. Technical innovations in donor hepatectomy, vascular and biliary reconstruction, and interventional radiology, perioperative management of transplant patients and development of associated specialties have enabled achievement of excellent results after both adult and pediatric LDLT. The establishment of rigorous protocols to withstand strict medico-legal scrutiny, combined with technical excellence has contributed to excellent surgical outcomes. The socioeconomic development of Taiwan and the first nationwide hepatitis B vaccination program in the world have also contributed to the decrease in disease burden and improvement of quality of healthcare. This article examines the factors enabling the development of LT in Taiwan, the innovations that have contributed to excellent outcomes, and indicates the future prospects of LDLT in Taiwan. PMID:27115009

  2. Living donor liver transplantation in Taiwan—challenges beyond surgery

    PubMed Central

    Pillai, Vinod G.

    2016-01-01

    Taiwan has a high prevalence of hepatitis B and C viral infections, and consequently a high burden of chronic liver diseases. Liver transplantation (LT) began in Taiwan in 1984, and living donor liver transplantation (LDLT) in 1994. Education and collaboration between physicians on a national and international scale were important factors in the development of transplantation in East Asia. Technical innovations in donor hepatectomy, vascular and biliary reconstruction, and interventional radiology, perioperative management of transplant patients and development of associated specialties have enabled achievement of excellent results after both adult and pediatric LDLT. The establishment of rigorous protocols to withstand strict medico-legal scrutiny, combined with technical excellence has contributed to excellent surgical outcomes. The socioeconomic development of Taiwan and the first nationwide hepatitis B vaccination program in the world have also contributed to the decrease in disease burden and improvement of quality of healthcare. This article examines the factors enabling the development of LT in Taiwan, the innovations that have contributed to excellent outcomes, and indicates the future prospects of LDLT in Taiwan. PMID:27115009

  3. Physical parameters affecting living cells in space.

    PubMed

    Langbein, D

    1986-01-01

    The question is posed: Why does a living cell react to the absence of gravity? What sensors may it have? Does it note pressure, sedimentation, convection, or other parameters? If somewhere in a liquid volume sodium ions are replaced by potassium ions, the density of the liquid changes locally: the heavier regions sink, the lighter regions rise. This may contribute to species transport, to the metabolism. Under microgravity this mechanism is strongly reduced. On the other hand, other reasons for convection like thermal and solutal interface convection are left. Do they affect species transport? Another important effect of gravity is the hydrostatic pressure. On the macroscopic side, the pressure between our head and feet changes by 0.35 atmospheres. On the microscopic level the hydrostatic pressure on the upper half of a cell membrane is lower than on the lower half. This, by affecting the ion transport through the membrane, may change the surrounding electric potential. It has been suggested to be one of the reasons for graviperception. Following the discussion of these and other effects possibly important in life sciences in space, an order of magnitude analysis of the residual accelerations tolerable during experiments in materials sciences is outlined. In the field of life sciences only rough estimates are available at present. PMID:11537842

  4. Kinase Activity Studied in Living Cells Using an Immunoassay

    ERIC Educational Resources Information Center

    Bavec, Aljos?a

    2014-01-01

    This laboratory exercise demonstrates the use of an immunoassay for studying kinase enzyme activity in living cells. The advantage over the classical method, in which students have to isolate the enzyme from cell material and measure its activity in vitro, is that enzyme activity is modulated and measured in living cells, providing a more…

  5. Delineating cooperative responses of processive motors in living cells

    PubMed Central

    Efremov, Artem K.; Radhakrishnan, Anand; Tsao, David S.; Bookwalter, Carol S.; Trybus, Kathleen M.; Diehl, Michael R.

    2014-01-01

    Characterizing the collective functions of cytoskeletal motors is critical to understanding mechanisms that regulate the internal organization of eukaryotic cells as well as the roles various transport defects play in human diseases. Though in vitro assays using synthetic motor complexes have generated important insights, dissecting collective motor functions within living cells still remains challenging. Here, we show that the protein heterodimerization switches FKBP-rapalog-FRB can be harnessed in engineered COS-7 cells to compare the collective responses of kinesin-1 and myosinVa motors to changes in motor number and cargo size. The dependence of cargo velocities, travel distances, and position noise on these parameters suggests that multiple myosinVa motors can cooperate more productively than collections of kinesins in COS-7 cells. In contrast to observations with kinesin-1 motors, the velocities and run lengths of peroxisomes driven by multiple myosinVa motors are found to increase with increasing motor density, but are relatively insensitive to the higher loads associated with transporting large peroxisomes in the viscoelastic environment of the COS-7 cell cytoplasm. Moreover, these distinctions appear to be derived from the different sensitivities of kinesin-1 and myosinVa velocities and detachment rates to forces at the single-motor level. The collective behaviors of certain processive motors, like myosinVa, may therefore be more readily tunable and have more substantial roles in intracellular transport regulatory mechanisms compared with those of other cytoskeletal motors. PMID:24402168

  6. Live Cell Optical Sensing for High Throughput Applications

    NASA Astrophysics Data System (ADS)

    Fang, Ye

    Live cell optical sensing employs label-free optical biosensors to non-invasively measure stimulus-induced dynamic mass redistribution (DMR) in live cells within the sensing volume of the biosensor. The resultant DMR signal is an integrated cellular response, and reflects cell signaling mediated through the cellular target(s) with which the stimulus intervenes. This article describes the uses of live cell optical sensing for probing cell biology and ligand pharmacology, with an emphasis of resonant waveguide grating biosensor cellular assays for high throughput applications.

  7. Summary: present and future challenges for stem cell research.

    PubMed

    Hogan, B L M

    2008-01-01

    Stem cell research is being driven forward at an intense pace by creative interactions among scientists working in different fields. These include developmental and reproductive biology, regeneration, genomics, live cell imaging, RNA biology, and cancer biology, to name a few. Numerous model systems and techniques are being exploited, and lab scientists are teaming up with bioengineers and clinicians. The ferment of ideas that makes the field so exciting was in full evidence throughout the Symposium. However, many challenges still need to be overcome to translate basic discoveries into therapeutic outcomes that will save lives and fulfill the promises that have been made. This chapter summarizes some of the highlights of the Symposium and indicates future directions that are being taken by leaders in the field. PMID:19329577

  8. Challenges in the detection of long lived particles: the Hidden Valley Scenario

    SciTech Connect

    Sidoti, Antonio

    2008-11-23

    Neutral particles with long decay paths and many particles in the final state represent, from an experimental point of view, a challenge both for the trigger and for the reconstruction capabilities of the ATLAS detector. The Hidden Valley scenario is an excellent framework to explore the challenges posed by long-lived neutral particles. In this paper we present strategies to select such events, in particular for the Higgs boson decays, with special attention to trigger level problems.

  9. Resisting and challenging stigma in Uganda: the role of support groups of people living with HIV

    PubMed Central

    Mburu, Gitau; Ram, Mala; Skovdal, Morten; Bitira, David; Hodgson, Ian; Mwai, Grace W; Stegling, Christine; Seeley, Janet

    2013-01-01

    Introduction Global scale up of antiretroviral therapy is changing the context of HIV-related stigma. However, stigma remains an ongoing concern in many countries. Groups of people living with HIV can contribute to the reduction of stigma. However, the pathways through which they do so are not well understood. Methods This paper utilizes data from a qualitative study exploring the impact of networked groups of people living with HIV in Jinja and Mbale districts of Uganda. Participants were people living with HIV (n=40), members of their households (n=10) and their health service providers (n=15). Data were collected via interviews and focus group discussions in 2010, and analyzed inductively to extract key themes related to the approaches and outcomes of the groups’ anti-stigma activities. Results Study participants reported that HIV stigma in their communities had declined as a result of the collective activities of groups of people living with HIV. However, they believed that stigma remained an ongoing challenge. Gender, family relationships, social and economic factors emerged as important drivers of stigma. Challenging stigma collectively transcended individual experiences and united people living with HIV in a process of social renegotiation to achieve change. Groups of people living with HIV provided peer support and improved the confidence of their members, which ultimately reduced self-stigma and improved their ability to deal with external stigma when it was encountered. Conclusions Antiretroviral therapy and group-based approaches in the delivery of HIV services are opening up new avenues for the collective participation of people living with HIV to challenge HIV stigma and act as agents of social change. Interventions for reducing HIV stigma should be expanded beyond those that aim to increase the resilience and coping mechanisms of individuals, to those that build the capacity of groups to collectively cope with and challenge HIV stigma. Such

  10. Fluorogenic Probes for Multicolor Imaging in Living Cells.

    PubMed

    Lukinavičius, Gražvydas; Reymond, Luc; Umezawa, Keitaro; Sallin, Olivier; D'Este, Elisa; Göttfert, Fabian; Ta, Haisen; Hell, Stefan W; Urano, Yasuteru; Johnsson, Kai

    2016-08-01

    Here we present a far-red, silicon-rhodamine-based fluorophore (SiR700) for live-cell multicolor imaging. SiR700 has excitation and emission maxima at 690 and 715 nm, respectively. SiR700-based probes for F-actin, microtubules, lysosomes, and SNAP-tag are fluorogenic, cell-permeable, and compatible with superresolution microscopy. In conjunction with probes based on the previously introduced carboxy-SiR650, SiR700-based probes permit multicolor live-cell superresolution microscopy in the far-red, thus significantly expanding our capacity for imaging living cells. PMID:27420907

  11. Cross-correlation analysis for live-cell image trajectory

    NASA Astrophysics Data System (ADS)

    Cheng, Chih-Ming; Chang, Yu-Fen; Wu, Chien-ming

    2013-08-01

    In cell motility, researchers are usually used fluorescence microscopy, confocal microscopy, or total internal reflection microscopy to track a fluorescent labeled particle and reveal the dynamic trajectory in living. Because all fluorescent dyes have cell toxicity, quantum dots and gold nanoparticles can influence the structures and physical properties of biomolecules which they have labeled, to develop another label-free image approach becomes an important issue. We present here a Fourier-based cross-correlation process to analyze images of adhering living cell, including cell motility and single vesicle trajectory. We treated adhering MG-63 cell with 66 nM Epidermal growth factor (EGF) and observed its dynamic effect on cell motility based on the velocity fields of consecutive cell images. We also used crosscorrelation to track single vesicles in living cells. We found that EGF could rapidly activate the motility of adhering MG- 63 cell, and the vesicle exhibits either directed or diffusive motion.

  12. Nanoscale Label-free Bioprobes to Detect Intracellular Proteins in Single Living Cells

    NASA Astrophysics Data System (ADS)

    Hong, Wooyoung; Liang, Feng; Schaak, Diane; Loncar, Marko; Quan, Qimin

    2014-08-01

    Fluorescent labeling techniques have been widely used in live cell studies; however, the labeling processes can be laborious and challenging for use in non-transfectable cells, and labels can interfere with protein functions. While label-free biosensors have been realized by nanofabrication, a method to track intracellular protein dynamics in real-time, in situ and in living cells has not been found. Here we present the first demonstration of label-free detection of intracellular p53 protein dynamics through a nanoscale surface plasmon-polariton fiber-tip-probe (FTP).

  13. Direct Force Measurements of Receptor-Ligand Interactions on Living Cells

    NASA Astrophysics Data System (ADS)

    Eibl, Robert H.

    The characterization of cell adhesion between two living cells at the level of single receptor-ligand bonds is an experimental challenge. This chapter describes how the extremely sensitive method of atomic force microscopy (AFM) based force spectroscopy can be applied to living cells in order to probe for cell-to-cell or cell-to-substrate interactions mediated by single pairs of adhesion receptors. In addition, it is outlined how single-molecule AFM force spectroscopy can be used to detect physiologic changes of an adhesion receptor in a living cell. This force spectroscopy allows us to detect in living cells rapidly changing, chemokine SDF-1 triggered activation states of single VLA-4 receptors. This recently developed AFM application will allow for the detailed investigation of the integrin-chemokine crosstalk of integrin activation mechanisms and on how other adhesion receptors are modulated in health and disease. As adhesion molecules, living cells and even bacteria can be studied by single-molecule AFM force spectroscopy, this method is set to become a powerful tool that can not only be used in biophysics, but in cell biology as well as in immunology and cancer research.

  14. Challenges Experienced in Teaching Daily Living Skills to Learners with Mental Retardation

    ERIC Educational Resources Information Center

    Ruteere, Rosallin Kananu; Mutia, Jacob Mpekethu; Mwoma, Teresa; Runo, Mary

    2015-01-01

    The aim of this study was to establish the challenges encountered when teaching daily living skills (DLS) to learners with mental retardation (MR). The study used purposive sampling to select the sub-county, special units, learners and teachers. The target population in this study was eighty four respondents. The sample for the study was the same…

  15. Strategies for Living with the Challenges of HIV and Antiretroviral Use in Zambia

    ERIC Educational Resources Information Center

    Jones, Deborah; Zulu, Isaac; Mumbi, Miriam; Chitalu, Ndashi; Vamos, Szonja; Gomez, Jacqueline; Weiss, Stephen M.

    2009-01-01

    This study sought to identify strategies for living with the challenges of HIV and antiretroviral (ARV) use among new medication users in urban Zambia. Participants (n = 160) were recruited from urban Lusaka, Zambia. Qualitative Data was drawn from monthly ARV treatment education intervention groups addressing HIV and antiretroviral use. Themes…

  16. Builders Challenge High Performance Builder Spotlight - Masco Environments for Living, Las Vegas, Nevada

    SciTech Connect

    2009-01-01

    Building America Builders Challenge fact sheet on Masco’s Environments for Living Certified Green demo home at the 2009 International Builders Show in Las Vegas. The home has a Home Energy Rating System (HERS) index score of 44, a right-sized air conditi

  17. The forgotten ones: challenges and needs of children living with disabling parental chronic pain.

    PubMed

    Umberger, Wendy A; Risko, Judy; Covington, Edward

    2015-01-01

    A qualitative study explored the challenges and needs of children living with parental chronic pain. Young adult children (n=30) of parents with chronic pain were interviewed. Parents (n=20) with chronic pain participated in four focus groups. Content analysis yielded five categories of child challenges: (a) understanding the big picture; (b) enduring hardships; (c) grieving losses; (d) communicating with parent, and; (e) isolating self from peers. Three categories of child needs emerged: (a) knowledge; (b) skills, and; (c) supervised interaction. Understanding these challenges and needs is a vital step in the process of developing evidence-based interventions for this at-risk group. PMID:25557986

  18. Targeting endogenous nuclear antigens by electrotransfer of monoclonal antibodies in living cells

    PubMed Central

    Freund, Guillaume; Sibler, Annie-Paule; Desplancq, Dominique; Oulad-Abdelghani, Mustapha; Vigneron, Marc; Gannon, Julian; Van Regenmortel, Marc H.; Weiss, Etienne

    2013-01-01

    Antibodies are valuable tools for functional studies in vitro, but their use in living cells remains challenging because they do not naturally cross the cell membrane. Here, we present a simple and highly efficient method for the intracytoplasmic delivery of any antibody into cultured cells. By following the fate of monoclonal antibodies that bind to nuclear antigens, it was possible to image endogenous targets and to show that inhibitory antibodies are able to induce cell growth suppression or cell death. Our electrotransfer system allowed the cancer cells we studied to be transduced without loss of viability and may have applications for a variety of intracellular immuno-interventions. PMID:23765067

  19. Early Potent Protection against Heterologous SIVsmE660 Challenge Following Live Attenuated SIV Vaccination in Mauritian Cynomolgus Macaques

    PubMed Central

    Berry, Neil; Ham, Claire; Mee, Edward T.; Rose, Nicola J.; Mattiuzzo, Giada; Jenkins, Adrian; Page, Mark; Elsley, William; Robinson, Mark; Smith, Deborah; Ferguson, Deborah; Towers, Greg; Almond, Neil; Stebbings, Richard

    2011-01-01

    Background Live attenuated simian immunodeficiency virus (SIV) vaccines represent the most effective means of vaccinating macaques against pathogenic SIV challenge. However, thus far, protection has been demonstrated to be more effective against homologous than heterologous strains. Immune correlates of vaccine-induced protection have also been difficult to identify, particularly those measurable in the peripheral circulation. Methodology/Principal Findings Here we describe potent protection in 6 out of 8 Mauritian-derived cynomolgus macaques (MCM) against heterologous virus challenge with the pathogenic, uncloned SIVsmE660 viral stock following vaccination with live attenuated SIVmac251/C8. MCM provided a characterised host genetic background with limited Major Histocompatibility Complex (MHC) and TRIM5α allelic diversity. Early protection, observed as soon as 3 weeks post-vaccination, was comparable to that of 20 weeks vaccination. Recrudescence of vaccine virus was most pronounced in breakthrough cases where simultaneous identification of vaccine and challenge viruses by virus-specific PCR was indicative of active co-infection. Persistence of the vaccine virus in a range of lymphoid tissues was typified by a consistent level of SIV RNA positive cells in protected vaccinates. However, no association between MHC class I /II haplotype or TRIM5α polymorphism and study outcome was identified. Conclusion/Significance This SIV vaccine study, conducted in MHC-characterised MCM, demonstrated potent protection against the pathogenic, heterologous SIVsmE660 challenge stock after only 3 weeks vaccination. This level of protection against this viral stock by intravenous challenge has not been hitherto observed. The mechanism(s) of protection by vaccination with live attenuated SIV must account for the heterologous and early protection data described in this study, including those which relate to the innate immune system. PMID:21853072

  20. Cell nucleus targeting for living cell extraction of nucleic acid associated proteins with intracellular nanoprobes of magnetic carbon nanotubes.

    PubMed

    Zhang, Yi; Hu, Zhengyan; Qin, Hongqiang; Liu, Fangjie; Cheng, Kai; Wu, Ren'an; Zou, Hanfa

    2013-08-01

    Since nanoparticles could be ingested by cells naturally and target at a specific cellular location as designed, the extraction of intracellular proteins from living cells for large-scale analysis by nanoprobes seems to be ideally possible. Nucleic acid associated proteins (NAaP) take the crucial position during biological processes in maintaining and regulating gene structure and gene related behaviors, yet there are still challenges during the global investigation of intracellular NAaP, especially from living cells. In this work, a strategy to extract intracellular proteins from living cells with the magnetic carbon nanotube (oMWCNT@Fe3O4) as an intracellular probe is developed, to achieve the high throughput analysis of NAaP from living human hepatoma BEL-7402 cells with a mass spectrometry-based proteomic approach. Due to the specific intracellular localization of the magnetic carbon nanotubes around nuclei and its strong interaction with nucleic acids, the highly efficient extraction was realized for cellular NAaP from living cells, with the capability of identifying 2383 intracellular NAaP from only ca. 10,000 living cells. This method exhibited potential applications in dynamic and in situ analysis of intracellular proteins. PMID:23815738

  1. Visualization of RNA-Quadruplexes in Live Cells.

    PubMed

    Laguerre, Aurélien; Hukezalie, Kyle; Winckler, Pascale; Katranji, Fares; Chanteloup, Gaëtan; Pirrotta, Marc; Perrier-Cornet, Jean-Marie; Wong, Judy M Y; Monchaud, David

    2015-07-01

    Visualization of DNA and RNA quadruplex formation in human cells was demonstrated recently with different quadruplex-specific antibodies. Despite the significant interest in these immunodetection approaches, dynamic detection of quadruplex in live cells remains elusive. Here, we report on NaphthoTASQ (N-TASQ), a next-generation quadruplex ligand that acts as a multiphoton turn-on fluorescent probe. Single-step incubation of human and mouse cells with N-TASQ enables the direct detection of RNA-quadruplexes in untreated cells (no fixation, permeabilization or mounting steps), thus offering a unique, unbiased visualization of quadruplexes in live cells. PMID:26056849

  2. Chemically tunable mucin chimeras assembled on living cells

    PubMed Central

    Kramer, Jessica R.; Onoa, Bibiana; Bustamante, Carlos; Bertozzi, Carolyn R.

    2015-01-01

    Mucins are a family of secreted and transmembrane glycoproteins characterized by a massive domain of dense O-glycosylation on serine and threonine residues. Mucins are intimately involved in immunity and cancer, yet elucidation of the biological roles of their glycodomains has been complicated by their massive size, domain polymorphisms, and variable glycosylation patterns. Here we developed a synthetic route to a library of compositionally defined, high-molecular weight, dual end-functionalized mucin glycodomain constructs via N-carboxyanhydride polymerization. These glycopolypeptides are the first synthetic analogs to our knowledge to feature the native α-GalNAc linkage to serine with molecular weights similar to native mucins, solving a nearly 50-year synthetic challenge. Physical characterization of the mimics revealed insights into the structure and properties of mucins. The synthetic glycodomains were end-functionalized with an optical probe and a tetrazine moiety, which allowed site-specific bioorthogonal conjugation to an engineered membrane protein on live mammalian cells. This strategy in protein engineering will open avenues to explore the biological roles of cell surface mucins. PMID:26420872

  3. Anti-Fading Media for Live Cell GFP Imaging

    PubMed Central

    Bogdanov, Alexey M.; Kudryavtseva, Elena I.; Lukyanov, Konstantin A.

    2012-01-01

    Photostability is one of the most important characteristic of a dye for fluorescence microscopy. Recently we demonstrated that vitamins present in imaging media dramatically accelerate photobleaching of Enhanced Green Fluorescent Protein (EGFP) and many other green fluorescent and photoactivatable proteins. Here we tested all vitamins of commonly used media (such as Dulbecco's Modified Eagle Medium, DMEM) one-by-one and found that only two vitamins, riboflavin and pyridoxal, decrease photostability of EGFP. Thus, DMEM without riboflavin and pyridoxal can be used as an imaging medium, which ensures high photostability of GFPs at the expense of minimal biochemical disturbance. Then, we tested some antioxidants and found that a plant flavonoid rutin greatly enhances photostability of EGFP during live cell microscopy. In complete DMEM, rutin increased EGFP photostability up to the level of vitamin-depleted DMEM. Moreover, being added to vitamin-depleted DMEM, rutin was able to further suppress EGFP photobleaching. Potentially, new medium formulations can be widely used for fluorescence microscopy of GFP-expressing cells and model multicellular organisms in a variety of imaging applications, where photostability represents a challenge. PMID:23285248

  4. Living kidney donor assessment: challenges, uncertainties and controversies among transplant nephrologists and surgeons.

    PubMed

    Tong, A; Chapman, J R; Wong, G; Craig, J C

    2013-11-01

    The assessment of living kidney donors presents unique ethical challenges and complex psychosocial implications. This study aimed to ascertain the perspectives of transplant nephrologists and surgeons on living kidney donor assessment. Semi-structured, face-to-face interviews were conducted with 110 transplant nephrologists and surgeons from 43 transplant units in 12 countries from Europe, Australasia and North America. The challenge of defining acceptable risk to the donor was central to five themes identified: burden of responsibility (personal accountability, policing morality, democratic decision making, meeting legal obligations, optimizing outcomes and innovation, relinquished control); medical protectiveness (prognostic uncertainty, skepticism of donor risk perception, avoidance of undue coercion, concerns for dubious motivations and coercion, safeguard donor well-being, ethical information disclosure); respecting donor autonomy (facilitate informed-decision making, concede to donor risk acceptance, benefit of the doubt, donor mandate to maintain health, acceptable altruism); driving ideologies (preserving equity, championing living donation, cognizance of anti-paternalism) and contextual pressures (evolving donor demographic, resource limitations). Living kidney donor assessment involves complex interactions between safeguarding the donors' welfare and respecting their autonomy. In our opinion, authoritative and well-described transplant unit, hospital and public policy positions that make explicit the considerations that are often implicit may reduce the uncertainty within which living donors are assessed today. PMID:24020905

  5. Live single-cell laser tag

    PubMed Central

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

    2016-01-01

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

  6. Live single-cell laser tag.

    PubMed

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

    2016-01-01

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

  7. Ion-Selective Detection with Glass Nanopipette for Living Cells

    NASA Astrophysics Data System (ADS)

    Takami, T.; Son, J. W.; Kang, E. J.; Deng, X. L.; Kawai, T.; Lee, S.-W.; Park, B. H.

    2013-05-01

    We developed a method to probe local ion concentration with glass nanopipette in which poly(vinyl chloride) membrane containing ionophore for separate ion detection is prepared. Here we demonstrate how ion-selective detections are available for living cells such as HeLa cell, rat vascular myocyte, and neuron cell.

  8. Live cell imaging of the cytoskeleton and cell wall enzymes in plant cells.

    PubMed

    Sampathkumar, Arun; Wightman, Raymond

    2015-01-01

    The use of live imaging techniques to visualize the dynamic changes and interactions within plant cells has given us detailed information on the function and organization of the cytoskeleton and cell wall associated proteins. This information has grown with the constant improvement in imaging hardware and molecular tools. In this chapter, we describe the procedure for the preparation and live visualization of fluorescent protein fusions associated with the cytoskeleton and the cell wall in Arabidopsis. PMID:25408450

  9. Micropatterning tractional forces in living cells

    NASA Technical Reports Server (NTRS)

    Wang, Ning; Ostuni, Emanuele; Whitesides, George M.; Ingber, Donald E.

    2002-01-01

    Here we describe a method for quantifying traction in cells that are physically constrained within micron-sized adhesive islands of defined shape and size on the surface of flexible polyacrylamide gels that contain fluorescent microbeads (0.2-microm diameter). Smooth muscle cells were plated onto square (50 x 50 microm) or circular (25- or 50-microm diameter) adhesive islands that were created on the surface of the gels by applying a collagen coating through microengineered holes in an elastomeric membrane that was later removed. Adherent cells spread to take on the size and shape of the islands and cell tractions were quantitated by mapping displacement fields of the fluorescent microbeads within the gel. Cells on round islands did not exhibit any preferential direction of force application, but they exerted their strongest traction at sites where they formed protrusions. When cells were confined to squares, traction was highest in the corners both in the absence and presence of the contractile agonist, histamine, and cell protrusions were also observed in these regions. Quantitation of the mean traction exerted by cells cultured on the different islands revealed that cell tension increased as cell spreading was promoted. These results provide a mechanical basis for past studies that demonstrated a similar correlation between spreading and growth within various anchorage-dependent cells. This new approach for analyzing the spatial distribution of mechanical forces beneath individual cells that are experimentally constrained to defined sizes and shapes may provide additional insight into the biophysical basis of cell regulation. Copyright 2002 Wiley-Liss, Inc.

  10. African American kidney transplant patients’ perspectives on challenges in the living donation process

    PubMed Central

    Sieverdes, John C.; Nemeth, Lynne S.; Magwood, Gayenell S.; Baliga, Prabhakar K.; Chavin, Kenneth D.; Ruggiero, Ken J.; Treiber, Frank A.

    2015-01-01

    Context The increasing shortage of deceased donor kidneys suitable for African Americans highlights the critical need to increase living donations among African Americans. Little research has addressed African American transplant recipients’ perspectives on challenges and barriers related to the living donation process. Objective To understand the perspectives of African American recipients of deceased and living donor kidney transplants on challenges, barriers, and educational needs related to pursuing such transplants. Participants and Design A mixed-method design involved 27 African American kidney recipients (13 male) in 4 focus groups (2 per recipient type: 16 African American deceased donor and 11 living donor recipients) and questionnaires. Focus group transcripts were evaluated with NVivo 10.0 (QSR, International) by using inductive and deductive qualitative methods along with crystallization to develop themes of underlying barriers to the living donor kidney transplant process and were compared with the questionnaires. Results Four main themes were identified from groups: concerns, knowledge and learning, expectations of support, and communication. Many concerns for the donor were identified (eg, process too difficult, financial burden, effect on relationships). A general lack of knowledge about the donor process and lack of behavioral skills on how to approach others was noted. The latter was especially evident among deceased donor recipients. Findings from the questionnaires on myths and perceptions supported the lack of knowledge in a variety of domains, including donors’ surgical outcomes risks, costs of surgery, and impact on future health. Participants thought that an educational program led by an African American recipient of a living donor kidney transplant, including practice in approaching others, would increase the likelihood of transplant-eligible patients pursuing living donor kidney transplant. PMID:26107278

  11. Nanosurgery in live cells using ultrashort laser pulses

    NASA Astrophysics Data System (ADS)

    Heisterkamp, Alexander; Maxwell, Iva Z.; Kumar, Sanjay; Underwood, J. M.; Nickerson, J. A.; Ingber, Donald E.; Mazur, Eric

    2005-04-01

    We selectively disrupted the cytoskeletal network of fixed and live bovine capillary endothelial cell using ultrashort laser pulses. We image the microtubules in the cytoskeleton of the cultured cells using green fluorescent protein. The cells are placed on a custom-built inverted fluorescence microscope setup, using a 1.4 NA oil-immersion objective to both image the cell and focus the laser radiation into the cell samples. The laser delivers 100-fs laser pulses centered at 800 nm at a repetition rate of 1 kHz; the typical energy delivered at the sample is 1-5nJ. The fluorescent image of the cell is captured with a CCD-camera at one frame per second. To determine the spatial discrimination of the laser cutting we ablated microtubules and actin fibers in fixed cells. At pulse energies below 2 nJ we obtain an ablation size of 200 nm. This low pulse energy and high spatial discrimination enable the application of this technique to live cells. We severed a single microtubule inside the live cells without affecting the cell's viability. The targeted microtubule snaps and depolymerizes after the cutting. This nanosurgery technique will further the understanding and modeling of stress and compression in the cytoskeletal network of live cells.

  12. Therapeutic challenges in renal cell carcinoma

    PubMed Central

    Penticuff, Justin C; Kyprianou, Natasha

    2015-01-01

    Renal cell carcinoma (RCC) is a malignancy that in advanced disease, is highly resistant to systemic therapies. Elucidation of the angiogenesis pathways and their intrinsic signaling interactions with the genetic and metabolic disturbances within renal cell carcinoma variants has ushered in the era of “targeted therapies”. Advanced surgical interventions and novel drugs targeting VEGF and mTOR, have improved patient survival and prolonged clinically stable-disease states. This review discusses the current understanding of diagnostic challenges and the mechanism-based clinical evidence on therapeutic management of advanced RCC. PMID:26309897

  13. Correlates of Immunity to Influenza as Determined by Challenge of Children with Live, Attenuated Influenza Vaccine.

    PubMed

    Wright, Peter F; Hoen, Anne G; Ilyushina, Natalia A; Brown, Eric P; Ackerman, Margaret E; Wieland-Alter, Wendy; Connor, Ruth I; Jegaskanda, Sinthujan; Rosenberg-Hasson, Yael; Haynes, Brenda C; Luke, Catherine J; Subbarao, Kanta; Treanor, John J

    2016-04-01

    Background.  The efficacy of live, attenuated live attenuated influenza vaccine(LAIV) and inactivated influenza vaccine(IIV) is poorly explained by either single or composite immune responses to vaccination. Protective biomarkers were therefore studied in response to LAIV or IIV followed by LAIV challenge in children. Methods.  Serum and mucosal responses to LAIV or IIV were analyzed using immunologic assays to assess both quantitative and functional responses. Cytokines and chemokines were measured in nasal washes collected before vaccination, on days 2, 4, and 7 after initial LAIV, and again after LAIV challenge using a 63-multiplex Luminex panel. Results.  Patterns of immunity induced by LAIV and IIV were significantly different. Serum responses induced by IIV, including hemagglutination inhibition, did not correlate with detection or quantitation of LAIV on subsequent challenge. Modalities that induced sterilizing immunity seen after LAIV challenge could not be defined by any measurements of mucosal or serum antibodies induced by the initial LAIV immunization. No single cytokine or chemokine was predictive of protection. Conclusions.  The mechanism of protective immunity observed after LAIV could not be defined, and traditional measurements of immunity to IIV did not correlate with protection against an LAIV challenge. PMID:27419180

  14. Correlates of Immunity to Influenza as Determined by Challenge of Children with Live, Attenuated Influenza Vaccine

    PubMed Central

    Wright, Peter F.; Hoen, Anne G.; Ilyushina, Natalia A.; Brown, Eric P.; Ackerman, Margaret E.; Wieland-Alter, Wendy; Connor, Ruth I.; Jegaskanda, Sinthujan; Rosenberg-Hasson, Yael; Haynes, Brenda C.; Luke, Catherine J.; Subbarao, Kanta; Treanor, John J.

    2016-01-01

    Background. The efficacy of live, attenuated live attenuated influenza vaccine(LAIV) and inactivated influenza vaccine(IIV) is poorly explained by either single or composite immune responses to vaccination. Protective biomarkers were therefore studied in response to LAIV or IIV followed by LAIV challenge in children. Methods. Serum and mucosal responses to LAIV or IIV were analyzed using immunologic assays to assess both quantitative and functional responses. Cytokines and chemokines were measured in nasal washes collected before vaccination, on days 2, 4, and 7 after initial LAIV, and again after LAIV challenge using a 63-multiplex Luminex panel. Results. Patterns of immunity induced by LAIV and IIV were significantly different. Serum responses induced by IIV, including hemagglutination inhibition, did not correlate with detection or quantitation of LAIV on subsequent challenge. Modalities that induced sterilizing immunity seen after LAIV challenge could not be defined by any measurements of mucosal or serum antibodies induced by the initial LAIV immunization. No single cytokine or chemokine was predictive of protection. Conclusions. The mechanism of protective immunity observed after LAIV could not be defined, and traditional measurements of immunity to IIV did not correlate with protection against an LAIV challenge. PMID:27419180

  15. Living Composites of Electrospun Yeast Cells for Bioremediation and Ethanol Production.

    PubMed

    Letnik, Ilya; Avrahami, Ron; Rokem, J Stefan; Greiner, Andreas; Zussman, Eyal; Greenblatt, Charles

    2015-10-12

    The preparation of composites of living functional cells and polymers is a major challenge. We have fabricated such "living composites" by preparation of polymeric microtubes that entrap yeast cells. Our approach was the process of coaxial electrospinning in which a core containing the yeast was "spun" within a shell of nonbiodegradable polymer. We utilized the yeast Candida tropicalis, which was isolated from olive water waste. It is particularly useful since it degrades phenol and other natural polyphenols, and it is capable of accumulating ethanol. The electrospun yeast cells showed significant activity of bioremediation of phenol and produced ethanol, and, in addition, the metabolic processes remained active for a prolonged period. Comparison of electrospun cells to planktonic cells showed decreased cell activity; however, the olive water waste after treatment by the yeast was no longer toxic for Escherichia coli, suggesting that detoxification and prolonged viability and activity may outweigh the reduction of efficiency. PMID:26351729

  16. Introduction of genes into living cells

    NASA Astrophysics Data System (ADS)

    Nishimura, E.; Nagai, A.; Tomimasu, T.; Kina, T.; Fujimoto, S.; Katsura, Y.

    1998-02-01

    One of our main subjects is an application of the FEL to gene therapy of genetic diseases, immunodeficiency syndromes and cancer. In this study, using the FEL, we tried to establish a model system for introducing genes into the stem cells from which all blood cells are derived. Our aim is to specifically mark the stem cells with monoclonal antibodies which are conjugated with efficient FEL absorbers. Cells are then irradiated with FEL at a wavelength corresponding to the absorption energy of the absorber. We speculate that the gap formation of cell membrane will occur, caused by the thermal shock due to the absorption of the FEL energy. As an animal model for gene therapy, we tried to transfer the RAG-2 genes into hematopoietic stem cells from RAG-2 deficient mice, which have severe immunodeficiency because of the lack of RAG-2 gene required for lymphocyte development. As the results by this construct, the infant lymphocytes (T and B cells) could be observed in the thymus of the RAG-2 deficient mice 2 weeks post-operative.

  17. Biocomputers: from test tubes to live cells.

    PubMed

    Benenson, Yaakov

    2009-07-01

    Biocomputers are man-made biological networks whose goal is to probe and control biological hosts--cells and organisms--in which they operate. Their key design features, informed by computer science and engineering, are programmability, modularity and versatility. While still a work in progress, biocomputers will eventually enable disease diagnosis and treatment with single-cell precision, lead to "designer" cell functions for biotechnology, and bring about a new generation of biological measurement tools. This review describes the intellectual foundation of the "biocomputer" concept as well as surveys the state of the art in the field. PMID:19562106

  18. High efficiency labeling of glycoproteins on living cells

    PubMed Central

    Zeng, Ying; Ramya, T. N. C.; Dirksen, Anouk; Dawson, Philip E.; Paulson, James C.

    2010-01-01

    We describe a simple method for efficiently labeling cell surface glycans on virtually any living animal cell. The method employs mild Periodate oxidation to generate an aldehyde on sialic acids, followed by Aniline-catalyzed oxime Ligation with a suitable tag (PAL). Aniline catalysis dramatically accelerates oxime ligation, allowing use of low concentrations of aminooxy-biotin at neutral pH to label the majority of cell surface glycoproteins while maintaining high cell viability. PMID:19234450

  19. Isolation of Live Premature Senescent Cells Using FUCCI Technology.

    PubMed

    Wang, Danli; Lu, Ping; Liu, Yang; Chen, Li; Zhang, Rui; Sui, Weihao; Dumitru, Alexandru George; Chen, Xiaowen; Wen, Feiqiu; Ouyang, Hong-Wei; Ji, Junfeng

    2016-01-01

    Cellular senescence plays an important role in diverse biological processes such as tumorigenesis and organismal aging. However, lack of methods to specifically identify and isolate live senescent cells hampers the precise understanding of the molecular mechanisms regulating cellular senescence. Here, we report that utilization of fluorescent ubiquitination-based cell cycle indicator (FUCCI) technology allows isolation of live premature senescent cells induced by doxorubicin treatment. Exposure of human foreskin fibroblasts (HFFs) to a low dose of doxorubicin led to cellular senescent phenotypes including formation of γ-H2AX and 53BP1 foci indicative of DNA damage, decreased cell proliferation and increased senescence-associated β-galactosidase (SA-β-gal) activity. Importantly, doxorubicin-induced senescent cells were arrested at S/G2/M phases of cell cycle which can be reported by a construct encoding a fragment of hGeminin fused with monomeric Azami-Green (mAG-hGeminin). Flow cytometric sorting of GFP(+) cells from doxorubicin-treated HFFs carrying mAG-hGeminin reporter enabled isolation and enrichment of live senescent cells in the culture. Our study develops a novel method to identify and isolate live premature senescent cells, thereby providing a new tool to study cellular senescence. PMID:27503759

  20. Isolation of Live Premature Senescent Cells Using FUCCI Technology

    PubMed Central

    Wang, Danli; Lu, Ping; Liu, Yang; Chen, Li; Zhang, Rui; Sui, Weihao; Dumitru, Alexandru George; Chen, Xiaowen; Wen, Feiqiu; Ouyang, Hong-Wei; Ji, Junfeng

    2016-01-01

    Cellular senescence plays an important role in diverse biological processes such as tumorigenesis and organismal aging. However, lack of methods to specifically identify and isolate live senescent cells hampers the precise understanding of the molecular mechanisms regulating cellular senescence. Here, we report that utilization of fluorescent ubiquitination-based cell cycle indicator (FUCCI) technology allows isolation of live premature senescent cells induced by doxorubicin treatment. Exposure of human foreskin fibroblasts (HFFs) to a low dose of doxorubicin led to cellular senescent phenotypes including formation of γ-H2AX and 53BP1 foci indicative of DNA damage, decreased cell proliferation and increased senescence-associated β-galactosidase (SA-β-gal) activity. Importantly, doxorubicin-induced senescent cells were arrested at S/G2/M phases of cell cycle which can be reported by a construct encoding a fragment of hGeminin fused with monomeric Azami-Green (mAG-hGeminin). Flow cytometric sorting of GFP+ cells from doxorubicin-treated HFFs carrying mAG-hGeminin reporter enabled isolation and enrichment of live senescent cells in the culture. Our study develops a novel method to identify and isolate live premature senescent cells, thereby providing a new tool to study cellular senescence. PMID:27503759

  1. Single-molecule fluorescence imaging to quantify membrane protein dynamics and oligomerization in living plant cells.

    PubMed

    Wang, Xiaohua; Li, Xiaojuan; Deng, Xin; Luu, Doan-Trung; Maurel, Christophe; Lin, Jinxing

    2015-12-01

    Measuring the mobility and interactions of proteins is key to understanding cellular signaling mechanisms; however, quantitative analysis of protein dynamics in living plant cells remains a major challenge. Here we describe an automated, single-molecule protocol based on total internal reflection fluorescence microscopy (TIRFM) imaging that allows protein tracking and subunit counting in living plant cells. This protocol uses TIRFM to image transgenic plant tissues expressing fluorescently tagged proteins that are localized to the plasma membrane. Next, a tracking algorithm quantifies dynamic changes in fluorescent protein motion types, temporary particle displacement and protein photobleaching steps. This protocol allows researchers to study the kinetic characteristics of heterogeneously distributed proteins. The approach has potential applications for studies of protein dynamics and subunit stoichiometry for a wide variety of plasma membrane and intracellular proteins in living plant cells and other biological specimens visualized by TIRFM or other fluorescence imaging techniques. The whole protocol can be completed in 5-6 h. PMID:26584445

  2. A microfluidic live cell assay to study anthrax toxin induced cell lethality assisted by conditioned medium

    PubMed Central

    Shen, Jie; Cai, Changzu; Yu, Zhilong; Pang, Yuhong; Zhou, Ying; Qian, Lili; Wei, Wensheng; Huang, Yanyi

    2015-01-01

    It is technically challenging to investigate the function of secreted protein in real time by supply of conditioned medium that contains secreted protein of interest. The internalization of anthrax toxin is facilitated by a secreted protein Dickkopf-1 (DKK1) and its receptor, and eventually leads to cell lethality. To monitor the dynamic interplay between these components in live cells, we use an integrated microfluidic device to perform the cell viability assays with real-time controlled culture microenvironment in parallel. Conditioned medium, which contains the secreted proteins from specific cell lines, can be continuously pumped towards the cells that exposed to toxin. The exogenous DKK1 secreted from distant cells is able to rescue the sensitivity to toxin for those DKK1-knocked-down cells. This high-throughput assay allows us to precisely quantify the dynamic interaction between key components that cause cell death, and provide independent evidence of the function of DKK1 in the complex process of anthrax toxin internalization. PMID:25731605

  3. Nanometre-scale thermometry in a living cell.

    PubMed

    Kucsko, G; Maurer, P C; Yao, N Y; Kubo, M; Noh, H J; Lo, P K; Park, H; Lukin, M D

    2013-08-01

    Sensitive probing of temperature variations on nanometre scales is an outstanding challenge in many areas of modern science and technology. In particular, a thermometer capable of subdegree temperature resolution over a large range of temperatures as well as integration within a living system could provide a powerful new tool in many areas of biological, physical and chemical research. Possibilities range from the temperature-induced control of gene expression and tumour metabolism to the cell-selective treatment of disease and the study of heat dissipation in integrated circuits. By combining local light-induced heat sources with sensitive nanoscale thermometry, it may also be possible to engineer biological processes at the subcellular level. Here we demonstrate a new approach to nanoscale thermometry that uses coherent manipulation of the electronic spin associated with nitrogen-vacancy colour centres in diamond. Our technique makes it possible to detect temperature variations as small as 1.8 mK (a sensitivity of 9 mK Hz(-1/2)) in an ultrapure bulk diamond sample. Using nitrogen-vacancy centres in diamond nanocrystals (nanodiamonds), we directly measure the local thermal environment on length scales as short as 200 nanometres. Finally, by introducing both nanodiamonds and gold nanoparticles into a single human embryonic fibroblast, we demonstrate temperature-gradient control and mapping at the subcellular level, enabling unique potential applications in life sciences. PMID:23903748

  4. Silicon chips detect intracellular pressure changes in living cells

    NASA Astrophysics Data System (ADS)

    Gómez-Martínez, Rodrigo; Hernández-Pinto, Alberto M.; Duch, Marta; Vázquez, Patricia; Zinoviev, Kirill; de La Rosa, Enrique J.; Esteve, Jaume; Suárez, Teresa; Plaza, José A.

    2013-07-01

    The ability to measure pressure changes inside different components of a living cell is important, because it offers an alternative way to study fundamental processes that involve cell deformation. Most current techniques such as pipette aspiration, optical interferometry or external pressure probes use either indirect measurement methods or approaches that can damage the cell membrane. Here we show that a silicon chip small enough to be internalized into a living cell can be used to detect pressure changes inside the cell. The chip, which consists of two membranes separated by a vacuum gap to form a Fabry-Pérot resonator, detects pressure changes that can be quantified from the intensity of the reflected light. Using this chip, we show that extracellular hydrostatic pressure is transmitted into HeLa cells and that these cells can endure hypo-osmotic stress without significantly increasing their intracellular hydrostatic pressure.

  5. Brownian Motion and the Temperament of Living Cells

    NASA Astrophysics Data System (ADS)

    Tsekov, Roumen; Lensen, Marga C.

    2013-07-01

    The migration of living cells usually obeys the laws of Brownian motion. While the latter is due to the thermal motion of the surrounding matter, the locomotion of cells is generally associated with their vitality. We study what drives cell migration and how to model memory effects in the Brownian motion of cells. The concept of temperament is introduced as an effective biophysical parameter driving the motion of living biological entities in analogy with the physical parameter of temperature, which dictates the movement of lifeless physical objects. The locomemory of cells is also studied via the generalized Langevin equation. We explore the possibility of describing cell locomemory via the Brownian self-similarity concept. An heuristic expression for the diffusion coefficient of cells on structured surfaces is derived.

  6. Living on a flammable planet: interdisciplinary, cross-scalar and varied cultural lessons, prospects and challenges.

    PubMed

    Roos, Christopher I; Scott, Andrew C; Belcher, Claire M; Chaloner, William G; Aylen, Jonathan; Bird, Rebecca Bliege; Coughlan, Michael R; Johnson, Bart R; Johnston, Fay H; McMorrow, Julia; Steelman, Toddi

    2016-06-01

    Living with fire is a challenge for human communities because they are influenced by socio-economic, political, ecological and climatic processes at various spatial and temporal scales. Over the course of 2 days, the authors discussed how communities could live with fire challenges at local, national and transnational scales. Exploiting our diverse, international and interdisciplinary expertise, we outline generalizable properties of fire-adaptive communities in varied settings where cultural knowledge of fire is rich and diverse. At the national scale, we discussed policy and management challenges for countries that have diminishing fire knowledge, but for whom global climate change will bring new fire problems. Finally, we assessed major fire challenges that transcend national political boundaries, including the health burden of smoke plumes and the climate consequences of wildfires. It is clear that to best address the broad range of fire problems, a holistic wildfire scholarship must develop common agreement in working terms and build across disciplines. We must also communicate our understanding of fire and its importance to the media, politicians and the general public.This article is part of the themed issue 'The interaction of fire and mankind'. PMID:27216517

  7. Live Cell Imaging of a Fluorescent Gentamicin Conjugate

    PubMed Central

    Escobedo, Jorge O.; Chu, Yu-Hsuan; Wang, Qi; Steyger, Peter S.; Strongin, Robert M.

    2012-01-01

    Understanding cellular mechanisms of ototoxic and nephrotoxic drug uptake, intracellular distribution, and molecular trafficking across cellular barrier systems aids the study of potential uptake blockers that preserve sensory and renal function during critical life-saving therapy. Herein we report the design, synthesis characterization and evaluation of a fluorescent conjugate of the aminoglycoside antibiotic gentamicin. Live cell imaging results show the potential utility of this new material. Related gentamicin conjugates studied to date quench in live kindney cells, and have been largely restricted to use in fixed (delipidated) cells. PMID:22545403

  8. Understanding dynamic changes in live cell adhesion with neutron reflectometry

    DOE PAGESBeta

    Junghans, Ann; Waltman, Mary Jo; Smith, Hillary L.; Pocivavsek, Luka; Zebda, Noureddine; Birukov, Konstantin; Viapiano, Mariano; Majewski, Jaroslaw

    2014-12-10

    In this study, neutron reflectometry (NR) was used to examine various live cells' adhesion to quartz substrates under different environmental conditions, including flow stress. To the best of our knowledge, these measurements represent the first successful visualization and quantization of the interface between live cells and a substrate with sub-nanometer resolution. In our first experiments, we examined live mouse fibroblast cells as opposed to past experiments using supported lipids, proteins, or peptide layers with no associated cells. We continued the NR studies of cell adhesion by investigating endothelial monolayers and glioblastoma cells under dynamic flow conditions. We demonstrated that neutronmore » reflectometry is a powerful tool to study the strength of cellular layer adhesion in living tissues, which is a key factor in understanding the physiology of cell interactions and conditions leading to abnormal or disease circumstances. Continuative measurements, such as investigating changes in tumor cell — surface contact of various glioblastomas, could impact advancements in tumor treatments. In principle, this can help us to identify changes that correlate with tumor invasiveness. Pursuit of these studies can have significant medical impact on the understanding of complex biological problems and their effective treatment, e.g. for the development of targeted anti-invasive therapies.« less

  9. Understanding dynamic changes in live cell adhesion with neutron reflectometry

    NASA Astrophysics Data System (ADS)

    Junghans, Ann; Waltman, Mary Jo; Smith, Hillary L.; Pocivavsek, Luka; Zebda, Noureddine; Birukov, Konstantin; Viapiano, Mariano; Majewski, Jaroslaw

    2014-12-01

    Neutron reflectometry (NR) was used to examine various live cells' adhesion to quartz substrates under different environmental conditions, including flow stress. To the best of our knowledge, these measurements represent the first successful visualization and quantization of the interface between live cells and a substrate with sub-nanometer resolution. In our first experiments, we examined live mouse fibroblast cells as opposed to past experiments using supported lipids, proteins, or peptide layers with no associated cells. We continued the NR studies of cell adhesion by investigating endothelial monolayers and glioblastoma cells under dynamic flow conditions. We demonstrated that neutron reflectometry is a powerful tool to study the strength of cellular layer adhesion in living tissues, which is a key factor in understanding the physiology of cell interactions and conditions leading to abnormal or disease circumstances. Continuative measurements, such as investigating changes in tumor cell — surface contact of various glioblastomas, could impact advancements in tumor treatments. In principle, this can help us to identify changes that correlate with tumor invasiveness. Pursuit of these studies can have significant medical impact on the understanding of complex biological problems and their effective treatment, e.g. for the development of targeted anti-invasive therapies.

  10. Understanding dynamic changes in live cell adhesion with neutron reflectometry

    SciTech Connect

    Junghans, Ann; Waltman, Mary Jo; Smith, Hillary L.; Pocivavsek, Luka; Zebda, Noureddine; Birukov, Konstantin; Viapiano, Mariano; Majewski, Jaroslaw

    2014-12-10

    In this study, neutron reflectometry (NR) was used to examine various live cells' adhesion to quartz substrates under different environmental conditions, including flow stress. To the best of our knowledge, these measurements represent the first successful visualization and quantization of the interface between live cells and a substrate with sub-nanometer resolution. In our first experiments, we examined live mouse fibroblast cells as opposed to past experiments using supported lipids, proteins, or peptide layers with no associated cells. We continued the NR studies of cell adhesion by investigating endothelial monolayers and glioblastoma cells under dynamic flow conditions. We demonstrated that neutron reflectometry is a powerful tool to study the strength of cellular layer adhesion in living tissues, which is a key factor in understanding the physiology of cell interactions and conditions leading to abnormal or disease circumstances. Continuative measurements, such as investigating changes in tumor cell — surface contact of various glioblastomas, could impact advancements in tumor treatments. In principle, this can help us to identify changes that correlate with tumor invasiveness. Pursuit of these studies can have significant medical impact on the understanding of complex biological problems and their effective treatment, e.g. for the development of targeted anti-invasive therapies.

  11. From personal tragedy to personal challenge: responses to stigma among sober living home residents and operators

    PubMed Central

    Heslin, Kevin C.; Singzon, Trudy; Aimiuwu, Otaren; Sheridan, Dave; Hamilton, Alison

    2011-01-01

    Sober living homes for people attempting to maintain abstinence from alcohol and drugs can act as a buffer against the high rates of substance misuse that are endemic to many urban environments. Sober living homes and other group homes for people with disabilities have faced persistent opposition from neighborhood associations, which raises the question of stigma. This article describes the responses of sober living home residents and operators to the threat of stigma across a diverse set of neighborhoods. Ten focus groups were conducted with 68 residents and operators of 35 sober living homes in Los Angeles County, California, between January 2009 and March 2010. Results showed that few residents reported experiences of blatant stigmatization by neighbors; however, they were well aware of the stereotypes that could be ascribed to them. Despite this potential stigma, residents developed valued identities as helpers in their communities, providing advice to neighbors whose family or friends had substance use problems, and organizing community service activities to improve the appearance of their neighborhoods. With their attention to local context, sober living home residents and operators challenge the personal tragedy approach of much traditional advocacy on health-related stigma. PMID:21707663

  12. Bioenergetics and Diffusion in the Crowded Milieu of Living Cells

    NASA Astrophysics Data System (ADS)

    Heikal, Ahmed

    2014-06-01

    Intracellular nicotinamide adenine dinucleotide (NADH) is a key cofactor in energy metabolism pathways and a myriad of oxidation-reduction reactions in living cells. The crowded milieu of these cells with organelles and macromolecules influences many biological processes such as biomolecular diffusion, protein-protein and protein-substrate interactions, and protein folding. In this contribution, I will highlight our recent findings on the role of macromolecular crowding on biochemical reaction between NADH and selected dehydrogenases in both living cells and in controlled macromolecules-rich environment. In addition, multiscale diffusion (rotational and translational) of a small fluorophore will be used to understand the role of non-specific binding, heterogeneity in microenvironmental viscosity in crowded solutions. Our experimental approach is a combination of fluorescence lifetime imaging microscopy, time-resolved anisotropy and fluorescence correlation spectroscopy. The broader impacts of these results will be discussed within the context of energy metabolism and biophysics in the crowded milieu of living cells.

  13. Challenges for fuel cells in transport applications

    NASA Astrophysics Data System (ADS)

    Chalk, Steven G.; Miller, James F.; Wagner, Fred W.

    The US Department of Energy (DOE) and the US automotive industry are working cooperatively under the auspices of the Partnership for a New Generation of Vehicles (PNGV) to develop a six-passenger automobile that can achieve up to 80 miles/gal. These partners are continuing to invest heavily in research and development of polymer electrolyte membrane (PEM) fuel cells as a clean and efficient alternative technology for transport applications. In the past few years, US automakers have made significant advances in fuel cell technology and have announced plans to put fuel cell vehicles on the market by 2004. DOE is working with industry suppliers to address some of the biggest remaining challenges, which include fuel processing and lowering the cost of fuel cell systems. The refueling infrastructure necessary to support fuel cell vehicles presents additional unresolved issues. This paper provides a status report on the PNGV program and provides an overview of the technical accomplishments and future plans of the DOE Fuel Cells for Transportation Program.

  14. The living cells in four dimensions

    SciTech Connect

    Paillotin, G.

    1991-01-01

    The 47th international conference of the American Institute of Physics and the Societe Francaise de Chimie was held jointly at Gif Sur Yvette, France. The main focus of this interdisciplinary conference was the structure and function of the cell cytoskeleton. This has been a very active area of research in biology in recent, but it has received much less attention from physicists and physical chemists. Thus, it was thought appropriate hold this meeting to concentrate on biophysical issues in this area. Talks from the three main symposia are presented, as well as questions and answers following the presentations. The symposia were: general aspects of cell architecture and dynamics; properties of some cytoskeletal components (microtubules, microfilalaments, spectrin and other cytoskeletal membrane proteins); and physico-chemical model systems. Individual papers are cataloged separately.

  15. Can we see living structure in a cell?

    PubMed

    Ling, G N

    1992-06-01

    Colloid chemistry (kappa o lambda lambda alpha: glue, or gelatin) was introduced in 1861 after the discovery of protoplasm which exhibits gelatin-like properties. Some 80 years later, colloid chemistry (and with it, the concept of protoplasm) was largely abandoned. The membrane (pump) theory, according to which cell water and cell solute like K+ are free as in a dilute KCl solution, became dominant. Later studies revealed that rejecting the protoplasmic approach to cell physiology was not justified. Evidence against the membrane (pump) theory, on the other hand, has stood the test of time. In a new theory of the living cell called the association-induction (AI) hypothesis, the three major components of the living cell (water, proteins and K+) are closely associated; together they exist in a high-(negative)-energy-low entropy state called the living state. The bulk of cell water is adsorbed as polarized multilayers on some fully extended protein chains, and K+ is adsorbed singly on beta- and gamma-carboxyl groups carried on aspartic and glutamic residues of cell proteins. Extensive evidence in support of the AI hypothesis is reviewed. From an extension of the basic concepts of the AI hypothesis and the new knowledge on primary structure of the proteins, one begins to understand at long last what distinguishes gelatin from other proteins; in this new light, new definitions of protoplasm and of colloid chemistry have been introduced. With the return of the concept of protoplasm, living structure takes on renewed significance, linking cell anatomy to cell physiology. Finally, evidence is presented showing that electron microscopists have come close to seeing cell structure in its living state. PMID:1462129

  16. Can we see living structure in a cell?

    PubMed

    Ling, Gilbert N

    2014-01-01

    Colloid chemistry (κολλα: glue, or gelatin) was introduced in 1861 after the discovery of protoplasm, which exhibits gelatin-like properties. Some 80 years later, colloid chemistry (and with it, the concept of protoplasm) was largely abandoned. The membrane (pump) theory, according to which cell water and cell solute like K+ are free as in a dilute KC1 solution, became dominant. Later studies revealed that rejecting the protoplasmic approach to cell physiology was not justified. Evidence against the membrane (pump) theory, on the other hand, has stood the test of time. In a new theory of the living cell called the association-induction (AI) hypothesis, the three major components of the living cell (water, proteins and K+) are closely associated; together they exist in a high- (negative)-energy-low entropy state called the living state. The bulk of cell water is adsorbed as polarized multilayers on some fully extended protein chains, and K+ is adsorbed singly on β- and γ-carboxyl groups carried on aspartic and glutamic residues of cell proteins. Extensive evidence in support of the AI hypothesis is reviewed. From an extension of the basic concepts of the AI hypothesis and the new knowledge on primary structure of the proteins, one begins to understand at long last what distinguishes gelatin from other proteins; in this new light, new definitions of protoplasm and of colloid chemistry have been introduced. With the return of the concept of protoplasm, living structure takes on renewed significance, linking cell anatomy to cell physiology. Finally, evidence is presented showing that electron microscopists have come close to seeing cell structure in its living state. PMID:25854101

  17. Mechanical behavior in living cells consistent with the tensegrity model

    NASA Technical Reports Server (NTRS)

    Wang, N.; Naruse, K.; Stamenovic, D.; Fredberg, J. J.; Mijailovich, S. M.; Tolic-Norrelykke, I. M.; Polte, T.; Mannix, R.; Ingber, D. E.

    2001-01-01

    Alternative models of cell mechanics depict the living cell as a simple mechanical continuum, porous filament gel, tensed cortical membrane, or tensegrity network that maintains a stabilizing prestress through incorporation of discrete structural elements that bear compression. Real-time microscopic analysis of cells containing GFP-labeled microtubules and associated mitochondria revealed that living cells behave like discrete structures composed of an interconnected network of actin microfilaments and microtubules when mechanical stresses are applied to cell surface integrin receptors. Quantitation of cell tractional forces and cellular prestress by using traction force microscopy confirmed that microtubules bear compression and are responsible for a significant portion of the cytoskeletal prestress that determines cell shape stability under conditions in which myosin light chain phosphorylation and intracellular calcium remained unchanged. Quantitative measurements of both static and dynamic mechanical behaviors in cells also were consistent with specific a priori predictions of the tensegrity model. These findings suggest that tensegrity represents a unified model of cell mechanics that may help to explain how mechanical behaviors emerge through collective interactions among different cytoskeletal filaments and extracellular adhesions in living cells.

  18. Acoustic propulsion of nanorod motors inside living cells.

    PubMed

    Wang, Wei; Li, Sixing; Mair, Lamar; Ahmed, Suzanne; Huang, Tony Jun; Mallouk, Thomas E

    2014-03-17

    The ultrasonic propulsion of rod-shaped nanomotors inside living HeLa cells is demonstrated. These nanomotors (gold rods about 300 nm in diameter and about 3 mm long) attach strongly to the external surface of the cells, and are readily internalized by incubation with the cells for periods longer than 24 h. Once inside the cells, the nanorod motors can be activated by resonant ultrasound operating at 4 MHz, and show axial propulsion as well as spinning. The intracellular propulsion does not involve chemical fuels or high-power ultrasound and the HeLa cells remain viable. Ultrasonic propulsion of nanomotors may thus provide a new tool for probing the response of living cells to internal mechanical excitation, for controllably manipulating intracellular organelles, and for biomedical applications. PMID:24677393

  19. Acoustic Propulsion of Nanorod Motors Inside Living Cells**

    PubMed Central

    Wang, Wei; Li, Sixing; Mair, Lamar; Ahmed, Suzanne

    2014-01-01

    We demonstrate the ultrasonic propulsion of rod-shaped nanomotors inside living HeLa cells. These nanomotors (gold rods ~ 300 nm in diameter and ~ 3 μm long) attach strongly to the external surface of the cells, and are readily internalized by incubation with the cells for periods longer than 24 h. Once inside the cells, the nanorod motors can be activated by resonant ultrasound operating at ~ 4 MHz, and show axial propulsion as well as spinning. The intracellular propulsion does not involve chemical fuels or high power ultrasound and the HeLa cells remain viable. Ultrasonic propulsion of nanomotors may thus provide a new tool for probing the response of living cells to internal mechanical excitation, for controllably manipulating intracellular organelles, and for biomedical applications. PMID:24677393

  20. Integrated nanoscale tools for interrogating living cells

    NASA Astrophysics Data System (ADS)

    Jorgolli, Marsela

    and fabricated a new hybrid chip that combines a front-side nanowire-based interface for neuronal recording with backside complementary metal oxide semiconductor (CMOS) circuits for on-chip multiplexing, voltage control for stimulation, signal amplification, and signal processing. Individual chips contain 1024 stimulation/recording sites enabling large-scale interfacing of neuronal networks with single cell resolution. Through electrical and electrochemical characterization of the devices, we demonstrated their enhanced functionality at a massively parallel scale. In our initial cell experiments, we achieved intracellular stimulations and recordings of changes in the membrane potential in a variety of cells including: HEK293T, cardiomyocytes, and rat cortical neurons. This demonstrated the device capability for single-cell-resolution recording/stimulation which when extended to a large number of neurons in a massively parallel fashion will enable the functional mapping of a complex neuronal network.

  1. Imaging the coordination of multiple signaling activities in living cells

    PubMed Central

    Welch, Christopher M.; Elliott, Hunter; Danuser, Gaudenz; Hahn, Klaus M.

    2013-01-01

    Preface Cellular signal transduction occurs in complex and redundant interaction networks that are best examined at the level of single cells by simultaneously monitoring the activation dynamics of multiple components. Recent advances in biosensor technology have made it possible to visualize and quantify the activation of multiple network nodes in the same living cell. The precision and scope of this approach has been greatly extended by novel computational approaches to determine the relationships between different networks, studied in separate cells. PMID:22016058

  2. Continuous-Wave Stimulated Emission Depletion Microscope for Imaging Actin Cytoskeleton in Fixed and Live Cells.

    PubMed

    Neupane, Bhanu; Jin, Tao; Mellor, Liliana F; Loboa, Elizabeth G; Ligler, Frances S; Wang, Gufeng

    2015-01-01

    Stimulated emission depletion (STED) microscopy provides a new opportunity to study fine sub-cellular structures and highly dynamic cellular processes, which are challenging to observe using conventional optical microscopy. Using actin as an example, we explored the feasibility of using a continuous wave (CW)-STED microscope to study the fine structure and dynamics in fixed and live cells. Actin plays an important role in cellular processes, whose functioning involves dynamic formation and reorganization of fine structures of actin filaments. Frequently used confocal fluorescence and STED microscopy dyes were employed to image fixed PC-12 cells (dyed with phalloidin- fluorescein isothiocyante) and live rat chondrosarcoma cells (RCS) transfected with actin-green fluorescent protein (GFP). Compared to conventional confocal fluorescence microscopy, CW-STED microscopy shows improved spatial resolution in both fixed and live cells. We were able to monitor cell morphology changes continuously; however, the number of repetitive analyses were limited primarily by the dyes used in these experiments and could be improved with the use of dyes less susceptible to photobleaching. In conclusion, CW-STED may disclose new information for biological systems with a proper characteristic length scale. The challenges of using CW-STED microscopy to study cell structures are discussed. PMID:26393614

  3. Continuous-Wave Stimulated Emission Depletion Microscope for Imaging Actin Cytoskeleton in Fixed and Live Cells

    PubMed Central

    Neupane, Bhanu; Jin, Tao; Mellor, Liliana F.; Loboa, Elizabeth G.; Ligler, Frances S.; Wang, Gufeng

    2015-01-01

    Stimulated emission depletion (STED) microscopy provides a new opportunity to study fine sub-cellular structures and highly dynamic cellular processes, which are challenging to observe using conventional optical microscopy. Using actin as an example, we explored the feasibility of using a continuous wave (CW)-STED microscope to study the fine structure and dynamics in fixed and live cells. Actin plays an important role in cellular processes, whose functioning involves dynamic formation and reorganization of fine structures of actin filaments. Frequently used confocal fluorescence and STED microscopy dyes were employed to image fixed PC-12 cells (dyed with phalloidin- fluorescein isothiocyante) and live rat chondrosarcoma cells (RCS) transfected with actin-green fluorescent protein (GFP). Compared to conventional confocal fluorescence microscopy, CW-STED microscopy shows improved spatial resolution in both fixed and live cells. We were able to monitor cell morphology changes continuously; however, the number of repetitive analyses were limited primarily by the dyes used in these experiments and could be improved with the use of dyes less susceptible to photobleaching. In conclusion, CW-STED may disclose new information for biological systems with a proper characteristic length scale. The challenges of using CW-STED microscopy to study cell structures are discussed. PMID:26393614

  4. Raman spectroscopy: an evolving technique for live cell studies.

    PubMed

    Smith, Rachael; Wright, Karen L; Ashton, Lorna

    2016-06-21

    One of the most exciting developments in Raman spectroscopy in the last decade has been its application to cells and tissues for diagnostic and pharmaceutical applications, and in particular its use in the analysis of cellular dynamics. Raman spectroscopy is rapidly advancing as a cell imaging method that overcomes many of the limitations of current techniques and is earning its place as a routine tool in cell biology. In this review we focus on important developments in Raman spectroscopy that have evolved into the exciting technique of live-cell Raman microscopy and highlight some of the most recent and significant applications to cell biology. PMID:27072718

  5. Dynamic friction measurements on living HeLa cells

    NASA Astrophysics Data System (ADS)

    Goulet, Marc-Antoni; Colbert, Marie-Josée; Dalnoki-Veress, Kari

    2008-03-01

    The interaction of cells with various interfaces, and especially man-made surfaces, is an active field of research. In our experiment we use a micropipette to measure both the friction and normal force as a cell slides across a surface. A thin substrate, coated with Poly-L-Lysine is brought into contact with a HeLa cell. The adjustable substrate motion is used to study the response of the cell at various normal forces and speeds. Analysis of the micropipette provides dynamic measurements of both the friction and normal force. With our novel setup we are able to probe the attachment/detachment process of living cells.

  6. Imaging the division process in living tissue culture cells

    PubMed Central

    Khodjakov, Alexey; Rieder, Conly L.

    2008-01-01

    We detail some of the pitfalls encountered when following live cultured somatic cells by light microscopy during mitosis. Principle difficulties in this methodology arise from the necessity to compromise between maintaining the health of the cell while achieving the appropriate temporal and spatial resolutions required for the study. Although the quality of the data collected from fixed cells is restricted only by the quality of the imaging system and the optical properties of the specimen, the major limiting factor when viewing live cells is radiation damage induced during illumination. We discuss practical considerations for minimizing this damage, and for maintaining the general health of the cell, while it is being followed by multi-mode or multi-dimensional light microscopy. PMID:16343936

  7. Multiplexing Bioluminescent and Fluorescent Reporters to Monitor Live Cells

    PubMed Central

    Haugwitz, Michael; Nourzaie, Omar; Garachtchenko, Tatiana; Hu, Lanrong; Gandlur, Suvarna; Olsen, Cathy; Farmer, Andrew; Chaga, Grigoriy; Sagawa, Hiroaki

    2008-01-01

    Reporter proteins are valuable tools to monitor promoter activities and characterize signal transduction pathways. Many of the currently available promoter reporters have drawbacks that compromise their performance. Enzyme-based reporter systems using cytosolic luciferases are highly sensitive, but require a cell lysis step that prevents their use in long-term monitoring. By contrast, secreted bioluminescent reporters like Metridia luciferase and Secreted Alkaline Phosphatase can be assayed repeatedly, using supernatant from the same live cell population to produce many sets of data over time. This is crucial for studies with limited amounts of cells, as in the case of stem cells. The use of secreted bioluminescent reporters also enables broader applications to provide more detailed information using live cells; for example, multiplexing with fluorescent proteins. Here, data is presented describing the characteristics of secreted Metridia luciferase and its use in multiplexing applications with either Secreted Alkaline Phosphatase or a fluorescent protein. PMID:20161823

  8. Multiplexing bioluminescent and fluorescent reporters to monitor live cells.

    PubMed

    Haugwitz, Michael; Nourzaie, Omar; Garachtchenko, Tatiana; Hu, Lanrong; Gandlur, Suvarna; Olsen, Cathy; Farmer, Andrew; Chaga, Grigoriy; Sagawa, Hiroaki

    2008-01-01

    Reporter proteins are valuable tools to monitor promoter activities and characterize signal transduction pathways. Many of the currently available promoter reporters have drawbacks that compromise their performance. Enzyme-based reporter systems using cytosolic luciferases are highly sensitive, but require a cell lysis step that prevents their use in long-term monitoring. By contrast, secreted bioluminescent reporters like Metridia luciferase and Secreted Alkaline Phosphatase can be assayed repeatedly, using supernatant from the same live cell population to produce many sets of data over time. This is crucial for studies with limited amounts of cells, as in the case of stem cells. The use of secreted bioluminescent reporters also enables broader applications to provide more detailed information using live cells; for example, multiplexing with fluorescent proteins. Here, data is presented describing the characteristics of secreted Metridia luciferase and its use in multiplexing applications with either Secreted Alkaline Phosphatase or a fluorescent protein. PMID:20161823

  9. Tomographic phase microscopy of living three-dimensional cell cultures.

    PubMed

    Kuś, Arkadiusz; Dudek, Michał; Kemper, Björn; Kujawińska, Małgorzata; Vollmer, Angelika

    2014-04-01

    A successful application of self-interference digital holographic microscopy in combination with a sample-rotation-based tomography module for three-dimensional (3-D) label-free quantitative live cell imaging with subcellular resolution is demonstrated. By means of implementation of a hollow optical fiber as the sample cuvette, the observation of living cells in different 3-D matrices is enabled. The fiber delivers a stable and accurate rotation of a cell or cell cluster, providing quantitative phase data for tomographic reconstruction of the 3-D refractive index distribution with an isotropic spatial resolution. We demonstrate that it is possible to clearly distinguish and quantitatively analyze several cells grouped in a "3-D cluster" as well as subcellular organelles like the nucleoli and local internal refractive index changes. PMID:24723114

  10. Imaging cell biology in live animals: Ready for prime time

    PubMed Central

    Porat-Shliom, Natalie; Amornphimoltham, Panomwat

    2013-01-01

    Time-lapse fluorescence microscopy is one of the main tools used to image subcellular structures in living cells. Yet for decades it has been applied primarily to in vitro model systems. Thanks to the most recent advancements in intravital microscopy, this approach has finally been extended to live rodents. This represents a major breakthrough that will provide unprecedented new opportunities to study mammalian cell biology in vivo and has already provided new insight in the fields of neurobiology, immunology, and cancer biology. PMID:23798727

  11. Single Molecule Detection and Imaging in Single Living Cells

    NASA Astrophysics Data System (ADS)

    Nie, Shuming

    2002-03-01

    Direct observation of single molecules and single molecular events inside living cells could dramatically improve our understanding of basic cellular processes (e.g., signal transduction and gene transcription) as well as improving our knowledge on the intracellular transport and fate of therapeutic agents (e.g., antisense RNA and gene therapy vectors). This talk will focus on using single-molecule fluorescence and luminescent quantum dots to examine the dynamics and spatial distribution of RNA and proteins inside living cells and on the surface membrane surface. These single-molecule studies yield a detailed description of molecular events and cellular structures under physiological conditions.

  12. Uncertainty principle of genetic information in a living cell

    PubMed Central

    Strippoli, Pierluigi; Canaider, Silvia; Noferini, Francesco; D'Addabbo, Pietro; Vitale, Lorenza; Facchin, Federica; Lenzi, Luca; Casadei, Raffaella; Carinci, Paolo; Zannotti, Maria; Frabetti, Flavia

    2005-01-01

    Background Formal description of a cell's genetic information should provide the number of DNA molecules in that cell and their complete nucleotide sequences. We pose the formal problem: can the genome sequence forming the genotype of a given living cell be known with absolute certainty so that the cell's behaviour (phenotype) can be correlated to that genetic information? To answer this question, we propose a series of thought experiments. Results We show that the genome sequence of any actual living cell cannot physically be known with absolute certainty, independently of the method used. There is an associated uncertainty, in terms of base pairs, equal to or greater than μs (where μ is the mutation rate of the cell type and s is the cell's genome size). Conclusion This finding establishes an "uncertainty principle" in genetics for the first time, and its analogy with the Heisenberg uncertainty principle in physics is discussed. The genetic information that makes living cells work is thus better represented by a probabilistic model rather than as a completely defined object. PMID:16197549

  13. Quantitative phase microscopy and synthetic aperture tomography of live cells

    NASA Astrophysics Data System (ADS)

    Lue, Niyom

    For more than a decade MIT's George R. Harrison Spectroscopy Laboratory has been developing quantitative phase microscopy (QPM) for biological study. Measurements of a point field were made in the mid 90s, then extended to the full 2D field, and recently, to 3D by using tomography. In the first part of this thesis improvements in the techniques of Fourier Phase Microscopy (FPM) and Hilbert Phase Microscopy (HPM) and their applications to characterize cells and tissues are reported. Tomographic phase microscopy (TPM) provides quantitative information and highly detailed structural information about a live cell, but in its current form it can only examine one cell at a time. Many biological applications including statistical analysis of a large collection of cells such as flow cytometry need a tomography technique that can measure many cells at a time. For the second part of this thesis we have developed a new tomography technique that can measure many cells continuously. In this study we demonstrate the new technique by translating a live cell across a focused beam. This beam is composed of many angular plane waves, and by applying a so-called synthetic aperture algorithm we retrieve individual wave components of the focused beam. We demonstrate for the first time that we can retrieve the field of the focused beam and synthesize any arbitrary angular plane wave. We then construct a 3D map of the variations of the refractive index in a live cell from a series of these synthesized angular plane waves. This new technique is the first step needed to analyze cells flowing through a beam to provide a high-throughput 3D refractive index tomograms that can be used as a new kind of statistical optical assay of living cells.

  14. Truly Individualized Supported Living: Utilizing Currently Available Resources To Facilitate Community Living for Persons with Challenging Behavior.

    ERIC Educational Resources Information Center

    Marone, Frank J.

    This paper discusses the concept of individualized, supported living arrangements for individuals with severe disabilities. The supported living arrangements are intended to enable people with disabilities to live in the community in a manner of their choice that most closely approximates the experience of people without disabilities, with support…

  15. Vinblastine suppresses dynamics of individual microtubules in living interphase cells.

    PubMed Central

    Dhamodharan, R; Jordan, M A; Thrower, D; Wilson, L; Wadsworth, P

    1995-01-01

    We have characterized the effects of vinblastine on the dynamic instability behavior of individual microtubules in living BS-C-1 cells microinjected with rhodamine-labeled tubulin and have found that at low concentrations (3-64 nM), vinblastine potently suppresses dynamic instability without causing net microtubule depolymerization. Vinblastine suppressed the rates of microtubule growth and shortening, and decreased the frequency of transitions from growth or pause to shortening, also called catastrophe. In vinblastine-treated cells, both the average duration of a pause (a state of attenuated dynamics where neither growth nor shortening could be detected) and the percentage of total time spent in pause were significantly increased. Vinblastine potently decreased dynamicity, a measure of the overall dynamic activity of microtubules, reducing this parameter by 75% at 32 nM. The present work, consistent with earlier in vitro studies, demonstrates that vinblastine kinetically caps the ends of microtubules in living cells and supports the hypothesis that the potent chemotherapeutic action of vinblastine as an antitumor drug is suppression of mitotic spindle microtubule dynamics. Further, the results indicate that molecules that bind to microtubule ends can regulate microtubule dynamic behavior in living cells and suggest that endogenous regulators of microtubule dynamics that work by similar mechanisms may exist in living cells. Images PMID:8534917

  16. Photothermal modification of optical microscope for noninvasive living cell monitoring

    NASA Astrophysics Data System (ADS)

    Lapotko, Dmitry; Romanovskaya, Tat'yana; Zharov, Vladimir P.

    2001-06-01

    Photothermal method was applied to improve sensing and imaging capabilities of a light microscope in cell studies. We describe the methods, technical details and testing results of cytometric application of Laser Photothermal Phase Microscope (LPPM). The merits of the proposed approach include living single cell monitoring capability, quantitative measurement of cell functional features through the use of cell natural chromophores as the sensors. Such intracellular sensors are activated by the laser pulse and transform an absorbed energy into the heat. The latter causes thermal and mechanical loads to a cell and its components. The second stage of the process includes the reaction of the cell as integral system or of its components to such loads. This reaction is caused by the changes of cell functional and structural state and includes alterations of cell optical properties. Both processes are monitored for a single cell non-invasively with probe laser beam. Pulsed phase contrast dual beam illumination scheme with acquisition of several laser images at different stages of cell-laser interaction was introduced. An acquired cell image is considered as spatially and temporally resolved cell response to non-specific load that is induced in a cell with a pump laser. This method eliminates any cell staining and allows to monitor cell viability and cell reaction to the environmental factors. Also LPPM offers further improvement of spatial and temporal resolution of optical microscope: with pulsed probe laser monitoring we can detect components with the size down to 50 nm and temporal resolution of 10 ns. In our set up the cell is pumped by pulsed laser at 532 nm, 10 ns , 0.01-0.4 mJ. The source of probe beam is a pulsed dye laser (630 nm, 10 nJ, 10 ns) which forms cell phase image. The results obtained with living cells such as drug impact control, single cell dosimetry, immune action of light on a cell demonstrate basic features of LPPM as the tool for the study of the

  17. Live-cell analysis of plant reproduction: live-cell imaging, optical manipulation, and advanced microscopy technologies.

    PubMed

    Kurihara, Daisuke; Hamamura, Yuki; Higashiyama, Tetsuya

    2013-05-01

    Sexual reproduction ensures propagation of species and enhances genetic diversity within populations. In flowering plants, sexual reproduction requires complicated and multi-step cell-to-cell communications among male and female cells. However, the confined nature of plant reproduction processes, which occur in the female reproductive organs and several cell layers of the pistil, limits our ability to observe these events in vivo. In this review, we discuss recent live-cell imaging in in vitro systems and the optical manipulation techniques that are used to capture the dynamic mechanisms representing molecular and cellular communications in sexual plant reproduction. PMID:23438900

  18. Spray-Dried Multiscale Nano-biocomposites Containing Living Cells.

    PubMed

    Johnson, Patrick E; Muttil, Pavan; MacKenzie, Debra; Carnes, Eric C; Pelowitz, Jennifer; Mara, Nathan A; Mook, William M; Jett, Stephen D; Dunphy, Darren R; Timmins, Graham S; Brinker, C Jeffrey

    2015-07-28

    Three-dimensional encapsulation of cells within nanostructured silica gels or matrices enables applications as diverse as biosensors, microbial fuel cells, artificial organs, and vaccines; it also allows the study of individual cell behaviors. Recent progress has improved the performance and flexibility of cellular encapsulation, yet there remains a need for robust scalable processes. Here, we report a spray-drying process enabling the large-scale production of functional nano-biocomposites (NBCs) containing living cells within ordered 3D lipid-silica nanostructures. The spray-drying process is demonstrated to work with multiple cell types and results in dry powders exhibiting a unique combination of properties including highly ordered 3D nanostructure, extended lipid fluidity, tunable macromorphologies and aerodynamic diameters, and unexpectedly high physical strength. Nanoindentation of the encasing nanostructure revealed a Young's modulus and hardness of 13 and 1.4 GPa, respectively. We hypothesized this high strength would prevent cell growth and force bacteria into viable but not culturable (VBNC) states. In concordance with the VBNC state, cellular ATP levels remained elevated even over eight months. However, their ability to undergo resuscitation and enter growth phase greatly decreased with time in the VBNC state. A quantitative method of determining resuscitation frequencies was developed and showed that, after 36 weeks in a NBC-induced VBNC, less than 1 in 10,000 cells underwent resuscitation. The NBC platform production of large quantities of VBNC cells is of interest for research in bacterial persistence and screening of drugs targeting such cells. NBCs may also enable long-term preservation of living cells for applications in cell-based sensing and the packaging and delivery of live-cell vaccines. PMID:26083188

  19. Transverse mechanical properties of cell walls of single living plant cells probed by laser-generated acoustic waves.

    PubMed

    Gadalla, Atef; Dehoux, Thomas; Audoin, Bertrand

    2014-05-01

    Probing the mechanical properties of plant cell wall is crucial to understand tissue dynamics. However, the exact symmetry of the mechanical properties of this anisotropic fiber-reinforced composite remains uncertain. For this reason, biologically relevant measurements of the stiffness coefficients on individual living cells are a challenge. For this purpose, we have developed the single-cell optoacoustic nanoprobe (SCOPE) technique, which uses laser-generated acoustic waves to probe the stiffness, thickness and viscosity of live single-cell subcompartments. This all-optical technique offers a sub-micrometer lateral resolution, nanometer in-depth resolution, and allows the non-contact measurement of the mechanical properties of live turgid tissues without any assumption of mechanical symmetry. SCOPE experiments reveal that single-cell wall transverse stiffness in the direction perpendicular to the epidermis layer of onion cells is close to that of cellulose. This observation demonstrates that cellulose microfibrils are the main load-bearing structure in this direction, and suggests strong bonding of microfibrils by hemicelluloses. Altogether our measurement of the viscosity at high frequencies suggests that the rheology of the wall is dominated by glass-like dynamics. From a comparison with literature, we attribute this behavior to the influence of the pectin matrix. SCOPE's ability to unravel cell rheology and cell anisotropy defines a new class of experiments to enlighten cell nano-mechanics. PMID:24615232

  20. Imaging the action of antimicrobial peptides on living bacterial cells

    PubMed Central

    Gee, Michelle L.; Burton, Matthew; Grevis-James, Alistair; Hossain, Mohammed Akhter; McArthur, Sally; Palombo, Enzo A.; Wade, John D.; Clayton, Andrew H. A.

    2013-01-01

    Antimicrobial peptides hold promise as broad-spectrum alternatives to conventional antibiotics. The mechanism of action of this class of peptide is a topical area of research focused predominantly on their interaction with artificial membranes. Here we compare the interaction mechanism of a model antimicrobial peptide with single artificial membranes and live bacterial cells. The interaction kinetics was imaged using time-lapse fluorescence lifetime imaging of a fluorescently-tagged melittin derivative. Interaction with the synthetic membranes resulted in membrane pore formation. In contrast, the interaction with bacteria led to transient membrane disruption and corresponding leakage of the cytoplasm, but surprisingly with a much reduced level of pore formation. The discovery that pore formation is a less significant part of lipid-peptide interaction in live bacteria highlights the mechanistic complexity of these interactions in living cells compared to simple artificial systems. PMID:23532056

  1. Neurotransmitter imaging in living cells based on native fluorescence detection

    SciTech Connect

    Tan, W.; Yeung, E.S. |; Parpura, V.; Haydon, P.G.

    1995-08-01

    A UV laser-based optical microscope and CCD detection system with high sensitivity has been developed to image neurotransmitters in living cells. We demonstrate the detection of serotonin that has been taken up into individual living glial cells (astrocytes) based on its native fluorescence. We found that the fluorescence intensity of astrocytes increased by up to 10 times after serotonin uptake. The temporal resolution of this detection system at 10{sup -4} M serotonin is as fast as 50 ms, and the spatial resolution is diffraction limited. This UV laser microscope imaging system shows promise for studies of spatial-temporal dynamics of neurotransmitter levels in living neurons and glia. 19 refs., 5 figs., 1 tab.

  2. Biosynthesis of a Fully Functional Cyclotide inside Living Bacterial Cells

    SciTech Connect

    Camarero, J A; Kimura, R H; Woo, Y; Cantor, J; Shekhtman, A

    2007-04-05

    The cyclotide MCoTI-II is a powerful trypsin inhibitor recently isolated from the seeds of Momordica cochinchinensis, a plant member of cucurbitaceae family. We report for the first time the in vivo biosynthesis of natively-folded MCoTI-II inside live E. coli cells. Our biomimetic approach involves the intracellular backbone cyclization of a linear cyclotide-intein fusion precursor mediated by a modified protein splicing domain. The cyclized peptide then spontaneously folds into its native conformation. The use of genetically engineered E. coli cells containing mutations in the glutathione and thioredoxin reductase genes considerably improves the production of folded MCoTI-II in vivo. Biochemical and structural characterization of the recombinant MCoTI-II confirmed its identity. Biosynthetic access to correctly-folded cyclotides allows the possibility of generating cell-based combinatorial libraries that can be screened inside living cells for their ability to modulate or inhibit cellular processes.

  3. Efficient Immobilization and Patterning of Live Bacterial Cells

    PubMed Central

    Suo, Zhiyong; Avci, Recep; Yang, Xinghong; Pascual, David W.

    2008-01-01

    A monolayer of live bacterial cells has been patterned onto substrates through the interaction between CFA/I fimbriae and the corresponding antibody. Patterns of live bacteria have been prepared with cellular resolution on silicon and gold substrates for Salmonella enterica serovar Typhimurium as a model with high specificity and efficiency. The immobilized cells are capable of dividing in growth medium to form a self-sustaining bacterial monolayer on the patterned areas. Interestingly, the immobilized cells can alter their orientation on the substrate, from lying-down to standing-up, as a response to the cell density increase during incubation. This method was successfully used to sort a targeted bacterial species from a mixed culture within 2 h. PMID:18321142

  4. Live-cell thermometry with nitrogen vacancy centers in nanodiamonds

    NASA Astrophysics Data System (ADS)

    Jayakumar, Harishankar; Fedder, Helmut; Chen, Andrew; Yang, Liudi; Li, Chenghai; Wrachtrup, Joerg; Wang, Sihong; Meriles, Carlos

    The ability to measure temperature is typically affected by a tradeoff between sensitivity and spatial resolution. Good thermometers tend to be bulky systems and hence are ill-suited for thermal sensing with high spatial localization. Conversely, the signal resulting from nanoscale temperature probes is often impacted by noise to a level where the measurement precision becomes poor. Adding to the microscopist toolbox, the nitrogen vacancy (NV) center in diamond has recently emerged as a promising platform for high-sensitivity nanoscale thermometry. Of particular interest are applications in living cells because diamond nanocrystals are biocompatible and can be chemically functionalized to target specific organelles. Here we report progress on the ability to probe and compare temperature within and between living cells using nanodiamond-hosted NV thermometry. We focus our study on cancerous cells, where atypical metabolic pathways arguably lead to changes in the way a cell generates heat, and thus on its temperature profile.

  5. Autologous cell therapies: challenges in US FDA regulation.

    PubMed

    McAllister, Todd N; Audley, David; L'Heureux, Nicolas

    2012-11-01

    Cell-based therapies (CBTs) have been hailed for the last two decades as the next pillar of healthcare, yet the clinical and commercial potential of regenerative medicine has yet to live up to the hype. While recent analysis has suggested that regenerative medicine is maturing into a multibillion dollar industry, examples of clinical and commercial success are still relatively rare. With 30 years of laboratory and clinical efforts fueled by countless billions in public and private funding, one must contemplate why CBTs have not made a greater impact. The current regulatory environment, with its zero-risk stance, stymies clinical innovation while fueling a potentially risky medical tourism industry. Here, we highlight the challenges the US FDA faces and present talking points for an improved regulatory framework for autologous CBTs. PMID:23210819

  6. Active Cellular Mechanics and Information Processing in the Living Cell

    NASA Astrophysics Data System (ADS)

    Rao, M.

    2014-07-01

    I will present our recent work on the organization of signaling molecules on the surface of living cells. Using novel experimental and theoretical approaches we have found that many cell surface receptors are organized as dynamic clusters driven by active currents and stresses generated by the cortical cytoskeleton adjoining the cell surface. We have shown that this organization is optimal for both information processing and computation. In connecting active mechanics in the cell with information processing and computation, we bring together two of the seminal works of Alan Turing.

  7. The preservation of living cells with biocompatible microparticles

    NASA Astrophysics Data System (ADS)

    Yang, Jing; Zhu, Yingnan; Xu, Tong; Pan, Chao; Cai, Nana; Huang, He; Zhang, Lei

    2016-07-01

    Biomedical applications of living cells have rapidly expanded in many fields such as toxic detection, drug screening, and regenerative medicine, etc. Efficient methods to support cell survival and maintain activity in vitro have become increasingly important. However, traditional cryopreservation for living cell-based applications is limited by several problems. Here, we report that magnetic hydrogel microparticles can physically assemble into a 3D environment for efficient cell preservation in physiological conditions, avoiding any chemical reactions that would damage the cells. Two representative cell lines (loosely and firmly adherent) were tested to evaluate the versatility of this method. The results showed that cell longevity was significantly extended to at least 15 days, while the control cell samples without microparticles quickly died within 3 days. Moreover, after preservation, cells can be easily retrieved by applying a magnet to separate the magnetic particles. This strategy can also inhibit cell over-proliferation while avoiding the use of temperature extremes or toxic cryoprotectants that are essential in cryopreservation.

  8. The preservation of living cells with biocompatible microparticles.

    PubMed

    Yang, Jing; Zhu, Yingnan; Xu, Tong; Pan, Chao; Cai, Nana; Huang, He; Zhang, Lei

    2016-07-01

    Biomedical applications of living cells have rapidly expanded in many fields such as toxic detection, drug screening, and regenerative medicine, etc. Efficient methods to support cell survival and maintain activity in vitro have become increasingly important. However, traditional cryopreservation for living cell-based applications is limited by several problems. Here, we report that magnetic hydrogel microparticles can physically assemble into a 3D environment for efficient cell preservation in physiological conditions, avoiding any chemical reactions that would damage the cells. Two representative cell lines (loosely and firmly adherent) were tested to evaluate the versatility of this method. The results showed that cell longevity was significantly extended to at least 15 days, while the control cell samples without microparticles quickly died within 3 days. Moreover, after preservation, cells can be easily retrieved by applying a magnet to separate the magnetic particles. This strategy can also inhibit cell over-proliferation while avoiding the use of temperature extremes or toxic cryoprotectants that are essential in cryopreservation. PMID:27189861

  9. Dynamic membrane patterning, signal localization and polarity in living cells.

    PubMed

    Zamparo, M; Chianale, F; Tebaldi, C; Cosentino-Lagomarsino, M; Nicodemi, M; Gamba, A

    2015-02-01

    We review the molecular and physical aspects of the dynamic localization of signaling molecules on the plasma membranes of living cells. At the nanoscale, clusters of receptors and signaling proteins play an essential role in the processing of extracellular signals. At the microscale, "soft" and highly dynamic signaling domains control the interaction of individual cells with their environment. At the multicellular scale, individual polarity patterns control the forces that shape multicellular aggregates and tissues. PMID:25563791

  10. Towards Probing Living Cell Function with NV Centers in Nanodiamonds

    NASA Astrophysics Data System (ADS)

    Sushkov, Alexander; Lovchinsky, Igor; Chisholm, Nicholas; Hunger, David; Akimov, Alexey; Lo, Peggy; Sutton, Amy; Robinson, Jacob; Yao, Norman; Bennett, Steven; Park, Hongkun; Lukin, Mikhail

    2012-02-01

    We report on recent progress in using the nitrogen-vacancy (NV) center in nanodiamonds as a local probe of paramagnetic free radical concentrations in living cells. The ability to monitor the local magnetic environment within the cell provides us a new tool to study organelle function during normal operation or in response to applied stimuli. Our approach involving biologically inert, robust sensor of local magnetic fields with nanoscale resolution opens up a new interface between quantum and biological sciences.

  11. Live-cell CLEM of subcellular targets: an optimized procedure for polymer-based imaging substrates.

    PubMed

    Padman, Benjamin S; Ramm, Georg

    2014-01-01

    Live-cell correlative light and electron microscopy permits the visualization of ultrastructure details associated with dynamic biological processes. On the optical level, fluorescence microscopy can be further combined with functional studies of intracellular processes and manipulation of biological samples using laser light. However, the major challenge is to relocate intracellular compartments in three dimensions after the sample has undergone an extensive EM sample preparation process. Here, we describe a detailed protocol for live-cell CLEM that provides easy guidance for 3D relocalization. Based on the use of the novel polymer film TOPAS as direct imaging substrate, we provide a setup that uses highly visible toner particles for tracking the region of interest in 2D and fiducial markers for the 3D relocation of intracellular structures. An example is given where a single mitochondria is targeted by laser microirradiation in live-cell fluorescence microscopy. After relocating the same structure in 3D in serial EM sections, the changes to the mitochondrial ultrastructure are observed by TEM. The method is suitable for correlation of live-cell microscopy of cells and can be performed using any inverted optical microscope. PMID:25287846

  12. From surface to intracellular non-invasive nanoscale study of living cells impairments

    SciTech Connect

    Ewald, Dr. Maxime; Tetard, Laurene; Elie-Caille, Dr. Cecile; Nicod, Laurence; Passian, Ali; Bourillot, Dr. Eric; Lesniewska, Prof. Eric

    2014-01-01

    Among the enduring challenges in nanoscience, subsurface characterization of live cells holds major stakes. Developments in nanometrology for soft matter thriving on the sensitivity and high resolution benefits of atomic force microscopy have enabled detection of subsurface structures at the nanoscale (1,2,3). However, measurements in liquid environments remain complex (4,5,6,7), in particular in the subsurface domain. Here we introduce liquid-Mode Synthesizing Atomic Force Microscopy (l-MSAFM) to study both the inner structures and the chemically induced intracellular impairments of living cells. Specifically, we visualize the intracellular stress effects of glyphosate on living keratinocytes skin cells. This new approach for living cell nanoscale imaging, l-MSAFM, in their physiological environment or in presence of a chemical stress agent confirmed the loss of inner structures induced by glyphosate. The ability to monitor the cell's inner response to external stimuli, non-destructively and in real time, has the potential to unveil critical nanoscale mechanisms of life science.

  13. Live cell opto-injection by femtosecond laser pulses

    NASA Astrophysics Data System (ADS)

    Baumgart, J.; Bintig, W.; Ngezahayo, A.; Ertmer, W.; Lubatschowski, H.; Heisterkamp, A.

    2007-02-01

    Fluorescence imaging of cells and cell organelles requires labeling by fluorophores. The labeling of living cells is often done by transfection of fluorescent proteins. Viral vectors are transferring the DNA into the cell. To avoid the use of viruses, it is possible to perforate the cell membrane for example by electro-shocks, the so called electroporation, so that the fluorescent proteins can diffuse into the cell. This method causes cell death in up to 50% of the treated cells because the damage of the outer membrane is too large. A less lethal perforation of the cell membrane with high efficiency can be realized by femtosecond (fs) laser pulses. Transient pores are created by focusing the laser beam for some milliseconds on the membrane. Through this pore, the proteins can enter into the cell. This was demonstrated in a proof of principle experiment for a few cells, but it is essential to develop an opto-perforation system for large numbers of cells in order to obtain statistically significant samples for biological experiments. The relationship between pulse energy, irradiation time, repetition rate and efficacy of the transfer of a chromophor into the cells as well as the viability of the cells was analysed. The cell viability was observed up to 90 minutes after manipulation.

  14. Exploring dynamics in living cells by tracking single particles.

    PubMed

    Levi, Valeria; Gratton, Enrico

    2007-01-01

    In the last years, significant advances in microscopy techniques and the introduction of a novel technology to label living cells with genetically encoded fluorescent proteins revolutionized the field of Cell Biology. Our understanding on cell dynamics built from snapshots on fixed specimens has evolved thanks to our actual capability to monitor in real time the evolution of processes in living cells. Among these new tools, single particle tracking techniques were developed to observe and follow individual particles. Hence, we are starting to unravel the mechanisms driving the motion of a wide variety of cellular components ranging from organelles to protein molecules by following their way through the cell. In this review, we introduce the single particle tracking technology to new users. We briefly describe the instrumentation and explain some of the algorithms commonly used to locate and track particles. Also, we present some common tools used to analyze trajectories and illustrate with some examples the applications of single particle tracking to study dynamics in living cells. PMID:17703064

  15. Functional live cell imaging of the pulmonary neuroepithelial body microenvironment.

    PubMed

    De Proost, Ian; Pintelon, Isabel; Brouns, Inge; Kroese, Alfons B A; Riccardi, Daniela; Kemp, Paul J; Timmermans, Jean-Pierre; Adriaensen, Dirk

    2008-08-01

    Pulmonary neuroepithelial bodies (NEBs) are densely innervated groups of neuroendocrine cells invariably accompanied by Clara-like cells. Together with NEBs, Clara-like cells form the so-called "NEB microenvironment," which recently has been assigned a potential pulmonary stem cell niche. Conclusive data on the nature of physiological stimuli for NEBs are lacking. This study aimed at developing an ex vivo mouse lung vibratome slice model for confocal live cell imaging of physiological reactions in identified NEBs and surrounding epithelial cells. Immunohistochemistry of fixed slices demonstrated that NEBs are almost completely shielded from the airway lumen by tight junction-linked Clara-like cells. Besides the unambiguous identification of NEBs, the fluorescent dye 4-Di-2-ASP allowed microscopic identification of ciliated cells, Clara cells, and Clara-like cells in live lung slices. Using the mitochondrial uncoupler FCCP and a mitochondrial membrane potential indicator, JC-1, increases in 4-Di-2-ASP fluorescence in NEB cells and ciliated cells were shown to represent alterations in mitochondrial membrane potential. Changes in the intracellular free calcium concentration ([Ca2+](i)) in NEBs and surrounding airway epithelial cells were simultaneously monitored using the calcium indicator Fluo-4. Application (5 s) of 50 mM extracellular potassium ([K+](o)) evoked a fast and reproducible [Ca2+](i) increase in NEB cells, while Clara-like cells displayed a delayed (+/- 4 s) [Ca2+](i) increase, suggestive of an indirect, NEB-mediated activation. The presented approach opens interesting new perspectives for unraveling the functional significance of pulmonary NEBs in control lungs and disease models, and for the first time allows direct visualization of local interactions within the NEB microenvironment. PMID:18367726

  16. Animals In Synchrotrons: Overcoming Challenges For High-Resolution, Live, Small-Animal Imaging

    SciTech Connect

    Donnelley, Martin; Parsons, David; Morgan, Kaye; Siu, Karen

    2010-07-23

    Physiological studies in small animals can be complicated, but the complexity is increased dramatically when performing live-animal synchrotron X-ray imaging studies. Our group has extensive experience in high-resolution live-animal imaging at the Japanese SPring-8 synchrotron, primarily examining airways in two-dimensions. These experiments normally image an area of 1.8 mmx1.2 mm at a pixel resolution of 0.45 {mu}m and are performed with live, intact, anaesthetized mice.There are unique challenges in this experimental setting. Importantly, experiments must be performed in an isolated imaging hutch not specifically designed for small-animal imaging. This requires equipment adapted to remotely monitor animals, maintain their anesthesia, and deliver test substances while collecting images. The horizontal synchrotron X-ray beam has a fixed location and orientation that limits experimental flexibility. The extremely high resolution makes locating anatomical regions-of-interest slow and can result in a high radiation dose, and at this level of magnification small animal movements produce motion-artifacts that can render acquired images unusable. Here we describe our experimental techniques and how we have overcome several challenges involved in performing live mouse synchrotron imaging.Experiments have tested different mouse strains, with hairless strains minimizing overlying skin and hair artifacts. Different anesthetics have also be trialed due to the limited choices available at SPring-8. Tracheal-intubation methods have been refined and controlled-ventilation is now possible using a specialized small-animal ventilator. With appropriate animal restraint and respiratory-gating, motion-artifacts have been minimized. The animal orientation (supine vs. head-high) also appears to affect animal physiology, and can alter image quality. Our techniques and image quality at SPring-8 have dramatically improved and in the near future we plan to translate this experience to the

  17. Animals In Synchrotrons: Overcoming Challenges For High-Resolution, Live, Small-Animal Imaging

    NASA Astrophysics Data System (ADS)

    Donnelley, Martin; Parsons, David; Morgan, Kaye; Siu, Karen

    2010-07-01

    Physiological studies in small animals can be complicated, but the complexity is increased dramatically when performing live-animal synchrotron X-ray imaging studies. Our group has extensive experience in high-resolution live-animal imaging at the Japanese SPring-8 synchrotron, primarily examining airways in two-dimensions. These experiments normally image an area of 1.8 mm×1.2 mm at a pixel resolution of 0.45 μm and are performed with live, intact, anaesthetized mice. There are unique challenges in this experimental setting. Importantly, experiments must be performed in an isolated imaging hutch not specifically designed for small-animal imaging. This requires equipment adapted to remotely monitor animals, maintain their anesthesia, and deliver test substances while collecting images. The horizontal synchrotron X-ray beam has a fixed location and orientation that limits experimental flexibility. The extremely high resolution makes locating anatomical regions-of-interest slow and can result in a high radiation dose, and at this level of magnification small animal movements produce motion-artifacts that can render acquired images unusable. Here we describe our experimental techniques and how we have overcome several challenges involved in performing live mouse synchrotron imaging. Experiments have tested different mouse strains, with hairless strains minimizing overlying skin and hair artifacts. Different anesthetics have also be trialed due to the limited choices available at SPring-8. Tracheal-intubation methods have been refined and controlled-ventilation is now possible using a specialized small-animal ventilator. With appropriate animal restraint and respiratory-gating, motion-artifacts have been minimized. The animal orientation (supine vs. head-high) also appears to affect animal physiology, and can alter image quality. Our techniques and image quality at SPring-8 have dramatically improved and in the near future we plan to translate this experience to the

  18. Using a nano-flare probe to detect RNA in live donor cells prior to somatic cell nuclear transfer.

    PubMed

    Fu, Bo; Ren, Liang; Liu, Di; Ma, Jian-Zhang; An, Tie-Zhu; Yang, Xiu-Qin; Ma, Hong; Guo, Zhen-Hua; Zhu, Meng; Bai, Jing

    2016-01-01

    Many transgenes are silenced in mammalian cells (donor cells used for somatic cell nuclear transfer [SCNT]). Silencing correlated with a repressed chromatin structure or suppressed promoter, and it impeded the production of transgenic animals. Gene transcription studies in live cells are challenging because of the drawbacks of reverse-transcription polymerase chain reaction and fluorescence in situ hybridization. Nano-flare probes provide an effective approach to detect RNA in living cells. We used 18S RNA, a housekeeping gene, as a reference gene. This study aimed to establish a platform to detect RNA in single living donor cells using a Nano-flare probe prior to SCNT and to verify the safety and validity of the Nano-flare probe in order to provide a technical foundation for rescuing silenced transgenes in transgenic cloned embryos. We investigated cytotoxic effect of the 18S RNA-Nano-flare probe on porcine fetal fibroblasts, characterized the distribution of the 18S RNA-Nano-flare probe in living cells and investigated the effect of the 18S RNA-Nano-flare probe on the development of cloned embryos after SCNT. The cytotoxic effect of the 18S RNA-Nano-flare probe on porcine fetal fibroblasts was dose-dependent, and 18S RNA was detected using the 18S RNA-Nano-flare probe. In addition, treating donor cells with 500 pM 18S RNA-Nano-flare probe did not have adverse effects on the development of SCNT embryos at the pre-implantation stage. In conclusion, we established a preliminary platform to detect RNA in live donor cells using a Nano-flare probe prior to SCNT. PMID:26109144

  19. Live Cell Interferometry Quantifies Dynamics of Biomass Partitioning during Cytokinesis

    PubMed Central

    Zangle, Thomas A.; Teitell, Michael A.; Reed, Jason

    2014-01-01

    The equal partitioning of cell mass between daughters is the usual and expected outcome of cytokinesis for self-renewing cells. However, most studies of partitioning during cell division have focused on daughter cell shape symmetry or segregation of chromosomes. Here, we use live cell interferometry (LCI) to quantify the partitioning of daughter cell mass during and following cytokinesis. We use adherent and non-adherent mouse fibroblast and mouse and human lymphocyte cell lines as models and show that, on average, mass asymmetries present at the time of cleavage furrow formation persist through cytokinesis. The addition of multiple cytoskeleton-disrupting agents leads to increased asymmetry in mass partitioning which suggests the absence of active mass partitioning mechanisms after cleavage furrow positioning. PMID:25531652

  20. Radioimmunoimaging with longer-lived positron-emitting radionuclides: potentials and challenges

    PubMed Central

    Nayak, Tapan K.; Brechbiel, Martin W.

    2012-01-01

    Radioimmunoimaging and therapy has been an area of interest for several decades. Steady progress has been made towards clinical translation of radiolabeled monoclonal antibodies for diagnosis and treatment of diseases. Tremendous advances have been made in imaging technologies such as positron emission tomography (PET). However, these advances have so far eluded routine translation into clinical radioimmunoimaging applications due to the mismatch between the short half-lives of routinely used positron-emitting radionuclides such as 18F versus the pharmacokinetics of most intact monoclonal antibodies of interest. The lack of suitable positron-emitting radionuclides that match the pharmacokinetics of intact antibodies has generated interest in exploring the use of longer-lived positron emitters that are more suitable for radioimmunoimaging and dosimetry applications with intact monoclonal antibodies. In this review, we examine the opportunities and challenges of radioimmunoimaging with select longer-lived positron-emitting radionuclides such as 124I, 89Zr and 86Y with respect to radionuclide production, ease of radiolabeling intact antibodies, imaging characteristics, radiation dosimetry and clinical translation potential. PMID:19125647

  1. Consensus, Dilemmas, and Challenges in Living Donor Liver Transplantation in Latin America.

    PubMed

    Salvalaggio, Paolo R; Seda Neto, João; Alves, Jefferson Andre; Fonseca, Eduardo A; Carneiro de Albuquerque, Luiz; Andraus, Wellington; Massarollo, Paulo B; Duro Garcia, Valter; Maurette, Rafael J; Ruf, Andrés E; Pacheco-Moreira, Lucio F; Caicedo Rusca, Luis A; Osorio, Veronica Botero; Matamoros, Maria Amalia; Varela-Fascinetto, Gustavo; Jarufe, Nicolas P

    2016-06-01

    We reviewed the history, volume, outcomes, uniqueness, and challenges of living donor liver transplantation (LDLT) in Latin America. We used the data from the Latin American and Caribbean Transplant Society, local transplant societies, and opinions from local transplant experts. There are more than 160 active liver transplant teams in Latin America, but only 30 centers have used LDLT in the past 2 years. In 2014, 226 LDLTs were done in the region (8.5% of liver transplant activities). Living donor liver transplantation is mainly restricted to pediatric patients. Adult-to-adult LDLT activities decreased after the implementation of the model for end-stage liver disease score and a concomitant increase on the rate of deceased donors per million population. Posttransplant outcome analysis is not mandatory, transparent or regulated in most countries. More experienced teams have outcomes comparable to international expert centers, but donor and recipient morbidity might be underreported. Latin America lags behind in terms of the number of adult LDLT and the rate of living donor utilization in comparison with other continents with similar donation rates. Local alliances and collaborations with major transplant centers in the developed world will contribute to the development of LDLT in Latin America. PMID:27203583

  2. Detection of LacZ-Positive Cells in Living Tissue with Single-Cell Resolution.

    PubMed

    Doura, Tomohiro; Kamiya, Mako; Obata, Fumiaki; Yamaguchi, Yoshifumi; Hiyama, Takeshi Y; Matsuda, Takashi; Fukamizu, Akiyoshi; Noda, Masaharu; Miura, Masayuki; Urano, Yasuteru

    2016-08-01

    The LacZ gene, which encodes Escherichia coli β-galactosidase, is widely used as a marker for cells with targeted gene expression or disruption. However, it has been difficult to detect lacZ-positive cells in living organisms or tissues at single-cell resolution, limiting the utility of existing lacZ reporters. Herein we present a newly developed fluorogenic β-galactosidase substrate suitable for labeling live cells in culture, as well as in living tissues. This precisely functionalized fluorescent probe exhibited dramatic activation of fluorescence upon reaction with the enzyme, remained inside cells by anchoring itself to intracellular proteins, and provided single-cell resolution. Neurons labeled with this probe preserved spontaneous firing, which was enhanced by application of ligands of receptors expressed in the cells, suggesting that this probe would be applicable to investigate functions of targeted cells in living tissues and organisms. PMID:27400827

  3. Materials Challenges for Automotive PEM Fuel Cells

    NASA Astrophysics Data System (ADS)

    Gasteiger, Hubert

    2004-03-01

    Over the past few years, significant R efforts aimed at meeting the challenging cost and performance targets required for the use of Polymer Electrolyte Membrane (PEM) fuel cells in automotive applications. Besides engineering advances in bipolar plate materials and design, the optimization of membrane-electrode assemblies (MEAs) was an important enabler in reducing the cost and performance gaps towards commercial viability for the automotive market. On the one hand, platinum loadings were reduced from several mgPt/cm2MEA [1] to values of 0.5-0.6 mgPt/cm2MEA in current applications and loadings as low as 0.25 mgPt/cm2MEA have been demonstrated on the research level [2]. On the other hand, implementation of thin membranes (20-30 micrometer) [3, 4] as well as improvements in diffusion medium materials, essentially doubled the achievable power density of MEAs to ca. 0.9 W/cm2MEA (at 0.65 V) [5], thereby not only reducing the size of a PEMFC fuel cell system, but also reducing its overall materials cost (controlled to a large extent by membrane and Pt-catalyst cost). While this demonstrated a clear path towards automotive applications, a renewed focus of R efforts is now required to develop materials and fundamental materials understanding to assure long-term durability of PEM fuel cells. This presentation therefore will discuss the state-of-the-art knowledge of catalyst, catalyst-support, and membrane degradation mechanisms. In the area of Pt-catalysts, experience with phosphoric acid fuel cells (PAFCs) has shown that platinum sintering leads to long-term performance losses [6]. While this is less critical at the lower PEMFC operating temperatures (<100C) compared to PAFCs (>200C), very little is known about the dependence of Pt-sintering on temperature, cell voltage, and catalyst type (i.e., Pt versus Pt-alloys) and will be discussed here. Similarly, carbon-support corrosion can contribute significantly to voltage degradation in PAFCs [7], and even in the PEMFC

  4. A method to rapidly create protein aggregates in living cells

    PubMed Central

    Miyazaki, Yusuke; Mizumoto, Kota; Dey, Gautam; Kudo, Takamasa; Perrino, John; Chen, Ling-chun; Meyer, Tobias; Wandless, Thomas J.

    2016-01-01

    The accumulation of protein aggregates is a common pathological hallmark of many neurodegenerative diseases. However, we do not fully understand how aggregates are formed or the complex network of chaperones, proteasomes and other regulatory factors involved in their clearance. Here, we report a chemically controllable fluorescent protein that enables us to rapidly produce small aggregates inside living cells on the order of seconds, as well as monitor the movement and coalescence of individual aggregates into larger structures. This method can be applied to diverse experimental systems, including live animals, and may prove valuable for understanding cellular responses and diseases associated with protein aggregates. PMID:27229621

  5. Self-organization and entropy reduction in a living cell

    PubMed Central

    Davies, Paul C.W.; Rieper, Elisabeth; Tuszynski, Jack A.

    2012-01-01

    In this paper we discuss the entropy and information aspects of a living cell. Particular attention is paid to the information gain on assembling and maintaining a living state. Numerical estimates of the information and entropy reduction are given and discussed in the context of the cell’s metabolic activity. We discuss a solution to an apparent paradox that there is less information content in DNA than in the proteins that are assembled based on the genetic code encrypted in DNA. When energy input required for protein synthesis is accounted for, the paradox is clearly resolved. Finally, differences between biological information and instruction are discussed. PMID:23159919

  6. A Stretching Device for High Resolution Live-Cell Imaging

    PubMed Central

    Huang, Lawrence; Mathieu, Pattie S.; Helmke, Brian P.

    2012-01-01

    Several custom-built and commercially available devices are available to investigate cellular responses to substrate strain. However, analysis of structural dynamics by microscopy in living cells during stretch is not readily feasible. We describe a novel stretch device optimized for high-resolution live-cell imaging. The unit assembles onto standard inverted microscopes and applies constant magnitude or cyclic stretch at physiological magnitudes to cultured cells on elastic membranes. Interchangeable modular indenters enable delivery of equibiaxial and uniaxial stretch profiles. Strain analysis performed by tracking fluorescent microspheres adhered onto the substrate demonstrated reproducible application of stretch profiles. In endothelial cells transiently expressing EGFP-vimentin and paxillin-DsRed2 and subjected to constant magnitude equibiaxial stretch, the 2-D strain tensor demonstrated efficient transmission through the extracellular matrix and focal adhesions. Decreased transmission to the intermediate filament network was measured, and a heterogeneous spatial distribution of maximum stretch magnitude revealed discrete sites of strain focusing. Spatial correlation of vimentin and paxillin displacement vectors provided an estimate of the extent of mechanical coupling between the structures. Interestingly, switching the spatial profile of substrate strain reveals that actin-mediated edge ruffling is not desensitized to repeated mechano-stimulation. These initial observations show that the stretch device is compatible with live-cell microscopy and is a novel tool for measuring dynamic structural remodeling under mechanical strain. PMID:20195762

  7. Local viscoelasticity of living cells measured by rotational magnetic spectroscopy

    NASA Astrophysics Data System (ADS)

    Berret, J.-F.

    2016-01-01

    When submitted to a magnetic field, micron-size wires with superparamagnetic properties behave as embedded rheometers and represent interesting sensors for microrheology. Here we use rotational magnetic spectroscopy to measure the shear viscosity of the cytoplasm of living cells. We address the question of whether the cytoplasm is a viscoelastic liquid or an elastic gel. The main result of the study is the observation of a rotational instability between a synchronous and an asynchronous regime of rotation, found for murine fibroblasts and human cancer cells. For wires of susceptibility 3.6, the transition occurs in the range 0.01-1 rad s-1. The determination of the shear viscosity (10-100 Pa s) and elastic modulus (5-20 Pa) confirms the viscoelastic character of the cytoplasm. In contrast to earlier studies, it is concluded that the interior of living cells can be described as a viscoelastic liquid, and not as an elastic gel.

  8. Local viscoelasticity of living cells measured by rotational magnetic spectroscopy.

    PubMed

    Berret, J-F

    2016-01-01

    When submitted to a magnetic field, micron-size wires with superparamagnetic properties behave as embedded rheometers and represent interesting sensors for microrheology. Here we use rotational magnetic spectroscopy to measure the shear viscosity of the cytoplasm of living cells. We address the question of whether the cytoplasm is a viscoelastic liquid or an elastic gel. The main result of the study is the observation of a rotational instability between a synchronous and an asynchronous regime of rotation, found for murine fibroblasts and human cancer cells. For wires of susceptibility 3.6, the transition occurs in the range 0.01-1 rad s(-1). The determination of the shear viscosity (10-100 Pa s) and elastic modulus (5-20 Pa) confirms the viscoelastic character of the cytoplasm. In contrast to earlier studies, it is concluded that the interior of living cells can be described as a viscoelastic liquid, and not as an elastic gel. PMID:26729062

  9. Synthetic mixed-signal computation in living cells.

    PubMed

    Rubens, Jacob R; Selvaggio, Gianluca; Lu, Timothy K

    2016-01-01

    Living cells implement complex computations on the continuous environmental signals that they encounter. These computations involve both analogue- and digital-like processing of signals to give rise to complex developmental programs, context-dependent behaviours and homeostatic activities. In contrast to natural biological systems, synthetic biological systems have largely focused on either digital or analogue computation separately. Here we integrate analogue and digital computation to implement complex hybrid synthetic genetic programs in living cells. We present a framework for building comparator gene circuits to digitize analogue inputs based on different thresholds. We then demonstrate that comparators can be predictably composed together to build band-pass filters, ternary logic systems and multi-level analogue-to-digital converters. In addition, we interface these analogue-to-digital circuits with other digital gene circuits to enable concentration-dependent logic. We expect that this hybrid computational paradigm will enable new industrial, diagnostic and therapeutic applications with engineered cells. PMID:27255669

  10. Glycoarray Technologies: Deciphering Interactions from Proteins to Live Cell Responses

    PubMed Central

    Puvirajesinghe, Tania M.; Turnbull, Jeremy. E.

    2016-01-01

    Microarray technologies inspired the development of carbohydrate arrays. Initially, carbohydrate array technology was hindered by the complex structures of glycans and their structural variability. The first designs of glycoarrays focused on the HTP (high throughput) study of protein–glycan binding events, and subsequently more in-depth kinetic analysis of carbohydrate–protein interactions. However, the applications have rapidly expanded and now achieve successful discrimination of selective interactions between carbohydrates and, not only proteins, but also viruses, bacteria and eukaryotic cells, and most recently even live cell responses to immobilized glycans. Combining array technology with other HTP technologies such as mass spectrometry is expected to allow even more accurate and sensitive analysis. This review provides a broad overview of established glycoarray technologies (with a special focus on glycosaminoglycan applications) and their emerging applications to the study of complex interactions between glycans and whole living cells. PMID:27600069

  11. Live cell imaging of endosomal trafficking in fungi.

    PubMed

    Baumann, Sebastian; Takeshita, Norio; Grün, Nathalie; Fischer, Reinhard; Feldbrügge, Michael

    2015-01-01

    Endosomes are multipurpose membranous carriers important for endocytosis and secretion. During membrane trafficking, endosomes transport lipids, proteins, and even RNAs. In highly polarized cells such as fungal hyphae, they shuttle bidirectionally along microtubules mediated by molecular motors like kinesins and dynein. For in vivo studies of these highly dynamic protein/membrane complexes, advanced fluorescence microscopy is instrumental. In this chapter, we describe live cell imaging of endosomes in two distantly related fungal model systems, the basidiomycete Ustilago maydis and the ascomycete Aspergillus nidulans. We provide insights into live cell imaging of dynamic endosomal proteins and RNA, dual-color detection for colocalization studies, as well as fluorescence recovery after photobleaching (FRAP) for quantification and photo-activated localization microscopy (PALM) for super-resolution. These methods described in two well-studied fungal model systems are applicable to a broad range of other organisms. PMID:25702128

  12. RNA imaging in living cells – methods and applications

    PubMed Central

    Urbanek, Martyna O; Galka-Marciniak, Paulina; Olejniczak, Marta; Krzyzosiak, Wlodzimierz J

    2014-01-01

    Numerous types of transcripts perform multiple functions in cells, and these functions are mainly facilitated by the interactions of the RNA with various proteins and other RNAs. Insight into the dynamics of RNA biosynthesis, processing and cellular activities is highly desirable because this knowledge will deepen our understanding of cell physiology and help explain the mechanisms of RNA-mediated pathologies. In this review, we discuss the live RNA imaging systems that have been developed to date. We highlight information on the design of these systems, briefly discuss their advantages and limitations and provide examples of their numerous applications in various organisms and cell types. We present a detailed examination of one application of RNA imaging systems: this application aims to explain the role of mutant transcripts in human disease pathogenesis caused by triplet repeat expansions. Thus, this review introduces live RNA imaging systems and provides a glimpse into their various applications. PMID:25483044

  13. Synthetic mixed-signal computation in living cells

    PubMed Central

    Rubens, Jacob R.; Selvaggio, Gianluca; Lu, Timothy K.

    2016-01-01

    Living cells implement complex computations on the continuous environmental signals that they encounter. These computations involve both analogue- and digital-like processing of signals to give rise to complex developmental programs, context-dependent behaviours and homeostatic activities. In contrast to natural biological systems, synthetic biological systems have largely focused on either digital or analogue computation separately. Here we integrate analogue and digital computation to implement complex hybrid synthetic genetic programs in living cells. We present a framework for building comparator gene circuits to digitize analogue inputs based on different thresholds. We then demonstrate that comparators can be predictably composed together to build band-pass filters, ternary logic systems and multi-level analogue-to-digital converters. In addition, we interface these analogue-to-digital circuits with other digital gene circuits to enable concentration-dependent logic. We expect that this hybrid computational paradigm will enable new industrial, diagnostic and therapeutic applications with engineered cells. PMID:27255669

  14. Glycoarray Technologies: Deciphering Interactions from Proteins to Live Cell Responses.

    PubMed

    Puvirajesinghe, Tania M; Turnbull, Jeremy E

    2016-01-01

    Microarray technologies inspired the development of carbohydrate arrays. Initially, carbohydrate array technology was hindered by the complex structures of glycans and their structural variability. The first designs of glycoarrays focused on the HTP (high throughput) study of protein-glycan binding events, and subsequently more in-depth kinetic analysis of carbohydrate-protein interactions. However, the applications have rapidly expanded and now achieve successful discrimination of selective interactions between carbohydrates and, not only proteins, but also viruses, bacteria and eukaryotic cells, and most recently even live cell responses to immobilized glycans. Combining array technology with other HTP technologies such as mass spectrometry is expected to allow even more accurate and sensitive analysis. This review provides a broad overview of established glycoarray technologies (with a special focus on glycosaminoglycan applications) and their emerging applications to the study of complex interactions between glycans and whole living cells. PMID:27600069

  15. Microfluidic Devices for Terahertz Spectroscopy of Live Cells Toward Lab-on-a-Chip Applications.

    PubMed

    Tang, Qi; Liang, Min; Lu, Yi; Wong, Pak Kin; Wilmink, Gerald J; Zhang, Donna; Xin, Hao

    2016-01-01

    THz spectroscopy is an emerging technique for studying the dynamics and interactions of cells and biomolecules, but many practical challenges still remain in experimental studies. We present a prototype of simple and inexpensive cell-trapping microfluidic chip for THz spectroscopic study of live cells. Cells are transported, trapped and concentrated into the THz exposure region by applying an AC bias signal while the chip maintains a steady temperature at 37 °C by resistive heating. We conduct some preliminary experiments on E. coli and T-cell solution and compare the transmission spectra of empty channels, channels filled with aqueous media only, and channels filled with aqueous media with un-concentrated and concentrated cells. PMID:27049392

  16. Microfluidic Devices for Terahertz Spectroscopy of Live Cells Toward Lab-on-a-Chip Applications

    PubMed Central

    Tang, Qi; Liang, Min; Lu, Yi; Wong, Pak Kin; Wilmink, Gerald J.; D. Zhang, Donna; Xin, Hao

    2016-01-01

    THz spectroscopy is an emerging technique for studying the dynamics and interactions of cells and biomolecules, but many practical challenges still remain in experimental studies. We present a prototype of simple and inexpensive cell-trapping microfluidic chip for THz spectroscopic study of live cells. Cells are transported, trapped and concentrated into the THz exposure region by applying an AC bias signal while the chip maintains a steady temperature at 37 °C by resistive heating. We conduct some preliminary experiments on E. coli and T-cell solution and compare the transmission spectra of empty channels, channels filled with aqueous media only, and channels filled with aqueous media with un-concentrated and concentrated cells. PMID:27049392

  17. Towards Probing Living Cell Function with NV Centers in Nanodiamonds

    NASA Astrophysics Data System (ADS)

    Lovchinsky, Igor; Chisholm, Nicholas; Sushkov, Alex; Lo, Peggy; Sutton, Amy; Robinson, Jacob; Yao, Norman; Bennett, Steven; Park, Hongkun; Lukin, Mikhail

    2012-06-01

    We report on recent progress in using nitrogen-vacancy (NV) centers in nanodiamonds as local probes of radical concentrations in living cells. Nanodiamonds are biologically inert, and NV centers within them are robust and can sense local magnetic fields with nanoscale resolution. The ability to monitor the local magnetic environment within the cell would provide a new tool to study organelle function during normal operation or in response to applied stimuli. In addition, radical concentrations have been linked to cancer, aging, and signaling between cells, thus proving to be of significant importance to the biological and medical sciences.

  18. Long live the stem cell: the use of stem cells isolated from post mortem tissues for translational strategies.

    PubMed

    Hodgetts, Stuart I; Stagg, Kelda; Sturm, Marian; Edel, Michael; Blancafort, Pilar

    2014-11-01

    The "stem cell" has become arguably one of the most important biological tools in the arsenal of translational research directed at regeneration and repair. It remains to be seen whether every tissue has its own stem cell niche, although relatively recently a large amount of research has focused on isolating and characterizing tissue-specific stem cell populations, as well as those that are able to be directed to transdifferentiate into a variety of different lineages. Traditionally, stem cells are isolated from the viable tissue of embryonic, fetal, or adult living hosts; from "fresh" donated tissues that have been surgically or otherwise removed (biopsies), or obtained directly from tissues within minutes to several hours post mortem (PM). These human progenitor/stem cell sources remain potentially highly controversial, since they are accompanied by various still-unresolved ethical, social, moral and legal challenges. Due to the limited number of "live" donors, the small amount of material obtained from biopsies and difficulties during purification processes, harvesting from cadaveric material presents itself as an alternative strategy that could provide a hitherto untapped source of stem cells. However, PM stem cells are not without their own unique set of limitations including difficulty of obtaining samples, limited supply of material, variations in delay between death and sample collection, possible lack of medication history and suboptimal retrospective assignment of diagnostic and demographic data. This article is part of a Directed Issue entitled: Regenerative Medicine: The challenge of translation. PMID:25300917

  19. From surface to intracellular non-invasive nanoscale study of living cells impairments

    NASA Astrophysics Data System (ADS)

    Ewald, M.; Tetard, L.; Elie-Caille, C.; Nicod, L.; Passian, A.; Bourillot, E.; Lesniewska, E.

    2014-07-01

    Among the enduring challenges in nanoscience, subsurface characterization of living cells holds major stakes. Developments in nanometrology for soft matter thriving on the sensitivity and high resolution benefits of atomic force microscopy have enabled detection of subsurface structures at the nanoscale. However, measurements in liquid environments remain complex, in particular in the subsurface domain. Here we introduce liquid-mode synthesizing atomic force microscopy (l-MSAFM) to study both the inner structures and the chemically induced intracellular impairments of living cells. Specifically, we visualize the intracellular stress effects of glyphosate on living keratinocytes skin cells. This new approach, l-MSAFM, for nanoscale imaging of living cell in their physiological environment or in presence of a chemical stress agent could resolve the loss of inner structures induced by glyphosate, the main component of a well-known pesticide (RoundUp™). This firsthand ability to monitor the cell’s inner response to external stimuli non-destructively and in liquid, has the potential to unveil critical nanoscale mechanisms of life science.

  20. Materials Challenges for Automotive PEM Fuel Cells

    NASA Astrophysics Data System (ADS)

    Gasteiger, Hubert

    2004-03-01

    Over the past few years, significant R efforts aimed at meeting the challenging cost and performance targets required for the use of Polymer Electrolyte Membrane (PEM) fuel cells in automotive applications. Besides engineering advances in bipolar plate materials and design, the optimization of membrane-electrode assemblies (MEAs) was an important enabler in reducing the cost and performance gaps towards commercial viability for the automotive market. On the one hand, platinum loadings were reduced from several mgPt/cm2MEA [1] to values of 0.5-0.6 mgPt/cm2MEA in current applications and loadings as low as 0.25 mgPt/cm2MEA have been demonstrated on the research level [2]. On the other hand, implementation of thin membranes (20-30 micrometer) [3, 4] as well as improvements in diffusion medium materials, essentially doubled the achievable power density of MEAs to ca. 0.9 W/cm2MEA (at 0.65 V) [5], thereby not only reducing the size of a PEMFC fuel cell system, but also reducing its overall materials cost (controlled to a large extent by membrane and Pt-catalyst cost). While this demonstrated a clear path towards automotive applications, a renewed focus of R efforts is now required to develop materials and fundamental materials understanding to assure long-term durability of PEM fuel cells. This presentation therefore will discuss the state-of-the-art knowledge of catalyst, catalyst-support, and membrane degradation mechanisms. In the area of Pt-catalysts, experience with phosphoric acid fuel cells (PAFCs) has shown that platinum sintering leads to long-term performance losses [6]. While this is less critical at the lower PEMFC operating temperatures (<100C) compared to PAFCs (>200C), very little is known about the dependence of Pt-sintering on temperature, cell voltage, and catalyst type (i.e., Pt versus Pt-alloys) and will be discussed here. Similarly, carbon-support corrosion can contribute significantly to voltage degradation in PAFCs [7], and even in the PEMFC

  1. Lived experiences and challenges of older surgical patients during hospitalization for cancer: an ethnographic fieldwork.

    PubMed

    Uhrenfeldt, Lisbeth; Høybye, Mette Terp

    2014-01-01

    This paper explores the lived experiences of older surgical patients' (aged 74 years and older) experienced challenges during a brief admission to hospital. Age, gender, polypharmacy, and the severity of illness are also factors known to affect the hospitalization process. For an ethnographic study using participant observation and interviews, surgical cancer patients (n = 9, aged 74 years and older) were recruited during admission to a Danish teaching hospital. Using ethnographic strategies of participant observation and interviews, each patient was followed through the course of 1 day during their stay at the hospital. Interviews were carried out with all patients during this time. Three areas of concern were identified as prominent in the patients' experiences and challenges during their short hospital stay: teeth and oral cavity, eating in a hospital setting, and medication during hospitalization. Short-term hospitalization requires focused collaboration between staff and patient concerning individual challenges from their teeth and oral cavity as support of nutritional needs during surgical treatment for cancer. PMID:24559546

  2. Comparison of Two Commercial Type 1 Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) Modified Live Vaccines against Heterologous Type 1 and Type 2 PRRSV Challenge in Growing Pigs

    PubMed Central

    Kim, Taeyeon; Park, Changhoon; Choi, Kyuhyung; Jeong, Jiwoon; Kang, Ikjae; Park, Su-Jin

    2015-01-01

    The objective of the present study was to compare the efficacy of two commercial type 1 porcine reproductive and respiratory syndrome virus (PRRSV) modified live vaccines against heterologous type 1 and type 2 PRRSV challenge in growing pigs. Vaccination with a type 1 PRRSV vaccine reduced the level of viremia after type 1 PRRSV challenge but did not reduce the level of viremia after the type 2 PRRSV challenge in pigs. Increased levels of interleukin-10 (IL-10) stimulated by type 2 PRRSV coincided with the low numbers of type 2 PRRSV-specific interferon gamma-secreting cells (IFN-γ-SC) in vaccinated pigs after type 2 PRRSV challenge, whereas low levels of IL-10 stimulated by type 1 PRRSV coincided with high numbers of type 1 PRRSV-specific IFN-γ-SC in vaccinated pigs after type 1 PRRSV challenge. Additionally, vaccination with the type 1 PRRSV vaccine effectively reduced the lung lesions and type 1 PRRSV nucleic acids in type 1 PRRSV-challenged pigs but did not reduce lung lesions and type 2 PRRSV nucleic acids in type 2 PRRSV-challenged pigs. There were no significant differences between two commercial type 1 PRRSV vaccines against type 1 and type 2 PRRSV challenge based on virological results, immunological responses, and pathological outcomes. This study demonstrates that vaccinating pigs with the type 1 PRRSV vaccine provides partial protection against respiratory disease with heterologous type 1 PRRSV challenge but no protection with heterologous type 2 PRRSV challenge. PMID:25855554

  3. Bimolecular Fluorescence Complementation to Assay the Interactions of Ubiquitylation Enzymes in Living Yeast Cells.

    PubMed

    Blaszczak, Ewa; Prigent, Claude; Rabut, Gwenaël

    2016-01-01

    Ubiquitylation is a versatile posttranslational protein modification catalyzed through the concerted action of ubiquitin-conjugating enzymes (E2s) and ubiquitin ligases (E3s). These enzymes form transient complexes with each other and their modification substrates and determine the nature of the ubiquitin signals attached to their substrates. One challenge in the field of protein ubiquitylation is thus to identify the E2-E3 pairs that function in the cell. In this chapter, we describe the use of bimolecular fluorescence complementation to assay E2-E3 interactions in living cells, using budding yeast as a model organism. PMID:27613039

  4. A drug-compatible and temperature-controlled microfluidic device for live-cell imaging

    PubMed Central

    Chen, Tong; Gomez-Escoda, Blanca; Munoz-Garcia, Javier; Babic, Julien; Griscom, Laurent; Wu, Pei-Yun Jenny

    2016-01-01

    Monitoring cellular responses to changes in growth conditions and perturbation of targeted pathways is integral to the investigation of biological processes. However, manipulating cells and their environment during live-cell-imaging experiments still represents a major challenge. While the coupling of microfluidics with microscopy has emerged as a powerful solution to this problem, this approach remains severely underexploited. Indeed, most microdevices rely on the polymer polydimethylsiloxane (PDMS), which strongly absorbs a variety of molecules commonly used in cell biology. This effect of the microsystems on the cellular environment hampers our capacity to accurately modulate the composition of the medium and the concentration of specific compounds within the microchips, with implications for the reliability of these experiments. To overcome this critical issue, we developed new PDMS-free microdevices dedicated to live-cell imaging that show no interference with small molecules. They also integrate a module for maintaining precise sample temperature both above and below ambient as well as for rapid temperature shifts. Importantly, changes in medium composition and temperature can be efficiently achieved within the chips while recording cell behaviour by microscopy. Compatible with different model systems, our platforms provide a versatile solution for the dynamic regulation of the cellular environment during live-cell imaging. PMID:27512142

  5. A drug-compatible and temperature-controlled microfluidic device for live-cell imaging.

    PubMed

    Chen, Tong; Gomez-Escoda, Blanca; Munoz-Garcia, Javier; Babic, Julien; Griscom, Laurent; Wu, Pei-Yun Jenny; Coudreuse, Damien

    2016-08-01

    Monitoring cellular responses to changes in growth conditions and perturbation of targeted pathways is integral to the investigation of biological processes. However, manipulating cells and their environment during live-cell-imaging experiments still represents a major challenge. While the coupling of microfluidics with microscopy has emerged as a powerful solution to this problem, this approach remains severely underexploited. Indeed, most microdevices rely on the polymer polydimethylsiloxane (PDMS), which strongly absorbs a variety of molecules commonly used in cell biology. This effect of the microsystems on the cellular environment hampers our capacity to accurately modulate the composition of the medium and the concentration of specific compounds within the microchips, with implications for the reliability of these experiments. To overcome this critical issue, we developed new PDMS-free microdevices dedicated to live-cell imaging that show no interference with small molecules. They also integrate a module for maintaining precise sample temperature both above and below ambient as well as for rapid temperature shifts. Importantly, changes in medium composition and temperature can be efficiently achieved within the chips while recording cell behaviour by microscopy. Compatible with different model systems, our platforms provide a versatile solution for the dynamic regulation of the cellular environment during live-cell imaging. PMID:27512142

  6. Plasma needle: treatment of living cells and tissues

    NASA Astrophysics Data System (ADS)

    Stoffels, Eva

    2003-10-01

    Non-thermal plasmas are capable of refined treatment of heat sensitive surfaces. Recently, many non-thermal sources working under atmospheric pressure have been constructed. Their main applications are various surface treatments: cleaning, etching, changing the wettability/adhesion, and bacterial decontamination. A new research at the Eindhoven University of Technology focuses on in vivo treatment by means of a novel non-thermal plasma source (the plasma needle). At present, a fundamental study has been undertaken to identify all possible responses of living objects exposed to the plasma. Plasma treatment does not lead to cell death (necrosis), which is a cause of inflammation. On the contrary, we observe various sophisticated reactions of mammalian cells, e.g. cell detachment (loss of cell contact) and programmed cell death (apoptosis). Moreover, under certain conditions the plasma is capable of killing bacteria, while eukaryotic cells remain unharmed. These findings may result in development of new techniques, like bacterial sterilization of infected (living) tissues or removal of cells without inflammatory response, and on a longer time scale to new methods in the health care. Possible applications include treatment of skin ailments, aiding wound healing and sterilization of dental cavities.

  7. Scanning Ion Conductance Microscopy for living cell membrane potential measurement

    NASA Astrophysics Data System (ADS)

    Panday, Namuna

    Recently, the existence of multiple micro-domains of extracellular potential around individual cells have been revealed by voltage reporter dye using fluorescence microscopy. One hypothesis is that these long lasting potential patterns play a vital role in regulating important cell activities such as embryonic patterning, regenerative repair and reduction of cancerous disorganization. We used multifunctional Scanning Ion Conductance Microscopy (SICM) to study these extracellular potential patterns of single cell with higher spatial resolution. To validate this novel technique, we compared the extracellular potential distribution on the fixed HeLa cell surface and Polydimethylsiloxane (PDMS) surface and found significant difference. We then measured the extracellular potential distributions of living melanocytes and melanoma cells and found both the mean magnitude and spatial variation of extracellular potential of the melanoma cells are bigger than those of melanocytes. As compared to the voltage reporter dye based fluorescence microscope method, SICM can achieve quantitative potential measurements of non-labeled living cell membranes with higher spatial resolution.

  8. Foundations and Emerging Paradigms for Computing in Living Cells.

    PubMed

    Ma, Kevin C; Perli, Samuel D; Lu, Timothy K

    2016-02-27

    Genetic circuits, composed of complex networks of interacting molecular machines, enable living systems to sense their dynamic environments, perform computation on the inputs, and formulate appropriate outputs. By rewiring and expanding these circuits with novel parts and modules, synthetic biologists have adapted living systems into vibrant substrates for engineering. Diverse paradigms have emerged for designing, modeling, constructing, and characterizing such artificial genetic systems. In this paper, we first provide an overview of recent advances in the development of genetic parts and highlight key engineering approaches. We then review the assembly of these parts into synthetic circuits from the perspectives of digital and analog logic, systems biology, and metabolic engineering, three areas of particular theoretical and practical interest. Finally, we discuss notable challenges that the field of synthetic biology still faces in achieving reliable and predictable forward-engineering of artificial biological circuits. PMID:26908220

  9. Measurement of UV absorption of single living cell for cell manipulation using NIR femtosecond laser

    NASA Astrophysics Data System (ADS)

    Cho, Sung-Hak; Chang, Won-Seok; Kim, Kwang-Ryul; Hong, Jong Wook

    2009-02-01

    Optical UV absorption of single human living cells ranging from 200 nm to 360 nm was measured in situ for the study of cell manipulation using the near-infrared (NIR) femtosecond laser . Human breast living cells of MCF-10A, MCF-7, and MDA-MB-231 were used in this experiment. The selective photo-disruptions of single living cell and its sub-organelle (nucleus) were also demonstrated using the tightly focused 790 nm wavelength femtosecond laser with pulse duration of 110 fs. It was found that each living cell has its own absorption spectrum in UV wavelength ranges. It was also inferred that intrinsic absorption spectrum is attributed to the amount of DNA and protein of living cell. For the study of photo-disruption of single cell using the multi-photon absorption excited by the NIR femtosecond laser pulse, the origin UV absorption spectrum of targeted living cell is important and fundamental information to understand nonlinear interaction between NIR ultrashort, high-intensity laser light and transparent living cell.

  10. The dynamic lives of T cells: new approaches and themes

    PubMed Central

    Yamanaka, Yvonne J.; Gierahn, Todd M.; Love, J. Christopher

    2012-01-01

    Activated T cells have classically been thought to progress unidirectionally through discrete phenotypic states and differentiate into static lineages. It is increasingly evident, however, that T cells exhibit much more complex and flexible dynamic behaviors than initially appreciated, and that these behaviors influence the efficacy of T cell responses to immunological challenges. In this review, we discuss how new technologies for monitoring the dynamics of T cells are enhancing the resolution of the fine phenotypic and functional heterogeneity within populations of T cells and revealing how individual T cells transition among a continuum of states. Such insights into the dynamic properties of T cells should improve immune monitoring and inform strategies for therapeutic interventions. PMID:23200626

  11. Monitoring protein synthesis in single live cancer cells.

    PubMed

    Tu, Chengyi; Santo, Loredana; Mishima, Yuko; Raje, Noopur; Smilansky, Zeev; Zoldan, Janet

    2016-05-16

    Protein synthesis is generally under sophisticated and dynamic regulation to meet the ever-changing demands of a cell. Global up or down-regulation of protein synthesis and the shift of protein synthesis location (as shown, for example, during cellular stress or viral infection) are recognized as cellular responses to environmental changes such as nutrient/oxygen deprivation or to alterations such as pathological mutations in cancer cells. Monitoring protein synthesis in single live cells can be a powerful tool for cancer research. Here we employed a microfluidic platform to perform high throughput delivery of fluorescent labeled tRNAs into multiple myeloma cells with high transfection efficiency (∼45%) and high viability (>80%). We show that the delivered tRNAs were actively recruited to the ER for protein synthesis and that treatment with puromycin effectively disrupted this process. Interestingly, we observed the scattered distribution of tRNAs in cells undergoing mitosis, which has not been previously reported. Fluorescence lifetime analysis detected extensive FRET signals generated from tRNAs labeled as FRET pairs, further confirming that the delivered tRNAs were used by active ribosomes for protein translation. Our work demonstrates that the microfluidic delivery of FRET labeled tRNAs into living cancer cells can provide new insights into basic cancer metabolism and has the potential to serve as a platform for drug screening, diagnostics, or personalized medication. PMID:26956582

  12. Live Imaging of Adult Neural Stem Cells in Rodents

    PubMed Central

    Ortega, Felipe; Costa, Marcos R.

    2016-01-01

    The generation of cells of the neural lineage within the brain is not restricted to early development. New neurons, oligodendrocytes, and astrocytes are produced in the adult brain throughout the entire murine life. However, despite the extensive research performed in the field of adult neurogenesis during the past years, fundamental questions regarding the cell biology of adult neural stem cells (aNSCs) remain to be uncovered. For instance, it is crucial to elucidate whether a single aNSC is capable of differentiating into all three different macroglial cell types in vivo or these distinct progenies constitute entirely separate lineages. Similarly, the cell cycle length, the time and mode of division (symmetric vs. asymmetric) that these cells undergo within their lineage progression are interesting questions under current investigation. In this sense, live imaging constitutes a valuable ally in the search of reliable answers to the previous questions. In spite of the current limitations of technology new approaches are being developed and outstanding amount of knowledge is being piled up providing interesting insights in the behavior of aNSCs. Here, we will review the state of the art of live imaging as well as the alternative models that currently offer new answers to critical questions. PMID:27013941

  13. Nucleoplasmic viscosity of living cells investigated by fluorescence correlation spectroscopy

    NASA Astrophysics Data System (ADS)

    Liang, Lifang; Xing, Da; Chen, Tongshen; Pei, Yihui

    2007-11-01

    Fluorescence correlation spectroscopy (FCS) is a new kind of real-time, high-speed and single-molecule technique. It is used to detect the kinetic characteristics of fluorescent dye such as diffusion coefficient in the aqueous solution. Combined with confocal microscope optics, it has been now widely applied in cell biological research. Through a time correlation analysis of spontaneous intensity fluctuations, this technique with EGFP as a probe is capable of determining viscosity of fluids according to Stokes-Einstein equation. Nucleoplasmic viscosity is an important physical parameter to quantify the rheological characteristics of the nucleoplasm. Investigation on nucleoplasmic viscosity plays an important role in further understanding intranuclear environment. In this paper, FCS is introduced to noninvasively investigate nucleoplasmic viscosity of living cells. The results show that nucleoplasmic viscosity of lung adenocarcinoma (ASTC-a-1) cells is 2.55+/-0.61 cP and nucleoplasmic viscosity is larger than cytoplasmic viscosity at 37 °C (pH 7.4). In addition, significant changes in nucleoplasmic viscosity are detected by FCS when cells are exposed to hyper or hypotonic medium. Our study suggests that FCS can be used to detect the kinetic characteristics of biomolecules in living cells and thus helps to investigate the dynamic changes of the microenvironment in the cell.

  14. Mapping eGFP Oligomer Mobility in Living Cell Nuclei

    PubMed Central

    Zwerger, Monika; Müller, Gabriele; Waldeck, Waldemar; Langowski, Jörg

    2009-01-01

    Movement of particles in cell nuclei can be affected by viscosity, directed flows, active transport, or the presence of obstacles such as the chromatin network. Here we investigate whether the mobility of small fluorescent proteins is affected by the chromatin density. Diffusion of inert fluorescent proteins was studied in living cell nuclei using fluorescence correlation spectroscopy (FCS) with a two-color confocal scanning detection system. We first present experiments exposing FCS-specific artifacts encountered in live cell studies as well as strategies to prevent them, in particular those arising from the choice of the fluorophore used for calibration of the focal volume, as well as temperature and acquisition conditions used for fluorescence fluctuation measurements. After defining the best acquisition conditions, we show for various human cell lines that the mobility of GFP varies significantly within the cell nucleus, but does not correlate with chromatin density. The intranuclear diffusional mobility strongly depends on protein size: in a series of GFP-oligomers, used as free inert fluorescent tracers, the diffusion coefficient decreased from the monomer to the tetramer much more than expected for molecules free in aqueous solution. Still, the entire intranuclear chromatin network is freely accessible for small proteins up to the size of eGFP-tetramers, regardless of the chromatin density or cell line. Even the densest chromatin regions do not exclude free eGFP-monomers or multimers. PMID:19347038

  15. Secondary metabolite localization by autofluorescence in living plant cells.

    PubMed

    Talamond, Pascale; Verdeil, Jean-Luc; Conéjéro, Geneviève

    2015-01-01

    Autofluorescent molecules are abundant in plant cells and spectral images offer means for analyzing their spectra, yielding information on their accumulation and function. Based on their fluorescence characteristics, an imaging approach using multiphoton microscopy was designed to assess localization of the endogenous fluorophores in living plant cells. This method, which requires no previous treatment, provides an effective experimental tool for discriminating between multiple naturally-occurring fluorophores in living-tissues. Combined with advanced Linear Unmixing, the spectral analysis extends the possibilities and enables the simultaneous detection of fluorescent molecules reliably separating overlapping emission spectra. However, as with any technology, the possibility for artifactual results does exist. This methodological article presents an overview of the applications of tissular and intra-cellular localization of these intrinsic fluorophores in leaves and fruits (here for coffee and vanilla). This method will provide new opportunities for studying cellular environments and the behavior of endogenous fluorophores in the intracellular environment. PMID:25808147

  16. Engineered Upconversion Nanoparticles for Resolving Protein Interactions inside Living Cells.

    PubMed

    Drees, Christoph; Raj, Athira Naduviledathu; Kurre, Rainer; Busch, Karin B; Haase, Markus; Piehler, Jacob

    2016-09-12

    Upconversion nanoparticles (UCNPs) convert near-infrared into visible light at much lower excitation densities than those used in classic two-photon absorption microscopy. Here, we engineered <50 nm UCNPs for application as efficient lanthanide resonance energy transfer (LRET) donors inside living cells. By optimizing the dopant concentrations and the core-shell structure for higher excitation densities, we observed enhanced UCNP emission as well as strongly increased sensitized acceptor fluorescence. For the application of these UCNPs in complex biological environments, we developed a biocompatible surface coating functionalized with a nanobody recognizing green fluorescent protein (GFP). Thus, rapid and specific targeting to GFP-tagged fusion proteins in the mitochondrial outer membrane and detection of protein interactions by LRET in living cells was achieved. PMID:27510808

  17. Tensile Strength of Cell Walls of Living Cells 1

    PubMed Central

    Carpita, Nicholas C.

    1985-01-01

    A gas decompression technique was used to determine the breaking strength of cell walls of single cells. Breaking strengths of the bacterium Salmonella typhimurium and the unicellular green alga Chlamydomonas eugametos were 100 and 95 atmospheres, respectively, while those of sporophytes of the water mold Blastocladiella emersonii were 65 atmospheres, and those of suspension cultured cells of carrot were only 30 atmospheres. Estimation of wall tensile stress based on breaking pressures, cell radii, and estimation of wall thickness, indicates that microfibrillar walls are not necessarily stronger than walls of primitive organisms. Hence, alternative hypotheses for their evolution must be considered. PMID:16664436

  18. Automatic Detection of Single Fluorophores in Live Cells

    PubMed Central

    Mashanov, G. I.; Molloy, J. E.

    2007-01-01

    Recent developments in light microscopy enable individual fluorophores to be observed in aqueous conditions. Biological molecules, labeled with a single fluorophore, can be localized as isolated spots of light when viewed by optical microscopy. Total internal reflection fluorescence microscopy greatly reduces background fluorescence and allows single fluorophores to be observed inside living cells. This advance in live-cell imaging means that the spatial and temporal dynamics of individual molecules can be measured directly. Because of the stochastic nature of single molecule behavior a statistically meaningful number of individual molecules must be detected and their separate trajectories in space and time stored and analyzed. Here, we describe digital image processing methods that we have devised for automatic detection and tracking of hundreds of molecules, observed simultaneously, in vitro and within living cells. Using this technique we have measured the diffusive behavior of pleckstrin homology domains bound to phosphoinositide phospholipids at the plasma membrane of live cultured mammalian cells. We found that mobility of these membrane-bound protein domains is dominated by mobility of the lipid molecule to which they are attached and is highly temperature dependent. Movement of PH domains isolated from the tail region of myosin-10 is consistent with a simple random walk, whereas, diffusion of intact PLC-δ1 shows behavior inconsistent with a simple random walk. Movement is rapid over short timescales but much slower at longer timescales. This anomalous behavior can be explained by movement being restricted to membrane regions of 0.7 μm diameter. PMID:17208981

  19. Live Attenuated Borrelia burgdorferi Targeted Mutants in an Infectious Strain Background Protect Mice from Challenge Infection.

    PubMed

    Hahn, Beth L; Padmore, Lavinia J; Ristow, Laura C; Curtis, Michael W; Coburn, Jenifer

    2016-08-01

    Borrelia burgdorferi, B. garinii, and B. afzelii are all agents of Lyme disease in different geographic locations. If left untreated, Lyme disease can cause significant and long-term morbidity, which may continue after appropriate antibiotic therapy has been administered and live bacteria are no longer detectable. The increasing incidence and geographic spread of Lyme disease are renewing interest in the vaccination of at-risk populations. We took the approach of vaccinating mice with two targeted mutant strains of B. burgdorferi that, unlike the parental strain, are avirulent in mice. Mice vaccinated with both strains were protected against a challenge with the parental strain and a heterologous B. burgdorferi strain by either needle inoculation or tick bite. In ticks, the homologous strain was eliminated but the heterologous strain was not, suggesting that the vaccines generated a response to antigens that are produced by the bacteria both early in mammalian infection and in the tick. Partial protection against B. garinii infection was also conferred. Protection was antibody mediated, and reactivity to a variety of proteins was observed. These experiments suggest that live attenuated B. burgdorferi strains may be informative regarding the identification of protective antigens produced by the bacteria and recognized by the mouse immune system in vivo Further work may illuminate new candidates that are effective and safe for the development of Lyme disease vaccines. PMID:27335385

  20. Delivery of optical contrast agents using Triton-X100, part 1: reversible permeabilization of live cells for intracellular labeling

    NASA Astrophysics Data System (ADS)

    van de Ven, Anne L.; Adler-Storthz, Karen; Richards-Kortum, Rebecca

    2009-03-01

    Effective delivery of optical contrast agents into live cells remains a significant challenge. We sought to determine whether Triton-X100, a detergent commonly used for membrane isolation and protein purification, could be used to effectively and reversibly permeabilize live cells for delivery of targeted optical contrast agents. Although Triton-X100 is widely recognized as a good cell permeabilization agent, no systematic study has evaluated the efficiency, reproducibility, and reversibility of Triton-X100-mediated permeabilization in live mammalian cells. We report a series of studies to characterize macromolecule delivery in cells following Triton-X100 treatment. Using this approach, we demonstrate that molecules ranging from 1 to 150 kDa in molecular weight can be reproducibly delivered into live cells by controlling the moles of Triton-X100 relative to the number of cells to be treated. When Triton-X100 is administered at or near the minimum effective concentration, cell permeabilization is generally reversed within 24 h, and treated cells continue to proliferate and show metabolic activity during the restoration of membrane integrity. We conclude that Triton-X100 is a promising permeabilization agent for efficient and reproducible delivery of optical contrast agents into live mammalian cells.

  1. Novel cell identification: markerfree and suitable for living cells

    NASA Astrophysics Data System (ADS)

    Koch, S.; Walles, H.; Krause, K. H.; Baquié, M.; Hansmann, M. L.; Schuetze, K.

    2013-06-01

    Raman spectroscopy increasingly becomes a valuable analytical tool in biomedicine. A novel Raman microscope designed for biomedical applications was used to discriminate viability states and cell types of Hodgkin's disease as well as different neural and invading glioblastoma cells within a human engineered neural tissue (ENT).

  2. Catalytic Molecular Imaging of MicroRNA in Living Cells by DNA-Programmed Nanoparticle Disassembly.

    PubMed

    He, Xuewen; Zeng, Tao; Li, Zhi; Wang, Ganglin; Ma, Nan

    2016-02-24

    Molecular imaging is an essential tool for disease diagnostics and treatment. Direct imaging of low-abundance nucleic acids in living cells remains challenging because of the relatively low sensitivity and insufficient signal-to-background ratio of conventional molecular imaging probes. Herein, we report a class of DNA-templated gold nanoparticle (GNP)-quantum dot (QD) assembly-based probes for catalytic imaging of cancer-related microRNAs (miRNA) in living cells with signal amplification capacity. We show that a single miRNA molecule could catalyze the disassembly of multiple QDs with the GNP through a DNA-programmed thermodynamically driven entropy gain process, yielding significantly amplified QD photoluminescence (PL) for miRNA imaging. By combining the robust PL of QDs with the catalytic amplification strategy, three orders of magnitude improvement in detection sensitivity is achieved in comparison with non-catalytic imaging probe, which enables facile and accurate differentiation between cancer cells and normal cells by miRNA imaging in living cells. PMID:26694689

  3. Single-Molecule Studies of Integrins by AFM-Based Force Spectroscopy on Living Cells

    NASA Astrophysics Data System (ADS)

    Eibl, Robert H.

    The characterization of cell adhesion between two living cells at the single-molecule level, i.e., between one adhesion receptor and its counter-receptor, appears to be an experimental challenge. Atomic force microscopy (AFM) can be used in its force spectroscopy mode to determine unbinding forces of a single pair of adhesion receptors, even with a living cell as a probe. This chapter provides an overview of AFM force measurements of the integrin family of cell adhesion receptors and their ligands. A focus is given to major integrins expressed on leukocytes, such as lymphocyte function-associated antigen 1 (LFA-1) and very late antigen 4 (VLA-4). These receptors are crucial for leukocyte trafficking in health and disease. LFA-1 and VLA-1 can be activated within the bloodstream from a low-affinity to a high-affinity receptor by chemokines in order to adhere strongly to the vessel wall before the receptor-bearing leukocytes extravasate. The experimental considerations needed to provide near-physiological conditions for a living cell and to be able to measure adequate forces at the single-molecule level are discussed in detail. AFM technology has been developed into a modern and extremely sensitive tool in biomedical research. It appears now that AFM force spectroscopy could enter, within a few years, medical applications in diagnosis and therapy of cancer and autoimmune diseases.

  4. Nanomolar pyrophosphate detection and nucleus staining in living cells with simple terpyridine-Zn(II) complexes.

    PubMed

    Chao, Duobin; Ni, Shitan

    2016-01-01

    Great efforts have been made to develop fluorescent probes for pyrophosphate (PPi) detection. Nucleus staining with fluorescence microscopy has been also widely investigated. But fluorescent probes for PPi detection with high sensitivity in water medium and nucleus staining with low-cost non-precious metal complexes in living cells are still challenging. Herein, we report simple terpyridine-Zn(II) complexes for selective nanomolar PPi detection over ATP and ADP in water based on aggregation induced emission (AIE) and intramolecular charge transfer (ICT). In addition, these terpyridine-Zn(II) complexes were successfully employed for nucleus staining in living cells. These results demonstrated simply obtained terpyridine-Zn(II) complexes are powerful tool for PPi detection and the development of PPi-related studies. PMID:27198968

  5. Nanomolar pyrophosphate detection and nucleus staining in living cells with simple terpyridine–Zn(II) complexes

    NASA Astrophysics Data System (ADS)

    Chao, Duobin; Ni, Shitan

    2016-05-01

    Great efforts have been made to develop fluorescent probes for pyrophosphate (PPi) detection. Nucleus staining with fluorescence microscopy has been also widely investigated. But fluorescent probes for PPi detection with high sensitivity in water medium and nucleus staining with low–cost non–precious metal complexes in living cells are still challenging. Herein, we report simple terpyridine–Zn(II) complexes for selective nanomolar PPi detection over ATP and ADP in water based on aggregation induced emission (AIE) and intramolecular charge transfer (ICT). In addition, these terpyridine–Zn(II) complexes were successfully employed for nucleus staining in living cells. These results demonstrated simply obtained terpyridine–Zn(II) complexes are powerful tool for PPi detection and the development of PPi–related studies.

  6. Development and application of 2-color live-cell STED nanoscopy (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Allgeyer, Edward S.; Bottanelli, Francesca; Kromann, Emil B.; Hao, Xiang; Bewersdorf, Joerg

    2016-02-01

    Stimulated emission depletion (STED) microscopy has been established as an important technique for imaging below the diffraction limit facilitating new discoveries in an array of biological systems. In STED microscopy a "donut-shaped" laser focus is super-imposed onto the diffraction-limited focus of an excitation laser. The dounut-shaped beam suppresses fluorescence in the periphery of the excitation spot, reducing the effective point spread function to a sub-diffraction size. However, the application of multicolor STED microscopy in living cells poses a number of challenges. Here we detail a novel STED system specifically designed for two-color STED applications. Our system employs FPGA-based gated detection and fast beam scanning to reduce pixel dwell time and photobleaching. We demonstrate the instrument's capability with two-color continuous imaging of intracellular targets below the diffraction limit allowing observation of rare events within live-cells.

  7. Nanomolar pyrophosphate detection and nucleus staining in living cells with simple terpyridine–Zn(II) complexes

    PubMed Central

    Chao, Duobin; Ni, Shitan

    2016-01-01

    Great efforts have been made to develop fluorescent probes for pyrophosphate (PPi) detection. Nucleus staining with fluorescence microscopy has been also widely investigated. But fluorescent probes for PPi detection with high sensitivity in water medium and nucleus staining with low–cost non–precious metal complexes in living cells are still challenging. Herein, we report simple terpyridine–Zn(II) complexes for selective nanomolar PPi detection over ATP and ADP in water based on aggregation induced emission (AIE) and intramolecular charge transfer (ICT). In addition, these terpyridine–Zn(II) complexes were successfully employed for nucleus staining in living cells. These results demonstrated simply obtained terpyridine–Zn(II) complexes are powerful tool for PPi detection and the development of PPi–related studies. PMID:27198968

  8. Selective, rapid and optically switchable regulation of protein function in live mammalian cells

    NASA Astrophysics Data System (ADS)

    Tsai, Yu-Hsuan; Essig, Sebastian; James, John R.; Lang, Kathrin; Chin, Jason W.

    2015-07-01

    The rapid and selective regulation of a target protein within living cells that contain closely related family members is an outstanding challenge. Here we introduce genetically directed bioorthogonal ligand tethering (BOLT) and demonstrate selective inhibition (iBOLT) of protein function. In iBOLT, inhibitor-conjugate/target protein pairs are created where the target protein contains a genetically encoded unnatural amino acid with bioorthogonal reactivity and the inhibitor conjugate contains a complementary bioorthogonal group. iBOLT enables the first rapid and specific inhibition of MEK isozymes, and introducing photoisomerizable linkers in the inhibitor conjugate enables reversible, optical regulation of protein activity (photo-BOLT) in live mammalian cells. We demonstrate that a pan kinase inhibitor conjugate allows selective and rapid inhibition of the lymphocyte specific kinase, indicating the modularity and scalability of BOLT. We anticipate that BOLT will enable the rapid and selective regulation of diverse proteins for which no selective small-molecule ligands exist.

  9. Single mRNA Tracking in Live Cells

    PubMed Central

    Park, Hye Yoon; Buxbaum, Adina R.; Singer, Robert H.

    2011-01-01

    Asymmetric distribution of mRNA is a prevalent phenomenon observed in diverse cell types. The posttranscriptional movement and localization of mRNA provides an important mechanism to target certain proteins to specific cytoplasmic regions of their function. Recent technical advances have enabled real-time visualization of single mRNA molecules in living cells. Studies analyzing the motion of individual mRNAs have shed light on the complex RNA transport system. This chapter presents an overview of general approaches for single particle tracking and some methodologies that are used for single mRNA detection. PMID:20580973

  10. Th17 memory cells: live long and proliferate.

    PubMed

    McGeachy, Mandy J

    2013-11-01

    The development of immune memory is a double-edged sword, helping to maintain health by preventing repeated infections but also driving chronic inflammation when dysregulated. Th17 cells are now well-known as major drivers of autoimmune disease but also play roles in protective immune responses against pathogens. This mini-review will focus on the recent evidence for long-lived, robust Th17 memory cell populations in mouse models and humans, and their functional roles in mediating host protection and chronic disease states. PMID:24006508

  11. Photoacoustic recovery after photothermal bleaching in living cells

    PubMed Central

    Li, Chiye; Zhang, Chi; Gao, Liang; Garcia-Uribe, Alejandro

    2013-01-01

    Abstract. We present an innovative method, photoacoustic recovery after photothermal bleaching (PRAP), for studying particle dynamics at micron scale via photoacoustic imaging. As an intuitive way to visualize and quantify dynamic processes, PRAP is demonstrated first in a simple phantom study and then in a more complex measurement involving live cells. Compared with the conventional fluorescence-based approach, PRAP provides high signal-to-noise ratio (SNR) imaging with minimal bleaching-induced artifacts during the recovery stage, ideal for monitoring the diffusive and kinetic processes inside a cell. PMID:24089253

  12. A Novel Ex Vivo Method for Visualizing Live-Cell Calcium Response Behavior in Intact Human Tumors

    PubMed Central

    Sosa, Julie A.

    2016-01-01

    The functional impact of intratumoral heterogeneity has been difficult to assess in the absence of a means to interrogate dynamic, live-cell biochemical events in the native tissue context of a human tumor. Conventional histological methods can reveal morphology and static biomarker expression patterns but do not provide a means to probe and evaluate tumor functional behavior and live-cell responsiveness to experimentally controlled stimuli. Here, we describe an approach that couples vibratome-mediated viable tissue sectioning with live-cell confocal microscopy imaging to visualize human parathyroid adenoma tumor cell responsiveness to extracellular calcium challenge. Tumor sections prepared as 300 micron-thick tissue slices retain viability throughout a >24 hour observation period and retain the native architecture of the parental tumor. Live-cell observation of biochemical signaling in response to extracellular calcium challenge in the intact tissue slices reveals discrete, heterogeneous kinetic waveform categories of calcium agonist reactivity within each tumor. Plotting the proportion of maximally responsive tumor cells as a function of calcium concentration yields a sigmoid dose-response curve with a calculated calcium EC50 value significantly elevated above published reference values for wild-type calcium-sensing receptor (CASR) sensitivity. Subsequent fixation and immunofluorescence analysis of the functionally evaluated tissue specimens allows alignment and mapping of the physical characteristics of individual cells within the tumor to specific calcium response behaviors. Evaluation of the relative abundance of intracellular PTH in tissue slices challenged with variable calcium concentrations demonstrates that production of the hormone can be dynamically manipulated ex vivo. The capability of visualizing live human tumor tissue behavior in response to experimentally controlled conditions opens a wide range of possibilities for personalized ex vivo

  13. Lived challenges and getting through them: Alaska Native youth narratives as a way to understand resilience.

    PubMed

    Wexler, Lisa; Jernigan, Kasey; Mazzotti, Janet; Baldwin, Elizabeth; Griffin, Megan; Joule, Linda; Garoutte, Joe

    2014-01-01

    Because of imposed rapid social change, Alaska Native youth are growing up in a context different from their elders and suffering far worse health and behavioral outcomes. This research seeks to understand (a) their everyday struggles and life challenges, (b) the practices and resources they rely on to get through challenges, and (c) the meaning they make from these experiences. Data were generated from interviews with 20 Alaska Native youth between the ages of 11 and 18 years, balanced by gender and age-group (early and late adolescence). Purposive sampling identified participants with a broad range of experiences. Following a semistructured guide, youth participated in face-to-face, audio-recorded interviews, transcribed verbatim. A codebook was developed using an iterative process and transcripts were coded using ATLAS.ti. The most commonly identified stressors were relationship loss, "not being there for me," nonsupportive/hostile experiences, transitioning into adulthood, and boredom. Resilience strategies included developing and maintaining relationships with others, being responsible, creating systems of reciprocity, practicing subsistence living, and giving back to family and the community. These opportunities allowed youth to gain a sense of competence and mastery. When difficult experiences align with opportunities for being responsible and competent, youth are most likely to exhibit resilience. PMID:23431127

  14. Cationic nanoparticles induce nanoscale disruption in living cell plasma membranes.

    PubMed

    Chen, Jiumei; Hessler, Jessica A; Putchakayala, Krishna; Panama, Brian K; Khan, Damian P; Hong, Seungpyo; Mullen, Douglas G; Dimaggio, Stassi C; Som, Abhigyan; Tew, Gregory N; Lopatin, Anatoli N; Baker, James R; Holl, Mark M Banaszak; Orr, Bradford G

    2009-08-13

    It has long been recognized that cationic nanoparticles induce cell membrane permeability. Recently, it has been found that cationic nanoparticles induce the formation and/or growth of nanoscale holes in supported lipid bilayers. In this paper, we show that noncytotoxic concentrations of cationic nanoparticles induce 30-2000 pA currents in 293A (human embryonic kidney) and KB (human epidermoid carcinoma) cells, consistent with a nanoscale defect such as a single hole or group of holes in the cell membrane ranging from 1 to 350 nm(2) in total area. Other forms of nanoscale defects, including the nanoparticle porating agents adsorbing onto or intercalating into the lipid bilayer, are also consistent; although the size of the defect must increase to account for any reduction in ion conduction, as compared to a water channel. An individual defect forming event takes 1-100 ms, while membrane resealing may occur over tens of seconds. Patch-clamp data provide direct evidence for the formation of nanoscale defects in living cell membranes. The cationic polymer data are compared and contrasted with patch-clamp data obtained for an amphiphilic phenylene ethynylene antimicrobial oligomer (AMO-3), a small molecule that is proposed to make well-defined 3.4 nm holes in lipid bilayers. Here, we observe data that are consistent with AMO-3 making approximately 3 nm holes in living cell membranes. PMID:19606833

  15. Live cell imaging with chemical specificity using dual frequency CARS microscopy.

    PubMed

    Pope, Iestyn; Langbein, Wolfgang; Borri, Paola; Watson, Peter

    2012-01-01

    Live cell microscopy using fluorescent proteins and small fluorescent probes is a well-established and essential tool for cell biology; however, there is a considerable need for noninvasive techniques able to study tissue and cell dynamics without the need to introduce chemical or genetically encoded probes. Coherent anti-Stokes Raman scattering (CARS) microscopy is an emerging tool for cell biologists to examine live cell dynamics with chemical specificity in a label-free, noninvasive way. CARS is a multiphoton process offering intrinsic three-dimensional submicron resolution, where the image contrast is obtained from light inelastically scattered by the vibrations of endogenous chemical bonds. CARS is particularly well suited to study lipid biology, since the CARS signal of localized lipids (exhibiting a large amount of identical bonds in the focal volume) is very strong. Conversely, photostable, lipid-specific markers for fluorescence microscopy are difficult to produce and the process of labeling often affects lipid localization and function, making imaging lipids in live cells challenging, and accurate quantification often impossible. Here, we describe in detail the principles behind our experimental setup for performing CARS microscopy of lipid droplets on live cells. Since typical vibrational resonances in liquid have coherence times in the picosecond range, CARS is preferably implemented with picosecond lasers which are however expensive and less efficient than femtosecond lasers, which could also be used for other multiphoton techniques such as two-photon fluorescence. In our setup, we show that femtosecond lasers can be spectrally focused in a simple, alignment insensitive, and cost-effective way to achieve a vibrational excitation similar to picosecond lasers. This opens the way to integrate CARS and two-photon fluorescence in a single multimodal instrument for its widespread application. We also describe our dual frequency CARS system which eliminates

  16. Increased influenza pneumonia mortality of mice adoptively immunized with node and spleen cells sensitized by inactivated but not live virus.

    PubMed Central

    Cate, T R; Mold, N G

    1975-01-01

    Syngeneic mice adoptively immunized intravenously with 25 million washed node and spleen cells from donors vaccinated subcutaneously with formolized influenza A PR8 had a higher mortality with influenza pneumonia after challenge with homologous virus than occurred in recipients of similar cells from unsensitized donors, and this increased mortality was prevented by treatment of the sensitized cells with antithymocyte serum. Mice adoptively immunized with cells from donors vaccinated with formolized influenza A PR8 also had a higher mortality than recipients of unsensitized cells after challenge with heterologous influenza B Lee. Mice who received PR8-sensitized cells and survived challenge with influenza B Lee developed antibody only to the challenge virus, and serum antibody titers to the challenge virus in surviving recipients of sensitized cells were similar to those of recipients of unsensitized cells in all studies. Influenza mortality of recipients of antibody-containing mouse serum after homologous virus challenge was similar to that of recipients of antibody-free mouse serum in this model. Washed node and spleen cells from donor mice who had survived respiratory infection or received subcutaneous vaccination with live influenza A PR8 and those from donor mice given typhoid vaccine subcutaneously all failed to alter mortality from that observed in recipients of unsensitized cells after challenge with influenza A PR8. These results suggest that subcutaneous vaccination with inactivated influenza establishes a reactivity of the cell-mediated immunologic system which can increase the severity of influenza infection of the respiratory tract under certain conditions, and that sensitization by live influenza fails to produce this effect. PMID:47313

  17. Real-time transposable element activity in individual live cells

    PubMed Central

    Lee, Gloria; Martini, K. Michael

    2016-01-01

    The excision and reintegration of transposable elements (TEs) restructure their host genomes, generating cellular diversity involved in evolution, development, and the etiology of human diseases. Our current knowledge of TE behavior primarily results from bulk techniques that generate time and cell ensemble averages, but cannot capture cell-to-cell variation or local environmental and temporal variability. We have developed an experimental system based on the bacterial TE IS608 that uses fluorescent reporters to directly observe single TE excision events in individual cells in real time. We find that TE activity depends upon the TE’s orientation in the genome and the amount of transposase protein in the cell. We also find that TE activity is highly variable throughout the lifetime of the cell. Upon entering stationary phase, TE activity increases in cells hereditarily predisposed to TE activity. These direct observations demonstrate that real-time live-cell imaging of evolution at the molecular and individual event level is a powerful tool for the exploration of genome plasticity in stressed cells. PMID:27298350

  18. Real-time transposable element activity in individual live cells.

    PubMed

    Kim, Neil H; Lee, Gloria; Sherer, Nicholas A; Martini, K Michael; Goldenfeld, Nigel; Kuhlman, Thomas E

    2016-06-28

    The excision and reintegration of transposable elements (TEs) restructure their host genomes, generating cellular diversity involved in evolution, development, and the etiology of human diseases. Our current knowledge of TE behavior primarily results from bulk techniques that generate time and cell ensemble averages, but cannot capture cell-to-cell variation or local environmental and temporal variability. We have developed an experimental system based on the bacterial TE IS608 that uses fluorescent reporters to directly observe single TE excision events in individual cells in real time. We find that TE activity depends upon the TE's orientation in the genome and the amount of transposase protein in the cell. We also find that TE activity is highly variable throughout the lifetime of the cell. Upon entering stationary phase, TE activity increases in cells hereditarily predisposed to TE activity. These direct observations demonstrate that real-time live-cell imaging of evolution at the molecular and individual event level is a powerful tool for the exploration of genome plasticity in stressed cells. PMID:27298350

  19. Live cell imaging of membrane / cytoskeleton interactions and membrane topology

    NASA Astrophysics Data System (ADS)

    Chierico, Luca; Joseph, Adrian S.; Lewis, Andrew L.; Battaglia, Giuseppe

    2014-09-01

    We elucidate the interaction between actin and specific membrane components, using real time live cell imaging, by delivering probes that enable access to components, that cannot be accessed genetically. We initially investigated the close interplay between Phosphatidylinositol 4,5-bisphosphate (PIP2) and the F-actin network. We show that, during the early stage of cell adhesion, PIP2 forms domains within the filopodia membrane. We studied these domains alongside cell spreading and observed that these very closely follow the actin tread-milling. We show that this mechanism is associated with an active transport of PIP2 rich organelles from the cell perinuclear area to the edge, along actin fibers. Finally, mapping other phospholipids and membrane components we observed that the PIP2 domains formation is correlated with sphingosine and cholesterol rafts.

  20. Miniaturized biological and electrochemical fuel cells: challenges and applications.

    PubMed

    Yang, Jie; Ghobadian, Sasan; Goodrich, Payton J; Montazami, Reza; Hashemi, Nastaran

    2013-09-14

    This paper discusses the fundamentals and developments of miniaturized fuel cells, both biological and electrochemical. An overview of microfluidic fuel cells, miniaturized microbial fuel cells, enzymatic biofuel cells, and implanted biofuel cells in an attempt to provide green energy and to power implanted microdevices is provided. Also, the challenges and applications of each type of fuel cell are discussed in detail. Most recent developments in fuel cell technologies such as novel catalysts, compact designs, and fabrication methods are reviewed. PMID:23503374

  1. Simulations of Living Cell Origins Using a Cellular Automata Model

    NASA Astrophysics Data System (ADS)

    Ishida, Takeshi

    2014-04-01

    Understanding the generalized mechanisms of cell self-assembly is fundamental for applications in various fields, such as mass producing molecular machines in nanotechnology. Thus, the details of real cellular reaction networks and the necessary conditions for self-organized cells must be elucidated. We constructed a 2-dimensional cellular automata model to investigate the emergence of biological cell formation, which incorporated a looped membrane and a membrane-bound information system (akin to a genetic code and gene expression system). In particular, with an artificial reaction system coupled with a thermal system, the simultaneous formation of a looped membrane and an inner reaction process resulted in a more stable structure. These double structures inspired the primitive biological cell formation process from chemical evolution stage. With a model to simulate cellular self-organization in a 2-dimensional cellular automata model, 3 phenomena could be realized: (1) an inner reaction system developed as an information carrier precursor (akin to DNA); (2) a cell border emerged (akin to a cell membrane); and (3) these cell structures could divide into 2. This double-structured cell was considered to be a primary biological cell. The outer loop evolved toward a lipid bilayer membrane, and inner polymeric particles evolved toward precursor information carriers (evolved toward DNA). This model did not completely clarify all the necessary and sufficient conditions for biological cell self-organization. Further, our virtual cells remained unstable and fragile. However, the "garbage bag model" of Dyson proposed that the first living cells were deficient; thus, it would be reasonable that the earliest cells were more unstable and fragile than the simplest current unicellular organisms.

  2. Visualizing dopamine released from living cells using a nanoplasmonic probe

    NASA Astrophysics Data System (ADS)

    Qin, W. W.; Wang, S. P.; Li, J.; Peng, T. H.; Xu, Y.; Wang, K.; Shi, J. Y.; Fan, C. H.; Li, D.

    2015-09-01

    We report the development of an ultrasensitive nanoplasmonic probe for discriminative detection and imaging of dopamine released from living cells. The sensing mechanism is based on the dopamine-induced seeded-growth of Au nanoparticles (Au NPs) that leads to the shift of the plasmon band. This platform allows for the detection of dopamine with a detection limit down to 0.25 pM within 1 min. This nanoplasmonic assay is further applied to visualize the release of dopamine from living rat pheochromocytoma (PC12) cells under ATP-stimulation with dark-field microscopy (DFM). The DFM results together with real time fluorescence imaging of PC12 cells stained with the Fluo calcium indicator, suggested that ATP stimulated-release of dopamine is concomitant with the Ca2+ influx, and the influx of Ca2+ is through ATP-activated channels instead of the voltage-gated Ca2+ channel (VGC).We report the development of an ultrasensitive nanoplasmonic probe for discriminative detection and imaging of dopamine released from living cells. The sensing mechanism is based on the dopamine-induced seeded-growth of Au nanoparticles (Au NPs) that leads to the shift of the plasmon band. This platform allows for the detection of dopamine with a detection limit down to 0.25 pM within 1 min. This nanoplasmonic assay is further applied to visualize the release of dopamine from living rat pheochromocytoma (PC12) cells under ATP-stimulation with dark-field microscopy (DFM). The DFM results together with real time fluorescence imaging of PC12 cells stained with the Fluo calcium indicator, suggested that ATP stimulated-release of dopamine is concomitant with the Ca2+ influx, and the influx of Ca2+ is through ATP-activated channels instead of the voltage-gated Ca2+ channel (VGC). Electronic supplementary information (ESI) available: Fig. S1-S4 and Table S1. See DOI: 10.1039/c5nr04433b

  3. Visualization and targeted disruption of protein interactions in living cells

    PubMed Central

    Herce, Henry D.; Deng, Wen; Helma, Jonas; Leonhardt, Heinrich; Cardoso, M. Cristina

    2013-01-01

    Protein–protein interactions are the basis of all processes in living cells, but most studies of these interactions rely on biochemical in vitro assays. Here we present a simple and versatile fluorescent-three-hybrid (F3H) strategy to visualize and target protein–protein interactions. A high-affinity nanobody anchors a GFP-fusion protein of interest at a defined cellular structure and the enrichment of red-labelled interacting proteins is measured at these sites. With this approach, we visualize the p53–HDM2 interaction in living cells and directly monitor the disruption of this interaction by Nutlin 3, a drug developed to boost p53 activity in cancer therapy. We further use this approach to develop a cell-permeable vector that releases a highly specific peptide disrupting the p53 and HDM2 interaction. The availability of multiple anchor sites and the simple optical readout of this nanobody-based capture assay enable systematic and versatile analyses of protein–protein interactions in practically any cell type and species. PMID:24154492

  4. Visualization and targeted disruption of protein interactions in living cells.

    PubMed

    Herce, Henry D; Deng, Wen; Helma, Jonas; Leonhardt, Heinrich; Cardoso, M Cristina

    2013-01-01

    Protein-protein interactions are the basis of all processes in living cells, but most studies of these interactions rely on biochemical in vitro assays. Here we present a simple and versatile fluorescent-three-hybrid (F3H) strategy to visualize and target protein-protein interactions. A high-affinity nanobody anchors a GFP-fusion protein of interest at a defined cellular structure and the enrichment of red-labelled interacting proteins is measured at these sites. With this approach, we visualize the p53-HDM2 interaction in living cells and directly monitor the disruption of this interaction by Nutlin 3, a drug developed to boost p53 activity in cancer therapy. We further use this approach to develop a cell-permeable vector that releases a highly specific peptide disrupting the p53 and HDM2 interaction. The availability of multiple anchor sites and the simple optical readout of this nanobody-based capture assay enable systematic and versatile analyses of protein-protein interactions in practically any cell type and species. PMID:24154492

  5. Practical fabrication of microfluidic platforms for live-cell microscopy.

    PubMed

    Lorusso, Daniel; Nikolov, Hristo N; Milner, Jaques S; Ochotny, Noelle M; Sims, Stephen M; Dixon, S Jeffrey; Holdsworth, David W

    2016-10-01

    We describe a simple fabrication technique - targeted towards non-specialists - that allows for the production of leak-proof polydimethylsiloxane (PDMS) microfluidic devices that are compatible with live-cell microscopy. Thin PDMS base membranes were spin-coated onto a glass-bottom cell culture dish and then partially cured via microwave irradiation. PDMS chips were generated using a replica molding technique, and then sealed to the PDMS base membrane by microwave irradiation. Once a mold was generated, devices could be rapidly fabricated within hours. Fibronectin pre-treatment of the PDMS improved cell attachment. Coupling the device to programmable pumps allowed application of precise fluid flow rates through the channels. The transparency and minimal thickness of the device enabled compatibility with inverted light microscopy techniques (e.g. phase-contrast, fluorescence imaging, etc.). The key benefits of this technique are the use of standard laboratory equipment during fabrication and ease of implementation, helping to extend applications in live-cell microfluidics for scientists outside the engineering and core microdevice communities. PMID:27523472

  6. Visualizing light-triggered release of molecules inside living cells

    PubMed Central

    Barhoumi, Aoune; Halas, Naomi J.

    2013-01-01

    The light-triggered release of deoxyribonucleic acid (DNA) from gold nanoparticle-based, plasmon resonant vectors, such as nanoshells, shows great promise for gene delivery in living cells. Here we show that intracellular light-triggered release can be performed on molecules that associate with the DNA in a DNA host-guest complex bound to nanoshells. DAPI (4′,6-diamidino-2-phenylindole), a bright blue fluorescent molecule that binds reversibly to double-stranded DNA, was chosen to visualize this intracellular light-induced release process. Illumination of nanoshell-dsDNA-DAPI complexes at their plasmon resonance wavelength dehybridizes the DNA, releasing the DAPI molecules within living cells, where they diffuse to the nucleus and associate with the cell's endogenous DNA. The low laser power and irradiation times required for molecular release do not compromise cell viability. This highly controlled co-release of nonbiological molecules accompanying the oligonucleotides could have broad applications in the study of cellular processes and in the development of intracellular targeted therapies. PMID:20857946

  7. Intravital live cell triggered imaging system reveals monocyte patrolling and macrophage migration in atherosclerotic arteries

    PubMed Central

    McArdle, Sara; Chodaczek, Grzegorz; Ray, Nilanjan; Ley, Klaus

    2015-01-01

    Abstract. Intravital multiphoton imaging of arteries is technically challenging because the artery expands with every heartbeat, causing severe motion artifacts. To study leukocyte activity in atherosclerosis, we developed the intravital live cell triggered imaging system (ILTIS). This system implements cardiac triggered acquisition as well as frame selection and image registration algorithms to produce stable movies of myeloid cell movement in atherosclerotic arteries in live mice. To minimize tissue damage, no mechanical stabilization is used and the artery is allowed to expand freely. ILTIS performs multicolor high frame-rate two-dimensional imaging and full-thickness three-dimensional imaging of beating arteries in live mice. The external carotid artery and its branches (superior thyroid and ascending pharyngeal arteries) were developed as a surgically accessible and reliable model of atherosclerosis. We use ILTIS to demonstrate Cx3cr1GFP monocytes patrolling the lumen of atherosclerotic arteries. Additionally, we developed a new reporter mouse (Apoe−/−Cx3cr1GFP/+Cd11cYFP) to image GFP+ and GFP+YFP+ macrophages “dancing on the spot” and YFP+ macrophages migrating within intimal plaque. ILTIS will be helpful to answer pertinent open questions in the field, including monocyte recruitment and transmigration, macrophage and dendritic cell activity, and motion of other immune cells. PMID:25710308

  8. Intravital live cell triggered imaging system reveals monocyte patrolling and macrophage migration in atherosclerotic arteries

    NASA Astrophysics Data System (ADS)

    McArdle, Sara; Chodaczek, Grzegorz; Ray, Nilanjan; Ley, Klaus

    2015-02-01

    Intravital multiphoton imaging of arteries is technically challenging because the artery expands with every heartbeat, causing severe motion artifacts. To study leukocyte activity in atherosclerosis, we developed the intravital live cell triggered imaging system (ILTIS). This system implements cardiac triggered acquisition as well as frame selection and image registration algorithms to produce stable movies of myeloid cell movement in atherosclerotic arteries in live mice. To minimize tissue damage, no mechanical stabilization is used and the artery is allowed to expand freely. ILTIS performs multicolor high frame-rate two-dimensional imaging and full-thickness three-dimensional imaging of beating arteries in live mice. The external carotid artery and its branches (superior thyroid and ascending pharyngeal arteries) were developed as a surgically accessible and reliable model of atherosclerosis. We use ILTIS to demonstrate Cx3cr1GFP monocytes patrolling the lumen of atherosclerotic arteries. Additionally, we developed a new reporter mouse (Apoe-/-Cx3cr1GFP/+Cd11cYFP) to image GFP+ and GFP+YFP+ macrophages "dancing on the spot" and YFP+ macrophages migrating within intimal plaque. ILTIS will be helpful to answer pertinent open questions in the field, including monocyte recruitment and transmigration, macrophage and dendritic cell activity, and motion of other immune cells.

  9. Fourier-transform infrared spectroscopy for rapid screening and live-cell monitoring: application to nanotoxicology

    SciTech Connect

    Sundaram, S. K.; Sacksteder, Colette A.; Weber, T. J.; Riley, Brian J.; Addleman, Raymond S.; Harrer, Bruce J.; Peterman, John W.

    2013-01-01

    A significant challenge to realize the full potential of nanotechnology for therapeutic and diagnostic applications is to understand and evaluate how live-cells interact with an external stimulus, e.g., a nanosized particle (NSP), and the toxicity and broad risk associated with these stimuli. NSPs are increasingly used in medicine with largely undetermined hazards in complex cell dynamics and environments. It is difficult to capture the complexity and dynamics of these interactions by following an omics-based approach exclusively, which are expensive and time-consuming. Additionally, this approach needs destructive sampling methods. Live-cell attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectrometry is well suited to provide noninvasive approach to provide rapid screening of cellular responses to potentially toxic NSPs or any stimuli. Herein we review the technical basis of the approach, the instrument configuration and interface with the biological media, and various effects that impact the data, data analysis, and toxicity. Our preliminary results on live-cell monitoring show promise for rapid screening the NSPs.

  10. Intravital live cell triggered imaging system reveals monocyte patrolling and macrophage migration in atherosclerotic arteries.

    PubMed

    McArdle, Sara; Chodaczek, Grzegorz; Ray, Nilanjan; Ley, Klaus

    2015-02-01

    Intravital multiphoton imaging of arteries is technically challenging because the artery expands with every heartbeat, causing severe motion artifacts. To study leukocyte activity in atherosclerosis, we developed the intravital live cell triggered imaging system (ILTIS). This system implements cardiac triggered acquisition as well as frame selection and image registration algorithms to produce stable movies of myeloid cell movement in atherosclerotic arteries in live mice. To minimize tissue damage, no mechanical stabilization is used and the artery is allowed to expand freely. ILTIS performs multicolor high frame-rate two-dimensional imaging and full-thickness three-dimensional imaging of beating arteries in live mice. The external carotid artery and its branches (superior thyroid and ascending pharyngeal arteries) were developed as a surgically accessible and reliable model of atherosclerosis. We use ILTIS to demonstrate Cx3cr1GFP monocytes patrolling the lumen of atherosclerotic arteries. Additionally, we developed a new reporter mouse (Apoe−/−Cx3cr1GFP/+Cd11cYFP) to image GFP+ and GFP+YFP + macrophages “dancing on the spot” and YFP+ macrophages migrating within intimal plaque. ILTIS will be helpful to answer pertinent open questions in the field, including monocyte recruitment and transmigration, macrophage and dendritic cell activity, and motion of other immune cells. PMID:25710308