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Sample records for live cell movies

  1. Teachable, high-content analytics for live-cell, phase contrast movies.

    PubMed

    Alworth, Samuel V; Watanabe, Hirotada; Lee, James S J

    2010-09-01

    CL-Quant is a new solution platform for broad, high-content, live-cell image analysis. Powered by novel machine learning technologies and teach-by-example interfaces, CL-Quant provides a platform for the rapid development and application of scalable, high-performance, and fully automated analytics for a broad range of live-cell microscopy imaging applications, including label-free phase contrast imaging. The authors used CL-Quant to teach off-the-shelf universal analytics, called standard recipes, for cell proliferation, wound healing, cell counting, and cell motility assays using phase contrast movies collected on the BioStation CT and BioStation IM platforms. Similar to application modules, standard recipes are intended to work robustly across a wide range of imaging conditions without requiring customization by the end user. The authors validated the performance of the standard recipes by comparing their performance with truth created manually, or by custom analytics optimized for each individual movie (and therefore yielding the best possible result for the image), and validated by independent review. The validation data show that the standard recipes' performance is comparable with the validated truth with low variation. The data validate that the CL-Quant standard recipes can provide robust results without customization for live-cell assays in broad cell types and laboratory settings. PMID:20639505

  2. Interdisciplinarity, Debate And Movie Clips As Highly Motivating Factors In Live Shows - Five Years Of Success

    NASA Astrophysics Data System (ADS)

    Stengler, E.; Sirera, J. M.

    2011-09-01

    A live show on any subject that includes experiments and continuous interaction with the audience is a well known approach for EPO activities that many are carrying out all over. We present such an initiative with some added ingredients such as interdisciplinarity, the use of movie clips, and especially the debate between the two presenters, a debate that is all the more attractive to the public if it not fully staged but closely represents their actual points of view. José Montesinos, from the "Orotava" Canarian Foundation for the History of Science, is and plays the role of the more mature math professor who has grown weary of the overrated value given in science to mathematics and its consequences. This poses a constant challenge to his colleague, Erik Stengler, from the Science Museum of Tenerife, the young down-to-earth hands-on scientist, who defends the usual view that science and technology are to be judged by their achievements, which have brought about the advancement of modern society. With this approach and as a collaboration between our institutions, we have produced and toured highly successful activities on: Einstein and Relativity (from 2005 to 2008, "Einstein Goes To School," including a theatre play); circularity, the number π, forces of inertia and the Newtonian revolution (in 2008/2009, "The Tension Between Circularity and The Straight Line"); and the foundations of modern astronomy (in 2009/2010 "Kepler and Galileo, Messengers of the Stars"). Audiences were very varied - students, adult students, general public, prison inmates, teachers - and all appreciated the presentations as fun, thought-provoking and highly motivating, and valued especially the interdisciplinary character of the activity. Movie clips have shown to be especially useful to recover the attention of the young when they lose the thread due to the short attention spans they presently have.

  3. 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.

  4. Visual pattern discrimination by population retinal ganglion cells' activities during natural movie stimulation.

    PubMed

    Zhang, Ying-Ying; Wang, Ru-Bin; Pan, Xiao-Chuan; Gong, Hai-Qing; Liang, Pei-Ji

    2014-02-01

    In the visual system, neurons often fire in synchrony, and it is believed that synchronous activities of group neurons are more efficient than single cell response in transmitting neural signals to down-stream neurons. However, whether dynamic natural stimuli are encoded by dynamic spatiotemporal firing patterns of synchronous group neurons still needs to be investigated. In this paper we recorded the activities of population ganglion cells in bullfrog retina in response to time-varying natural images (natural scene movie) using multi-electrode arrays. In response to some different brief section pairs of the movie, synchronous groups of retinal ganglion cells (RGCs) fired with similar but different spike events. We attempted to discriminate the movie sections based on temporal firing patterns of single cells and spatiotemporal firing patterns of the synchronous groups of RGCs characterized by a measurement of subsequence distribution discrepancy. The discrimination performance was assessed by a classification method based on Support Vector Machines. Our results show that different movie sections of the natural movie elicited reliable dynamic spatiotemporal activity patterns of the synchronous RGCs, which are more efficient in discriminating different movie sections than the temporal patterns of the single cells' spike events. These results suggest that, during natural vision, the down-stream neurons may decode the visual information from the dynamic spatiotemporal patterns of the synchronous group of RGCs' activities. PMID:24465283

  5. 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

  6. 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

  7. 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

  8. 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.

  9. p53 as Batman: using a movie plot to understand control of the cell cycle.

    PubMed

    Gadi, Nikhita; Foley, Sage E; Nowey, Mark; Plopper, George E

    2013-01-01

    This Teaching Resource provides and describes a two-part classroom exercise to help students understand control of the cell cycle, with a focus on the transcription factor p53, the E3 ubiquitin ligase Mdm2, the Mdm2 inhibitor ARF, the kinases ATM and ATR, the kinase Chk2, and the cell cycle inhibitor p21(Cip1). Students use characters and scenes from the movie The Dark Knight to represent elements of the cell cycle control machinery, then they apply these characters and scenes to translate a primary research article on p53 function into a new movie scene in the "Batman universe." This exercise is appropriate for college-level courses in cell biology and cancer biology and requires students to have a background in introductory cell biology. Explicit learning outcomes and associated assessment methods are provided, as well as slides, student assignments, the primary research article, and an instructor's guide for the exercise. PMID:23592841

  10. 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.

  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. Making Movies

    ERIC Educational Resources Information Center

    Crompton, Zoe; Davies, Emma

    2012-01-01

    Children enjoy making movies but can it help them to understand science? In this article, the authors discuss how creating stop-frame animations of salt dissolving can deepen children's understanding of this process. (Contains 1 figure.)

  13. 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

  14. 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

  15. 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

  16. Tvashtar Movie

    NASA Technical Reports Server (NTRS)

    2007-01-01

    [figure removed for brevity, see original site] Click on the image for QuickTime movie of Tvashtar Movie

    Using its Long Range Reconnaissance Imager (LORRI), the New Horizons spacecraft captured the two frames in this 'movie' of the 330-kilometer (200-mile) high Tvashtar volcanic eruption plume on Jupiter's moon Io on February 28, 2007, from a range of 2.7 million kilometers (1.7 million miles). The two images were taken 50 minutes apart, at 03:50 and 04:40 Universal Time, and because particles in the plume take an estimated 30 minutes to fall back to the surface after being ejected by the central volcano, each image likely shows an entirely different set of particles. The details of the plume structure look quite different in each frame, though the overall brightness and size of the plume remain constant.

    Surface details on the nightside of Io, faintly illuminated by Jupiter, show the 5-degree change in Io's central longitude, from 22 to 27 degrees west, between the two frames.

  17. ``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.

  18. Movies in Chemistry Education

    ERIC Educational Resources Information Center

    Pekdag, Bulent; Le Marechal, Jean-Francois

    2010-01-01

    This article reviews numerous studies on chemistry movies. Movies, or moving pictures, are important elements of multimedia and signify a privileged or motivating means of presenting knowledge. Studies on chemistry movies show that the first movie productions in this field were devoted to university lectures or documentaries. Shorter movies were…

  19. Movie Books: A Bibliography.

    ERIC Educational Resources Information Center

    Top of the News, 1984

    1984-01-01

    This 22-item annotated bibliography lists books dealing with various aspects of children's films: monsters, television production, filmmaking, kids of the movies, animation, movie stunts, magic, movie animals, and photography. Publisher, publication date, and intended grade level are included. (EJS)

  20. Millikan Movies

    NASA Astrophysics Data System (ADS)

    Zou, Xueli; Dietz, Eric; McGuire, Trevor; Fox, Louise; Norris, Tiara; Diamond, Brendan; Chavez, Ricardo; Cheng, Stephen

    2008-09-01

    Since Robert Millikan discovered the quantization of electric charge and measured its fundamental value over 90 years ago, his oil-drop experiment has become essential in physics laboratory classes at both the high school and college level. As physics instructors, however, many of us have used the traditional setup and experienced the tedium of collecting data and the frustration of students who obtain disappointing results for the charges on individual oil drops after two or three hours of hard work. Some novel approaches have been developed to make the data collection easier and more accurate. One method is to attach a CCD (charge coupled device) camera to the microscope of the traditional setup.1,2 Through the CCD camera, the motion of an oil drop can be displayed on a TV monitor and/or on a computer.2 This allows several students to view the image of a droplet simultaneously instead of taking turns squinting through the tiny microscope eyepiece on the traditional setup. Furthermore, the motion of an oil drop can be captured and analyzed using software such as VideoPoint,3 which enhances the accuracy of the measurement of the charge on each oil drop.2 While these innovative methods improve the convenience and efficiency with which data can be collected, an instructor has to invest a considerable amount of money and time so as to adapt the new techniques to his or her own classroom. In this paper, we will report on the QuickTime movies we made, which can be used to analyze the motions of 16 selected oil drops. These digital videos are available on the web4 for teachers to download and use with their own students. We will also share the procedure for analyzing the videos using Logger Pro,5 as well as our results for the charges on the oil drops and some pedagogical aspects of using the movies with students.

  1. 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

  2. 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.

  3. 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)

  4. Diabetes Movie (For Parents)

    MedlinePlus Videos and Cool Tools

    ... Story" 5 Things to Know About Zika & Pregnancy Diabetes Movie KidsHealth > For Parents > Diabetes Movie Print A A A Text Size Kids who have diabetes have trouble taking energy from food and delivering ...

  5. Electromagnetism in the Movies.

    ERIC Educational Resources Information Center

    Everitt, Lori R.; Patterson, Evelyn T.

    1999-01-01

    Describes how the authors used portions of popular movies to help students review concepts related to electromagnetism. Movies used and concepts covered in the review are listed, and a sample activity is described. (WRM)

  6. Movies in America.

    ERIC Educational Resources Information Center

    Kuhns, William

    Two main themes of motion picture development in America are presented in this comprehensive historical guide to movies. The sophistication and broadening of the movies as an art form and the complex relationships between a period and the movies of that period are fully explored. Particular emphasis has been placed on the role of the director.…

  7. 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

  8. 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

  9. 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

  10. 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

  11. 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

  12. 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

  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. 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.

  16. 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.

  17. 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.

  18. Live Imaging of Border Cell Migration in Drosophila.

    PubMed

    Dai, Wei; Montell, Denise J

    2016-01-01

    Border cells are a cluster of cells that migrate from the anterior tip of the Drosophila egg chamber to the border of the oocyte in stage 9. They serve as a useful model to study collective cell migration in a native tissue environment. Here we describe a protocol for preparing ex vivo egg chamber cultures from transgenic flies expressing fluorescent proteins in the border cells, and using confocal microscopy to take a multi-positional time-lapse movie. We include an image analysis method for tracking border cell cluster dynamics as well as tracking individual cell movements. PMID:27271901

  19. 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.

  20. 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

  1. 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.

  2. 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.

  3. Real-time visualization of prion transport in single live cells using quantum dots

    SciTech Connect

    Luo, Kan; Li, Shu; Xie, Min; Wu, Di; Wang, WenXi; Chen, Rui; Huang, Liqin; Huang, Tao; Pang, Daiwen; Xiao, Gengfu

    2010-04-09

    Prion diseases are fatal neurodegenerative disorders resulting from structural conversion of the cellular isoform of PrP{sup C} to the infectious scrapie isoform PrP{sup Sc}. It is believed that such structural alteration may occur within the internalization pathway. However, there is no direct evidence to support this hypothesis. Employing quantum dots (QDs) as a probe, we have recorded a real-time movie demonstrating the process of prion internalization in a living cell for the first time. The entire internalization process can be divided into four discrete but connected stages. In addition, using methyl-beta-cyclodextrin to disrupt cell membrane cholesterol, we show that lipid rafts play an important role in locating cellular PrP{sup C} to the cell membrane and in initiating PrP{sup C} endocytosis.

  4. 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.

  5. 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

  6. 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

  7. "Life" in Movies

    ERIC Educational Resources Information Center

    Berumen, Michael L.

    2008-01-01

    As biology teachers, we should embrace the ever-increasing appearance of biology in movies and other media as an opportunity to engage students in active learning and to facilitate critical-thinking and investigative skills in the classroom. In this article, the author provides examples and strategies from his experience using popular movies in…

  8. SteroMoviePlayer

    SciTech Connect

    Hodson, Steve; Pugmire, Dave

    2005-03-14

    StereoMoviePlayer StereoMoviePlayer (SMP) is a software package for creating and displaying stereo movies on a variety of computer architectures and display configuations. SMP is capable of running in serial, or in parallel to facilitate multiple computers driving a collection of display surfaces. SMP utilizes the standatd openGL gaphics library for display of both monoscopic and stereoscopic images and MPI for parallel communication and sychronization between multiple computers. SMP uses standard IO methods for loading movie files into memory and, when compressed movies are being displayed uses zLIB (which is standard in most Linux/Unix;/IRIX distributions) for decompression. A movie file is simply a concatenation of each frame. Each frame is a raw red/green/blue encoding. For stereoscopic movies, concatenation is left followed by right, as follows; Frame0-Left, Frame0-Right, Frame1-Left, Frame1-Right .... FrameN-Left, FrameN-Right To enhance performance, this concatenation of frames can be compressed using the aforementioned zLib compression/decompression library. ConvertMovie is a utility that converts between compressed and uncompressed movie formats. ConvertMovie uses zLib, which is included in most standard Linux/Unix/IRIX distributions for compression and decompression. StereoMoviePlayer consists of 3 main parts: 1-Initialization. Information is parsed from a configuration script that specifies machines on which to run, the movie file and the parameters for each graphics display. MPI is then used to instantiate a movie player on each specified computer. 2-Per-node initialization. Each parallel node creates 2 threads of execution, an IO thread and a display and communication thread. 3-Execution: The IO thread reads movie frames from disk, decompresses if necessary and places the frames in main memory. The display thread copies fromes from main memory to the graphics card for display. The display thread also handles synchronization among the other nodes

  9. 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.

  10. 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

  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. 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

  14. 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

  15. SteroMoviePlayer

    Energy Science and Technology Software Center (ESTSC)

    2005-03-14

    StereoMoviePlayer StereoMoviePlayer (SMP) is a software package for creating and displaying stereo movies on a variety of computer architectures and display configuations. SMP is capable of running in serial, or in parallel to facilitate multiple computers driving a collection of display surfaces. SMP utilizes the standatd openGL gaphics library for display of both monoscopic and stereoscopic images and MPI for parallel communication and sychronization between multiple computers. SMP uses standard IO methods for loading moviemore » files into memory and, when compressed movies are being displayed uses zLIB (which is standard in most Linux/Unix;/IRIX distributions) for decompression. A movie file is simply a concatenation of each frame. Each frame is a raw red/green/blue encoding. For stereoscopic movies, concatenation is left followed by right, as follows; Frame0-Left, Frame0-Right, Frame1-Left, Frame1-Right .... FrameN-Left, FrameN-Right To enhance performance, this concatenation of frames can be compressed using the aforementioned zLib compression/decompression library. ConvertMovie is a utility that converts between compressed and uncompressed movie formats. ConvertMovie uses zLib, which is included in most standard Linux/Unix/IRIX distributions for compression and decompression. StereoMoviePlayer consists of 3 main parts: 1-Initialization. Information is parsed from a configuration script that specifies machines on which to run, the movie file and the parameters for each graphics display. MPI is then used to instantiate a movie player on each specified computer. 2-Per-node initialization. Each parallel node creates 2 threads of execution, an IO thread and a display and communication thread. 3-Execution: The IO thread reads movie frames from disk, decompresses if necessary and places the frames in main memory. The display thread copies fromes from main memory to the graphics card for display. The display thread also handles synchronization among the other

  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. Lost moon, saved lives: using the movie Apollo 13 as a video primer in behavioral skills for simulation trainees and instructors.

    PubMed

    Halamek, Louis P

    2010-10-01

    Behavioral skills such as effective communication, teamwork, and leadership are critically important to successful outcomes in patient care, especially in resuscitation situations where correct decisions must be made rapidly. However, historically, these important skills have rarely been specifically addressed in learning programs directed at healthcare professionals. Not only have most healthcare professionals had little or no formal education and training in applying behavioral skills to their patient care activities but also many of those serving as instructors and content experts for training programs have few resources available that clearly illustrate what these skills are and how they may be used in the context of real clinical situations. This represents a serious shortcoming in the education and training of healthcare professionals and stands in distinct contrast to other industries.Aerospace, similar to other high-consequence industries, has a long history of the use of simulation to improve human performance and reduce risk: astronauts and the engineers in Mission Control spend hundreds of hours in simulated flight in preparation for every mission. The value of time spent in the simulator was clearly illustrated during the flight of Apollo 13, the third mission to land men on the moon. The Apollo 13 crew had to overcome a number of life-threatening technical and medical problems, and it was their simulation-based training that allowed them to display the teamwork, ingenuity, and determination needed to return to earth safely.The movie Apollo 13 depicts in a highly realistic manner the events that occurred during the flight, including the actions of the crew in space and those in Mission Control in Houston. Three scenes from this movie are described in this article; each serves as a useful example for healthcare professionals of the importance of simulation-based learning and the application of behavioral skills to successful resolution of crises. This

  18. 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

  19. 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.

  20. 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

  1. 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

  2. Collaborative Movie Annotation

    NASA Astrophysics Data System (ADS)

    Zad, Damon Daylamani; Agius, Harry

    In this paper, we focus on metadata for self-created movies like those found on YouTube and Google Video, the duration of which are increasing in line with falling upload restrictions. While simple tags may have been sufficient for most purposes for traditionally very short video footage that contains a relatively small amount of semantic content, this is not the case for movies of longer duration which embody more intricate semantics. Creating metadata is a time-consuming process that takes a great deal of individual effort; however, this effort can be greatly reduced by harnessing the power of Web 2.0 communities to create, update and maintain it. Consequently, we consider the annotation of movies within Web 2.0 environments, such that users create and share that metadata collaboratively and propose an architecture for collaborative movie annotation. This architecture arises from the results of an empirical experiment where metadata creation tools, YouTube and an MPEG-7 modelling tool, were used by users to create movie metadata. The next section discusses related work in the areas of collaborative retrieval and tagging. Then, we describe the experiments that were undertaken on a sample of 50 users. Next, the results are presented which provide some insight into how users interact with existing tools and systems for annotating movies. Based on these results, the paper then develops an architecture for collaborative movie annotation.

  3. Displaying Data As Movies

    NASA Technical Reports Server (NTRS)

    Moore, Judith G.

    1992-01-01

    NMSB Movie computer program displays large sets of data (more than million individual values). Presentation dynamic, rapidly displaying sequential image "frames" in main "movie" window. Any sequence of two-dimensional sets of data scaled between 0 and 255 (1-byte resolution) displayed as movie. Time- or slice-wise progression of data illustrated. Originally written to present data from three-dimensional ultrasonic scans of damaged aerospace composite materials, illustrates data acquired by thermal-analysis systems measuring rates of heating and cooling of various materials. Developed on Macintosh IIx computer with 8-bit color display adapter and 8 megabytes of memory using Symantec Corporation's Think C, version 4.0.

  4. 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

  5. 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

  6. The Lexical Coverage of Movies

    ERIC Educational Resources Information Center

    Webb, Stuart; Rodgers, Michael P. H.

    2009-01-01

    The scripts of 318 movies were analyzed in this study to determine the vocabulary size necessary to understand 95% and 98% of the words in movies. The movies consisted of 2,841,887 running words and had a total running time of 601 hours and 33 minutes. The movies were classified as either American or British, and then put into the following…

  7. 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

  8. 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

  9. Politics and the Movie

    ERIC Educational Resources Information Center

    Funderburk, Charles

    1978-01-01

    Explains how the use of feature-length motion pictures, combined with interesting readings, can generate enthusiasm, discussion, and analysis of basic political ideas, concepts, and values. Reviews costs and identifies specific movies and readings on various political topics. (AV)

  10. 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

  11. 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

  12. 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

  13. 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

  14. 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

  15. 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.

  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. 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

  19. Galileo and the Movies

    NASA Astrophysics Data System (ADS)

    Olivotto, Cristina; Testa, Antonella

    2010-12-01

    We analyze the character of Galileo Galilei (1564-1642), one of the most famous scientists of all time, as portrayed in three significant movies: Luigi Maggi's Galileo Galilei (1909), Liliana Cavani's Galileo (1968), and Joseph Losey's Galileo (1975), the last one of which was based upon Bertolt Brecht's drama, Das Leben des Galilei (1947). We investigate the relationships between the main characteristics of these fictional Galileos and the most important twentieth-century Galilean historiographic models. We also analyze the veracity of the plots of these three movies and the role that historical and scientific consultants played in producing them. We conclude that connections between these three movies and Galilean historiographic models are far from evident, that other factors deeply influenced the representation of Galileo on the screen.

  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. [Allotransplantation, literature and movie].

    PubMed

    Glicenstein, J

    2007-10-01

    Writers and movie makers have always dreamed of creating a human being, changing completely a face or giving new hands. The legend of Saint Come and Saint Damien is the first example of miraculous allotransplantation. Mary Shelley's Frankenstein is considered as founder work of modern science fiction. In the 19th and 20th century, authors used the advances in medicine to imagine diabolic practitioners or brilliant surgeons to transplant entire faces or hands. Cinema uses special effects to show spectacular operations. The author presents examples of books and movies treating directly or indirectly with composite allotransplantations. PMID:17850947

  2. 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

  3. 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…

  4. Tobacco and the movie industry.

    PubMed

    Charlesworth, Annemarie; Glantz, Stanton A

    2006-01-01

    Despite the tobacco industry's voluntary restrictions and its agreement with the state attorneys general prohibiting direct and indirect cigarette marketing to youth and paid product placement, tobacco use remains prevalent in movies. Extensive research provides strong and consistent evidence that smoking in the movies promotes smoking. This article summarizes the evidence on the nature and effect of smoking in the movies on adolescents (and others) and proposes several solutions to reduce adolescent exposure to movie smoking and subsequent smoking. PMID:16446255

  5. Movies and Literary Elements.

    ERIC Educational Resources Information Center

    Keller, Rodney D.

    Showing ten-minute movie clips can be an effective way to motivate students to read literature and to teach elements of fiction, namely plot, character, setting, symbol, irony, and theme. A clip from "And Then There Were None" may be used to teach various elements of plot, including conflict and the four types of conflict (man vs. man, man vs.…

  6. 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

  7. A Collection of The Movies

    NASA Technical Reports Server (NTRS)

    1991-01-01

    This video contains computer-generated animation made from still data sets processed by computer to give the illusion of flying around the objects. 'Earth - The Movie' uses cloud data from satellites and geographical data from maps. 'L.A. - The Movie' was taken from Landsat data of the Los Angeles area. This was the first experimental demonstration of the technology. 'Mars - The Movie' was taken from Viking orbiter data. 'Miranda - The Movie' was made from a mosaic of 9 frames taken by Voyager of the Uranium moon, Miranda. The last movie is 'Monterey - The Bay.'

  8. 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.

  9. Satellite Rings Movie

    NASA Technical Reports Server (NTRS)

    2000-01-01

    This brief movie clip (of which the release image is a still frame), taken by NASA's Cassini spacecraft as it approached Jupiter, shows the motions, over a 16 hour-period, of two satellites embedded in Jupiter's ring. The moon Adrastea is the fainter of the two, and Metis the brighter. Images such as these will be used to refine the orbits of the two bodies.

    The movie was made from images taken during a 40-hour sequence of the Jovian ring on December 11, 2000.

    Cassini is a cooperative mission of NASA, the European Space Agency and the Italian Space Agency. JPL, a division of the California Institute of Technology in Pasadena, manages Cassini for NASA's Office of Space Science, Washington, D.C.

  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. Voyager 1 'Blue Movie'

    NASA Technical Reports Server (NTRS)

    2000-01-01

    This is the original Voyager 'Blue Movie' (so named because it was built from Blue filter images). It records the approach of Voyager 1 during a period of over 60 Jupiter days. Notice the difference in speed and direction of the various zones of the atmosphere. The interaction of the atmospheric clouds and storms shows how dynamic the Jovian atmosphere is.

    As Voyager 1 approached Jupiter in 1979, it took images of the planet at regular intervals. This sequence is made from 66 images taken once every Jupiter rotation period (about 10 hours). This time-lapse movie uses images taken every time Jupiter longitude 68W passed under the spacecraft. These images were acquired in the Blue filter from Jan. 6 to Feb. 3 1979. The spacecraft flew from 58 million kilometers to 31 million kilometers from Jupiter during that time.

    This time-lapse movie was produced at JPL by the Image Processing Laboratory in 1979.

  12. Psychiatry and movies.

    PubMed

    Damjanović, Aleksandar; Vuković, Olivera; Jovanović, Aleksandar A; Jasović-Gasić, Miroslava

    2009-06-01

    As one of the most potent and substantial form of mass communication, film exercises a very significant influence upon the perceptions of the audience, especially in relation to mental illness issues, and that perception is very much blurred with populists' misinterpretation and lack of awareness regarding problems faced by persons suffering from mental disorders. Movies such as "Psycho", "One Flew Over Cuckoo's Nest", "Exorcist", despite being valuable in an artistic sense, corroborated and encouraged confusion and undermined the clarity and certainty concerning the fine line separating mental health from mental illness. Modern film makers and movie theoreticians try to overcome these limitations which are often generated by exploitation of stereotypes and myths referring to mentally ill people. This paper defines and discusses the most frequent thematic stereotypes seen in movies which are perpetuating stigmatization of mentally ill people. They are: free-spirited rebel, maniac on a killing spree, seducer, enlightened member of society, narcissistic parasite, beastly person (stereotype of animal sort). Psychiatry and cinematography are linked inseparably not only because they creatively complement each other, but also as an opportunity of mutual influences blending into didactical categories and professional driving forces, benefiting both the filmmakers' and the psychiatrists' professions. PMID:19556954

  13. 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.

  14. 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

  15. Jupiter Polar Winds Movie

    NASA Technical Reports Server (NTRS)

    2001-01-01

    Bands of eastward and westward winds on Jupiter appear as concentric rotating circles in this movie composed of Cassini spacecraft images that have been re-projected as if the viewer were looking down at Jupiter's north pole and the planet were flattened.

    The sequence covers 70 days, from October 1 to December 9, 2000. Cassini's narrow-angle camera captured the images of Jupiter's atmosphere in the near-infrared region of the spectrum.

    What is surprising in this view is the coherent nature of the high-latitude flows, despite the very chaotic, mottled and non-banded appearance of the planet's polar regions. This is the first extended movie sequence to show the coherence and longevity of winds near the pole and the features blown around the planet by them.

    There are thousands of spots, each an active storm similar to the size to the largest of storms on Earth. Large terrestrial storms usually last only a week before they dissolve and are replaced by other storms. But many of the Jovian storms seen here, while occasionally changing latitude or merging with each other, persist for the entire 70 days. Until now, the lifetime of the high-latitude features was unknown. Their longevity is a mystery of Jovian weather.

    Cassini collected images of Jupiter for months before and after it passed the planet on December 30, 2000. Six or more images of the planet in each of several spectral filters were taken at evenly spaced intervals over the course of Jupiter's 10-hour rotation period. The entire sequence was repeated generally every other Jupiter rotation, yielding views of every sector of the planet at least once every 20 hours.

    The images used for the movie shown here were taken every 20 hours through a filter centered at a wavelength of 756 nanometers, where there are almost no absorptions in the planet's atmosphere. The images covering each rotation were mosaiced together to form a cylindrical map extending from 75 degrees north to 75 degrees south in

  16. Superconductor as movie star

    SciTech Connect

    Pool, R.

    1993-12-03

    Japanese researchers have succeeded in producing a movie of changes in the magnetic flux lattice of a high-Tc superconductor as it is warmed. They used a technique called electron holography, in which electrons are passed through a superconductor, and flux lines are visualized as interference patterns induced by the electrons as they undergo a phase change as they pass to one side or another of the flux lines. The technique will have application in designing superconductors so that they do not lose their superconductivity when exposed to magnetic fields.

  17. Red Spot Movie

    NASA Technical Reports Server (NTRS)

    2000-01-01

    This brief movie shows counterclockwise atmospheric motion around Jupiter's Great Red Spot. The clip was made from blue-filter images taken with the narrow-angle camera on NASA's Cassini spacecraft during seven separate rotations of Jupiter between Oct. 1 and Oct. 5, 2000.

    The clip also shows the eastward and westward motion of the zonal jets, seen as the horizontal stripes flowing in opposite directions. The zonal jets circle the planet. As far as can be determined from both Earth-based and spacecraft measurements, the positions and speeds of the jets have not changed for 100 years. Since Jupiter is a fluid planet without a solid boundary, the jet speeds are measured relative to Jupiter's magnetic field, which rotates, wobbling like a top because of its tilt, every 9 hours 55.5 minutes. The movie shows motions in the magnetic reference frame, so winds to the west correspond to features that are rotating a little slower than the magnetic field, and eastward winds correspond to features rotating a little faster.

    Because the Red Spot is in the southern hemisphere, the direction of motion indicates it is a high-pressure center. Small bright clouds appear suddenly to the west of the Great Red Spot. Scientists suspect these small white features are lightning storms. The storms eventually merge with the Red Spot and surrounding jets, and may be the main energy source for the large-scale features.

    The smallest features in the movie are about 500 kilometers (about 300 miles) across. The spacing of the movie frames in time is not uniform; some consecutive images are separated by two Jupiter rotations, and some by one. The images have been re-projected using a simple cylindrical map projection. They show an area from 50 degrees north of Jupiter's equator to 50 degrees south, extending 100 degrees east-west, about one quarter of Jupiter's circumference.

    Cassini is a cooperative project of NASA, the European Space Agency and the Italian Space Agency. The Jet

  18. 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

  19. 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

  20. 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.

  1. Jupiter Polar Winds Movie Blowup

    NASA Technical Reports Server (NTRS)

    2001-01-01

    Persistent polar storms and zonal winds on Jupiter can be seen in this magnified quadrant from a movie projecting images from NASA's Cassini spacecraft as if the viewer were looking down at Jupiter's north pole and the planet were flattened.

    The sequence covers 70 days, from October 1 to December 9, 2000. Cassini's narrow-angle camera captured the images of Jupiter's atmosphere in the near-infrared region of the spectrum.

    Like the accompanying full-circle movie of polar winds, this zoomed-inversion shows that the polar region has coherent flows, despite its chaotic, mottled appearance. There are thousands of spots, each an active storm similar in size to the largest storms on Earth. The spots occasionally change latitude or merge with each other, but usually they last for the entire 70 days. Until now, the lifetime of those storms was unknown.

    The mystery of Jupiter's weather is why the storms last so long. Storms on Earth last for a week before they break up and are replaced by other storms. This movie heightens the mystery because it shows long-lived storms at the highest latitudes, where the weather patterns are more disorganized than at low latitudes.

    Cassini collected images of Jupiter for months before and after it passed the planet on December 30, 2000. Six images or more of the planet in each of several spectral filters were taken at evenly spaced intervals over the course of Jupiter's 10-hour rotation period. The entire sequence was repeated generally every other Jupiter rotation, yielding views of every sector of the planet at least once every 20 hours.

    The images used for the movie shown here were taken every 20 hours through a filter centered at a wavelength of 756 nanometers, where there are almost no absorptions in the planet's atmosphere. Images from each rotation were assembled first into a cylindrical map. The 84 resulting cylindrical maps, spanning 70 Earth days or 168 Jupiter rotations, were transformed to polar stereographic

  2. 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

  3. Movie smoking, movie horror, and urge to smoke.

    PubMed

    Sargent, James D; Maruska, Karin; Morgenstern, Matthis; Isensee, Barbara; Hanewinkel, Reiner

    2009-01-01

    It is known that exposure to smoking cues increases urge to smoke (UTS), but little is known about other media factors that might also increase UTS. We hypothesized that horror/ thriller movies might also increase UTS by increasing negative affect. We surveyed 536 movie patrons who were smokers aged 18 years or older. Subjects had exited 26 movies, of which 12 contained smoking and two were horrorfilms, one with and one without smoking. We used random effects regression to assess the association between exposure to movie smoking, movie horror, both and UTS, controlling for confounding factors. Median age was 26 years and 52% were female. Mean UTS was 5.9, 6.6, 6.6, and 8.7 for smokers exiting movies without smoking, with smoking, horror without smoking and horror with smoking respectively. Smoking in movies was associated with a significantly higher UTS (0.63 [95% CI 0.31-0.94]). Horror with smoking increased UTS by 2.8 points (95% C.I. 2.3, 3.5); the horror without smoking estimate was 0.88, but not statistically significant. This short report offers preliminary evidence that movie horror as one factor besides visual smoking cues that could increase UTS in a community setting. PMID:20301876

  4. 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.

  5. 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.

  6. Europa Tide Movie

    NASA Technical Reports Server (NTRS)

    2007-01-01

    [figure removed for brevity, see original site] Click on the image for Europa Tide Movie

    In this movie Europa is seen in a cutaway view through two cycles of its 3.5 day orbit about the giant planet Jupiter. Like Earth, Europa is thought to have an iron core, a rocky mantle and a surface ocean of salty water. Unlike on Earth, however, this ocean is deep enough to cover the whole moon, and being far from the sun, the ocean surface is globally frozen over. Europa's orbit is eccentric, which means as it travels around Jupiter, large tides, raised by Jupiter, rise and fall. Jupiter's position relative to Europa is also seen to librate, or wobble, with the same period. This tidal kneading causes frictional heating within Europa, much in the same way a paper clip bent back and forth can get hot to the touch, as illustrated by the red glow in the interior of Europa's rocky mantle and in the lower, warmer part of its ice shell. This tidal heating is what keeps Europa's ocean liquid and could prove critical to the survival of simple organisms within the ocean, if they exist.

  7. Movie Palaces: Renaissance and Reuse.

    ERIC Educational Resources Information Center

    Valerio, Joseph M.; Friedman, Daniel

    This book explores the potential of U.S. movie theaters as an important national asset. Each of the 4,000 movie palaces constructed during Hollywood's Golden Age, as well as the countless smaller theaters modeled after the grander showcases, has a role to play in the life of today's cities. The first section of the book explores the social and…

  8. 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.

  9. 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

  10. All-optical encrypted movie.

    PubMed

    Mosso, Fabian; Barrera, John Fredy; Tebaldi, Myrian; Bolognini, Néstor; Torroba, Roberto

    2011-03-14

    We introduce for the first time the concept of an all-optical encrypted movie. This movie joints several encrypted frames corresponding to a time evolving situation employing the same encoding mask. Thanks to a multiplexing operation we compact the encrypted movie information into a single package. But the decryption of this single package implies the existence of cross-talk if we do not adequately pre-process the encoded information before multiplexing. In this regard, we introduce a grating modulation to each encoded image, and then we proceed to multiplexing. After appropriate filtering and synchronizing procedures applied to the multiplexing, we are able to decrypt and to reproduce the movie. This movie is only properly decoded when in possession of the right decoding key. The concept development is carried-out in virtual optical systems, both for the encrypting and the filtering-decrypting stages. Experimental results are shown to confirm our approach. PMID:21445211

  11. Characteristics of DNA-AuNP networks on cell membranes and real-time movies for viral infection.

    PubMed

    Li, Chunmei; Zheng, Linling; Yang, Xiaoxi; Wan, Xiaoyan; Wu, Wenbi; Zhen, Shujun; Li, Yuanfang; Luo, Lingfei; Huang, Chengzhi

    2016-03-01

    This data article provides complementary data for the article entitled "DNA-AuNP networks on cell membranes as a protective barrier to inhibit viral attachment, entry and budding" Li et al. (2016) [1]. The experimental methods for the preparation and characterization of DNA-conjugated nanoparticle networks on cell membranes were described. Confocal fluorescence images, agarose gel electrophoresis images and hydrodynamic diameter of DNA-conjugated gold nanoparticle (DNA-AuNP) networks were presented. In addition, we have prepared QDs-labeled RSV (QDs-RSV) to real-time monitor the RSV infection on HEp-2 cells in the absence and presence of DNA-AuNP networks. Finally, the cell viability of HEp-2 cells coated by six types of DNA-nanoparticle networks was determined after RSV infection. PMID:26909382

  12. 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.

  13. 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

  14. 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

  15. 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

  16. Planetwide Color Movie

    NASA Technical Reports Server (NTRS)

    2000-01-01

    The first color movie of Jupiter from NASA's Cassini spacecraft shows what it would look like to peel the entire globe of Jupiter, stretch it out on a wall into the form of a rectangular map, and watch its atmosphere evolve with time.

    The brief movie clip spans 24 Jupiter rotations between Oct. 31 and Nov. 9, 2000.

    Various patterns of motion are apparent all across Jupiter at the cloudtop level seen here. The Great Red Spot shows its counterclockwise rotation, and the uneven distribution of its high haze is obvious. To the east (right) of the Red Spot, oval storms, like ball bearings, roll over and pass each other. Horizontal bands adjacent to each other move at different rates. Strings of small storms rotate around northern-hemisphere ovals. The large grayish-blue 'hot spots' at the northern edge of the white Equatorial Zone change over the course of time as they march eastward across the planet. Ovals in the north rotate counter to those in the south. Small, very bright features appear quickly and randomly in turbulent regions, candidates for lightning storms.

    The clip consists of 14 unevenly spaced timesteps, each a true color cylindrical projection of the complete circumference of Jupiter, from 60 degrees south to 60 degrees north. The maps are made by first assembling mosaics of six images taken by Cassini's narrow-angle camera in the same spectral filter over the course of one Jupiter rotation and, consequently, covering the whole planet. Three such global maps -- in red, green and blue filters -- are combined to make one color map showing Jupiter during one Jovian rotation. Fourteen such maps, spanning 24 Jovian rotations at uneven time intervals comprise the movie. The maps were reduced in scale by a factor of two to make them accessible on the Internet at reasonable rates. Occasional appearances of Io, Europa, and their shadows have not been removed.

    The smallest visible features at the equator are about 600 kilometers (about 370 miles

  17. 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.

  18. 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.

  19. 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

  20. 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

  1. 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.

  2. 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.

  3. 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.

  4. IO Rotation Movie

    NASA Technical Reports Server (NTRS)

    2000-01-01

    During its 1979 flyby, Voyager 2 observed Io only from a distance. However, the volcanic activity discovered by Voyager 1 months earlier was readily visible. This sequence of nine color images was collected using the Blue, Green and Orange filters from about 1.2 million kilometers. A 2.5 hour period is covered during which Io rotates 7 degrees.

    Rotating into view over the limb of Io are the plumes of the volcanoes Amirani (top) and Maui (lower). These plumes are very distinct against the black sky because they are being illuminated from behind. Notice that as Io rotates, the proportion of Io which is sunlit decreases greatly. This changing phase angle is because Io is moving between the spacecraft and the Sun.

    This time-lapse movie was produced at JPL by the Image Processing Laboratory in 1985.

  5. Droplet Combustion Experiment movie

    NASA Technical Reports Server (NTRS)

    2003-01-01

    The Droplet Combustion Experiment (DCE) was designed to investigate the fundamental combustion aspects of single, isolated droplets under different pressures and ambient oxygen concentrations for a range of droplet sizes varying between 2 and 5 mm. The DCE principal investigator was Forman Williams, University of California, San Diego. The experiment was part of the space research investigations conducted during the Microgravity Science Laboratory-1 mission (STS-83, April 4-8 1997; the shortened mission was reflown as MSL-1R on STS-94). Advanced combustion experiments will be a part of investigations plarned for the International Space Station. (1.1 MB, 12-second MPEG, screen 320 x 240 pixels; downlinked video, higher quality not available)A still JPG composite of this movie is available at http://mix.msfc.nasa.gov/ABSTRACTS/MSFC-0300164.html.

  6. Personalized Movie Recommendation

    NASA Astrophysics Data System (ADS)

    Lekakos, George; Charami, Matina; Caravelas, Petros

    This article proposes a movie recommender system, named MoRe, which follows a hybrid approach that combines content-based and collaborative filtering. MoR's performance is empirically evaluated upon the predictive accuracy of the algorithms as well as other important indicators such as the percentage of items that the system can actually predict (called prediction coverage) and the time required for generating predictions. The remainder of this article is organized as follows. The next section is devoted to the fundamental background of recommender systems describing the main recommendation techniques along with their advantages and limitations. Right after, we illustrate the MoRe system overview and in the section following, we describe in detail the algorithms implemented. The empirical evaluation results are then presented, while the final section provides a discussion about conclusions and future research.

  7. Wide Angle Movie

    NASA Technical Reports Server (NTRS)

    1999-01-01

    This brief movie illustrates the passage of the Moon through the Saturn-bound Cassini spacecraft's wide-angle camera field of view as the spacecraft passed by the Moon on the way to its closest approach with Earth on August 17, 1999. From beginning to end of the sequence, 25 wide-angle images (with a spatial image scale of about 14 miles per pixel (about 23 kilometers)were taken over the course of 7 and 1/2 minutes through a series of narrow and broadband spectral filters and polarizers, ranging from the violet to the near-infrared regions of the spectrum, to calibrate the spectral response of the wide-angle camera. The exposure times range from 5 milliseconds to 1.5 seconds. Two of the exposures were smeared and have been discarded and replaced with nearby images to make a smooth movie sequence. All images were scaled so that the brightness of Crisium basin, the dark circular region in the upper right, is approximately the same in every image. The imaging data were processed and released by the Cassini Imaging Central Laboratory for Operations (CICLOPS)at the University of Arizona's Lunar and Planetary Laboratory, Tucson, AZ.

    Photo Credit: NASA/JPL/Cassini Imaging Team/University of Arizona

    Cassini, launched in 1997, is a joint mission of NASA, the European Space Agency and Italian Space Agency. The mission is managed by NASA's Jet Propulsion Laboratory, Pasadena, CA, for NASA's Office of Space Science, Washington DC. JPL is a division of the California Institute of Technology, Pasadena, CA.

  8. "All The President's Men": How Two Journalists Brought Down a President and Lived To Tell about It. A Guide for Teaching the Movie and the Book in Your Classroom.

    ERIC Educational Resources Information Center

    Wilson, Bradley

    2002-01-01

    Explains how the news story surrounding the Watergate events led not only to the resignation of President Nixon but also to one of journalism's greatest triumphs. Presents a Watergate timeline with 25 events. Gives three exercises to accompany the study of the journalism side of the Watergate events and to compare the movie, "All The President's…

  9. 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.

  10. 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

  11. 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

  12. 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.

  13. 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

  14. 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

  15. 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.

  16. 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

  17. 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

  18. 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.

  19. 28 CFR 544.33 - Movies.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 28 Judicial Administration 2 2014-07-01 2014-07-01 false Movies. 544.33 Section 544.33 Judicial... Programs § 544.33 Movies. If there is a program to show movies, the Supervisor of Education shall ensure that X-rated movies are not shown....

  20. 28 CFR 544.33 - Movies.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 28 Judicial Administration 2 2011-07-01 2011-07-01 false Movies. 544.33 Section 544.33 Judicial... Programs § 544.33 Movies. If there is a program to show movies, the Supervisor of Education shall ensure that X-rated movies are not shown....

  1. 28 CFR 544.33 - Movies.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 28 Judicial Administration 2 2012-07-01 2012-07-01 false Movies. 544.33 Section 544.33 Judicial... Programs § 544.33 Movies. If there is a program to show movies, the Supervisor of Education shall ensure that X-rated movies are not shown....

  2. 28 CFR 544.33 - Movies.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 28 Judicial Administration 2 2013-07-01 2013-07-01 false Movies. 544.33 Section 544.33 Judicial... Programs § 544.33 Movies. If there is a program to show movies, the Supervisor of Education shall ensure that X-rated movies are not shown....

  3. 28 CFR 544.33 - Movies.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 28 Judicial Administration 2 2010-07-01 2010-07-01 false Movies. 544.33 Section 544.33 Judicial... Programs § 544.33 Movies. If there is a program to show movies, the Supervisor of Education shall ensure that X-rated movies are not shown....

  4. 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

  5. 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

  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. 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

  10. 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.

  11. 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

  12. 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.

  13. 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

  14. 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

  15. Still from Planetwide Movie

    NASA Technical Reports Server (NTRS)

    2000-01-01

    This single frame from a color movie of Jupiter from NASA's Cassini spacecraft shows what it would look like to unpeel the entire globe of Jupiter, stretch it out on a wall into the form of a rectangular map.

    The image is a color cylindrical projection of the complete circumference of Jupiter, from 60 degrees south to 60 degrees north. It was produced from six images taken by Cassini's narrow-band camera on Oct. 31, 2000, in each of three filters: red, green and blue.

    The smallest visible features at the equator are about 600 kilometers (about 370 miles) across. In a map of this type, the most extreme northern and southern latitudes are unnaturally stretched out.

    Cassini is a cooperative project of NASA, the European Space Agency and the Italian Space Agency. The Jet Propulsion Laboratory, a division of the California Institute of Technology in Pasadena, manages the Cassini mission for NASA's Office of Space Science, Washington, D.C.

  16. 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

  17. 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

  18. 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

  19. 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.

  20. 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.

  1. 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

  2. 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.

  3. 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

  4. 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.

  5. 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.

  6. 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

  7. Real time imaging of live cell ATP leaking or release events by chemiluminescence microscopy

    SciTech Connect

    Zhang, Yun

    2008-12-18

    The purpose of this research was to expand the chemiluminescence microscopy applications in live bacterial/mammalian cell imaging and to improve the detection sensitivity for ATP leaking or release events. We first demonstrated that chemiluminescence (CL) imaging can be used to interrogate single bacterial cells. While using a luminometer allows detecting ATP from cell lysate extracted from at least 10 bacterial cells, all previous cell CL detection never reached this sensitivity of single bacteria level. We approached this goal with a different strategy from before: instead of breaking bacterial cell membrane and trying to capture the transiently diluted ATP with the firefly luciferase CL assay, we introduced the firefly luciferase enzyme into bacteria using the modern genetic techniques and placed the CL reaction substrate D-luciferin outside the cells. By damaging the cell membrane with various antibacterial drugs including antibiotics such as Penicillins and bacteriophages, the D-luciferin molecules diffused inside the cell and initiated the reaction that produces CL light. As firefly luciferases are large protein molecules which are retained within the cells before the total rupture and intracellular ATP concentration is high at the millmolar level, the CL reaction of firefly luciferase, ATP and D-luciferin can be kept for a relatively long time within the cells acting as a reaction container to generate enough photons for detection by the extremely sensitive intensified charge coupled device (ICCD) camera. The result was inspiring as various single bacterium lysis and leakage events were monitored with 10-s temporal resolution movies. We also found a new way of enhancing diffusion D-luciferin into cells by dehydrating the bacteria. Then we started with this novel single bacterial CL imaging technique, and applied it for quantifying gene expression levels from individual bacterial cells. Previous published result in single cell gene expression quantification

  8. 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

  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 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.

  12. 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

  13. 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

  14. 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

  15. Movies of cellular and sub-cellular motion by digital holographic microscopy

    PubMed Central

    Mann, Christopher J; Yu, Lingfeng; Kim, Myung K

    2006-01-01

    Background Many biological specimens, such as living cells and their intracellular components, often exhibit very little amplitude contrast, making it difficult for conventional bright field microscopes to distinguish them from their surroundings. To overcome this problem phase contrast techniques such as Zernike, Normarsky and dark-field microscopies have been developed to improve specimen visibility without chemically or physically altering them by the process of staining. These techniques have proven to be invaluable tools for studying living cells and furthering scientific understanding of fundamental cellular processes such as mitosis. However a drawback of these techniques is that direct quantitative phase imaging is not possible. Quantitative phase imaging is important because it enables determination of either the refractive index or optical thickness variations from the measured optical path length with sub-wavelength accuracy. Digital holography is an emergent phase contrast technique that offers an excellent approach in obtaining both qualitative and quantitative phase information from the hologram. A CCD camera is used to record a hologram onto a computer and numerical methods are subsequently applied to reconstruct the hologram to enable direct access to both phase and amplitude information. Another attractive feature of digital holography is the ability to focus on multiple focal planes from a single hologram, emulating the focusing control of a conventional microscope. Methods A modified Mach-Zender off-axis setup in transmission is used to record and reconstruct a number of holographic amplitude and phase images of cellular and sub-cellular features. Results Both cellular and sub-cellular features are imaged with sub-micron, diffraction-limited resolution. Movies of holographic amplitude and phase images of living microbes and cells are created from a series of holograms and reconstructed with numerically adjustable focus, so that the moving object

  16. Narrow Angle movie

    NASA Technical Reports Server (NTRS)

    1999-01-01

    This brief three-frame movie of the Moon was made from three Cassini narrow-angle images as the spacecraft passed by the Moon on the way to its closest approach with Earth on August 17, 1999. The purpose of this particular set of images was to calibrate the spectral response of the narrow-angle camera and to test its 'on-chip summing mode' data compression technique in flight. From left to right, they show the Moon in the green, blue and ultraviolet regions of the spectrum in 40, 60 and 80 millisecond exposures, respectively. All three images have been scaled so that the brightness of Crisium basin, the dark circular region in the upper right, is the same in each image. The spatial scale in the blue and ultraviolet images is 1.4 miles per pixel (2.3 kilometers). The original scale in the green image (which was captured in the usual manner and then reduced in size by 2x2 pixel summing within the camera system) was 2.8 miles per pixel (4.6 kilometers). It has been enlarged for display to the same scale as the other two. The imaging data were processed and released by the Cassini Imaging Central Laboratory for Operations (CICLOPS) at the University of Arizona's Lunar and Planetary Laboratory, Tucson, AZ.

    Photo Credit: NASA/JPL/Cassini Imaging Team/University of Arizona

    Cassini, launched in 1997, is a joint mission of NASA, the European Space Agency and Italian Space Agency. The mission is managed by NASA's Jet Propulsion Laboratory, Pasadena, CA, for NASA's Office of Space Science, Washington DC. JPL is a division of the California Institute of Technology, Pasadena, CA.

  17. "I Caught It at the Movies": Reflections on Medical History, Movie Theaters, and the Cinema of Contagion.

    PubMed

    Wahlert, Lance

    2016-01-01

    Undertaking an examination of the precarious places of the movies and movie theaters in queer lives in the 20th century, this article takes up a series of anecdotal episodes and feature-length films to consider how the space-related stakes of LGBT health have been best understood in literal cinema houses and the narrative cinema projections inside of them. The author argues for an appreciation of LGBT-themed motion pictures as oscillating between perpetuator of queer pathology and its potential solution. PMID:26644058

  18. 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

  19. 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

  20. 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

  1. 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

  2. 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

  3. 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

  4. 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

  5. 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

  6. 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

  7. 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

  8. 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.

  9. 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

  10. 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.

  11. Cloning goes to the movies.

    PubMed

    Cormick, Craig

    2006-10-01

    Public attitude research conducted by Biotechnology Australia shows that one of the major sources of information on human reproductive cloning is movies. Traditionally, understanding of new and emerging technologies has come through the mass media but human cloning, being so widely addressed through the popular culture of movies, is more effectively defined by Hollywood than the news media or science media. But how well are the science and social issues of cloning portrayed in box office hits such as The Island, Multiplicity, Star Wars: Attack of the Clones and Jurassic Park? These movies have enormous reach and undoubted influence, and are therefore worth analyzing in some detail. This study looks at 33 movies made between 1971 and 2005 that address human reproductive cloning, and it categorizes the films based on their genre and potential influence. Yet rather than simply rating the quality of the science portrayed, the study compares the key messages in these movies with public attitudes towards cloning, to examine the correlations. PMID:17214211

  12. 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.

  13. 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.

  14. Using Science Fiction Movies in Introductory Physics

    NASA Astrophysics Data System (ADS)

    Dark, Marta L.

    2005-10-01

    This paper discusses the use of science fiction movies in introductory physics courses at Spelman College. There are several reasons to use these movies in the classroom environment. Movies are a visual learning aid. Introductory physics students show a strong interest in participating in movie-related activities compared to standard group problem-solving sessions. Finally, these activities encourage creative thinking and can be used to develop writing skills. The students involved with these movie-based activities have included biology and pre-medical majors taking general physics. In the introductory level courses, physics, chemistry, and engineering majors worked on movie-based activities.

  15. 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.

  16. Voyager 1 Red Spot Movie

    NASA Technical Reports Server (NTRS)

    2000-01-01

    This movie shows the portion of Jupiter around the Great Red Spot as it swirls through more than 60 Jupiter days. Notice the difference in speed and direction of the various zones of the atmosphere. The interaction of the atmospheric clouds and storm shows how dynamic the Jovian atmosphere is.

    As Voyager 1 approached Jupiter in 1979, it took images of the planet at regular intervals. This sequence is made from 66 images taken once every Jupiter rotation period (about 10 hours). This time-lapse movie uses images taken every time Jupiter longitude 68W passed under the spacecraft. These images were acquired in the Blue filter from Jan. 6 to Feb. 3 1979. The spacecraft flew from 58 million kilometers to 31 million kilometers from Jupiter during that time.

    This time-lapse movie was produced at JPL by the Image Processing Laboratory in 1979.

  17. 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

  18. 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.

  19. 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

  20. 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

  1. 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.

  2. 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

  3. 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

  4. 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

  5. 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

  6. 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

  7. 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).

  8. Movie Ratings and Their Effect on Movie Attendance.

    ERIC Educational Resources Information Center

    Austin, Bruce A.

    A study was conducted to examine how the motion picture Motion Picture Association of America (MPAA) rating system (G-PG-R-X) affects movie attendance. The study also tested the validity of two behavioral theories: (1) reactance theory, which predicts that when a behavioral freedom is restricted or eliminated an individual is motivated to restore…

  9. 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

  10. 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

  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. 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

  13. 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

  14. 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

  15. 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.

  16. Computer-Generated Movies for Mission Planning

    NASA Technical Reports Server (NTRS)

    Roberts, P. H., Jr.; vanDillen, S. L.

    1973-01-01

    Computer-generated movies help the viewer to understand mission dynamics and get quantitative details. Sample movie frames demonstrate the uses and effectiveness of movies in mission planning. Tools needed for movie-making include computer programs to generate images on film and film processing to give the desired result. Planning scenes to make an effective product requires some thought and experience. Viewpoints and timing are particularly important. Lessons learned so far and problems still encountered are discussed.

  17. 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.

  18. Beyond the Movie Screen: An Antarctic Adventure

    ERIC Educational Resources Information Center

    Cajigal, Aris Reynold V.; Chamrat, Suthida; Tippins, Deborah; Mueller, Mike; Thomson, Norman

    2011-01-01

    Movies depicting science-related issues often capture the attention of today's youth. As an instructional tool, movies can take us beyond the drama and action and thrilling scenes. In this article we share our experiences of using the movie "Eight Below" as a centerpiece for developing high school students' understanding of basic chemistry…

  19. A movie of RNA polymerase II transcription.

    PubMed

    Cheung, Alan C M; Cramer, Patrick

    2012-06-22

    We provide here a molecular movie that captures key aspects of RNA polymerase II initiation and elongation. To create the movie, we combined structural snapshots of the initiation-elongation transition and of elongation, including nucleotide addition, translocation, pausing, proofreading, backtracking, arrest, reactivation, and inhibition. The movie reveals open questions about the mechanism of transcription and provides a useful teaching tool. PMID:22726432

  20. VHS Movies: Perturbations for Morphogenesis.

    ERIC Educational Resources Information Center

    Holmes, Danny L.

    This paper discusses the concept of a family system in terms of an interactive system of interrelated, interdependent parts and suggests that VHS movies can act as perturbations, i.e., change promoting agents, for certain dysfunctional family systems. Several distinct characteristics of a family system are defined with particular emphasis on…

  1. Grasping the Social through Movies

    ERIC Educational Resources Information Center

    Kennedy, Nilgun Fehim; Senses, Nazli; Ayan, Pelin

    2011-01-01

    In Turkey, one of the major challenges that university education faces is the indifference of young people towards social issues. The aim of this article is to contribute to the "practice" of critical pedagogy by proposing that showing movies is an important critical teaching method with the power both to give pleasure to the students and to…

  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. Optical Control of Living Cells Electrical Activity by Conjugated Polymers.

    PubMed

    Martino, Nicola; Bossio, Caterina; Vaquero Morata, Susana; Lanzani, Guglielmo; Antognazza, Maria Rosa

    2016-01-01

    Hybrid interfaces between organic semiconductors and living tissues represent a new tool for in-vitro and in-vivo applications. In particular, conjugated polymers display several optimal properties as substrates for biological systems, such as good biocompatibility, excellent mechanical properties, cheap and easy processing technology, and possibility of deposition on light, thin and flexible substrates. These materials have been employed for cellular interfaces like neural probes, transistors for excitation and recording of neural activity, biosensors and actuators for drug release. Recent experiments have also demonstrated the possibility to use conjugated polymers for all-optical modulation of the electrical activity of cells. Several in-vitro study cases have been reported, including primary neuronal networks, astrocytes and secondary line cells. Moreover, signal photo-transduction mediated by organic polymers has been shown to restore light sensitivity in degenerated retinas, suggesting that these devices may be used for artificial retinal prosthesis in the future. All in all, light sensitive conjugated polymers represent a new approach for optical modulation of cellular activity. In this work, all the steps required to fabricate a bio-polymer interface for optical excitation of living cells are described. The function of the active interface is to transduce the light stimulus into a modulation of the cell membrane potential. As a study case, useful for in-vitro studies, a polythiophene thin film is used as the functional, light absorbing layer, and Human Embryonic Kidney (HEK-293) cells are employed as the biological component of the interface. Practical examples of successful control of the cell membrane potential upon stimulation with light pulses of different duration are provided. In particular, it is shown that both depolarizing and hyperpolarizing effects on the cell membrane can be achieved depending on the duration of the light stimulus. The reported

  8. Tracking and localization of calmodulin in live cells.

    PubMed

    Johnson, Carey K; Harms, Gregory S

    2016-08-01

    The calcium signaling protein calmodulin (CaM) interacts with many target proteins inside the cell to regulate a wide range of biological signals. CaM's availability to propagate signals depends on its mobility, which may be regulated by interactions with multiple target proteins. We detected single molecules of CaM labeled with a fluorescent dye and injected into living HEK 293 cells, and we used high-speed, wide-field, single-molecule imaging to track single CaM molecules. Single-molecule trajectories were analyzed to characterize the motions of individual CaM molecules. Single-molecule localization resolved CaM positions with a position accuracy of <100nm, permitting sub-diffraction imaging of features with localized CaM that form in response to increased free Ca(2+). Single-molecule tracking demonstrated the presence of a wide range of mobilities of individual calmodulin molecules in a cell, with diffusion coefficients ranging from <0.01μm(2)s(-1) to ~5μm(2) s(-1), whereas analysis by spatio-temporal image correlation spectroscopy revealed faster-moving components with diffusion coefficients of >10μm(2)s(-1). For molecules confined to small regions of the cell, super-resolved images of presumed signaling complexes were recovered. Individual trajectories were classified as normal diffusion, confined diffusion, or directed motion, and could suggest how the individual CaM molecules were bound in the cell. The results show that interactions of CaM with target proteins result in decreased translational mobilities of a significant fraction of CaM molecules inside cells. The work presented here illustrates methods that can characterize location, mobilities, and the availability of signaling molecules in live cells. PMID:27113857

  9. Trypanosoma cruzi: single cell live imaging inside infected tissues.

    PubMed

    Ferreira, Bianca Lima; Orikaza, Cristina Mary; Cordero, Esteban Mauricio; Mortara, Renato Arruda

    2016-06-01

    Although imaging the live Trypanosoma cruzi parasite is a routine technique in most laboratories, identification of the parasite in infected tissues and organs has been hindered by their intrinsic opaque nature. We describe a simple method for in vivo observation of live single-cell Trypanosoma cruzi parasites inside mammalian host tissues. BALB/c or C57BL/6 mice infected with DsRed-CL or GFP-G trypomastigotes had their organs removed and sectioned with surgical blades. Ex vivo organ sections were observed under confocal microscopy. For the first time, this procedure enabled imaging of individual amastigotes, intermediate forms and motile trypomastigotes within infected tissues of mammalian hosts. PMID:26639617

  10. Processed Movie of Zonal Jets

    NASA Technical Reports Server (NTRS)

    2000-01-01

    This movie is a manipulated sequence showing motions in Jupiter's atmosphere over the course of five days beginning Oct. 1, 2000, as seen by a camera on NASA's Cassini spacecraft, using a blue filter.

    Beginning with seven images taken at uneven time intervals, this sequence was made by using information on wind speeds derived from actual Jupiter images to create evenly spaced time steps throughout. The final result is a smooth movie sequence consisting of both real and false frames.

    The view is of the opposite side of the planet from Jupiter's Great Red Spot. The region shown reaches from 50 degrees north to 50 degrees south of Jupiter's equator, and extends 100 degrees east-to-west, about one-quarter of Jupiter's circumference. The smallest features are about 500 kilometers (about 300 miles) across.

    Towards the end of the sequence, a shadow appears from one of Jupiter's moons, Europa.

    The movie shows the remains of a historic merger that began several years ago, when three white oval storms that had existed for 60 years merged into two, then one. The resulting oval is visible in the lower left portion of the movie.

    The movie also shows zonal jets that circle the planet on constant latitudes. Winds seen moving toward the left (westward) correspond to features that are rotating a little slower than Jupiter's magnetic field, and winds moving the opposite direction correspond to features that are rotating a little faster than the magnetic field. Since Jupiter has no solid surface, the rotation of the magnetic field is the point of reference for the rotation of the planet.

    Cassini is a cooperative project of NASA, the European Space Agency and the Italian Space Agency. The Jet Propulsion Laboratory, a division of the California Institute of Technology in Pasadena, manages the Cassini mission for NASA's Office of Space Science, Washington, D.C.

  11. Classic “broken cell” techniques and newer live cell methods for cell cycle assessment

    PubMed Central

    Henderson, Lindsay; Bortone, Dante S.; Lim, Curtis

    2013-01-01

    Many common, important diseases are either caused or exacerbated by hyperactivation (e.g., cancer) or inactivation (e.g., heart failure) of the cell division cycle. A better understanding of the cell cycle is critical for interpreting numerous types of physiological changes in cells. Moreover, new insights into how to control it will facilitate new therapeutics for a variety of diseases and new avenues in regenerative medicine. The progression of cells through the four main phases of their division cycle [G0/G1, S (DNA synthesis), G2, and M (mitosis)] is a highly conserved process orchestrated by several pathways (e.g., transcription, phosphorylation, nuclear import/export, and protein ubiquitination) that coordinate a core cell cycle pathway. This core pathway can also receive inputs that are cell type and cell niche dependent. “Broken cell” methods (e.g., use of labeled nucleotide analogs) to assess for cell cycle activity have revealed important insights regarding the cell cycle but lack the ability to assess living cells in real time (longitudinal studies) and with single-cell resolution. Moreover, such methods often require cell synchronization, which can perturb the pathway under study. Live cell cycle sensors can be used at single-cell resolution in living cells, intact tissue, and whole animals. Use of these more recently available sensors has the potential to reveal physiologically relevant insights regarding the normal and perturbed cell division cycle. PMID:23392113

  12. Insertion of Vertically Aligned Nanowires into Living Cells by Inkjet Printing of Cells.

    PubMed

    Lee, Donggyu; Lee, Daehee; Won, Yulim; Hong, Hyeonaug; Kim, Yongjae; Song, Hyunwoo; Pyun, Jae-Chul; Cho, Yong Soo; Ryu, Wonhyoung; Moon, Jooho

    2016-03-01

    Effective insertion of vertically aligned nanowires (NWs) into cells is critical for bioelectrical and biochemical devices, biological delivery systems, and photosynthetic bioenergy harvesting. However, accurate insertion of NWs into living cells using scalable processes has not yet been achieved. Here, NWs are inserted into living Chlamydomonas reinhardtii cells (Chlamy cells) via inkjet printing of the Chlamy cells, representing a low-cost and large-scale method for inserting NWs into living cells. Jetting conditions and printable bioink composed of living Chlamy cells are optimized to achieve stable jetting and precise ink deposition of bioink for indentation of NWs into Chlamy cells. Fluorescence confocal microscopy is used to verify the viability of Chlamy cells after inkjet printing. Simple mechanical considerations of the cell membrane and droplet kinetics are developed to control the jetting force to allow penetration of the NWs into cells. The results suggest that inkjet printing is an effective, controllable tool for stable insertion of NWs into cells with economic and scale-related advantages. PMID:26800021

  13. Fluorescence-Activated Cell Sorting of Live Versus Dead Bacterial Cells and Spores

    NASA Technical Reports Server (NTRS)

    Bernardini, James N.; LaDuc, Myron T.; Diamond, Rochelle; Verceles, Josh

    2012-01-01

    This innovation is a coupled fluorescence-activated cell sorting (FACS) and fluorescent staining technology for purifying (removing cells from sampling matrices), separating (based on size, density, morphology, and live versus dead), and concentrating cells (spores, prokaryotic, eukaryotic) from an environmental sample.

  14. Biosensing based on surface plasmon resonance and living cells.

    PubMed

    Chabot, Vincent; Cuerrier, Charles M; Escher, Emanuel; Aimez, Vincent; Grandbois, Michel; Charette, Paul G

    2009-02-15

    We propose the combination of surface plasmon resonance (SPR) with living cells as a biosensing method. Our detection scheme is based on the premise that cellular activity induced by external agents is often associated with changes in cellular morphology, which in turn should lead to a variation of the effective refractive index at the interface between the cell membrane and the metal layer. We monitored surface plasmon resonance signals originating from a gold surface coated with cells on a custom apparatus after injection of various agents known to influence cellular activity and morphology. Specifically, we evaluated three types of stimulation: response to an endotoxin (lipopolysaccharides), a chemical toxin (sodium azide) and a physiological agonist (thrombin). A comparison with phase contrast microscopy reveals that SPR signal variations are associated with the induction of cell death for lipopolysaccharides treatment and a contraction of the cell body for sodium azide. Thrombin-induced cellular response shows a rapid decrease of the measured laser reflectance over 5min followed by a return to the original value. For this treatment, phase contrast micrographs relate the first phase of the SPR variation to cell contraction and increase of the intercellular gaps, whereas the recovery phase can be associated with a spreading of the cell on the sensing surface. Hence, the SPR signal is very consistent with the cellular response normally observed for these treatments. This confirms the validity of the biosensing method, which could be applied to a large variety of cellular responses involving shape remodeling induced by external agents. PMID:18845432

  15. Vertical nanowire probes for intracellular signaling of living cells

    PubMed Central

    2014-01-01

    The single living cell action potential was measured in an intracellular mode by using a vertical nanoelectrode. For intracellular interfacing, Si nanowires were vertically grown in a controlled manner, and optimum conditions, such as diameter, length, and nanowire density, were determined by culturing cells on the nanowires. Vertical nanowire probes were then fabricated with a complimentary metal-oxide-semiconductor (CMOS) process including sequential deposition of the passivation and electrode layers on the nanowires, and a subsequent partial etching process. The fabricated nanowire probes had an approximately 60-nm diameter and were intracellular. These probes interfaced with a GH3 cell and measured the spontaneous action potential. It successfully measured the action potential, which rapidly reached a steady state with average peak amplitude of approximately 10 mV, duration of approximately 140 ms, and period of 0.9 Hz. PMID:24484729

  16. Vertical nanowire probes for intracellular signaling of living cells

    NASA Astrophysics Data System (ADS)

    Lee, Ki-Young; Kim, Ilsoo; Kim, So-Eun; Jeong, Du-Won; Kim, Ju-Jin; Rhim, Hyewhon; Ahn, Jae-Pyeong; Park, Seung-Han; Choi, Heon-Jin

    2014-02-01

    The single living cell action potential was measured in an intracellular mode by using a vertical nanoelectrode. For intracellular interfacing, Si nanowires were vertically grown in a controlled manner, and optimum conditions, such as diameter, length, and nanowire density, were determined by culturing cells on the nanowires. Vertical nanowire probes were then fabricated with a complimentary metal-oxide-semiconductor (CMOS) process including sequential deposition of the passivation and electrode layers on the nanowires, and a subsequent partial etching process. The fabricated nanowire probes had an approximately 60-nm diameter and were intracellular. These probes interfaced with a GH3 cell and measured the spontaneous action potential. It successfully measured the action potential, which rapidly reached a steady state with average peak amplitude of approximately 10 mV, duration of approximately 140 ms, and period of 0.9 Hz.

  17. 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

  18. Confocal imaging of ionised calcium in living plant cells.

    PubMed

    Williams, D A; Cody, S H; Gehring, C A; Parish, R W; Harris, P J

    1990-04-01

    Laser-scanning confocal microscopy has been used in conjunction with Fluo-3, a highly fluorescent visible wavelength probe for Ca2+, to visualize Ca2(+)-dynamics in the function of living plant cells. This combination has overcome many of the problems that have limited the use of fluorescence imaging techniques in the study of the role of cations (Ca2+ and H+) in plant cell physiology and enables these processes to be studied in single cells within intact plant tissue preparations. Maize coleoptiles respond to application of ionophores and plant growth hormones with elevations in cytosolic Ca2+ that can be resolved with a high degree of spatial resolution and can be interpreted quantitatively. PMID:2113832

  19. Encapsulation system for the immunoisolation of living cells

    NASA Technical Reports Server (NTRS)

    Wang, Taylor G. (Inventor); Lacik, Igor (Inventor); Brissova, Marcela (Inventor); Anikumar, Amrutur V. (Inventor); Prokop, Ales (Inventor); Powers, Alvin C. (Inventor)

    1999-01-01

    The present invention is drawn to a composition of matter comprising high viscosity sodium alginate, cellulose sulfate and a multi-component polycation. Additionally, the present invention provides methods for making capsules, measuring capsule permeability to immunologically-relevant proteins and treating disease in an animal using encapsulated cells. Over one thousand combinations of polyanions and polycations were examined as polymer candidates suitable for encapsulation of living cells and thirty-three pairs were effective. The combination of sodium alginate, cellulose sulfate, poly(methylene-co-guanidine) hydrochloride, calcium chloride, and sodium chloride produced the most desirable results. Pancreatic islets encapsulated in this multicomponent capsule demonstrated glucose-stimulated insulin secretion in vitro and reversed diabetes without stimulating immune reaction in mice. The capsule formulation and system of the present invention allows independent adjustments of capsule size, wall thickness, mechanical strength and permeability, and offers distinct advantages for immunoisolating cells.

  20. Direct Measurement of Acetylesterase in Living Protist Cells1

    PubMed Central

    Medzon, Edward L.; Brady, Marilyn L.

    1969-01-01

    The fluorogenic acetylesterase (acetic ester hydrolase EC 3.1.1.6.) substrate, fluorescein diacetate, was used to measure enzyme activity in living protist cells. The visual enzyme assay was done by monitoring fluorochromasia by fluorescent microscopy. Quantitative fluorogenic assays were done by measuring the evolved fluorescein in a fluorometer. Of 59 strains of bacteria, 35 were fluorochromatically positive. Eight of the fluorochromatically negative strains were fluorogenically positive. Of 22 strains of slime molds and fungi, all were fluorochromatically positive. Three out of 12 different algae were fluorochromatically positive. Several unidentified protozoa were also fluorochromatically positive. Four out of six protozoa were fluorochromatically positive. Structures of special interest showing acetylesterase activity were: the growing hyphal tips of fungi, the vacuolated areas of yeast and protozoa, newly formed bacterial spores or immature fungal spores, “mesosome-like” bodies in Bacillus megaterium, and the cell membrane and nuclear region of green algae. Yeast protoplasts and bacterial protoplasts and spheroplasts were fluorochromatically positive when derived from positive cells and negative when derived from negative cells. There was no correlation between the possession of a capsule and acetylesterase activity. There was no effect on the viability of bacterial cells incubated in the presence of fluorescein diacetate. Paraoxon inhibited bacterial and yeast enzyme at 10−5m. Eserine (10−5m) and Paraoxon (10−7m) inhibited B. megaterium enzyme. Sodium acetate at 10−2m did not inhibit bacterial enzyme. The implications of these findings on the location and expression of esterase activity in living cells are discussed. Images PMID:4974398

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

    PubMed Central

    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

  2. Single-Molecule Ion Channel Conformational Dynamics in Living Cells

    NASA Astrophysics Data System (ADS)

    Lu, H. Peter

    2014-03-01

    Stochastic and inhomogeneous conformational changes regulate the function and dynamics of ion channels that are crucial for cell functions, neuronal signaling, and brain functions. Such complexity makes it difficult, if not impossible, to characterize ion channel dynamics using conventional electrical recording alone since that the measurement does not specifically interrogate the associated conformational changes but rather the consequences of the conformational changes. Recently, new technology developments on single-molecule spectroscopy, and especially, the combined approaches of using single ion channel patch-clamp electrical recording and single-molecule fluorescence imaging have provided us the capability of probing ion channel conformational changes simultaneously with the electrical single channel recording. By combining real-time single-molecule fluorescence imaging measurements with real-time single-channel electric current measurements in artificial lipid bilayers and in living cell membranes, we were able to probe single ion-channel-protein conformational changes simultaneously, and thus providing an understanding the dynamics and mechanism of ion-channel proteins at the molecular level. The function-regulating and site-specific conformational changes of ion channels are now measurable under physiological conditions in real-time, one molecule at a time. We will focus our discussion on the new development and results of real-time imaging of the dynamics of gramicidin, colicin, and NMDA receptor ion channels in lipid bilayers and living cells. Our results shed light on new perspectives of the intrinsic interplay of lipid membrane dynamics, solvation dynamics, and the ion channel functions.

  3. Small Molecule-Mediated Cleavage of RNA in Living Cells

    PubMed Central

    Guan, Lirui

    2013-01-01

    Antisense oligonucleotides and small interfering RNAs (siRNAs) control gene expression by triggering the degradation of a mRNA via recruitment of RNase H or the RNA-induced silencing complex (RISC), respectively.[1] These approaches are hampered, however, by the poor cellular permeability of oligonucleotides. A small molecule approach to cleave RNA targets could obviate uptake issues. Several compounds can induce RNA cleavage in vitro,[2] however, to the best of our knowledge no small molecules have been previously described to cleave RNA in living cells. Herein, we describe the development of a potentially general approach to design small molecules that specifically cleave an RNA in a living cell, affecting biological function. Specifically, a designed, modularly assembled small molecule that binds the RNA that causes myotonic dystrophy type 1 (DM1)[3] was appended with a moiety that generates hydroxyl radicals upon irradiation. Cleavage of the transcript improves DM1-associated defects in cell culture, and compounds are non-toxic at an efficacious dose as determined by a MTT viability assay. This approach may allow for the site-specific cleavage and inactivation of other cellular RNAs.[4] Compounds that bind to and cleave RNA have the potential to serve as chemical genetics probes of function or lead therapeutics with spatial and temporal control. PMID:23280953

  4. Tracking single mRNA molecules in live cells

    NASA Astrophysics Data System (ADS)

    Moon, Hyungseok C.; Lee, Byung Hun; Lim, Kiseong; Son, Jae Seok; Song, Minho S.; Park, Hye Yoon

    2016-06-01

    mRNAs inside cells interact with numerous RNA-binding proteins, microRNAs, and ribosomes that together compose a highly heterogeneous population of messenger ribonucleoprotein (mRNP) particles. Perhaps one of the best ways to investigate the complex regulation of mRNA is to observe individual molecules. Single molecule imaging allows the collection of quantitative and statistical data on subpopulations and transient states that are otherwise obscured by ensemble averaging. In addition, single particle tracking reveals the sequence of events that occur in the formation and remodeling of mRNPs in real time. Here, we review the current state-of-the-art techniques in tagging, delivery, and imaging to track single mRNAs in live cells. We also discuss how these techniques are applied to extract dynamic information on the transcription, transport, localization, and translation of mRNAs. These studies demonstrate how single molecule tracking is transforming the understanding of mRNA regulation in live cells.

  5. Localization of Mitochondria in Living Cells with Rhodamine 123

    NASA Astrophysics Data System (ADS)

    Johnson, Lincoln V.; Walsh, Marcia L.; Chen, Lan Bo

    1980-02-01

    The laser dye rhodamine 123 is shown to be a specific probe for the localization of mitochondria in living cells. By virtue of its selectivity for mitochondria and its fluorescent properties, the detectability of mitochondria stained with rhodamine 123 is significantly improved over that provided by conventional light microscopic techniques. With the use of rhodamine 123, it is possible to detect alterations in mitochondrial distribution following transformation by Rous sarcoma virus and changes in the shape and organization of mitochondria induced by colchicine treatment.

  6. Interaction of multi-functional silver nanoparticles with living cells

    NASA Astrophysics Data System (ADS)

    Sur, Ilknur; Cam, Dilek; Kahraman, Mehmet; Baysal, Asli; Culha, Mustafa

    2010-04-01

    Silver nanoparticles (AgNPs) are widely used in household products and in medicine due to their antibacterial and to wound healing properties. In recent years, there is also an effort for their use in biomedical imaging and photothermal therapy. The primary reason behind the effort for their utility in biomedicine and therapy is their unique plasmonic properties and easy surface chemistry for a variety of functionalizations. In this study, AgNPs modified with glucose, lactose, oligonucleotides and combinations of these ligands are investigated for their cytotoxicity and cellular uptake in living non-cancer (L929) and cancer (A549) cells. It is found that the chemical nature of the ligand strongly influences the toxicity and cellular uptake into the model cells. While the lactose-and glucose-modified AgNPs enter the L929 cells at about the same rate, a significant increase in the rate of lactose-modified AgNPs into the A549 cells is observed. The binding of oligonucleotides along with the carbohydrate on the AgNP surfaces influences the differential uptake rate pattern into the cells. The cytotoxicity study with the modified AgNPs reveals that only naked AgNPs influence the viability of the A549 cells. The findings of this study may provide the key to developing effective applications in medicine such as cancer therapy.

  7. Automated analysis of invadopodia dynamics in live cells

    PubMed Central

    Berginski, Matthew E.; Creed, Sarah J.; Cochran, Shelly; Roadcap, David W.

    2014-01-01

    Multiple cell types form specialized protein complexes that are used by the cell to actively degrade the surrounding extracellular matrix. These structures are called podosomes or invadopodia and collectively referred to as invadosomes. Due to their potential importance in both healthy physiology as well as in pathological conditions such as cancer, the characterization of these structures has been of increasing interest. Following early descriptions of invadopodia, assays were developed which labelled the matrix underneath metastatic cancer cells allowing for the assessment of invadopodia activity in motile cells. However, characterization of invadopodia using these methods has traditionally been done manually with time-consuming and potentially biased quantification methods, limiting the number of experiments and the quantity of data that can be analysed. We have developed a system to automate the segmentation, tracking and quantification of invadopodia in time-lapse fluorescence image sets at both the single invadopodia level and whole cell level. We rigorously tested the ability of the method to detect changes in invadopodia formation and dynamics through the use of well-characterized small molecule inhibitors, with known effects on invadopodia. Our results demonstrate the ability of this analysis method to quantify changes in invadopodia formation from live cell imaging data in a high throughput, automated manner. PMID:25071988

  8. Quantifying cell-generated mechanical forces within living embryonic tissues

    PubMed Central

    Campàs, Otger; Mammoto, Tadanori; Hasso, Sean; Sperling, Ralph A; O’Connell, Daniel; Bischof, Ashley G; Maas, Richard; Weitz, David A; Mahadevan, Lakshminarayanan; Ingber, Donald E

    2014-01-01

    Cell-generated mechanical forces play a critical role during tissue morphogenesis and organ formation in the embryo. However, little is known about how these forces shape embryonic organs, mainly because it has not been possible to measure cellular forces within developing three-dimensional (3D) tissues in vivo. Here we present a method to quantify cell-generated mechanical stresses that are exerted locally within living embryonic tissues using fluorescent, cell-sized, oil microdroplets with defined mechanical properties and coated with surface integrin or cadherin receptor ligands. After introducing a droplet between cells in a tissue, local stresses are determined from the droplet shape deformations, which are obtained via fluorescence microscopy and computerized image analysis. Using this method, we quantify the anisotropic stresses generated by mammary epithelial cells cultured within 3D aggregates and confirm that these stresses (3.4 nN/µm2) are dependent on myosin II activity and more than two-fold larger than the stresses generated by cells of embryonic tooth mesenchyme when analyzed within similar cultured aggregates or in developing whole mouse mandibles. PMID:24317254

  9. Rapid Actin-Dependent Viral Motility in Live Cells

    PubMed Central

    Vaughan, Joshua C.; Brandenburg, Boerries; Hogle, James M.; Zhuang, Xiaowei

    2009-01-01

    During the course of an infection, viruses take advantage of a variety of mechanisms to travel in cells, ranging from diffusion within the cytosol to active transport along cytoskeletal filaments. To study viral motility within the intrinsically heterogeneous environment of the cell, we have developed a motility assay that allows for the global and unbiased analysis of tens of thousands of virus trajectories in live cells. Using this assay, we discovered that poliovirus exhibits anomalously rapid intracellular movement that was independent of microtubules, a common track for fast and directed cargo transport. Such rapid motion, with speeds of up to 5 μm/s, allows the virus particles to quickly explore all regions of the cell with the exception of the nucleus. The rapid, microtubule-independent movement of poliovirus was observed in multiple human-derived cell lines, but appeared to be cargo-specific. Other cargo, including a closely related picornavirus, did not exhibit similar motility. Furthermore, the motility is energy-dependent and requires an intact actin cytoskeleton, suggesting an active transport mechanism. The speed of this microtubule-independent but actin-dependent movement is nearly an order of magnitude faster than the fastest speeds reported for actin-dependent transport in animal cells, either by actin polymerization or by myosin motor proteins. PMID:19751669

  10. Self-Control and the Effects of Movie Alcohol Portrayals on Immediate Alcohol Consumption in Male College Students

    PubMed Central

    Koordeman, Renske; Anschutz, Doeschka J.; Engels, Rutger C. M. E.

    2015-01-01

    Background: In movies, alcohol-related cues are frequently depicted and there is evidence for a link between movie alcohol cues and immediate alcohol consumption. Less is known about factors influencing immediate effects movie alcohol exposure on drinking. The exertion of self-control is thought to be important in avoiding or resisting certain temptations. Aims: The aim of the present study was to assess the immediate effects of movie alcohol portrayals on drinking of male social drinkers and to assess the moderating role of self-control in this relation. It was hypothesized that participants would drink more when exposed to movie alcohol portrayals and that especially participants with low self-control would be affected by these portrayals. Methods: A between-subjects design comparing two movie conditions (alcohol or no portrayal of alcohol) was used, in which 154 pairs of male friends (ages 18–30) watched a 1-h movie in a semi-naturalistic living room setting. Their alcohol consumption while watching was examined. Participants completed a questionnaire assessing self-control as well as their self-reported weekly alcohol use. A multivariate regression analysis was conducted to test the effects of movie condition on alcohol comsumption. Results: Self-control moderated the relation between movie condition and alcohol consumption. Assignment to the alcohol movie condition increased alcohol consumption during the movie for males with high self-control but not for males with low self-control. Conclusion: Viewing a movie with alcohol portrayals can lead to higher alcohol consumption in a specific sample of young men while watching a movie. PMID:25691873

  11. Long-lived intestinal tuft cells serve as colon cancer–initiating cells

    PubMed Central

    Westphalen, C. Benedikt; Asfaha, Samuel; Hayakawa, Yoku; Takemoto, Yoshihiro; Lukin, Dana J.; Nuber, Andreas H.; Brandtner, Anna; Setlik, Wanda; Remotti, Helen; Muley, Ashlesha; Chen, Xiaowei; May, Randal; Houchen, Courtney W.; Fox, James G.; Gershon, Michael D.; Quante, Michael; Wang, Timothy C.

    2014-01-01

    Doublecortin-like kinase 1 protein (DCLK1) is a gastrointestinal tuft cell marker that has been proposed to identify quiescent and tumor growth–sustaining stem cells. DCLK1+ tuft cells are increased in inflammation-induced carcinogenesis; however, the role of these cells within the gastrointestinal epithelium and their potential as cancer-initiating cells are poorly understood. Here, using a BAC-CreERT–dependent genetic lineage–tracing strategy, we determined that a subpopulation of DCLK1+ cells is extremely long lived and possesses rare stem cell abilities. Moreover, genetic ablation of Dclk1 revealed that DCLK1+ tuft cells contribute to recovery following intestinal and colonic injury. Surprisingly, conditional knockdown of the Wnt regulator APC in DCLK1+ cells was not sufficient to drive colonic carcinogenesis under normal conditions; however, dextran sodium sulfate–induced (DSS-induced) colitis promoted the development of poorly differentiated colonic adenocarcinoma in mice lacking APC in DCLK1+ cells. Importantly, colonic tumor formation occurred even when colitis onset was delayed for up to 3 months after induced APC loss in DCLK1+ cells. Thus, our data define an intestinal DCLK1+ tuft cell population that is long lived, quiescent, and important for intestinal homeostasis and regeneration. Long-lived DCLK1+ cells maintain quiescence even following oncogenic mutation, but are activated by tissue injury and can serve to initiate colon cancer. PMID:24487592

  12. Nanoparticle PEBBLE sensors in live cells and in vivo

    PubMed Central

    Smith, Ron

    2009-01-01

    Nanoparticle sensors have been developed for imaging and dynamic monitoring, in live cells and in vivo, of the molecular or ionic components, constructs, forces and dynamics, all in real time, during biological/chemical/physical processes. With their biocompatible small size and inert matrix, nanoparticle sensors have been successfully applied for non-invasive real-time measurements of analytes and fields in cells and rodents, with spatial, temporal, physical and chemical resolution. This review describes the diverse designs of nanoparticle sensors for ions and small molecules, physical fields and biological features, as well as the characterization, properties, and applications of these nanosensors to in vitro and in vivo measurements. Their floating as well as localization ability in biological media is captured by the acronym PEBBLE: photonic explorer for bioanalysis with biologically localized embedding. PMID:20098636

  13. RNA computers in live cells: results and prospects

    PubMed Central

    Benenson, Yaakov

    2009-01-01

    Summary Man-made molecular “computers” that operate inside live cells will enable unprecedented level of control over cellular physiology. A promising approach to building these computers uses RNA molecules and RNA-based regulation. RNA naturally lends itself to create “digital” molecular networks that embody standardized (normal) forms of logic functions. The network’s inputs, that may or may not be inverted by single-input NOT logic gates, feed into multi-input AND gates whose outputs are in turn integrated in a multi-input OR gate. Below I review recent steps that have been taken toward implementing these networks with allosteric riboswitches and ribozymes in bacteria and yeast, and RNAi in mammalian cells. I also propose how to co-opt recently-discovered additional RNA regulation mechanisms into future construction efforts. PMID:19720518

  14. Synthetic circuits integrating logic and memory in living cells.

    PubMed

    Siuti, Piro; Yazbek, John; Lu, Timothy K

    2013-05-01

    Logic and memory are essential functions of circuits that generate complex, state-dependent responses. Here we describe a strategy for efficiently assembling synthetic genetic circuits that use recombinases to implement Boolean logic functions with stable DNA-encoded memory of events. Application of this strategy allowed us to create all 16 two-input Boolean logic functions in living Escherichia coli cells without requiring cascades comprising multiple logic gates. We demonstrate long-term maintenance of memory for at least 90 cell generations and the ability to interrogate the states of these synthetic devices with fluorescent reporters and PCR. Using this approach we created two-bit digital-to-analog converters, which should be useful in biotechnology applications for encoding multiple stable gene expression outputs using transient inputs of inducers. We envision that this integrated logic and memory system will enable the implementation of complex cellular state machines, behaviors and pathways for therapeutic, diagnostic and basic science applications. PMID:23396014

  15. Amyloplast sedimentation and organelle saltation in living corn columella cells

    NASA Technical Reports Server (NTRS)

    Sack, F. D.; Suyemoto, M. M.; Leopold, A. C.

    1986-01-01

    Amyloplast sedimentation during gravistimulation and organelle movements was studied in living central rootcap cells of Zea mays L. cv. Merit. Cells from sectioned roots were viewed with a horizontally-mounted videomicroscope. The kinetics of gravity-induced amyloplast sedimentation were comparable to those calculated from experiments using fixed material. Individual amyloplasts fell at an average velocity of 5.5 micrometers min-1; the maximal velocity of fall measured was 18.0 micrometers min-1. Amyloplasts often rotated, sometimes rose in the cytoplasm, and occasionally underwent sudden rapid movements as fast as 58 micrometers min-1. Saltations of other organelles were frequently observed. This appears to be the first report of cytoplasmic streaming in the presumptive statocytes of roots.

  16. Automated quantification of cellular traffic in living cells.

    PubMed

    Broeke, Jurjen H P; Ge, Haifang; Dijkstra, Ineke M; Cemgil, Ali Taylan; Riedl, Jürgen A; Cornelisse, L Niels; Toonen, Ruud F; Verhage, Matthijs; Fitzgerald, William J

    2009-04-15

    Cellular traffic is a central aspect of cell function in health and disease. It is highly dynamic, and can be investigated at increasingly finer temporal and spatial resolution due to new imaging techniques and probes. Manual tracking of these data is labor-intensive and observer-biased and existing automation is only semi-automatic and requires near-perfect object detection and high-contrast images. Here, we describe a novel automated technique for quantifying cellular traffic. Using local intrinsic information from adjacent images in a sequence and a model for object characteristics, our approach detects and tracks multiple objects in living cells via Multiple Hypothesis Tracking and handles several confounds (merge/split, birth/death, and clutters), as reliable as expert observers. By replacing the related component (e.g. using a different appearance model) the method can be easily adapted for quantitative analysis of other biological samples. PMID:19146878

  17. 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

  18. TCSPC based approaches for multiparameter detection in living cells

    NASA Astrophysics Data System (ADS)

    Jahn, Karolina; Buschmann, Volker; Koberling, Felix; Hille, Carsten

    2014-03-01

    In living cells a manifold of processes take place simultaneously. This implies a precise regulation of intracellular ion homeostasis. In order to understand their spatio-temporal pattern comprehensively, the development of multiplexing concepts is essential. Due to the multidimensional characteristics of fluorescence dyes (absorption and emission spectra, decay time, anisotropy), the highly sensitive and non-invasive fluorescence microscopy is a versatile tool for realising multiplexing concepts. A prerequisite are analyte-specific fluorescence dyes with low cross-sensitivity to other dyes and analytes, respectively. Here, two approaches for multiparameter detection in living cells are presented. Insect salivary glands are well characterised secretory active tissues which were used as model systems to evaluate multiplexing concepts. Salivary glands secrete a KCl-rich or NaCl-rich fluid upon stimulation which is mainly regulated by intracellular Ca2+ as second messenger. Thus, pairwise detection of intracellular Na+, Cl- and Ca2+ with the fluorescent dyes ANG2, MQAE and ACR were tested. Therefore, the dyes were excited simultaneously (2-photon excitation) and their corresponding fluorescence decay times were recorded within two spectral ranges using time-correlated singlephoton counting (TCSPC). A second approach presented here is based on a new TCSPC-platform covering decay time detection from picoseconds to milliseconds. Thereby, nanosecond decaying cellular fluorescence and microsecond decaying phosphorescence of Ruthenium-complexes, which is quenched by oxygen, were recorded simultaneously. In both cases changes in luminescence decay times can be linked to changes in analyte concentrations. In consequence of simultaneous excitation as well as detection, it is possible to get a deeper insight into spatio-temporal pattern in living tissues.

  19. Probing mechanical properties of living cells by magnetopneumography.

    PubMed

    Möller, W; Takenaka, S; Rust, M; Stahlhofen, W; Heyder, J

    1997-01-01

    Magnetopneumography (MPG) has been used to study long-term particle clearance from human lungs as well as cellular motility of pulmonary macrophages (PMs). This study describes an extension of the method enabling the measurement of mechanical properties of PM cells in vivo. Ferromagnetic microparticles are inhaled and then retained in the alveolar region of the lungs, where they are phagocytized within hours by PMs. The magnetic particles can be rotated in weak magnetic fields, and the response to this twisting shear (force) is detected as a macroscopic magnetic field producing a measure of cytoskeletal mechanics. Cytoplasmic viscosity is very high compared with that of water and is strongly non-Newtonian. Under rotational stresses from 0.4 to 6.4 Pa, it acts like a pseudoplastic fluid showing a characteristic shear rate dependence. The viscosity as well as the stiffness of the cytoskeleton increases with increasing shear stress as seems typical for living tissue and evidence for an intact cytoskeletal matrix. The particle recoil as measured by the amount of recoverable strain following a short twisting force describes a cytoplasmic elasticity that depends on both level and duration of stress. These investigations on the mechanical properties of living human cells are promising and should lead to better understanding of cellular dysfunction in disease as well as pathways for drug administration. PMID:10174196

  20. Advances in radiation biology: Radiosensitization in DNA and living cells

    NASA Astrophysics Data System (ADS)

    Lacombe, S.; Sech, C. Le

    2009-06-01

    One fundamental goal of radiation biology is the evolution of concepts and methods for the elaboration of new approaches and protocols for the treatment of cancers. In this context, the use of fast ions as ionizing particles offers the advantage of optimizing cell killing inside the tumor whilst preserving the surrounding healthy tissues. One extremely promising strategy investigated recently is the addition of radiosensitizers in the targeted tissue. The optimization of radiotherapy with fast ions implies a multidisciplinary approach to ionizing radiation effects on complex living systems, ranging from studies on single molecules to investigations of entire organisms. In this article we review recent studies on ion induced damages in simple and complex biological systems, from DNA to living cells. The specific aspect of radiosensitization induced by metallic atoms is described. As a fundamental result, the addition of sensitizing compounds with ion irradiation may improve therapeutic index in cancer therapy. In conclusion, new perspectives are proposed based on the experience and contribution of different communities including Surface Sciences, to improve the development of radiation biology.

  1. Carotenoid Distribution in Living Cells of Haematococcus pluvialis (Chlorophyceae)

    SciTech Connect

    Collins, Aaron M.; Jones, Howland D. T.; Han, Danxiang; Hu, Qiang; Beechem, Thomas E.; Timlin, Jerilyn A.; Evens, Terence

    2011-09-06

    Haematococcus pluvialis is a freshwater unicellular green microalga belonging to the class Chlorophyceae and is of commercial interest for its ability to accumulate massive amounts of the red ketocarotenoid astaxanthin (3,3'-dihydroxy-β,β-carotene-4,4'-dione). Using confocal Raman microscopy and multivariate analysis, we demonstrate the ability to spectrally resolve resonance–enhanced Raman signatures associated with astaxanthin and β-carotene along with chlorophyll fluorescence. By mathematically isolating these spectral signatures, in turn, it is possible to locate these species independent of each other in living cells of H. pluvialis in various stages of the life cycle. Chlorophyll emission was found only in the chloroplast whereas astaxanthin was identified within globular and punctate regions of the cytoplasmic space. Moreover, we found evidence for β-carotene to be co-located with both the chloroplast and astaxanthin in the cytosol. These observations imply that β-carotene is a precursor for astaxanthin and the synthesis of astaxanthin occurs outside the chloroplast. Finally, our work demonstrates the broad utility of confocal Raman microscopy to resolve spectral signatures of highly similar chromophores in living cells.

  2. Labeling proteins on live mammalian cells using click chemistry.

    PubMed

    Nikić, Ivana; Kang, Jun Hee; Girona, Gemma Estrada; Aramburu, Iker Valle; Lemke, Edward A

    2015-05-01

    We describe a protocol for the rapid labeling of cell-surface proteins in living mammalian cells using click chemistry. The labeling method is based on strain-promoted alkyne-azide cycloaddition (SPAAC) and strain-promoted inverse-electron-demand Diels-Alder cycloaddition (SPIEDAC) reactions, in which noncanonical amino acids (ncAAs) bearing ring-strained alkynes or alkenes react, respectively, with dyes containing azide or tetrazine groups. To introduce ncAAs site specifically into a protein of interest (POI), we use genetic code expansion technology. The protocol can be described as comprising two steps. In the first step, an Amber stop codon is introduced--by site-directed mutagenesis--at the desired site on the gene encoding the POI. This plasmid is then transfected into mammalian cells, along with another plasmid that encodes an aminoacyl-tRNA synthetase/tRNA (RS/tRNA) pair that is orthogonal to the host's translational machinery. In the presence of the ncAA, the orthogonal RS/tRNA pair specifically suppresses the Amber codon by incorporating the ncAA into the polypeptide chain of the POI. In the second step, the expressed POI is labeled with a suitably reactive dye derivative that is directly supplied to the growth medium. We provide a detailed protocol for using commercially available ncAAs and dyes for labeling the insulin receptor, and we discuss the optimal surface-labeling conditions and the limitations of labeling living mammalian cells. The protocol involves an initial cloning step that can take 4-7 d, followed by the described transfections and labeling reaction steps, which can take 3-4 d. PMID:25906116

  3. Fluorescence resonance energy transfer-based stoichiometry in living cells.

    PubMed Central

    Hoppe, Adam; Christensen, Kenneth; Swanson, Joel A

    2002-01-01

    Imaging of fluorescence resonance energy transfer (FRET) between fluorescently labeled molecules can measure the timing and location of intermolecular interactions inside living cells. Present microscopic methods measure FRET in arbitrary units, and cannot discriminate FRET efficiency and the fractions of donor and acceptor in complex. Here we describe a stoichiometric method that uses three microscopic fluorescence images to measure FRET efficiency, the relative concentrations of donor and acceptor, and the fractions of donor and acceptor in complex in living cells. FRET stoichiometry derives from the concept that specific donor-acceptor complexes will give rise to a characteristic FRET efficiency, which, if measured, can allow stoichiometric discrimination of interacting components. A first equation determines FRET efficiency and the fraction of acceptor molecules in complex with donor. A second equation determines the fraction of donor molecules in complex by estimating the donor fluorescence lost due to energy transfer. This eliminates the need for acceptor photobleaching to determine total donor concentrations and allows for repeated measurements from the same cell. A third equation obtains the ratio of total acceptor to total donor molecules. The theory and method were confirmed by microscopic measurements of fluorescence from cyan fluorescent protein (CFP), citrine, and linked CFP-Citrine fusion protein, in solutions and inside cells. Together, the methods derived from these equations allow sensitive, rapid, and repeatable detection of donor-, acceptor-, and donor-acceptor complex stoichiometry at each pixel in an image. By accurately imaging molecular interactions, FRET stoichiometry opens new areas for quantitative study of intracellular molecular networks. PMID:12496132

  4. Voyager 2 Jupiter Eruption Movie

    NASA Technical Reports Server (NTRS)

    2000-01-01

    This movie records an eruptive event in the southern hemisphere of Jupiter over a period of 8 Jupiter days. Prior to the event, an undistinguished oval cloud mass cruised through the turbulent atmosphere. The eruption occurs over avery short time at the very center of the cloud. The white eruptive material is swirled about by the internal wind patterns of the cloud. As a result of the eruption, the cloud then becomes a type of feature seen elsewhere on Jupiter known as 'spaghetti bowls'.

    As Voyager 2 approached Jupiter in 1979, it took images of the planet at regular intervals. This sequence is made from 8 images taken once every Jupiter rotation period (about 10 hours). These images were acquired in the Violet filter around May 6, 1979. The spacecraft was about 50 million kilometers from Jupiter at that time.

    This time-lapse movie was produced at JPL by the Image Processing Laboratory in 1979.

  5. Live-cell protein labelling with nanometre precision by cell squeezing

    PubMed Central

    Kollmannsperger, Alina; Sharei, Armon; Raulf, Anika; Heilemann, Mike; Langer, Robert; Jensen, Klavs F.; Wieneke, Ralph; Tampé, Robert

    2016-01-01

    Live-cell labelling techniques to visualize proteins with minimal disturbance are important; however, the currently available methods are limited in their labelling efficiency, specificity and cell permeability. We describe high-throughput protein labelling facilitated by minimalistic probes delivered to mammalian cells by microfluidic cell squeezing. High-affinity and target-specific tracing of proteins in various subcellular compartments is demonstrated, culminating in photoinduced labelling within live cells. Both the fine-tuned delivery of subnanomolar concentrations and the minimal size of the probe allow for live-cell super-resolution imaging with very low background and nanometre precision. This method is fast in probe delivery (∼1,000,000 cells per second), versatile across cell types and can be readily transferred to a multitude of proteins. Moreover, the technique succeeds in combination with well-established methods to gain multiplexed labelling and has demonstrated potential to precisely trace target proteins, in live mammalian cells, by super-resolution microscopy. PMID:26822409

  6. Enhancing Soundtracks From Old Movies

    NASA Technical Reports Server (NTRS)

    Frazer, Robert E.

    1992-01-01

    Proposed system enhances soundtracks of old movies. Signal on optical soundtrack of film digitized and processed to reduce noise and improve quality; timing signals added, and signal recorded on compact disk. Digital comparator and voltage-controlled oscillator synchronizes speed of film-drive motor and compact disk motor. Frame-coded detector reads binary frame-identifying marks on film. Digital comparator generates error signal if marks on film do not match those on compact disk.

  7. Mechanodelivery of nanoparticles to the cytoplasm of living cells

    NASA Astrophysics Data System (ADS)

    Emerson, Nyssa T.; Hsia, Chih-Hao; Rafalska-Metcalf, Ilona U.; Yang, Haw

    2014-04-01

    Nanotechnology has opened up the opportunity to probe, sense, and manipulate the chemical environment of biological systems with an unprecedented level of spatiotemporal control. A major obstacle to the full realization of these novel technologies is the lack of a general, robust, and simple method for the delivery of arbitrary nanostructures to the cytoplasm of intact live cells. Here, we identify a new delivery modality, based on mechanical disruption of the plasma membrane, which efficiently mediates the delivery of nanoparticles to the cytoplasm of mammalian cells. We use two distinct execution modes, two adherent cell lines, and three sizes of semiconducting nanocrystals, or quantum dots, to demonstrate its applicability and effectiveness. As the underlying mechanism is purely physical, we anticipate that such ``mechanodelivery'' can be generalized to other modes of execution as well as to the cytoplasmic introduction of a structurally diverse array of functional nanomaterials.Nanotechnology has opened up the opportunity to probe, sense, and manipulate the chemical environment of biological systems with an unprecedented level of spatiotemporal control. A major obstacle to the full realization of these novel technologies is the lack of a general, robust, and simple method for the delivery of arbitrary nanostructures to the cytoplasm of intact live cells. Here, we identify a new delivery modality, based on mechanical disruption of the plasma membrane, which efficiently mediates the delivery of nanoparticles to the cytoplasm of mammalian cells. We use two distinct execution modes, two adherent cell lines, and three sizes of semiconducting nanocrystals, or quantum dots, to demonstrate its applicability and effectiveness. As the underlying mechanism is purely physical, we anticipate that such ``mechanodelivery'' can be generalized to other modes of execution as well as to the cytoplasmic introduction of a structurally diverse array of functional nanomaterials

  8. Enhanced live cell imaging via photonic crystal enhanced fluorescence microscopy.

    PubMed

    Chen, Weili; Long, Kenneth D; Yu, Hojeong; Tan, Yafang; Choi, Ji Sun; Harley, Brendan A; Cunningham, Brian T

    2014-11-21

    We demonstrate photonic crystal enhanced fluorescence (PCEF) microscopy as a surface-specific fluorescence imaging technique to study the adhesion of live cells by visualizing variations in cell-substrate gap distance. This approach utilizes a photonic crystal surface incorporated into a standard microscope slide as the substrate for cell adhesion, and a microscope integrated with a custom illumination source as the detection instrument. When illuminated with a monochromatic light source, angle-specific optical resonances supported by the photonic crystal enable efficient excitation of surface-confined and amplified electromagnetic fields when excited at an on-resonance condition, while no field enhancement occurs when the same photonic crystal is illuminated in an off-resonance state. By mapping the fluorescence enhancement factor for fluorophore-tagged cellular components between on- and off-resonance states and comparing the results to numerical calculations, the vertical distance of labelled cellular components from the photonic crystal substrate can be estimated, providing critical and quantitative information regarding the spatial distribution of the specific components of cells attaching to a surface. As an initial demonstration of the concept, 3T3 fibroblast cells were grown on fibronectin-coated photonic crystals with fluorophore-labelled plasma membrane or nucleus. We demonstrate that PCEF microscopy is capable of providing information about the spatial distribution of cell-surface interactions at the single-cell level that is not available from other existing forms of microscopy, and that the approach is amenable to large fields of view, without the need for coupling prisms, coupling fluids, or special microscope objectives. PMID:25265458

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

    NASA Astrophysics Data System (ADS)

    Junghans, Ann

    Understanding the structure and functionality of biological systems on a nanometer-resolution and short temporal scales is important for solving complex biological problems, developing innovative treatment, and advancing the design of highly functionalized biomimetic materials. For example, adhesion of cells to an underlying substrate plays a crucial role in physiology and disease development, and has been investigated with great interest for several decades. In the talk, we would like to highlight recent advances in utilizing neutron scattering to study bio-related structures in dynamic conditions (e . g . under the shear flow) including in-situ investigations of the interfacial properties of living cells. The strength of neutron reflectometry is its non-pertubative nature, the ability to probe buried interfaces with nanometer resolution and its sensitivity to light elements like hydrogen and carbon. That allows us to study details of cell - substrate interfaces that are not accessible with any other standard techniques. We studied the adhesion of human brain tumor cells (U251) to quartz substrates and their responses to the external mechanical forces. Such cells are isolated within the central nervous system which makes them difficult to reach with conventional therapies and therefore making them highly invasive. Our results reveal changes in the thickness and composition of the adhesion layer (a layer between the cell lipid membrane and the quartz substrate), largely composed of hyaluronic acid and associated proteoglycans, when the cells were subjected to shear stress. Further studies will allow us to determine more conditions triggering changes in the composition of the bio-material in the adhesion layer. This, in turn, can help to identify changes that correlate with tumor invasiveness, which can have significant medical impact for the development of targeted anti-invasive therapies.

  10. Enhanced live cell imaging via photonic crystal enhanced fluorescence microscopy†

    PubMed Central

    Chen, Weili; Long, Kenneth D.; Yu, Hojeong; Tan, Yafang; Choi, Ji Sun; Harley, Brendan A.; Cunningham, Brian T.

    2014-01-01

    We demonstrate photonic crystal enhanced fluorescence (PCEF) microscopy as a surface-specific fluorescence imaging technique to study the adhesion of live cells by visualizing variations in cell-substrate gap distance. This approach utilizes a photonic crystal surface incorporated into a standard microscope slide as the substrate for cell adhesion, and a microscope integrated with a custom illumination source as the detection instrument. When illuminated with a monochromatic light source, angle-specific optical resonances supported by the photonic crystal enable efficient excitation of surface-confined and amplified electromagnetic fields when excited at an on-resonance condition, while no field enhancement occurs when the same photonic crystal is illuminated in an off-resonance state. By mapping the fluorescence enhancement factor for fluorophore-tagged cellular components between on- and off-resonance states and comparing the results to numerical calculations, the vertical distance of labelled cellular components from the photonic crystal substrate can be estimated, providing critical and quantitative information regarding the spatial distribution of the specific components of cells attaching to a surface. As an initial demonstration of the concept, 3T3 fibroblast cells were grown on fibronectin-coated photonic crystals with fluorophore-labelled plasma membrane or nucleus. We demonstrate that PCEF microscopy is capable of providing information about the spatial distribution of cell-surface interactions at the single-cell level that is not available from other existing forms of microscopy, and that the approach is amenable to large fields of view, without the need for coupling prisms, coupling fluids, or special microscope objectives. PMID:25265458

  11. Imaging Cell Shape Change in Living Drosophila Embryos

    PubMed Central

    Figard, Lauren; Sokac, Anna Marie

    2011-01-01

    The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging. Cell shape changes in the fly are analogous to those in higher organisms, and they drive tissue morphogenesis. So, in many cases, their study has direct implications for understanding human disease (Table 1)1-5. On the sub-cellular scale, these cell shape changes are the product of activities ranging from gene expression to signal transduction, cell polarity, cytoskeletal remodeling and membrane trafficking. Thus, the Drosophila embryo provides not only the context to evaluate cell shape changes as they relate to tissue morphogenesis, but also offers a completely physiological environment to study the sub-cellular activities that shape cells. The protocol described here is designed to image a specific cell shape change called cellularization. Cellularization is a process of dramatic plasma membrane growth, and it ultimately converts the syncytial embryo into the cellular blastoderm. That is, at interphase of mitotic cycle 14, the plasma membrane simultaneously invaginates around each of ~6000 cortically anchored nuclei to generate a sheet of primary epithelial cells. Counter to previous suggestions, cellularization is not driven by Myosin-2 contractility6, but is instead fueled largely by exocytosis of membrane from internal stores7. Thus, cellularization is an excellent system for studying membrane trafficking during cell shape changes that require plasma membrane invagination or expansion, such as cytokinesis or transverse-tubule (T-tubule) morphogenesis in muscle. Note that this protocol is easily applied to the imaging of other cell shape changes in the fly embryo, and only requires slight adaptations such as changing the stage of embryo collection, or using "embryo glue" to mount the embryo in a specific orientation (Table 1)8-19. In all cases, the workflow is basically the same (Figure 1). Standard methods for cloning and

  12. Imaging cell shape change in living Drosophila embryos.

    PubMed

    Figard, Lauren; Sokac, Anna Marie

    2011-01-01

    The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging. Cell shape changes in the fly are analogous to those in higher organisms, and they drive tissue morphogenesis. So, in many cases, their study has direct implications for understanding human disease (Table 1)(1-5). On the sub-cellular scale, these cell shape changes are the product of activities ranging from gene expression to signal transduction, cell polarity, cytoskeletal remodeling and membrane trafficking. Thus, the Drosophila embryo provides not only the context to evaluate cell shape changes as they relate to tissue morphogenesis, but also offers a completely physiological environment to study the sub-cellular activities that shape cells. The protocol described here is designed to image a specific cell shape change called cellularization. Cellularization is a process of dramatic plasma membrane growth, and it ultimately converts the syncytial embryo into the cellular blastoderm. That is, at interphase of mitotic cycle 14, the plasma membrane simultaneously invaginates around each of ~6000 cortically anchored nuclei to generate a sheet of primary epithelial cells. Counter to previous suggestions, cellularization is not driven by Myosin-2 contractility(6), but is instead fueled largely by exocytosis of membrane from internal stores(7). Thus, cellularization is an excellent system for studying membrane trafficking during cell shape changes that require plasma membrane invagination or expansion, such as cytokinesis or transverse-tubule (T-tubule) morphogenesis in muscle. Note that this protocol is easily applied to the imaging of other cell shape changes in the fly embryo, and only requires slight adaptations such as changing the stage of embryo collection, or using "embryo glue" to mount the embryo in a specific orientation (Table 1)(8-19). In all cases, the workflow is basically the same (Figure 1). Standard methods for

  13. Enlightening intracellular complexity of living cells with quantitative phase microscopy

    NASA Astrophysics Data System (ADS)

    Martinez Torres, C.; Laperrousaz, B.; Berguiga, L.; Boyer Provera, E.; Elezgaray, J.; Nicolini, F. E.; Maguer-Satta, V.; Arneodo, A.; Argoul, F.

    2016-03-01

    The internal distribution of refractive indices (RIs) of a living cell is much more complex than usually admitted in multi-shell models. The reconstruction of RI maps from single phase images has rarely been achieved for several reasons: (i) we still have very little knowledge of the impact of internal macromolecular complexes on the local RI and (ii) phase changes produced by light propagation through the sample are mixed with diffraction effects by internal cell bodies. We propose the implementation a 2D wavelet-based contour chain detection method to distinguish internal boundaries thanks to their greatest optical path difference gradients. These contour chains correspond to the highest image phase contrast and follow the local RI inhomogeneities linked to the intracellular structural intricacy. Their statistics and spatial distribution are morphological indicators for distinguishing cells of different origins and to follow their transformation in pathologic situations. We use this method to compare non adherent blood cells from primary and laboratory culture origins, in healthy and pathological situations (chronic myelogenous leukaemia). In a second part of this presentation, we concentrate on the temporal dynamics of the phase contour chains and we discuss the spectral decomposition of their dynamics in both health and disease.

  14. Temperature dependence of protein folding kinetics in living cells

    PubMed Central

    Guo, Minghao; Xu, Yangfan; Gruebele, Martin

    2012-01-01

    We measure the stability and folding rate of a mutant of the enzyme phosphoglycerate kinase (PGK) inside bone tissue cells as a function of temperature from 38 to 48 °C. To facilitate measurement in individual living cells, we developed a rapid laser temperature stepping method capable of measuring complete thermal melts and kinetic traces in about two min. We find that this method yields improved thermal melts compared to heating a sample chamber or microscope stage. By comparing results for six cells with in vitro data, we show that the protein is stabilized by about 6 kJ/mole in the cytoplasm, but the temperature dependence of folding kinetics is similar to in vitro. The main difference is a slightly steeper temperature dependence of the folding rate in some cells that can be rationalized in terms of temperature-dependent crowding, local viscosity, or hydrophobicity. The observed rate coefficients can be fitted within measurement uncertainty by an effective two-state model, even though PGK folds by a multistate mechanism. We validate the effective two-state model with a three-state free energy landscape of PGK to illustrate that the effective fitting parameters can represent a more complex underlying free energy landscape. PMID:22665776

  15. Phasor FLIM metabolic mapping of stem cells and cancer cells in live tissues

    NASA Astrophysics Data System (ADS)

    Stringari, Chiara; Donovan, Peter; Gratton, Enrico

    2012-03-01

    We use the phasor approach to fluorescence lifetime imaging and intrinsic biochemical fluorescence biomarkers in conjunction with image segmentation and the concept of cell phasor for deriving metabolic maps of cells and living tissues in vivo. In issues we identify and separate intrinsic fluorophores such as collagen, retinol, retinoic acid, porphyrin, flavins, free and bound nicotinamide adenine dinucleotide (NADH). Metabolic signatures of tissues are obtained by calculating the phasor fingerprint of single cells and by mapping the relative concentration of metabolites. This method detects small changes in metabolic signatures and redox states of cells. Phasor fingerprints of stem cells cluster according to their differentiation state in a living tissue such as the C. elegans germ line and the crypt base of small intestine and colon. Phasor FLIM provides a label-free and fit-free sensitive method to identify metabolic states of cells and to classify stem cells, normal differentiated cells and cancer cells both in vitro and in a live tissue. Our method could identify symmetric and asymmetric divisions, predict cell fate and identify pre-cancer stages in vivo. This method is a promising non-invasive optical tool for monitoring metabolic pathways during differentiation and carcinogenesis, for cell sorting and high throughput screening.

  16. Imaging phosphatidylinositol 4-phosphate dynamics in living plant cells.

    PubMed

    Vermeer, Joop E M; Thole, Julie M; Goedhart, Joachim; Nielsen, Erik; Munnik, Teun; Gadella, Theodorus W J

    2009-01-01

    Polyphosphoinositides represent a minor group of phospholipids, accounting for less than 1% of the total. Despite their low abundance, these molecules have been implicated in various signalling and membrane trafficking events. Phosphatidylinositol 4-phosphate (PtdIns4P) is the most abundant polyphosphoinositide. (32)Pi-labelling studies have shown that the turnover of PtdIns4P is rapid, but little is known about where in the cell or plant this occurs. Here, we describe the use of a lipid biosensor that monitors PtdIns4P dynamics in living plant cells. The biosensor consists of a fusion between a fluorescent protein and a lipid-binding domain that specifically binds PtdIns4P, i.e. the pleckstrin homology domain of the human protein phosphatidylinositol-4-phosphate adaptor protein-1 (FAPP1). YFP-PH(FAPP1) was expressed in four plant systems: transiently in cowpea protoplasts, and stably in tobacco BY-2 cells, Medicago truncatula roots and Arabidopsis thaliana seedlings. All systems allowed YFP-PH(FAPP1) expression without detrimental effects. Two distinct fluorescence patterns were observed: labelling of motile punctate structures and the plasma membrane. Co-expression studies with organelle markers revealed strong co-labelling with the Golgi marker STtmd-CFP, but not with the endocytic/pre-vacuolar marker GFP-AtRABF2b. Co-expression with the Ptdins3P biosensor YFP-2 x FYVE revealed totally different localization patterns. During cell division, YFP-PH(FAPP1) showed strong labelling of the cell plate, but PtdIns3P was completely absent from the newly formed cell membrane. In root hairs of M. truncatula and A. thaliana, a clear PtdIns4P gradient was apparent in the plasma membrane, with the highest concentration in the tip. This only occurred in growing root hairs, indicating a role for PtdIns4P in tip growth. PMID:18785997

  17. Quantitative imaging of glutathione in live cells using a reversible reaction-based ratiometric fluorescent probe

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Glutathione (GSH) plays an important role in maintaining redox homeostasis inside cells. Currently, there are no methods available to quantitatively assess the GSH concentration in live cells. Live cell fluorescence imaging revolutionized the understanding of cell biology and has become an indispens...

  18. Influence of cell growth conditions and medium composition on EGFP photostability in live cells.

    PubMed

    Mamontova, Anastasia V; Bogdanov, Alexey M; Lukyanov, Konstantin A

    2015-05-01

    Photostability is a key characteristic of fluorescent proteins. It was recently demonstrated that green fluorescent protein (GFP) photobleaching in live cells can be suppressed by changes in medium composition. Here we show that Ham's F12 medium provides very high enhanced GFP (EGFP) photostability during fluorescence microscopy of live cells. This property of Ham's F12 medium is associated with decreased concentrations of riboflavin and pyridoxine, and increased concentrations of FeSO4, cyanocobalamine, lipoic acid, hypoxanthine, and thymidine compared with DMEM. We also found that the rate of EGFP photobleaching strongly depends on cell growth conditions such as cell density and the concentration of serum. We conclude that both imaging medium composition and the physiological state of the cells can strongly affect the photostability of fluorescent proteins. Thus, accurate comparison of the photostabilities of fluorescent proteins should be performed only in side-by-side analysis in identical cell growth conditions and media. PMID:25967905

  19. Dynamic cell culture: a microfluidic function generator for live cell microscopy.

    PubMed

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

    2009-01-01

    We present a microfluidic system for time-lapsed, live cell microscopy with the ability to control solution exchange via a dynamic flow controller. The application specific microfluidic plates are designed to maintain adherent and non-adherent cell types for multiple days with continuous medium perfusion. Upstream channels with flow controlled via custom software allow the delivery of unique exposure profiles to the cultured cells, such as square waves, step functions, ramps, etc. PMID:19209350

  20. Synthetic recombinase-based state machines in living cells.

    PubMed

    Roquet, Nathaniel; Soleimany, Ava P; Ferris, Alyssa C; Aaronson, Scott; Lu, Timothy K

    2016-07-22

    State machines underlie the sophisticated functionality behind human-made and natural computing systems that perform order-dependent information processing. We developed a recombinase-based framework for building state machines in living cells by leveraging chemically controlled DNA excision and inversion operations to encode states in DNA sequences. This strategy enables convenient readout of states (by sequencing and/or polymerase chain reaction) as well as complex regulation of gene expression. We validated our framework by engineering state machines in Escherichia coli that used one, two, or three chemical inputs to control up to 16 DNA states. These state machines were capable of recording the temporal order of all inputs and performing multi-input, multi-output control of gene expression. We also developed a computational tool for the automated design of gene regulation programs using recombinase-based state machines. Our scalable framework should enable new strategies for recording and studying how combinational and temporal events regulate complex cell functions and for programming sophisticated cell behaviors. PMID:27463678

  1. Visualization of the intracellular behavior of HIV in living cells

    PubMed Central

    McDonald, David; Vodicka, Marie A.; Lucero, Ginger; Svitkina, Tatyana M.; Borisy, Gary G.; Emerman, Michael; Hope, Thomas J.

    2002-01-01

    To track the behavior of human immunodeficiency virus (HIV)-1 in the cytoplasm of infected cells, we have tagged virions by incorporation of HIV Vpr fused to the GFP. Observation of the GFP-labeled particles in living cells revealed that they moved in curvilinear paths in the cytoplasm and accumulated in the perinuclear region, often near the microtubule-organizing center. Further studies show that HIV uses cytoplasmic dynein and the microtubule network to migrate toward the nucleus. By combining GFP fused to the NH2 terminus of HIV-1 Vpr tagging with other labeling techniques, it was possible to determine the state of progression of individual particles through the viral life cycle. Correlation of immunofluorescent and electron micrographs allowed high resolution imaging of microtubule-associated structures that are proposed to be reverse transcription complexes. Based on these observations, we propose that HIV uses dynein and the microtubule network to facilitate the delivery of the viral genome to the nucleus of the cell during early postentry steps of the HIV life cycle. PMID:12417576

  2. Optogenetics: optical control of a photoactivatable Rac in living cells.

    PubMed

    Yin, Taofei; Wu, Yi I

    2015-01-01

    Recent developments in optogenetics have extended optical control of signaling to intracellular proteins, including Rac, a small G protein in the Rho family. A blue light-sensing LOV (light, oxygen, or voltage) domain derived from Avena sativa (oat) phototropin was fused to the N-terminus of a constitutively active mutant of Rac, via an α-helix (Jα) that is conserved among plant phototropins. The fused LOV domain occluded binding of downstream effectors to Rac in the dark. Exposure to blue light caused a conformational change of the LOV domain and unwinding of the Jα helix, relieving steric inhibition. The LOV domain incorporates a flavin as the photon-absorbing cofactor and can be activated by light in a reversible and repeatable fashion. In cultured cells, global illumination with blue light rapidly activated Rac and led to cell spreading and membrane ruffling. Localized and pulsed illumination generated a gradient of Rac activity and induced directional migration. In this chapter, we will describe the techniques in detail and present some examples of applications of using photoactivatable Rac (PA-Rac) in living cells. PMID:25391805

  3. Bioluminescence microscopy: application to ATP measurements in single living cells

    NASA Astrophysics Data System (ADS)

    Brau, Frederic; Helle, Pierre; Bernengo, Jean C.

    1997-12-01

    Bioluminescence microscopy can be used to measure intracellular cofactors and ionic concentrations (Ca2+, K+, ATP, NADH), as an alternative to micro- spectrophotometry and micro-fluorimetry, due to the development of sensitive detectors (cooled photomultipliers tubes and CCD). The main limitation comes from the very small and brief intensity of the emitted light. Our instrumentation based on an inverted microscope, equipped with high aperture immersion lenses is presented. Light intensity measurements are carried out through a photomultiplier sorted for low dark current and cooled at -5 degree(s)C to reduce thermal noise. Our first aim is to quantify ATP on single living cells using the firefly luciferin-luciferase couple. Experimental and kinetic aspects are presented to emphasize the potentialities of the technique.

  4. Fluorescent ligand for human progesterone receptor imaging in live cells.

    PubMed

    Weinstain, Roy; Kanter, Joan; Friedman, Beth; Ellies, Lesley G; Baker, Michael E; Tsien, Roger Y

    2013-05-15

    We employed molecular modeling to design and then synthesize fluorescent ligands for the human progesterone receptor. Boron dipyrromethene (BODIPY) or tetramethylrhodamine were conjugated to the progesterone receptor antagonist RU486 (Mifepristone) through an extended hydrophilic linker. The fluorescent ligands demonstrated comparable bioactivity to the parent antagonist in live cells and triggered nuclear translocation of the receptor in a specific manner. The BODIPY labeled ligand was applied to investigate the dependency of progesterone receptor nuclear translocation on partner proteins and to show that functional heat shock protein 90 but not immunophilin FKBP52 activity is essential. A tissue distribution study indicated that the fluorescent ligand preferentially accumulates in tissues that express high levels of the receptor in vivo. The design and properties of the BODIPY-labeled RU486 make it a potential candidate for in vivo imaging of PR by positron emission tomography through incorporation of (18)F into the BODIPY core. PMID:23600997

  5. Probes for intracellular RNA imaging in live cells.

    PubMed

    Santangelo, Philip J; Alonas, Eric; Jung, Jeenah; Lifland, Aaron W; Zurla, Chiara

    2012-01-01

    RNA localization, dynamics, and regulation are becoming increasingly important to our basic understanding of gene expression and RNA virus pathogenesis. An improved understanding of these processes will be necessary in order to identify new drug targets, as well as to create models of gene expression networks. Much of this new understanding will likely come from imaging studies of RNA, which can generate the spatiotemporal information necessary to characterize RNA within the cellular milieu. Ideally, this would be performed imaging native, nonengineered RNAs, but the approaches for performing these experiments are still evolving. In order for them to reach their potential, it is critical that they have characteristics that allow for the tracking of RNA throughout their life cycle. This chapter presents an overview of RNA imaging methodologies, and focuses on a single RNA sensitive method, employing exogenous probes, for imaging, native, nonengineered RNA in live cells. PMID:22289464

  6. Lasing within Live Cells Containing Intracellular Optical Microresonators for Barcode-Type Cell Tagging and Tracking.

    PubMed

    Schubert, Marcel; Steude, Anja; Liehm, Philipp; Kronenberg, Nils M; Karl, Markus; Campbell, Elaine C; Powis, Simon J; Gather, Malte C

    2015-08-12

    We report on a laser that is fully embedded within a single live cell. By harnessing natural endocytosis of the cell, we introduce a fluorescent whispering gallery mode (WGM) microresonator into the cell cytoplasm. On pumping with nanojoule light pulses, green laser emission is generated inside the cells. Our approach can be applied to different cell types, and cells with microresonators remain viable for weeks under standard conditions. The characteristics of the lasing spectrum provide each cell with a barcode-type label which enables uniquely identifying and tracking individual migrating cells. Self-sustained lasing from cells paves the way to new forms of cell tracking, intracellular sensing, and adaptive imaging. PMID:26186167

  7. Multimodality imaging of reporter gene expression using a novel fusion vector in living cells and animals

    DOEpatents

    Gambhir, Sanjiv; Pritha, Ray

    2015-07-14

    Novel double and triple fusion reporter gene constructs harboring distinct imagable reporter genes are provided, as well as applications for the use of such double and triple fusion constructs in living cells and in living animals using distinct imaging technologies.

  8. Multimodality imaging of reporter gene expression using a novel fusion vector in living cells and animals

    DOEpatents

    Gambhir; Sanjiv , Pritha; Ray

    2009-04-28

    Novel double and triple fusion reporter gene constructs harboring distinct imageable reporter genes are provided, as well as applications for the use of such double and triple fusion constructs in living cells and in living animals using distinct imaging technologies.

  9. Multimodality imaging of reporter gene expression using a novel fusion vector in living cells and animals

    DOEpatents

    Gambhir, Sanjiv; Pritha, Ray

    2011-06-07

    Novel double and triple fusion reporter gene constructs harboring distinct imagable reporter genes are provided, as well as applications for the use of such double and triple fusion constructs in living cells and in living animals using distinct imaging technologies.

  10. Control of cell growth, division and death: information processing in living cells

    PubMed Central

    Tyson, John J.; Novak, Bela

    2014-01-01

    By way of surface receptor molecules and internal surveillance mechanisms, the living cell receives information about its external environment and internal state. In light of this information, the cell must determine its most appropriate course of action under the circumstances and initiate the relevant response pathways. Typical responses include growth and division, sexual reproduction, movement, differentiation and programmed cell death. Similar to a digital computer that uses bistable electrical switches to store and process information, the living cell uses bistable biochemical switches to implement its decision-making capabilities. In this review article, we describe some of the lines of thought that led, over the last 50 years, to our current understanding of cellular information processing, particularly related to cell growth, division and death. PMID:24904735

  11. Water Dynamics in Living Cells and Tumor Cell Migration in Confined Microenvironments

    NASA Astrophysics Data System (ADS)

    Sun, Sean

    More than 70% of the total mass in living cells is water. In most biological scenarios water serves as a passive medium responsible for solvation and proper functioning of proteins. However, it has been long recognized that there are situations where dynamic transport of water in cells is important. First, cells actively transport water in order to maintain its volume, and because cell volume directly influences cell shape and internal hydrostatic pressure, it is a critical aspect of cell mechanics. Furthermore, cell volume is coupled to protein synthesis which ultimately determines the cell size. Therefore water transport and cell volume dynamics ultimately impact cell growth and division. Second, epithelial cells in organs such as the eye and kidney actively transport water across the cell membrane and the epithelial layer. Indeed, water channels such as aquaporins increase water permeability of the membrane and facilitate this transport. Recent, we have shown that in confined microenvironments, active transport of water is responsible for actin-independent cell movement in confined spaces, especially for cancer cells. These results suggest that cells actively control its water content. The active regulation of water content is a crucial aspect of cell dynamics. We will discuss a theoretical model of cell pressure/volume control. Implications of this model for active cell dynamics in multi-cellular epithelial sheets will be discussed.

  12. Interaction of carbohydrate modified boron nitride nanotubes with living cells.

    PubMed

    Emanet, Melis; Şen, Özlem; Çobandede, Zehra; Çulha, Mustafa

    2015-10-01

    Boron nitride nanotubes (BNNTs) are composed of boron and nitrogen atoms and they show significantly different properties from their carbon analogues (carbon nanotubes, CNTs). Due to their unique properties including low electrical conductivity, and imaging contrast and neutron capture properties; they can be used in biomedical applications. When their use in biological fields is considered, the route of their toxic effect should be clarified. Therefore, the study of interactions between BNNTs and living systems is important in envisaging biological applications at both cellular and sub-cellular levels to fully gain insights of their potential adverse effects. In this study, BNNTs were modified with lactose, glucose and starch and tested for their cytotoxicity. First, the interactions and the behavior of BNNTs with bovine serum albumin (BSA), Dulbecco's Modified Eagle's Medium (DMEM) and DMEM/Nutrient Mixture F-12Ham were investigated. Thereafter, their cellular uptake and the cyto- and genotoxicity on human dermal fibroblasts (HDFs) and adenocarcinoma human alveolar basal epithelial cells (A549) were evaluated. HDFs and A549 cells internalized the modified and unmodified BNNTs, and BNNTs were found to not cause significant viability change and DNA damage. A higher uptake rate of BNNTs by A549 cells compared to HDFs was observed. Moreover, a concentration-dependent cytotoxicity was observed on A549 cells while they were safer for HDFs in the same concentration range. Based on these findings, it can be concluded that BNNTs and their derivatives made with biomacromolecules might be good candidates for several applications in medicine and biomedical applications. PMID:26222410

  13. Long-lived intestinal tuft cells serve as colon cancer-initiating cells.

    PubMed

    Westphalen, C Benedikt; Asfaha, Samuel; Hayakawa, Yoku; Takemoto, Yoshihiro; Lukin, Dana J; Nuber, Andreas H; Brandtner, Anna; Setlik, Wanda; Remotti, Helen; Muley, Ashlesha; Chen, Xiaowei; May, Randal; Houchen, Courtney W; Fox, James G; Gershon, Michael D; Quante, Michael; Wang, Timothy C

    2014-03-01

    Doublecortin-like kinase 1 protein (DCLK1) is a gastrointestinal tuft cell marker that has been proposed to identify quiescent and tumor growth-sustaining stem cells. DCLK1⁺ tuft cells are increased in inflammation-induced carcinogenesis; however, the role of these cells within the gastrointestinal epithelium and their potential as cancer-initiating cells are poorly understood. Here, using a BAC-CreERT-dependent genetic lineage-tracing strategy, we determined that a subpopulation of DCLK1⁺ cells is extremely long lived and possesses rare stem cell abilities. Moreover, genetic ablation of Dclk1 revealed that DCLK1⁺ tuft cells contribute to recovery following intestinal and colonic injury. Surprisingly, conditional knockdown of the Wnt regulator APC in DCLK1⁺ cells was not sufficient to drive colonic carcinogenesis under normal conditions; however, dextran sodium sulfate-induced (DSS-induced) colitis promoted the development of poorly differentiated colonic adenocarcinoma in mice lacking APC in DCLK1⁺ cells. Importantly, colonic tumor formation occurred even when colitis onset was delayed for up to 3 months after induced APC loss in DCLK1⁺ cells. Thus, our data define an intestinal DCLK1⁺ tuft cell population that is long lived, quiescent, and important for intestinal homeostasis and regeneration. Long-lived DCLK1⁺ cells maintain quiescence even following oncogenic mutation, but are activated by tissue injury and can serve to initiate colon cancer. PMID:24487592

  14. Live cell imaging of septin dynamics in Ustilago maydis.

    PubMed

    Baumann, S; Zander, S; Weidtkamp-Peters, S; Feldbrügge, M

    2016-01-01

    Septins are highly conserved cytoskeletal proteins involved in a variety of biological processes such as cell polarization and cytokinesis. In humans, functional defects in these proteins have been linked to cancer and neuronal diseases. In recent years, substantial progress has been made in studying the structure of septin subunits and the formation of defined heteromeric building blocks. These are assembled into higher-order structures at distinct subcellular sites. An important microscopic approach in studying septin assembly and dynamics is the use of septins tagged with fluorescent proteins. This revealed, eg, that septins form rings during cytokinesis and that septins build extended filaments partially colocalizing with actin cables and microtubules. Here, we describe extensive live cell imaging of septins in the model microorganism Ustilago maydis. We present techniques to study dynamic localization of protein and septin mRNA on shuttling endosomes as well as colocalization of proteins at these highly motile units. Moreover, FLIM-FRET experiments for analyzing local protein interactions are presented. Importantly, these imaging approaches transfer well to other fungal and animal model systems for in vivo analysis of septin dynamics. PMID:27473908

  15. 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

  16. Visual detection of Flavivirus RNA in living cells.

    PubMed

    Miorin, Lisa; Maiuri, Paolo; Marcello, Alessandro

    2016-04-01

    Flaviviruses include a wide range of important human pathogens delivered by insects or ticks. These viruses have a positive-stranded RNA genome that is replicated in the cytoplasm of the infected cell. The viral RNA genome is the template for transcription by the virally encoded RNA polymerase and for translation of the viral proteins. Furthermore, the double-stranded RNA intermediates of viral replication are believed to trigger the innate immune response through interaction with cytoplasmic cellular sensors. Therefore, understanding the subcellular distribution and dynamics of Flavivirus RNAs is of paramount importance to understand the interaction of the virus with its cellular host, which could be of insect, tick or mammalian, including human, origin. Recent advances on the visualization of Flavivirus RNA in living cells together with the development of methods to measure the dynamic properties of viral RNA are reviewed and discussed in this essay. In particular the application of bleaching techniques such as fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) are analysed in the context of tick-borne encephalitis virus replication. Conclusions driven by this approached are discussed in the wider context Flavivirus infection. PMID:26542763

  17. 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

  18. Glimpse of natural selection of long-lived T-cell clones in healthy life.

    PubMed

    Zhang, Baojun; Jia, Qingzhu; Bock, Cheryl; Chen, Gang; Yu, Haili; Ni, Qingshan; Wan, Ying; Li, Qijing; Zhuang, Yuan

    2016-08-30

    Homeostatic maintenance of T cells with broad clonal diversity is influenced by both continuing output of young T cells from the thymus and ongoing turnover of preexisting clones in the periphery. In the absence of infection, self and commensal antigens are thought to play important roles in selection and homeostatic maintenance of the T-cell pool. Most naïve T cells are short-lived due to lack of antigen encounter, whereas antigen-experienced T cells may survive and persist as long-lived clones. Thus far, little is known about the homeostasis, antigenic specificity, and clonal diversity of long-lived T-cell clones in peripheral lymphoid organs under healthy living conditions. To identify long-lived T-cell clones in mice, we designed a lineage-tracing method to label a wave of T cells produced in the thymus of young mice. After aging the mice for 1.5 y, we found that lineage-tracked T cells consisted of primarily memory-like T cells and T regulatory cells. T-cell receptor repertoire analysis revealed that the lineage-tracked CD4 memory-like T cells and T regulatory cells exhibited age-dependent enrichment of shared clonotypes. Furthermore, these shared clonotypes were found across different mice maintained in the same housing condition. These findings suggest that nonrandom and shared antigens are involved in controlling selection, retention, and immune tolerance of long-lived T-cell clones under healthy living conditions. PMID:27535935

  19. IR-Live: fabrication of a low-cost plastic microfluidic device for infrared spectromicroscopy of living cells.

    PubMed

    Birarda, G; Ravasio, A; Suryana, M; Maniam, S; Holman, H-Y N; Grenci, G

    2016-04-26

    Water is a strong mid-infrared absorber, which has hindered the full exploitation of label-free and non-invasive infrared (IR) spectromicroscopy techniques for the study of living biological samples. To overcome this barrier, many researchers have built sophisticated fluidic chambers or microfluidic chips wherein the depth of the liquid medium in the sample compartment is limited to 10 μm or less. Here we report an innovative and simple way to fabricate plastic devices with infrared transparent view-ports enabling infrared spectromicroscopy of living biological samples; therefore the device is named "IR-Live". Advantages of this approach include lower production costs, a minimal need to access a micro-fabrication facility, and unlimited mass or waste exchange for the living samples surrounding the view-port area. We demonstrate that the low-cost IR-Live in combination with microfluidic perfusion techniques enables long term (>60 h) cell culture, which broadens the capability of IR spectromicroscopy for studying living biological samples. To illustrate this, we first applied the device to study protein and lipid polarity in migrating REF52 fibroblasts by collecting 2-dimensional spectral chemical maps at a micrometer spatial resolution. Then, we demonstrated the suitability of our approach to study dynamic cellular events by collecting a time series of spectral maps of U937 monocytes during the early stage of cell attachment to a bio-compatible surface. PMID:27040369

  20. DNA From Dead Cancer Cells Induces TLR9-Mediated Invasion and Inflammation In Living Cancer Cells

    PubMed Central

    Tuomela, Johanna; Sandholm, Jouko; Kaakinen, Mika; Patel, Ankita; Kauppila, Joonas H.; Ilvesaro, Joanna; Chen, Dongquan; Harris, Kevin W.; Graves, David; Selander, Katri S.

    2014-01-01

    TLR9 is a cellular DNA-receptor, which is widely expressed in breast and other cancers. Although synthetic TLR9-ligands induce cancer cell invasion in vitro, the role of TLR9 in cancer pathophysiology has remained unclear. We show here that living cancer cells uptake DNA from chemotherapy-killed cancer cells. We discovered that such DNA induces TLR9- and cathepsin-mediated invasion in living cancer cells. To study whether this phenomenon contributes to treatment responses, triple negative, human MDA-MB-231 breast cancer cells stably expressing control or TLR9 siRNA were inoculated orthotopically into nude mice. The mice were treated with vehicle or doxorubicin. The tumor groups exhibited equal decreases in size in response to doxorubicin. However, while the weights of vehicle-treated mice were similar, mice bearing control siRNA tumors became significantly more cachectic in response to doxorubicin, as compared with similarly treated mice bearing TLR9 siRNA tumors, suggesting a TLR9-mediated inflammation at the site of the tumor. In conclusion, our findings propose that DNA released from chemotherapy-killed cancer cells has significant influence on TLR9-mediated biological effects in living cancer cells. Through these mechanisms, tumor TLR9 expression may affect treatment responses to chemotherapy. PMID:24212717

  1. Recent advances and future applications of microfluidic live-cell microarrays.

    PubMed

    Rothbauer, Mario; Wartmann, David; Charwat, Verena; Ertl, Peter

    2015-11-01

    Microfluidic live-cell microarrays show much promise as screening tools for biomedical research because they could shed light on key biological processes such as cell signaling and cell-to-cell and cell-to-substrate dynamic responses. While miniaturization reduces the need for expensive clinical grade reagents, the integration of functional components including micropumps, biosensors, actuators, mixers and gradient generators results in improved assay reliability, reproducibility and well-defined cell culture conditions. The present review addresses recent technological advances in microfluidic live-cell microarray technology with a special focus on the applications of microfluidic single-cell, multi-cell and 3D cell microarrays. PMID:26133396

  2. Synthetic biology of minimal living cells: primitive cell models and semi-synthetic cells.

    PubMed

    Stano, Pasquale

    2010-09-01

    This article summarizes a contribution presented at the ESF 2009 Synthetic Biology focused on the concept of the minimal requirement for life and on the issue of constructive (synthetic) approaches in biological research. The attempts to define minimal life within the framework of autopoietic theory are firstly described, and a short report on the development of autopoietic chemical systems based on fatty acid vesicles, which are relevant as primitive cell models is given. These studies can be used as a starting point for the construction of more complex systems, firstly being inspired by possible origins of life scenarioes (and therefore by considering primitive functions), then by considering an approach based on modern biomacromolecular-encoded functions. At this aim, semi-synthetic minimal cells are defined as those man-made vesicle-based systems that are composed of the minimal number of genes, proteins, biomolecules and which can be defined as living. Recent achievements on minimal sized semi-synthetic cells are then discussed, and the kind of information obtained is recognized as being distinctively derived by a constructive approach. Synthetic biology is therefore a fundamental tool for gaining basic knowledge about biosystems, and it should not be confined at all to the engineering side. PMID:21886680

  3. Cell compressibility studies utilizing noncontact hydrostatic pressure measurements on single living cells in a microchamber

    NASA Astrophysics Data System (ADS)

    Lin, L. A. G.; Liu, A. Q.; Yu, Y. F.; Zhang, C.; Lim, C. S.; Ng, S. H.; Yap, P. H.; Gao, H. J.

    2008-06-01

    A micro-optical-fluidic system (MOFS), which integrates a force generating device and an optical detector, is designed to measure the bulk modulus of a single living cell in real time under a controlled hydrostatic pressure. In this design, the accuracy of the bulk modulus measurement is improved because neither the force generating device nor the optical detector needs to be in contact with the cells. The MOFS device has been used to investigate the mechanotransduction of THP-1 human acute monocytic leukemia cells and the effects of the toxin lipopolysaccharide and colchicine on various properties of these cells.

  4. Dicer Regulates the Balance of Short-Lived Effector and Long-Lived Memory CD8 T Cell Lineages.

    PubMed

    Baumann, Florian M; Yuzefpolskiy, Yevgeniy; Sarkar, Surojit; Kalia, Vandana

    2016-01-01

    MicroRNAs constitute a major post-transcriptional mechanism for controlling protein expression, and are emerging as key regulators during T cell development and function. Recent reports of augmented CD8 T cell activation and effector differentiation, and aberrant migratory properties upon ablation of Dicer/miRNAs in naïve cells have established a regulatory role of miRNAs during priming. Whether miRNAs continue to exert similar functions or are dispensable during later stages of CD8 T cell expansion and memory differentiation remains unclear. Here, we report a critical role of Dicer/miRNAs in regulating the balance of long-lived memory and short-lived terminal effector fates during the post-priming stages when CD8 T cells undergo clonal expansion to generate a large cytotoxic T lymphocyte (CTL) pool and subsequently differentiate into a quiescent memory state. Conditional ablation of Dicer/miRNAs in early effector CD8 T cells following optimal activation and expression of granzyme B, using unique dicerfl/fl gzmb-cre mice, led to a strikingly diminished peak effector size relative to wild-type antigen-specific cells in the same infectious milieu. Diminished expansion of Dicer-ablated CD8 T cells was associated with lack of sustained antigen-driven proliferation and reduced accumulation of short-lived effector cells. Additionally, Dicer-ablated CD8 T cells exhibited more pronounced contraction after pathogen clearance and comprised a significantly smaller proportion of the memory pool, despite significantly higher proportions of CD127Hi memory precursors at the effector peak. Combined with previous reports of dynamic changes in miRNA expression as CD8 T cells differentiate from naïve to effector and memory states, these findings support distinct stage-specific roles of miRNA-dependent gene regulation during CD8 T cell differentiation. PMID:27627450

  5. Beyond Film: Exploring the Content of Movies

    ERIC Educational Resources Information Center

    Scacco, John

    2007-01-01

    This article looks at the use of movies in the language-learning classroom. The author promotes the use of the movie "To Kill a Mockingbird" due to its content, which involves poverty, racial inequality and mental illness, and to the availability of websites related to its use in English classrooms. The author highlights six scenes for…

  6. Machines and Human Beings in the Movies

    ERIC Educational Resources Information Center

    van der Laan, J. M.

    2006-01-01

    Over the years, many movies have presented on-screen a struggle between machines and human beings. Typically, the machines have come to rule and threaten the existence of humanity. They must be conquered to ensure the survival of and to secure the freedom of the human race. Although these movies appear to expose the dangers of an autonomous and…

  7. Substance Use in Popular Movies and Music.

    ERIC Educational Resources Information Center

    Roberts, Donald F.; Henriksen, Lisa; Christenson, Peter G.

    This study examines the frequency and nature of substance use in the most popular movie rentals and songs of 1996 and 1997. The intent was to determine the accuracy of public perceptions about extensive substance use in media popular among youth. Because teenagers are major consumers of movies and music, there is concern about the potential for…

  8. 8mm/16mm Movie-Making.

    ERIC Educational Resources Information Center

    Provisor, Henry

    The materials, techniques, and attitudes needed to make professional-quality movies using 8mm., super 8mm., and 16mm. amateur equipment are covered in this guide to movie-making. The pros and cons are discussed of the various makes and models of cameras and lenses. Other topics discussed are: exposure and lighting, choosing film, camera speed and…

  9. Technology and Terrorism in the Movie Brazil

    ERIC Educational Resources Information Center

    Stivers, Richard

    2006-01-01

    The movie "Brazil" calls attention to the relationship between technology and terrorism. Terrorism appears to be a threat to the order that technology creates. But terrorism forces technology to adapt and change so that technology perfects itself as a system. In the movie, terrorism is equated with any form of bureaucratic deviance so that…

  10. A Look at the Movies by Baldwin

    ERIC Educational Resources Information Center

    Bogle, Donald

    1976-01-01

    Notes that James Baldwin's new book--The Devil Finds Work--is a look by Baldwin at the movies, and that it is also a look by Baldwin at Baldwin, and the conflicting and contradictory effects the movies have had on his life and all of ours. (Author/AM)

  11. Using Movies to Teach Family Systems Concepts.

    ERIC Educational Resources Information Center

    Hudock, Anthony M., Jr.; Warden, Sherry A. Gallagher

    2001-01-01

    This article reflects a review of research relevant to family systems training and the use of films in the teaching of family systems theory. Advantages and disadvantages of using movies in an introductory-level graduate family therapy course are discussed. An outline of family therapy training objectives, as well as examples of a movie-based…

  12. Using Movies To Teach Students about Disabilities.

    ERIC Educational Resources Information Center

    Safran, Stephen P.

    2000-01-01

    This article discusses using movies to teach students about disabilities. It addresses considerations in choosing movies, gauging the accuracy of the portrayal, and identifying positive images and negative stereotypes. A checklist for evaluating positive and negative representations is provided, along with a format to assess disability portrayal…

  13. Making Movies Active: Lessons from Simulations

    ERIC Educational Resources Information Center

    Sunderland, Sheri; Rothermel, Jonathan C.; Lusk, Adam

    2009-01-01

    Movies have a long and distinguished history in the political science and international relations classrooms; they provide connections between abstract theories and concepts and concrete everyday practices. However, traditional approaches to teaching movies in the political science and international relations classrooms allow for passive student…

  14. The Possible Impact of Teachers and School Nurses on the Lives of Children Living with Sickle Cell Disease

    ERIC Educational Resources Information Center

    Knight-Madden, Jennifer M.; Lewis, Norma; Tyson, Esther; Reid, Marvin E.; MooSang, Michelle

    2011-01-01

    It is well recognized that for people living with a chronic disease, the largest impact on preserved health may come from persons other than medical professionals. This may be especially true for children for whom the actions of parents and school professionals have significant importance. Sickle cell disease (SCD) is one such disease. Although…

  15. [Methods of substances and organelles introduction in living cell for cell engineering technologies].

    PubMed

    Nikitin, V A

    2007-01-01

    We have presented the classification of more than 40 methods of genetic material, substances and organelles introduction into a living cell. Each of them has its characteristic advantages, disadvantages and limitations with respect to cell viability, transfer efficiency, general applicability, and technical requirements. It this article we have enlarged on the description of our developments of several new and improved approaches, methods and devices of the direct microinjection into a single cell and cell microsurgery with the help of glass micropipettes. The problem of low efficiency of mammalian cloning is discussed with emphasis on the necessity of expertizing of each step of single cell reconstruction to begin with microsurgical manipulations and necessity of the development of such methods of single cell resonstruction that could minimize the possible damage of the cell. PMID:17926558

  16. Processing of Cryo-EM Movie Data.

    PubMed

    Ripstein, Z A; Rubinstein, J L

    2016-01-01

    Direct detector device (DDD) cameras dramatically enhance the capabilities of electron cryomicroscopy (cryo-EM) due to their improved detective quantum efficiency (DQE) relative to other detectors. DDDs use semiconductor technology that allows micrographs to be recorded as movies rather than integrated individual exposures. Movies from DDDs improve cryo-EM in another, more surprising, way. DDD movies revealed beam-induced specimen movement as a major source of image degradation and provide a way to partially correct the problem by aligning frames or regions of frames to account for this specimen movement. In this chapter, we use a self-consistent mathematical notation to explain, compare, and contrast several of the most popular existing algorithms for computationally correcting specimen movement in DDD movies. We conclude by discussing future developments in algorithms for processing DDD movies that would extend the capabilities of cryo-EM even further. PMID:27572725

  17. Movies in education of psychiatry residents.

    PubMed

    Jukić, Vlado; Brecić, Petrana; Savić, Aleksandar

    2010-06-01

    Movies are a complex entity representing simultaneously an art form, a powerful industry, and a social phenomenon. The movie industry has always shown keen interest in physicians and medicine in general, and psychiatry in particular has often been in the spotlight. While there can be positive aspects of interaction of the movies and the psychiatry, stigmatization and negative public perception are also the results we often have to consider. Movies exploit psychiatric topics, at the same time portrayal of mental conditions, psychiatrists, and psychiatry on big screen could be used in different kinds of education in psychiatry. We present our initial experience with introducing movies in education of psychiatry residents in Psychiatric Hospital Vrapce. PMID:20562770

  18. Simultaneous Fluorescence and Phosphorescence Lifetime Imaging Microscopy in Living Cells.

    PubMed

    Jahn, Karolina; Buschmann, Volker; Hille, Carsten

    2015-01-01

    In living cells, there are always a plethora of processes taking place at the same time. Their precise regulation is the basis of cellular functions, since small failures can lead to severe dysfunctions. For a comprehensive understanding of intracellular homeostasis, simultaneous multiparameter detection is a versatile tool for revealing the spatial and temporal interactions of intracellular parameters. Here, a recently developed time-correlated single-photon counting (TCSPC) board was evaluated for simultaneous fluorescence and phosphorescence lifetime imaging microscopy (FLIM/PLIM). Therefore, the metabolic activity in insect salivary glands was investigated by recording ns-decaying intrinsic cellular fluorescence, mainly related to oxidized flavin adenine dinucleotide (FAD) and the μs-decaying phosphorescence of the oxygen-sensitive ruthenium-complex Kr341. Due to dopamine stimulation, the metabolic activity of salivary glands increased, causing a higher pericellular oxygen consumption and a resulting increase in Kr341 phosphorescence decay time. Furthermore, FAD fluorescence decay time decreased, presumably due to protein binding, thus inducing a quenching of FAD fluorescence decay time. Through application of the metabolic drugs antimycin and FCCP, the recorded signals could be assigned to a mitochondrial origin. The dopamine-induced changes could be observed in sequential FLIM and PLIM recordings, as well as in simultaneous FLIM/PLIM recordings using an intermediate TCSPC timing resolution. PMID:26390855

  19. Simultaneous Fluorescence and Phosphorescence Lifetime Imaging Microscopy in Living Cells

    NASA Astrophysics Data System (ADS)

    Jahn, Karolina; Buschmann, Volker; Hille, Carsten

    2015-09-01

    In living cells, there are always a plethora of processes taking place at the same time. Their precise regulation is the basis of cellular functions, since small failures can lead to severe dysfunctions. For a comprehensive understanding of intracellular homeostasis, simultaneous multiparameter detection is a versatile tool for revealing the spatial and temporal interactions of intracellular parameters. Here, a recently developed time-correlated single-photon counting (TCSPC) board was evaluated for simultaneous fluorescence and phosphorescence lifetime imaging microscopy (FLIM/PLIM). Therefore, the metabolic activity in insect salivary glands was investigated by recording ns-decaying intrinsic cellular fluorescence, mainly related to oxidized flavin adenine dinucleotide (FAD) and the μs-decaying phosphorescence of the oxygen-sensitive ruthenium-complex Kr341. Due to dopamine stimulation, the metabolic activity of salivary glands increased, causing a higher pericellular oxygen consumption and a resulting increase in Kr341 phosphorescence decay time. Furthermore, FAD fluorescence decay time decreased, presumably due to protein binding, thus inducing a quenching of FAD fluorescence decay time. Through application of the metabolic drugs antimycin and FCCP, the recorded signals could be assigned to a mitochondrial origin. The dopamine-induced changes could be observed in sequential FLIM and PLIM recordings, as well as in simultaneous FLIM/PLIM recordings using an intermediate TCSPC timing resolution.

  20. High-throughput screening of phosphodiesterase activity in living cells.

    PubMed

    Rich, Thomas C; Karpen, Jeffrey W

    2005-01-01

    Phosphodiesterases (PDEs) hydrolyze the second messengers cyclic adenosine monophosphate (cAMP) and cyclic guanosine 5'-monophosphate (cGMP) and play a crucial role in the termination and spatial segregation of cyclic nucleotide signals. Despite a wealth of molecular information, very little is known about how PDEs regulate cAMP and cGMP signals in living cells because conventional methods lack the necessary spatial and temporal resolution. We present here a sensitive optical method for monitoring cAMP levels and PDE activity near the membrane, using cyclic nucleotide-gated (CNG) ion channels as sensors. These channels are directly opened by the binding of cyclic nucleotides and allow cations to cross the membrane. The olfactory channel A subunit (CNGA2) has been genetically modified to improve its cAMP sensitivity and specificity. Channel activity is assessed by measuring Ca2+ influx using standard fluorometric techniques. In addition to studying PDEs in their native setting, the approach should be particularly useful in high-throughput screening assays to test for compounds that affect PDE activity, as well as the activities of the many G protein-coupled receptors that cause changes in intracellular cAMP. PMID:15988054

  1. Spirituality and Religiosity in Adolescents Living With Sickle Cell Disease.

    PubMed

    Clayton-Jones, Dora; Haglund, Kristin; Belknap, Ruth Ann; Schaefer, Jame; Thompson, Alexis A

    2016-06-01

    This study purports to address paucity in the literature regarding how adolescents with sickle cell disease (SCD) describe and experience spirituality and religiosity (S/R). This was a qualitative descriptive study. Two semi-structured interviews were conducted with nine adolescents (Mage = 16.2 years). Data were analyzed using a template analysis style and a concurrent analysis process of data reduction. Three major themes encompassed the participants' descriptions of the relationships between S/R, health and illness in their lives including S/R as sources for coping, influence of S/R beliefs on health and illness, and sharing S/R with Health Care Providers (HCPs). S/R as coping mechanisms included six threads: interconnecting with God, interconnecting with others, interconnecting with creative arts, scriptural metanarratives, transcendent experiences, and acceptance and finding meaning. Expectations of health providers included two threads: Religiosity is private/personal and sharing spiritual and religious beliefs is risky. S/R are particularly salient for adolescents with SCD. PMID:26792855

  2. Simultaneous Fluorescence and Phosphorescence Lifetime Imaging Microscopy in Living Cells

    PubMed Central

    Jahn, Karolina; Buschmann, Volker; Hille, Carsten

    2015-01-01

    In living cells, there are always a plethora of processes taking place at the same time. Their precise regulation is the basis of cellular functions, since small failures can lead to severe dysfunctions. For a comprehensive understanding of intracellular homeostasis, simultaneous multiparameter detection is a versatile tool for revealing the spatial and temporal interactions of intracellular parameters. Here, a recently developed time-correlated single-photon counting (TCSPC) board was evaluated for simultaneous fluorescence and phosphorescence lifetime imaging microscopy (FLIM/PLIM). Therefore, the metabolic activity in insect salivary glands was investigated by recording ns-decaying intrinsic cellular fluorescence, mainly related to oxidized flavin adenine dinucleotide (FAD) and the μs-decaying phosphorescence of the oxygen-sensitive ruthenium-complex Kr341. Due to dopamine stimulation, the metabolic activity of salivary glands increased, causing a higher pericellular oxygen consumption and a resulting increase in Kr341 phosphorescence decay time. Furthermore, FAD fluorescence decay time decreased, presumably due to protein binding, thus inducing a quenching of FAD fluorescence decay time. Through application of the metabolic drugs antimycin and FCCP, the recorded signals could be assigned to a mitochondrial origin. The dopamine-induced changes could be observed in sequential FLIM and PLIM recordings, as well as in simultaneous FLIM/PLIM recordings using an intermediate TCSPC timing resolution. PMID:26390855

  3. Quantitative Measurement of Protein Relocalization in Live Cells

    PubMed Central

    Bush, Alan; Colman-Lerner, Alejandro

    2013-01-01

    Microscope cytometry provides a powerful means to study signaling in live cells. Here we present a quantitative method to measure protein relocalization over time, which reports the absolute fraction of a tagged protein in each compartment. Using this method, we studied an essential step in the early propagation of the pheromone signal in Saccharomyces cerevisiae: recruitment to the membrane of the scaffold Ste5 by activated Gβγ dimers. We found that the dose response of Ste5 recruitment is graded (EC50 = 0.44 ± 0.08 nM, Hill coefficient = 0.8 ± 0.1). Then, we determined the effective dissociation constant (Kde) between Ste5 and membrane sites during the first few minutes when the negative feedback from the MAPK Fus3 is first activated. Kde changed during the first minutes from a high affinity of <0.65 nM to a steady-state value of 17 ± 9 nM. During the same period, the total number of binding sites decreased slightly, from 1940 ± 150 to 1400 ± 200. This work shows how careful quantification of a protein relocalization dynamic can give insight into the regulation mechanisms of a biological system. PMID:23442923

  4. 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

  5. Mechanics of Cellulose Synthase Complexes in Living Plant Cells

    NASA Astrophysics Data System (ADS)

    Zehfroosh, Nina; Liu, Derui; Ramos, Kieran P.; Yang, Xiaoli; Goldner, Lori S.; Baskin, Tobias I.

    The polymer cellulose is one of the major components of the world's biomass with unique and fascinating characteristics such as its high tensile strength, renewability, biodegradability, and biocompatibility. Because of these distinctive aspects, cellulose has been the subject of enormous scientific and industrial interest, yet there are still fundamental open questions about cellulose biosynthesis. Cellulose is synthesized by a complex of transmembrane proteins called ``Cellulose Synthase A'' (CESA) in the plasma membrane. Studying the dynamics and kinematics of the CESA complex will help reveal the mechanism of cellulose synthesis and permit the development and validation of models of CESA motility. To understand what drives these complexes through the cell membrane, we used total internal reflection fluorescence microscopy (TIRFM) and variable angle epi-fluorescence microscopy to track individual, fluorescently-labeled CESA complexes as they move in the hypocotyl and root of living plants. A mean square displacement analysis will be applied to distinguish ballistic, diffusional, and other forms of motion. We report on the results of these tracking experiments. This work was funded by NSF/PHY-1205989.

  6. Evaluating the efficacy of subcellular fractionation of blast cells using live cell labeling and 2D DIGE.

    PubMed

    Ho, Yin Ying; Penno, Megan; Perugini, Michelle; Lewis, Ian; Hoffmann, Peter

    2012-01-01

    Labeling of exposed cell surface proteins of live cells using CyDye DIGE fluor minimal dyes is an efficient strategy for cell surface proteome profiling and quantifying differentially expressed proteins in diseases. Here we describe a strategy to evaluate a two-step detergent-based protein fractionation method using live cell labeling followed by visualization of the fluorescently labeled cell surface proteins and fractionated proteins within a single 2D gel. PMID:22311770

  7. SiR–Hoechst is a far-red DNA stain for live-cell nanoscopy

    PubMed Central

    Lukinavičius, Gražvydas; Blaukopf, Claudia; Pershagen, Elias; Schena, Alberto; Reymond, Luc; Derivery, Emmanuel; Gonzalez-Gaitan, Marcos; D'Este, Elisa; Hell, Stefan W.; Wolfram Gerlich, Daniel; Johnsson, Kai

    2015-01-01

    Cell-permeable DNA stains are popular markers in live-cell imaging. Currently used DNA stains for live-cell imaging are either toxic, require illumination with blue light or are not compatible with super-resolution microscopy, thereby limiting their utility. Here we describe a far-red DNA stain, SiR–Hoechst, which displays minimal toxicity, is applicable in different cell types and tissues, and is compatible with super-resolution microscopy. The combination of these properties makes this probe a powerful tool for live-cell imaging. PMID:26423723

  8. SiR-Hoechst is a far-red DNA stain for live-cell nanoscopy.

    PubMed

    Lukinavičius, Gražvydas; Blaukopf, Claudia; Pershagen, Elias; Schena, Alberto; Reymond, Luc; Derivery, Emmanuel; Gonzalez-Gaitan, Marcos; D'Este, Elisa; Hell, Stefan W; Gerlich, Daniel Wolfram; Johnsson, Kai

    2015-01-01

    Cell-permeable DNA stains are popular markers in live-cell imaging. Currently used DNA stains for live-cell imaging are either toxic, require illumination with blue light or are not compatible with super-resolution microscopy, thereby limiting their utility. Here we describe a far-red DNA stain, SiR-Hoechst, which displays minimal toxicity, is applicable in different cell types and tissues, and is compatible with super-resolution microscopy. The combination of these properties makes this probe a powerful tool for live-cell imaging. PMID:26423723

  9. Transduction of peptides and proteins into live cells by cell penetrating peptides.

    PubMed

    Mussbach, Franziska; Franke, Martin; Zoch, Ansgar; Schaefer, Buerk; Reissmann, Siegmund

    2011-12-01

    Internalization of peptides and proteins into live cells is an essential prerequisite for studies on intracellular signal pathways, for treatment of certain microbial diseases and for signal transduction therapy, especially for cancer treatment. Cell penetrating peptides (CPPs) facilitate the transport of cargo-proteins through the cell membrane into live cells. CPPs which allow formation of non-covalent complexes with the cargo are used primarily in this study due to the relatively easy handling procedure. Efficiency of the protein uptake is estimated qualitatively by fluorescence microscopy and quantitatively by SDS-PAGE. Using the CPP cocktail JBS-Proteoducin, the intracellular concentrations of a secondary antibody and bovine serum albumin can reach the micromolar range. Internalization of antibodies allows mediation of intracellular pathways including knock down of signal transduction. The high specificity and affinity of antibodies makes them potentially more powerful than siRNA. Thus, CPPs represent a significant new possibility to study signal transduction processes in competition or in comparison to the commonly used other techniques. To estimate the highest attainable intracellular concentrations of cargo proteins, the CPPs are tested for cytotoxicity. Cell viability and membrane integrity relative to concentration of CPPs are investigated. Viability as estimated by the reductive activity of mitochondria (MTT-test) is more sensitive to higher concentrations of CPPs versus membrane integrity, as measured by the release of dead cell protease. Distinct differences in uptake efficiency and cytotoxic effects are found using six different CPPs and six different adhesion and suspension cell lines. PMID:21826709

  10. 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

  11. 78 FR 49528 - Consolidation of Wound Care Products Containing Live Cells

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-08-14

    ... HUMAN SERVICES Food and Drug Administration Consolidation of Wound Care Products Containing Live Cells...) is transferring oversight responsibilities for certain wound care products containing live cells from... scientific and regulatory activities between CDRH and CBER. FDA believes that as more wound care...

  12. 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%.

  13. Live-Cell Imaging of Vaccinia Virus Recombination

    PubMed Central

    Paszkowski, Patrick; Noyce, Ryan S.; Evans, David H.

    2016-01-01

    Recombination between co-infecting poxviruses provides an important mechanism for generating the genetic diversity that underpins evolution. However, poxviruses replicate in membrane-bound cytoplasmic structures known as factories or virosomes. These are enclosed structures that could impede DNA mixing between co-infecting viruses, and mixing would seem to be essential for this process. We hypothesize that virosome fusion events would be a prerequisite for recombination between co-infecting poxviruses, and this requirement could delay or limit viral recombination. We have engineered vaccinia virus (VACV) to express overlapping portions of mCherry fluorescent protein fused to a cro DNA-binding element. In cells also expressing an EGFP-cro fusion protein, this permits live tracking of virus DNA and genetic recombination using confocal microscopy. Our studies show that different types of recombination events exhibit different timing patterns, depending upon the relative locations of the recombining elements. Recombination between partly duplicated sequences is detected soon after post-replicative genes are expressed, as long as the reporter gene sequences are located in cis within an infecting genome. The same kinetics are also observed when the recombining elements are divided between VACV and transfected DNA. In contrast, recombination is delayed when the recombining sequences are located on different co-infecting viruses, and mature recombinants aren’t detected until well after late gene expression is well established. The delay supports the hypothesis that factories impede inter-viral recombination, but even after factories merge there remain further constraints limiting virus DNA mixing and recombinant gene assembly. This delay could be related to the continued presence of ER-derived membranes within the fused virosomes, membranes that may once have wrapped individual factories. PMID:27525721

  14. Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

    PubMed Central

    Joshi, Sagar D.; Davidson, Lance A.

    2010-01-01

    Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes in cell surface area and cell height, and where cells undergo mitosis and migrate. Furthermore, epithelial cells can also regulate morphogenetic movements in adjacent tissues1. A traditional method to study epithelial cells and tissues involve chemical fixation and histological methods to determine cell morphology or localization of particular proteins of interest. These approaches continue to be useful and provide "snapshots" of cell shapes and tissue architecture, however, much remains to be understood about how cells acquire specific shapes, how various proteins move or localize to specific positions, and what paths cells follow toward their final differentiated fate. High resolution live imaging complements traditional methods and also allows more direct investigation into the dynamic cellular processes involved in the formation, maintenance, and morphogenesis of multicellular epithelial sheets. Here we demonstrate experimental methods from the isolation of animal cap tissues from Xenopus laevis embryos to confocal imaging of epithelial cells and simple measurement approaches that together can augment molecular and cellular studies of epithelial morphogenesis. PMID:20498627

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

    PubMed Central

    Chen, Chen; van der Ende-Metselaar, Heidi; Wilschut, Jan; Zhuang, Xiaowei; Smit, Jolanda M.

    2008-01-01

    Dengue virus (DENV) is an enveloped RNA virus that causes the most common arthropod-borne infection worldwide. The mechanism by which DENV infects the host cell remains unclear. In this work, we used live-cell imaging and single-virus tracking to investigate the cell entry, endocytic trafficking, and fusion behavior of DENV. Simultaneous tracking of DENV particles and various endocytic markers revealed that DENV enters cells exclusively via clathrin-mediated endocytosis. The virus particles move along the cell surface in a diffusive manner before being captured by a pre-existing clathrin-coated pit. Upon clathrin-mediated entry, DENV particles are transported to Rab5-positive endosomes, which subsequently mature into late endosomes through acquisition of Rab7 and loss of Rab5. Fusion of the viral membrane with the endosomal membrane was primarily detected in late endosomal compartments. PMID:19096510

  16. Understanding the initiation of B cell signaling through live cell imaging

    PubMed Central

    Pierce, Susan K.

    2013-01-01

    Antibody responses are initiated by the binding of antigens to clonally distributed cell surface B cell receptors (BCRs) that trigger signaling cascades resulting in B cell activation. Using conventional biochemical approaches, the components of the downstream BCR signaling pathways have been described in considerable detail. However, far less is known about the early molecular events by which the binding of antigens to the BCRs initiates BCR signaling. With the recent advent of high-resolution, high-speed, live-cell and single-molecule imaging technologies, these events are just beginning to be elucidated. Understanding the molecular mechanisms underlying the initiation of BCR signaling may provide new targets for therapeutics to block dysregulated BCR signaling in systemic autoimmune diseases and in B cell tumors and to aid in the design of protein subunit vaccines. In this chapter we describe the general procedures for using these new imaging techniques to investigate the early events in the initiation of BCR signaling. PMID:22341229

  17. Scanning electrochemical microscopy of living cells: different redox activities of nonmetastatic and metastatic human breast cells.

    PubMed

    Liu, B; Rotenberg, S A; Mirkin, M V

    2000-08-29

    Electrochemical methods have been widely used to monitor physiologically important molecules in biological systems. This report describes the first application of the scanning electrochemical microscope (SECM) to probe the redox activity of individual living cells. The possibilities of measuring the rate and investigating the pathway of transmembrane charge transfer are demonstrated. By this approach, significant differences are detected in the redox responses given by nonmotile, nontransformed human breast epithelial cells, breast cells with a high level of motility (engendered by overexpression of protein kinase Calpha), and highly metastatic breast cancer cells. SECM analysis of the three cell lines reveals reproducible differences with respect to the kinetics of charge transfer by several redox mediators. PMID:10963658

  18. Scanning electrochemical microscopy of living cells: Different redox activities of nonmetastatic and metastatic human breast cells

    PubMed Central

    Liu, Biao; Rotenberg, Susan A.; Mirkin, Michael V.

    2000-01-01

    Electrochemical methods have been widely used to monitor physiologically important molecules in biological systems. This report describes the first application of the scanning electrochemical microscope (SECM) to probe the redox activity of individual living cells. The possibilities of measuring the rate and investigating the pathway of transmembrane charge transfer are demonstrated. By this approach, significant differences are detected in the redox responses given by nonmotile, nontransformed human breast epithelial cells, breast cells with a high level of motility (engendered by overexpression of protein kinase Cα), and highly metastatic breast cancer cells. SECM analysis of the three cell lines reveals reproducible differences with respect to the kinetics of charge transfer by several redox mediators. PMID:10963658

  19. Live-Cell, Temporal Gene Expression Analysis of Osteogenic Differentiation in Adipose-Derived Stem Cells

    PubMed Central

    Desai, Hetal V.; Voruganti, Indu S.; Jayasuriya, Chathuraka; Chen, Qian

    2014-01-01

    Adipose-derived stem cells (ASCs) are a widely investigated type of mesenchymal stem cells with great potential for musculoskeletal regeneration. However, the use of ASCs is complicated by their cellular heterogeneity, which exists at both the population and single-cell levels. This study demonstrates a live-cell assay to investigate gene expression in ASCs undergoing osteogenesis using fluorescently tagged DNA hybridization probes called molecular beacons. A molecular beacon was designed to target the mRNA sequence for alkaline phosphatase (ALPL), a gene characteristically expressed during early osteogenesis. The percentage of cells expressing this gene in a population was monitored daily to quantify the uniformity of the differentiation process. Differentiating ASC populations were repeatedly measured in a nondestructive fashion over a 10-day period to obtain temporal gene expression data. Results showed consistent expression patterns for the investigated osteogenic genes in response to induction medium. Peak signal level, indicating when the most cells expressed ALPL at once, was observed on days 3–5. The differentiation response of sample populations was generally uniform when assessed on a well-by-well basis over time. The expression of alkaline phosphatase is consistent with previous studies of osteogenic differentiation, suggesting that molecular beacons are a viable means of monitoring the spatiotemporal gene expression of live, differentiating ASCs. PMID:24367991

  20. Neurotoxin quantum dot conjugates detect endogenous targets expressed in live cancer cells.

    PubMed

    Orndorff, Rebecca L; Rosenthal, Sandra J

    2009-07-01

    High affinity peptide neurotoxins are effective agents for integrating technological advances with biological inquiries. Both chlorotoxin (CTX) and dendrotoxin-1 (DTX-1) are peptide neurotoxins demonstrated to bind targets expressed by glioma cancer cells and are suitable ligands for quantum dot (QD) live cell investigations. Here, we present dual labeling of endogenously expressed cellular proteins within living cells utilizing high affinity peptide neurotoxins conjugated to QDs. Multiplexing experiments reveal quantifiable evidence that CTX and DTX-1 conjugated QDs may potentially be used as a live assessment of markers toward identification of cancer cell presence. PMID:19507837

  1. Light-induced cell damage in live-cell super-resolution microscopy

    NASA Astrophysics Data System (ADS)

    Wäldchen, Sina; Lehmann, Julian; Klein, Teresa; van de Linde, Sebastian; Sauer, Markus

    2015-10-01

    Super-resolution microscopy can unravel previously hidden details of cellular structures but requires high irradiation intensities to use the limited photon budget efficiently. Such high photon densities are likely to induce cellular damage in live-cell experiments. We applied single-molecule localization microscopy conditions and tested the influence of irradiation intensity, illumination-mode, wavelength, light-dose, temperature and fluorescence labeling on the survival probability of different cell lines 20-24 hours after irradiation. In addition, we measured the microtubule growth speed after irradiation. The photo-sensitivity is dramatically increased at lower irradiation wavelength. We observed fixation, plasma membrane permeabilization and cytoskeleton destruction upon irradiation with shorter wavelengths. While cells stand light intensities of ~1 kW cm-2 at 640 nm for several minutes, the maximum dose at 405 nm is only ~50 J cm-2, emphasizing red fluorophores for live-cell localization microscopy. We also present strategies to minimize phototoxic factors and maximize the cells ability to cope with higher irradiation intensities.

  2. Light-induced cell damage in live-cell super-resolution microscopy.

    PubMed

    Wäldchen, Sina; Lehmann, Julian; Klein, Teresa; van de Linde, Sebastian; Sauer, Markus

    2015-01-01

    Super-resolution microscopy can unravel previously hidden details of cellular structures but requires high irradiation intensities to use the limited photon budget efficiently. Such high photon densities are likely to induce cellular damage in live-cell experiments. We applied single-molecule localization microscopy conditions and tested the influence of irradiation intensity, illumination-mode, wavelength, light-dose, temperature and fluorescence labeling on the survival probability of different cell lines 20-24 hours after irradiation. In addition, we measured the microtubule growth speed after irradiation. The photo-sensitivity is dramatically increased at lower irradiation wavelength. We observed fixation, plasma membrane permeabilization and cytoskeleton destruction upon irradiation with shorter wavelengths. While cells stand light intensities of ~1 kW cm(-2) at 640 nm for several minutes, the maximum dose at 405 nm is only ~50 J cm(-2), emphasizing red fluorophores for live-cell localization microscopy. We also present strategies to minimize phototoxic factors and maximize the cells ability to cope with higher irradiation intensities. PMID:26481189

  3. Light-induced cell damage in live-cell super-resolution microscopy

    PubMed Central

    Wäldchen, Sina; Lehmann, Julian; Klein, Teresa; van de Linde, Sebastian; Sauer, Markus

    2015-01-01

    Super-resolution microscopy can unravel previously hidden details of cellular structures but requires high irradiation intensities to use the limited photon budget efficiently. Such high photon densities are likely to induce cellular damage in live-cell experiments. We applied single-molecule localization microscopy conditions and tested the influence of irradiation intensity, illumination-mode, wavelength, light-dose, temperature and fluorescence labeling on the survival probability of different cell lines 20–24 hours after irradiation. In addition, we measured the microtubule growth speed after irradiation. The photo-sensitivity is dramatically increased at lower irradiation wavelength. We observed fixation, plasma membrane permeabilization and cytoskeleton destruction upon irradiation with shorter wavelengths. While cells stand light intensities of ~1 kW cm−2 at 640 nm for several minutes, the maximum dose at 405 nm is only ~50 J cm−2, emphasizing red fluorophores for live-cell localization microscopy. We also present strategies to minimize phototoxic factors and maximize the cells ability to cope with higher irradiation intensities. PMID:26481189

  4. Development of living cell microarrays using non-contact micropipette printing.

    PubMed

    Jonczyk, Rebecca; Timur, Suna; Scheper, Thomas; Stahl, Frank

    2016-01-10

    During the last 30 years cellular screening systems were unidirectional developed towards high throughput applications on single cell level. We developed living cell microarrays, which provide an in vivo-like microenvironment for an advanced method to measure cellular response to external stimuli. To print living cells on glass slides, the classic microarray equipment, which involves printer and scanner, was fully transferred to suspensions of living cells. The microarray production was optimized using a contact-free spotting procedure in order to enhanced cell adhesion and growth rates. The printed model cells, A-549 (lung cancer cell line), were analyzed with conventional cell staining assays like DAPI (cell nuclei staining), calcein acetoxymethyl ester (viable cell staining), and CellTiter-Blue(®) Cell Viability Assay. After optimization, a reproducible (spot-to-spot variation: ± 8.6 cells) printing method for small living cell amounts (1200 cells and fewer) was established that achieved cell viabilities of up to 88% for ≥ 0.6 μL and good proliferation characteristics. Hence, this method could be advantageous for use in biomedical and diagnostic applications. PMID:26603124

  5. Fate by Chance, not by Choice: Epidermal Stem Cells Go Live.

    PubMed

    Gonzalez-Celeiro, Meryem; Zhang, Bing; Hsu, Ya-Chieh

    2016-07-01

    The skin epidermis is constantly renewed by epidermal stem cells. In a recent Science paper, Rompolas et al. utilize live imaging to track epidermal stem cells over their lifetimes. Their findings provide new insights into epidermal stem cell behaviors and unravel how newly generated cells are integrated into pre-existing tissues. PMID:27392221

  6. Optoelectronic tweezers system for single cell manipulation and fluorescence imaging of live immune cells.

    PubMed

    Jeorrett, Abigail H; Neale, Steven L; Massoubre, David; Gu, Erdan; Henderson, Robert K; Millington, Owain; Mathieson, Keith; Dawson, Martin D

    2014-01-27

    A compact optoelectronic tweezers system for combined cell manipulation and analysis is presented. CMOS-controlled gallium nitride micro-LED arrays are used to provide simultaneous spatio-temporal control of dielectrophoresis traps within an optoelectronic tweezers device and fluorescence imaging of contrasting dye labelled cells. This capability provides direct identification, selection and controlled interaction of single T-lymphocytes and dendritic cells. The trap strength and profile for two emission wavelengths of micro-LED array have been measured and a maximum trapping force of 13.1 and 7.6 pN was achieved for projected micro-LED devices emitting at λmax 520 and 450 nm, respectively. A potential application in biological research is demonstrated through the controlled interaction of live immune cells where there is potential for this method of OET to be implemented as a compact device. PMID:24515144

  7. Visualization of the Nucleolus in Living Cells with Cell-Penetrating Fluorescent Peptides.

    PubMed

    Martin, Robert M; Herce, Henry D; Ludwig, Anne K; Cardoso, M Cristina

    2016-01-01

    The nucleolus is the hallmark of nuclear compartmentalization and has been shown to exert multiple roles in cellular metabolism besides its main function as the place of ribosomal RNA synthesis and assembly of ribosomes. The nucleolus plays also a major role in nuclear organization as the largest compartment within the nucleus. The prominent structure of the nucleolus can be detected using contrast light microscopy providing an approximate localization of the nucleolus, but this approach does not allow to determine accurately the three-dimensional structure of the nucleolus in cells and tissues. Immunofluorescence staining with antibodies specific to nucleolar proteins albeit very useful is time consuming, normally antibodies recognize their epitopes only within a small range of species and is applicable only in fixed cells. Here, we present a simple method to selectively and accurately label this ubiquitous subnuclear compartment in living cells of a large range of species using a fluorescently labeled cell-penetrating peptide. PMID:27576711

  8. Movies, Books, Music, and Adult Fantasy Life.

    ERIC Educational Resources Information Center

    McIlwraith, Robert D.; Josephson, Wendy L.

    1985-01-01

    Explores the relationship between fantasy and media use by examining the fantasy styles of college students and the kinds of movies they attend, recorded music they listen to, and books they read. (PD)

  9. Statistical Patterns in Movie Rating Behavior

    PubMed Central

    2015-01-01

    Currently, users and consumers can review and rate products through online services, which provide huge databases that can be used to explore people’s preferences and unveil behavioral patterns. In this work, we investigate patterns in movie ratings, considering IMDb (the Internet Movie Database), a highly visited site worldwide, as a source. We find that the distribution of votes presents scale-free behavior over several orders of magnitude, with an exponent very close to 3/2, with exponential cutoff. It is remarkable that this pattern emerges independently of movie attributes such as average rating, age and genre, with the exception of a few genres and of high-budget films. These results point to a very general underlying mechanism for the propagation of adoption across potential audiences that is independent of the intrinsic features of a movie and that can be understood through a simple spreading model with mean-field avalanche dynamics. PMID:26322899

  10. Statistical Patterns in Movie Rating Behavior.

    PubMed

    Ramos, Marlon; Calvão, Angelo M; Anteneodo, Celia

    2015-01-01

    Currently, users and consumers can review and rate products through online services, which provide huge databases that can be used to explore people's preferences and unveil behavioral patterns. In this work, we investigate patterns in movie ratings, considering IMDb (the Internet Movie Database), a highly visited site worldwide, as a source. We find that the distribution of votes presents scale-free behavior over several orders of magnitude, with an exponent very close to 3/2, with exponential cutoff. It is remarkable that this pattern emerges independently of movie attributes such as average rating, age and genre, with the exception of a few genres and of high-budget films. These results point to a very general underlying mechanism for the propagation of adoption across potential audiences that is independent of the intrinsic features of a movie and that can be understood through a simple spreading model with mean-field avalanche dynamics. PMID:26322899

  11. Development of living cell force sensors for the interrogation of cell surface interactions

    NASA Astrophysics Data System (ADS)

    Brown, Scott Chang

    The measurement of cell surface interactions, or cell interaction forces, are critical for the early diagnosis and prevention of disease, the design of targeted drug and gene delivery vehicles, the development of next-generation implant materials, and much more. However, the technologies and devices that are currently available are highly limited with respect to the dynamic force range over which they can measure cell-cell or cell-substratum interactions, and with their ability to adequately mimic biologically relevant systems. Consequently, research efforts that involve cell surface interactions have been limited. In this dissertation, existing tools for research at the nanoscale (i.e., atomic force microscopy microcantilevers) are modified to develop living cell force sensors that allow for the highly sensitive measurement of cell-mediated interactions over the entire range of forces expected in biotechnology (and nano-biotechnology) research (from a single to millions of receptor-ligand bonds). Several force sensor motifs have been developed that can be used to measure interactions using single adherent cells, single suspension culture cell, and cell monolayers (tissues) over a wide range of interaction conditions (e.g., approach velocity, shear rate, contact time) using a conventional atomic force microscope. This new tool has been applied to study the pathogenesis of spontaneous pneumothorax and the interaction of cells with 14 man-made interfaces. Consequently, a new hypothesis of the interactions that manifest spontaneous pneumothorax has been developed. Additionally, these findings have the potential to lead to the development of tools for data mining materials and surfaces for unique cell interactions that could have an immense societal impact.

  12. Still from Red Spot Movie

    NASA Technical Reports Server (NTRS)

    2000-01-01

    This image is one of seven from the narrow-angle camera on NASA's Cassini spacecraft assembled as a brief movie of cloud movements on Jupiter. It was taken with a blue filter. The smallest features visible are about 500 kilometers (about 300 miles) across.

    Small bright clouds appear suddenly to the west of the Great Red Spot. Based on data from NASA's Galileo spacecraft, scientists suspect that these small white features are lightning storms, where falling raindrops create an electrical charge. The lightning storms eventually merge with the Red Spot and surrounding jets, and may be the main energy source for these large-scale features. Imaging observations of the darkside of the planet in the weeks following Cassini's closest approach to Jupiter on Dec. 30, 2000 will search for lightning storms like these.

    This image was re-projected by cylindrical-map projection of an image taken in the first week of October 2000. It shows an area from 50 degrees north of Jupiter's equator to 50 degrees south, extending 100 degrees east west, about one quarter of Jupiter's circumference.

    Cassini is a cooperative project of NASA, the European Space Agency and the Italian Space Agency. The Jet Propulsion Laboratory, a division of the California Institute of Technology in Pasadena, manages the Cassini mission for NASA's Office of Space Science, Washington, D.C.

  13. Visual Categorization of Natural Movies by Rats

    PubMed Central

    Vinken, Kasper; Vermaercke, Ben

    2014-01-01

    Visual categorization of complex, natural stimuli has been studied for some time in human and nonhuman primates. Recent interest in the rodent as a model for visual perception, including higher-level functional specialization, leads to the question of how rodents would perform on a categorization task using natural stimuli. To answer this question, rats were trained in a two-alternative forced choice task to discriminate movies containing rats from movies containing other objects and from scrambled movies (ordinate-level categorization). Subsequently, transfer to novel, previously unseen stimuli was tested, followed by a series of control probes. The results show that the animals are capable of acquiring a decision rule by abstracting common features from natural movies to generalize categorization to new stimuli. Control probes demonstrate that they did not use single low-level features, such as motion energy or (local) luminance. Significant generalization was even present with stationary snapshots from untrained movies. The variability within and between training and test stimuli, the complexity of natural movies, and the control experiments and analyses all suggest that a more high-level rule based on more complex stimulus features than local luminance-based cues was used to classify the novel stimuli. In conclusion, natural stimuli can be used to probe ordinate-level categorization in rats. PMID:25100598

  14. 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

  15. Atomic Force Microscopy Measurements of the Mechanical Properties of Cell Walls on Living Bacterial Cells

    NASA Astrophysics Data System (ADS)

    Bailey, Richard; Mullin, Nic; Turner, Robert; Foster, Simon; Hobbs, Jamie

    2014-03-01

    Staphylococcus aureus is a major cause of infection in humans, including the Methicillin resistant strain, MRSA. However, very little is known about the mechanical properties of these cells. Our investigations use AFM to examine live S. aureus cells to quantify mechanical properties. These were explored using force spectroscopy with different trigger forces, allowing the properties to be extracted at different indentation depths. A value for the cell wall stiffness has been extracted, along with a second, higher value which is found upon indenting at higher forces. This higher value drops as the cells are exposed to high salt, sugar and detergent concentrations, implying that this measurement contains a contribution from the internal turgor pressure. We have monitored these properties as the cells progress through the cell cycle. Force maps were taken over the cells at different stages of the growth process to identify changes in the mechanics throughout the progression of growth and division. The effect of Oxacillin has also been studied, to better understand its mechanism of action. Finally mutant strains of S. aureus and a second species Bacillus subtilis have been used to link the mechanical properties of the cell walls with the chain lengths and substructures involved.

  16. Viscoelastic properties of cell walls of single living plant cells determined by dynamic nanoindentation

    PubMed Central

    Hayot, Céline M.; Forouzesh, Elham; Goel, Ashwani; Avramova, Zoya; Turner, Joseph A.

    2012-01-01

    Plant development results from controlled cell divisions, structural modifications, and reorganizations of the cell wall. Thereby, regulation of cell wall behaviour takes place at multiple length scales involving compositional and architectural aspects in addition to various developmental and/or environmental factors. The physical properties of the primary wall are largely determined by the nature of the complex polymer network, which exhibits time-dependent behaviour representative of viscoelastic materials. Here, a dynamic nanoindentation technique is used to measure the time-dependent response and the viscoelastic behaviour of the cell wall in single living cells at a micron or sub-micron scale. With this approach, significant changes in storage (stiffness) and loss (loss of energy) moduli are captured among the tested cells. The results reveal hitherto unknown differences in the viscoelastic parameters of the walls of same-age similarly positioned cells of the Arabidopsis ecotypes (Col 0 and Ws 2). The technique is also shown to be sensitive enough to detect changes in cell wall properties in cells deficient in the activity of the chromatin modifier ATX1. Extensive computational modelling of the experimental measurements (i.e. modelling the cell as a viscoelastic pressure vessel) is used to analyse the influence of the wall thickness, as well as the turgor pressure, at the positions of our measurements. By combining the nanoDMA technique with finite element simulations quantifiable measurements of the viscoelastic properties of plant cell walls are achieved. Such techniques are expected to find broader applications in quantifying the influence of genetic, biological, and environmental factors on the nanoscale mechanical properties of the cell wall. PMID:22291130

  17. Viscoelastic properties of cell walls of single living plant cells determined by dynamic nanoindentation.

    PubMed

    Hayot, Céline M; Forouzesh, Elham; Goel, Ashwani; Avramova, Zoya; Turner, Joseph A

    2012-04-01

    Plant development results from controlled cell divisions, structural modifications, and reorganizations of the cell wall. Thereby, regulation of cell wall behaviour takes place at multiple length scales involving compositional and architectural aspects in addition to various developmental and/or environmental factors. The physical properties of the primary wall are largely determined by the nature of the complex polymer network, which exhibits time-dependent behaviour representative of viscoelastic materials. Here, a dynamic nanoindentation technique is used to measure the time-dependent response and the viscoelastic behaviour of the cell wall in single living cells at a micron or sub-micron scale. With this approach, significant changes in storage (stiffness) and loss (loss of energy) moduli are captured among the tested cells. The results reveal hitherto unknown differences in the viscoelastic parameters of the walls of same-age similarly positioned cells of the Arabidopsis ecotypes (Col 0 and Ws 2). The technique is also shown to be sensitive enough to detect changes in cell wall properties in cells deficient in the activity of the chromatin modifier ATX1. Extensive computational modelling of the experimental measurements (i.e. modelling the cell as a viscoelastic pressure vessel) is used to analyse the influence of the wall thickness, as well as the turgor pressure, at the positions of our measurements. By combining the nanoDMA technique with finite element simulations quantifiable measurements of the viscoelastic properties of plant cell walls are achieved. Such techniques are expected to find broader applications in quantifying the influence of genetic, biological, and environmental factors on the nanoscale mechanical properties of the cell wall. PMID:22291130

  18. A quantum dot photoswitch for DNA detection, gene transfection, and live-cell imaging.

    PubMed

    Wu, Yuzhou; Eisele, Klaus; Doroshenko, Mikheil; Algara-Siller, Gerardo; Kaiser, Ute; Koynov, Kaloian; Weil, Tanja

    2012-11-19

    Quantum dots (QDs) coated with an albumin-derived copolymer shell exhibit significant photoresponsiveness to DNA loading and have great potential for investigating gene delivery processes. The QDs reported herein are positively charged, have attractive optical properties, and are noncytotoxic and notably stable in live cells. Their complex formation with plasmid DNA leads to proportionally decreased photoluminescence and efficient gene transfection is observed. Therefore, they are suitable for live-cell bioimaging and mechanistic studies of nonviral gene delivery. Fluorescence correlation spectroscopy is applied for the first time to investigate individual QDs diffusing in large endosomes inside living cells, and serves as a valuable tool to study the physical properties of QDs inside live cells. The data obtained in this study strongly support the notable stability of these QDs, even in cell endosomes. PMID:22915540

  19. A fluorimetric sensor for detection of one living cell

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Nowadays, studies of metabolic pathways and processes in living organisms cannot be easily done at the cellular level. That is why the development of a new analytical methods and approaches is needed, to allow detection of different biologically important species at very low concentrations levels an...

  20. Microfabricated Electrochemical Cell-Based Biosensors for Analysis of Living Cells In Vitro

    PubMed Central

    Wang, Jun; Wu, Chengxiong; Hu, Ning; Zhou, Jie; Du, Liping; Wang, Ping

    2012-01-01

    Cellular biochemical parameters can be used to reveal the physiological and functional information of various cells. Due to demonstrated high accuracy and non-invasiveness, electrochemical detection methods have been used for cell-based investigation. When combined with improved biosensor design and advanced measurement systems, the on-line biochemical analysis of living cells in vitro has been applied for biological mechanism study, drug screening and even environmental monitoring. In recent decades, new types of miniaturized electrochemical biosensor are emerging with the development of microfabrication technology. This review aims to give an overview of the microfabricated electrochemical cell-based biosensors, such as microelectrode arrays (MEA), the electric cell-substrate impedance sensing (ECIS) technique, and the light addressable potentiometric sensor (LAPS). The details in their working principles, measurement systems, and applications in cell monitoring are covered. Driven by the need for high throughput and multi-parameter detection proposed by biomedicine, the development trends of electrochemical cell-based biosensors are also introduced, including newly developed integrated biosensors, and the application of nanotechnology and microfluidic technology. PMID:25585708

  1. Vitellogenin Recognizes Cell Damage through Membrane Binding and Shields Living Cells from Reactive Oxygen Species*

    PubMed Central

    Havukainen, Heli; Münch, Daniel; Baumann, Anne; Zhong, Shi; Halskau, Øyvind; Krogsgaard, Michelle; Amdam, Gro V.

    2013-01-01

    Large lipid transfer proteins are involved in lipid transportation and diverse other molecular processes. These serum proteins include vitellogenins, which are egg yolk precursors and pathogen pattern recognition receptors, and apolipoprotein B, which is an anti-inflammatory cholesterol carrier. In the honey bee, vitellogenin acts as an antioxidant, and elevated vitellogenin titer is linked to prolonged life span in this animal. Here, we show that vitellogenin has cell and membrane binding activity and that it binds preferentially to dead and damaged cells. Vitellogenin binds directly to phosphatidylcholine liposomes and with higher affinity to liposomes containing phosphatidylserine, a lipid of the inner leaflet of cell membranes that is exposed in damaged cells. Vitellogenin binding to live cells, furthermore, improves cell oxidative stress tolerance. This study can shed more light on why large lipid transfer proteins have a well conserved α-helical domain, because we locate the lipid bilayer-binding ability of vitellogenin largely to this region. We suggest that recognition of cell damage and oxidation shield properties are two mechanisms that allow vitellogenin to extend honey bee life span. PMID:23897804

  2. Microtubules in Plant Cells: Strategies and Methods for Immunofluorescence, Transmission Electron Microscopy, and Live Cell Imaging.

    PubMed

    Celler, Katherine; Fujita, Miki; Kawamura, Eiko; Ambrose, Chris; Herburger, Klaus; Holzinger, Andreas; Wasteneys, Geoffrey O

    2016-01-01

    Microtubules (MTs) are required throughout plant development for a wide variety of processes, and different strategies have evolved to visualize and analyze them. This chapter provides specific methods that can be used to analyze microtubule organization and dynamic properties in plant systems and summarizes the advantages and limitations for each technique. We outline basic methods for preparing samples for immunofluorescence labeling, including an enzyme-based permeabilization method, and a freeze-shattering method, which generates microfractures in the cell wall to provide antibodies access to cells in cuticle-laden aerial organs such as leaves. We discuss current options for live cell imaging of MTs with fluorescently tagged proteins (FPs), and provide chemical fixation, high-pressure freezing/freeze substitution, and post-fixation staining protocols for preserving MTs for transmission electron microscopy and tomography. PMID:26498784

  3. Microtubules in Plant Cells: Strategies and Methods for Immunofluorescence, Transmission Electron Microscopy and Live Cell Imaging

    PubMed Central

    Celler, Katherine; Fujita, Miki; Kawamura, Eiko; Ambrose, Chris; Herburger, Klaus; Wasteneys, Geoffrey O.

    2016-01-01

    Microtubules are required throughout plant development for a wide variety of processes, and different strategies have evolved to visualize and analyze them. This chapter provides specific methods that can be used to analyze microtubule organization and dynamic properties in plant systems and summarizes the advantages and limitations for each technique. We outline basic methods for preparing samples for immunofluorescence labelling, including an enzyme-based permeabilization method, and a freeze-shattering method, which generates microfractures in the cell wall to provide antibodies access to cells in cuticle-laden aerial organs such as leaves. We discuss current options for live cell imaging of MTs with fluorescently tagged proteins (FPs), and provide chemical fixation, high pressure freezing/freeze substitution, and post-fixation staining protocols for preserving MTs for transmission electron microscopy and tomography. PMID:26498784

  4. 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.

  5. An automated tool for 3D tracking of single molecules in living cells

    NASA Astrophysics Data System (ADS)

    Gardini, L.; Capitanio, M.; Pavone, F. S.

    2015-03-01

    Since the behaviour of proteins and biological molecules is tightly related to cell's environment, more and more microscopy techniques are moving from in vitro to in living cells experiments. Looking at both diffusion and active transportation processes inside a cell requires three-dimensional localization over a few microns range, high SNR images and high temporal resolution. Since protein dynamics inside a cell involve all three dimensions, we developed an automated routine for 3D tracking of single fluorescent molecules inside living cells with nanometer accuracy, by exploiting the properties of the point-spread-function of out-of-focus Quantum Dots bound to the protein of interest.

  6. Noncontact microsurgery of living cell membrane using femtosecond laser pulses

    NASA Astrophysics Data System (ADS)

    Ilina, I. V.; Ovchinnikov, A. V.; Sitnikov, D. S.; Chefonov, O. V.; Agranat, M. B.; Mikaelyan, A. S.

    2013-06-01

    Near-infrared femtosecond laser pulses were applied to initiate reversible permeabilization of cell membrane and inject extrinsic substances into the target cells. Successful laser-based injection of a membrane impermeable dye, as well as plasmid DNA was demonstrated.

  7. Nanoscale optomechanical actuators for controlling mechanotransduction in living cells.

    PubMed

    Liu, Zheng; Liu, Yang; Chang, Yuan; Seyf, Hamid Reza; Henry, Asegun; Mattheyses, Alexa L; Yehl, Kevin; Zhang, Yun; Huang, Zhuangqun; Salaita, Khalid

    2016-02-01

    To control receptor tension optically at the cell surface, we developed an approach involving optomechanical actuator nanoparticles that are controlled with near-infrared light. Illumination leads to particle collapse, delivering piconewton forces to specific cell surface receptors with high spatial and temporal resolution. We demonstrate optomechanical actuation by controlling integrin-based focal adhesion formation, cell protrusion and migration, and T cell receptor activation. PMID:26657558

  8. Dielectrophoretic registration of living cells to a microelectrode array.

    PubMed

    Gray, Darren S; Tan, John L; Voldman, Joel; Chen, Christopher S

    2004-02-15

    We present a novel microfabricated device to simultaneously and actively trap thousands of single mammalian cells in alignment with a planar microelectrode array. Thousands of 3 micromdiameter trapping electrodes were fabricated within the bottom of a parallel-plate flow chamber. Cells were trapped on the electrodes and held against destabilizing fluid flows by dielectrophoretic forces generated in the device. In general, each electrode trapped only one cell. Adhesive regions were patterned onto the surface in alignment with the traps such that cells adhered to the array surface and remained in alignment with the electrodes. By driving the device with different voltages, we showed that trapped cells could be killed by stronger electric fields. However, with weaker fields, cells were not damaged during trapping, as indicated by the similar morphologies and proliferation rates of trapped cells versus controls. As a test of the device, we patterned approximately 20000 cells onto a 1cm(2) grid of rectangular adhesive regions, with two electrodes and thus two cells per rectangle. Our method obtained 70+/-1% fidelity versus 17+/-1% when using an existing cell-registration technique. By allowing the placement of desired numbers of cells at specified locations, this approach addresses many needs to manipulate and register cells to the surfaces of biosensors and other devices with high precision and fidelity. PMID:14709396

  9. Dielectrophoretic registration of living cells to a microelectrode array.

    PubMed

    Gray, Darren S; Tan, John L; Voldman, Joel; Chen, Christopher S

    2004-07-15

    We present a novel microfabricated device to simultaneously and actively trap thousands of single mammalian cells in alignment with a planar microelectrode array. Thousands of 3 Ipm diameter trapping electrodes were fabricated within the bottom of a parallel-plate flow chamber. Cells were trapped on the electrodes and held against destabilizing fluid flows by dielectrophoretic forces generated in the device. In general, each electrode trapped only one cell. Adhesive regions were patterned onto the surface in alignment with the traps such that cells adhered to the array surface and remained in alignment with the electrodes. By driving the device with different voltages, we showed that trapped cells could be killed by stronger electric fields. However, with weaker fields, cells were not damaged during trapping, as indicated by the similar morphologies and proliferation rates of trapped cells versus controls. As a test of the device, we patterned approximately 20,000 cells onto aI cm2 grid of rectangular adhesive regions, with two electrodes and thus two cells per rectangle. Our method obtained 70 +/- 1% fidelity versus 17 +/- 1% when using an existing cell-registration technique. By allowing the placement of desired numbers of cells at specified locations, this approach addresses many needs to manipulate and register cells to the surfaces of biosensors and other devices with high precision and fidelity. PMID:15198083

  10. Stopping biological time: The freezing of living cells

    SciTech Connect

    Mazur, P.

    1987-01-01

    The fundamental physical-chemical events that occur during the freezing and thawing of cells are outlined and the manner in which cell permeability determines the response of the cell to freezing is discussed both in terms of physical response and in terms of survival. 40 refs., 12 figs.

  11. Model system for plant cell biology: GFP imaging in living onion epidermal cells

    NASA Technical Reports Server (NTRS)

    Scott, A.; Wyatt, S.; Tsou, P. L.; Robertson, D.; Allen, N. S.

    1999-01-01

    The ability to visualize organelle localization and dynamics is very useful in studying cellular physiological events. Until recently, this has been accomplished using a variety of staining methods. However, staining can give inaccurate information due to nonspecific staining, diffusion of the stain or through toxic effects. The ability to target green fluorescent protein (GFP) to various organelles allows for specific labeling of organelles in vivo. The disadvantages of GFP thus far have been the time and money involved in developing stable transformants or maintaining cell cultures for transient expression. In this paper, we present a rapid transient expression system using onion epidermal peels. We have localized GFP to various cellular compartments (including the cell wall) to illustrate the utility of this method and to visualize dynamics of these compartments. The onion epidermis has large, living, transparent cells in a monolayer, making them ideal for visualizing GFP. This method is easy and inexpensive, and it allows for testing of new GFP fusion proteins in a living tissue to determine deleterious effects and the ability to express before stable transformants are attempted.

  12. A Filmography of Films About Movies and Movie-Making. Revised Edition.

    ERIC Educational Resources Information Center

    Parker, David L.

    More than 230 titles of films on the subject of movie-making are listed. The listed films illustrate many aspects of motion picture production, the history of cinema, general facts about movie film, and the nature of the film medium. The range of films included is wide. Selections deal with, among other subjects, the aesthetics of film, the…

  13. 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

  14. Smoking in Movies and Adolescent Smoking Initiation

    PubMed Central

    Morgenstern, Matthis; Sargent, James D.; Engels, Rutger C.M.E.; Scholte, Ron H.J.; Florek, Ewa; Hunt, Kate; Sweeting, Helen; Mathis, Federica; Faggiano, Fabrizio; Hanewinkel, Reiner

    2013-01-01

    Background Longitudinal studies from the U.S. suggest a causal relationship between exposure to images of smoking in movies and adolescent smoking onset. Purpose This study investigates whether adolescent smoking onset is predicted by the amount of exposure to smoking in movies across six European countries with various cultural and regulatory approaches to tobacco. Methods Longitudinal survey of 9987 adolescent never-smokers recruited in the years 2009–2010 (mean age 13.2 years) in 112 state-funded schools from Germany, Iceland, Italy, The Netherlands, Poland, and the United Kingdom (UK), and followed-up in 2011. Exposure to movie smoking was estimated from 250 top-grossing movies in each country. Multilevel mixed-effects Poisson regressions were performed in 2012 to assess the relationship between exposure at baseline and smoking status at follow-up. Results During the observation period (M=12 months), 17% of the sample initiated smoking. The estimated mean exposure to on-screen tobacco was 1560 occurrences. Overall, and after controlling for age; gender; family affluence; school performance; TVscreen time; personality characteristics; and smoking status of peers, parents, and siblings, exposure to each additional 1000 tobacco occurrences increased the adjusted relative risk for smoking onset by 13% (95% CI=8%, 17%, p<0.001). The crude relationship between movie smoking exposure and smoking initiation was significant in all countries; after covariate adjustment, the relationship remained significant in Germany, Iceland, The Netherlands, Poland, and UK. Conclusions Seeing smoking in movies is a predictor of smoking onset in various cultural contexts. The results confirm that limiting young people’s exposure to movie smoking might be an effective way to decrease adolescent smoking onset. PMID:23498098

  15. 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

  16. THE OSMOTIC PROPERTIES OF LIVING CELLS (EGGS OF ARBACIA PUNCTULATA).

    PubMed

    McCutcheon, M; Lucké, B; Hartline, H K

    1931-01-20

    WE HAVE ATTEMPTED TO ANSWER THE QUESTION: How nearly ideal, as an osmometer, is the unfertilized Arbacia egg? The following conclusion have been reached: 1. Volumes can be measured accurately over a wide range of pressures since the cell is in general spherical and does not suffer deformation from its own weight or other factors. 2. The product of volume and pressure is approximately constant, if allowance be made for osmotically inactive cell contents. It is computed that from 7 to 14 per cent of cell volume is occupied by osmotically inactive material. 3. Evidence is presented that no appreciable escape of cell contents occurs while the cell is in hypotonic sea water; that, therefore, the semipermeability of the membrane is approximately perfect, so long as injury to the cell is avoided. 4. In comparison with osmotic pressure the influence of other forces, such as elasticity or surface tension, on cell volume must in these experiments be slight. PMID:19872593

  17. Further observations on the phenomenon of secondary vacuolation in living cells.

    NASA Technical Reports Server (NTRS)

    Mahlberg, P.

    1972-01-01

    The dynamics of secondary vacuole movement is studied in living hair cells of Tradescantia virginiana. The pattern of movement of these vacuoles is found to be similar to that described by the author previously for organelles in cultured cells. Evidence is presented in support of the thesis that the occurrence and dynamics of secondary vacuoles is a common phenomenon for plant cells.

  18. Phosphorescent Imaging of Living Cells Using a Cyclometalated Iridium(III) Complex

    PubMed Central

    Ma, Dik-Lung; Zhong, Hai-Jing; Fu, Wai-Chung; Chan, Daniel Shiu-Hin; Kwan, Hiu-Yee; Fong, Wang-Fun; Chung, Lai-Hon; Wong, Chun-Yuen; Leung, Chung-Hang

    2013-01-01

    A cell permeable cyclometalated iridium(III) complex has been developed as a phosphorescent probe for cell imaging. The iridium(III) solvato complex [Ir(phq)2(H2O]2)] preferentially stains the cytoplasm of both live and dead cells with a bright luminescence. PMID:23457478

  19. Uniaxial cell stretching device for live-cell imaging of mechanosensitive cellular functions

    NASA Astrophysics Data System (ADS)

    Shao, Yue; Tan, Xinyu; Novitski, Roman; Muqaddam, Mishaal; List, Paul; Williamson, Laura; Fu, Jianping; Liu, Allen P.

    2013-11-01

    External mechanical stretch plays an important role in regulating cellular behaviors through intracellular mechanosensitive and mechanotransductive machineries such as the F-actin cytoskeleton (CSK) structures and focal adhesions (FAs) anchoring the F-actin CSK to the extracellular environment. Studying the mechanoresponsive behaviors of the F-actin CSK and FAs in response to cell stretch has great importance for further understanding mechanotransduction and mechanobiology. In this work, we developed a novel cell stretching device combining dynamic directional cell stretch with in situ subcellular live-cell imaging. Using a cam and follower mechanism and applying a standard mathematical model for cam design, we generated different dynamic stretch outputs. By examining stretch-mediated FA dynamics under step-function static stretch and the realignment of cell morphology and the F-actin CSK under cyclic stretch, we demonstrated successful applications of our cell stretching device for mechanobiology studies where external stretch plays an important role in regulating subcellular molecular dynamics and cellular phenotypes.

  20. Tracking Single Cells in Live Animals Using a Photoconvertible Near-Infrared Cell Membrane Label

    PubMed Central

    Wu, Juwell; Runnels, Judith M.; Turcotte, Raphaël; Celso, Cristina Lo; Scadden, David T.; Strom, Terry B.; Lin, Charles P.

    2013-01-01

    We describe a novel photoconversion technique to track individual cells in vivo using a commercial lipophilic membrane dye, DiR. We show that DiR exhibits a permanent fluorescence emission shift (photoconversion) after light exposure and does not reacquire the original color over time. Ratiometric imaging can be used to distinguish photoconverted from non-converted cells with high sensitivity. Combining the use of this photoconvertible dye with intravital microscopy, we tracked the division of individual hematopoietic stem/progenitor cells within the calvarium bone marrow of live mice. We also studied the peripheral differentiation of individual T cells by tracking the gain or loss of FoxP3-GFP expression, a marker of the immune suppressive function of CD4+ T cells. With the near-infrared photoconvertible membrane dye, the entire visible spectral range is available for simultaneous use with other fluorescent proteins to monitor gene expression or to trace cell lineage commitment in vivo with high spatial and temporal resolution. PMID:23990881

  1. Uniaxial cell stretching device for live-cell imaging of mechanosensitive cellular functions

    PubMed Central

    Shao, Yue; Tan, Xinyu; Novitski, Roman; Muqaddam, Mishaal; List, Paul; Williamson, Laura; Fu, Jianping; Liu, Allen P.

    2013-01-01

    External mechanical stretch plays an important role in regulating cellular behaviors through intracellular mechanosensitive and mechanotransductive machineries such as the F-actin cytoskeleton (CSK) structures and focal adhesions (FAs) anchoring the F-actin CSK to the extracellular environment. Studying the mechanoresponsive behaviors of the F-actin CSK and FAs in response to cell stretch has great importance for further understanding mechanotransduction and mechanobiology. In this work, we developed a novel cell stretching device combining dynamic directional cell stretch with in situ subcellular live-cell imaging. Using a cam and follower mechanism and applying a standard mathematical model for cam design, we generated different dynamic stretch outputs. By examining stretch-mediated FA dynamics under step-function static stretch and the realignment of cell morphology and the F-actin CSK under cyclic stretch, we demonstrated successful applications of our cell stretching device for mechanobiology studies where external stretch plays an important role in regulating subcellular molecular dynamics and cellular phenotypes. PMID:24289415

  2. Quantitative Imaging of Single mRNA Splice Variants in Living Cells

    PubMed Central

    Lee, Kyuwan; Cui, Yi

    2015-01-01

    Alternative mRNA splicing is a fundamental process of gene regulation via the precise control of the post-transcriptional step that occurs before mRNA translation. Errors in RNA splicing have been known to correlate with different diseases; however, a key limitation is the lack of technologies for live cell monitoring and quantification to understand the process of alternative splicing. Here, we report a spectroscopic strategy for quantitative imaging of mRNA splice variants in living cells, using nanoplasmonic dimer antennas. The spatial and temporal distribution of three selected splice variants of the breast cancer susceptibility gene, BRCA1 were monitored at single copy resolution by measuring the hybridization dynamics of nanoplasmonic antennas targeting complementary mRNA sequences in live cells. Our study provides valuable insights on RNA and its transport in living cells, which has the potential to enhance our understanding of cellular protein complex, pharmacogenomics, genetic diagnosis, and gene therapies. PMID:24747838

  3. Population of Vibrational State of Carotenoid Molecules in Living Cells of Chlorella

    NASA Astrophysics Data System (ADS)

    Kinoshita, Shuichi; Hirata, Kuniko; Kushida, Takashi

    1980-07-01

    Stokes and anti-Stokes Raman spectra have been measured in living cells of Chlorella vulgaris as well as in chloroform, toluene, benzene and β-carotene. Population in the vibrational state has been determined by taking account of resonance Raman effect. The result shows that this population is well explained by thermal distribution even in the case of living biological cells, contrary to recently reported observation of some population enhancement. Possible experimental artifacts are discussed.

  4. Nanoscale Optomechanical Actuators for Controlling Mechanotransduction in Living Cells

    PubMed Central

    Liu, Zheng; Liu, Yang; Chang, Yuan; Seyf, Hamid Reza; Henry, Asegun; Mattheyses, Alexa L.; Yehl, Kevin; Zhang, Yun; Huang, Zhuangqun; Salaita, Khalid

    2015-01-01

    Herein we develop an approach for optically controlling receptor tension. This is achieved using optomechanical actuator nanoparticles that are controlled with non-invasive near-infrared light. Illumination leads to particle collapse, delivering piconewton forces to specific cell surface receptors with high spatial and temporal resolution. As a proof-of-concept, we applied optomechanical actuation to trigger integrin-based focal adhesion formation, cell protrusion and migration, as well as T cell receptor activation. PMID:26657558

  5. Poly(ethylene glycol) hydrogel microstructures encapsulating living cells

    NASA Technical Reports Server (NTRS)

    Koh, Won-Gun; Revzin, Alexander; Pishko, Michael V.

    2002-01-01

    We present an easy and effective method for the encapsulation of cells inside PEG-based hydrogel microstructures fabricated using photolithography. High-density arrays of three-dimensional microstructures were created on substrates using this method. Mammalian cells were encapsulated in cylindrical hydrogel microstructures of 600 and 50 micrometers in diameter or in cubic hydrogel structures in microfluidic channels. Reducing lateral dimension of the individual hydrogel microstructure to 50 micrometers allowed us to isolate 1-3 cells per microstructure. Viability assays demonstrated that cells remained viable inside these hydrogels after encapsulation for up to 7 days.

  6. Effect of Enzymes on the Interaction of Enteroviruses with Living HeLa Cells1

    PubMed Central

    Zajac, Ihor; Crowell, Richard L.

    1965-01-01

    Zajac, Ihor (Hahnemann Medical College, Philadelphia, Pa.), and Richard L. Crowell. Effect of enzymes on the interaction of enteroviruses with living HeLa cells. J. Bacteriol. 89:574–582. 1965.—Eight crude enzyme preparations and two crystalline enzymes were tested for ability to inactivate coxsackie group B and poliovirus receptors on living HeLa cells. Receptor-destroying enzyme, erepsin, lysozyme, collagenase, proteinase, and cobra venom did not alter attachment of coxsackie B3 or poliovirus T1 to cells, whereas elastase prevented attachment of both viruses tested. Treatment of live cells with pancreatin or chymotrypsin rendered cells unable to attach group B coxsackie viruses, whereas cells treated with trypsin failed to attach poliovirus T1. In addition, chymotrypsin was found to release coxsackie B3 and poliovirus T1 from cell surfaces, whereas trypsin was unable to dissociate virus-cell union. These results indicate that cellular receptors for polioviruses differ from those for group B coxsackie-viruses. The finding that 1% solutions of enzymes will inactivate differentially the enteroviral receptors of HeLa cells, without altering cell viability, provides a useful approach for study of enterovirus receptors of live host cells. PMID:14273631

  7. Nonperturbative Imaging of Nucleoid Morphology in Live Bacterial Cells during an Antimicrobial Peptide Attack

    PubMed Central

    Bakshi, Somenath; Choi, Heejun; Rangarajan, Nambirajan; Barns, Kenneth J.; Bratton, Benjamin P.

    2014-01-01

    Studies of time-dependent drug and environmental effects on single, live bacterial cells would benefit significantly from a permeable, nonperturbative, long-lived fluorescent stain specific to the nucleoids (chromosomal DNA). The ideal stain would not affect cell growth rate or nucleoid morphology and dynamics, even during laser illumination for hundreds of camera frames. In this study, time-dependent, single-cell fluorescence imaging with laser excitation and a sensitive electron-multiplying charge-coupled-device (EMCCD) camera critically tested the utility of “dead-cell stains” (SYTOX orange and SYTOX green) and “live-cell stains” (DRAQ5 and SYTO 61) and also 4′,6-diamidino-2-phenylindole (DAPI). Surprisingly, the dead-cell stains were nearly ideal for imaging live Escherichia coli, while the live-cell stains and DAPI caused nucleoid expansion and, in some cases, cell permeabilization and the halting of growth. SYTOX orange performed well for both the Gram-negative E. coli and the Gram-positive Bacillus subtilis. In an initial application, we used two-color fluorescence imaging to show that the antimicrobial peptide cecropin A destroyed nucleoid-ribosome segregation over 20 min after permeabilization of the E. coli cytoplasmic membrane, reminiscent of the long-term effects of the drug rifampin. In contrast, the human cathelicidin LL-37, while similar to cecropin A in structure, length, charge, and the ability to permeabilize bacterial membranes, had no observable effect on nucleoid-ribosome segregation. Possible underlying causes are suggested. PMID:24907320

  8. Nanoscale bio-platforms for living cell interrogation: current status and future perspectives

    NASA Astrophysics Data System (ADS)

    Chang, Lingqian; Hu, Jiaming; Chen, Feng; Chen, Zhou; Shi, Junfeng; Yang, Zhaogang; Li, Yiwen; Lee, Ly James

    2016-02-01

    The living cell is a complex entity that dynamically responds to both intracellular and extracellular environments. Extensive efforts have been devoted to the understanding intracellular functions orchestrated with mRNAs and proteins in investigation of the fate of a single-cell, including proliferation, apoptosis, motility, differentiation and mutations. The rapid development of modern cellular analysis techniques (e.g. PCR, western blotting, immunochemistry, etc.) offers new opportunities in quantitative analysis of RNA/protein expression up to a single cell level. The recent entries of nanoscale platforms that include kinds of methodologies with high spatial and temporal resolution have been widely employed to probe the living cells. In this tutorial review paper, we give insight into background introduction and technical innovation of currently reported nanoscale platforms for living cell interrogation. These highlighted technologies are documented in details within four categories, including nano-biosensors for label-free detection of living cells, nanodevices for living cell probing by intracellular marker delivery, high-throughput platforms towards clinical current, and the progress of microscopic imaging platforms for cell/tissue tracking in vitro and in vivo. Perspectives for system improvement were also discussed to solve the limitations remains in current techniques, for the purpose of clinical use in future.

  9. Nanoscale bio-platforms for living cell interrogation: current status and future perspectives.

    PubMed

    Chang, Lingqian; Hu, Jiaming; Chen, Feng; Chen, Zhou; Shi, Junfeng; Yang, Zhaogang; Li, Yiwen; Lee, Ly James

    2016-02-14

    The living cell is a complex entity that dynamically responds to both intracellular and extracellular environments. Extensive efforts have been devoted to the understanding intracellular functions orchestrated with mRNAs and proteins in investigation of the fate of a single-cell, including proliferation, apoptosis, motility, differentiation and mutations. The rapid development of modern cellular analysis techniques (e.g. PCR, western blotting, immunochemistry, etc.) offers new opportunities in quantitative analysis of RNA/protein expression up to a single cell level. The recent entries of nanoscale platforms that include kinds of methodologies with high spatial and temporal resolution have been widely employed to probe the living cells. In this tutorial review paper, we give insight into background introduction and technical innovation of currently reported nanoscale platforms for living cell interrogation. These highlighted technologies are documented in details within four categories, including nano-biosensors for label-free detection of living cells, nanodevices for living cell probing by intracellular marker delivery, high-throughput platforms towards clinical current, and the progress of microscopic imaging platforms for cell/tissue tracking in vitro and in vivo. Perspectives for system improvement were also discussed to solve the limitations remains in current techniques, for the purpose of clinical use in future. PMID:26745513

  10. Microbeam studies of the sensitivity of structures within living cells

    NASA Technical Reports Server (NTRS)

    Braby, L. A.

    1992-01-01

    Determining the biological effects of low doses of radiation with high linear energy transfer (LET) is complicated by the stochastic nature of charged-particle interactions. Populations of cells exposed to very low radiation doses contain a few cells which have been hit by a charged particle, while the majority of the cells receive no radiation damage. At somewhat higher doses, a few cells receive two or more events. Because the effects of damage produced by separate events can interact in the cell, we have had to make assumptions about the nature of these interactions in order to interpret the results of the experiments. Many of those assumptions can be tested if we can be sure of the number of charged-particle events which occur in individual cells, and correlate this number with the biological effect. We have developed a special irradiation facility at Pacific Northwest Laboratory (PNL) to control the actual number of charged particle tracks that pass through cell nuclei. The beam from a 2 MeV tandem accelerator is collimated to approximately 5 microns. Cells, grown in special dishes with 1.5 microns thick plastic bottoms, are positioned so that the desired portion of the cell aligns with the collimator. A shutter in the beam line is opened and closed after the desired number of particle tracks has been counted. This approach can be used to investigate the effects of the interaction between irradiated and unirradiated cells in an organized system, as well as to study the effects of spatial and temporal distribution of radiation damage within single cells.(ABSTRACT TRUNCATED AT 250 WORDS).

  11. Question 7: construction of a semi-synthetic minimal cell: a model for early living cells.

    PubMed

    Murtas, Giovanni

    2007-10-01

    Using a Synthetic Biology approach we are building a semi-synthetic minimal cell. This represents an exercise to shape a minimal-cell model system recalling the simplicity of early living cells in early evolution. We have recently introduced into liposome compartments a minimal set of enzymes named "Puresystem" (PS) synthesizing EGFP proteins. To establish reproduction of the shell compartment with a minimal set of genes we have cloned the genes for the Fatty Acid Synthase (FAS) type I enzymes. These FAS genes introduced into liposomes, translated into FAS enzymes by PS and in the presence of precursors produce fatty acids. The resulting release of fatty acid molecules within liposome vesicles should promote vesicle growth and reproduction. The core reproduction of a minimal cell corresponding to the replication of the minimal genome will require a few genes for the DNA replication and the PS, and a minimum set of genes for the synthesis of t-RNAs. In future the reconstruction of a minimal ribosome will bring the number of genes for ribosomal proteins from 54 of an existing minimal genome down to 30-20 genes. A Synthetic Biology approach could bring the number of essential genes for a minimal cell down to 100 or less. PMID:17668286

  12. 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

  13. On-line tracking of living cell subjected to cyclic stretch.

    PubMed

    Huang, Wenjing; Ahmad, Belal; Kawahara, Tomohiro

    2015-08-01

    We propose a novel system for the observation of living cell exposed to cyclic stretch under dynamic conditions. The developed system is mainly composed of a laptop PC, a stretching unit with three motorized stages, and a microscope with a CCD camera. The design of the cell tracking system is based on the deformation characteristics of the elastic chamber and its performance was confirmed through the basic experiments. Finally, we succeeded in on-line imaging of living single cells under the microscope with a high magnification ratio. We believe that the developed system is a promising platform for studying the immediate responses of cells exposed to cyclic stretch. PMID:26737060

  14. Imaging translucent cell bodies in the living mouse retina without contrast agents.

    PubMed

    Guevara-Torres, A; Williams, D R; Schallek, J B

    2015-06-01

    The transparency of most retinal cell classes typically precludes imaging them in the living eye; unless invasive methods are used that deploy extrinsic contrast agents. Using an adaptive optics scanning light ophthalmoscope (AOSLO) and capitalizing on the large numerical aperture of the mouse eye, we enhanced the contrast from otherwise transparent cells by subtracting the left from the right half of the light distribution in the detector plane. With this approach, it is possible to image the distal processes of photoreceptors, their more proximal cell bodies and the mosaic of horizontal cells in the living mouse retina. PMID:26114032

  15. Probing protein complexes inside living cells using a silicon nanowire-based pull-down assay.

    PubMed

    Choi, Sojoong; Kim, Hyunju; Kim, So Yeon; Yang, Eun Gyeong

    2016-06-01

    Most proteins perform their functions as interacting complexes. Here we propose a novel method for capturing an intracellular protein and its interacting partner out of living cells by utilizing intracellular access of antibody modified vertical silicon nanowire arrays whose surface is covered with a polyethylene glycol layer to prevent strong cell adhesion. Such a feature facilitates the removal of cells by simple washing, enabling subsequent detection of a pulled-down protein and its interacting partner, and further assessment of a drug-induced change in the interacting complex. Our new SiNW-based tool is thus suitable for authentication of protein networks inside living cells. PMID:27198202

  16. Imaging translucent cell bodies in the living mouse retina without contrast agents

    PubMed Central

    Guevara-Torres, A.; Williams, D. R.; Schallek, J. B.

    2015-01-01

    The transparency of most retinal cell classes typically precludes imaging them in the living eye; unless invasive methods are used that deploy extrinsic contrast agents. Using an adaptive optics scanning light ophthalmoscope (AOSLO) and capitalizing on the large numerical aperture of the mouse eye, we enhanced the contrast from otherwise transparent cells by subtracting the left from the right half of the light distribution in the detector plane. With this approach, it is possible to image the distal processes of photoreceptors, their more proximal cell bodies and the mosaic of horizontal cells in the living mouse retina. PMID:26114032

  17. Optical micromanipulation of nanoparticles and cells inside living zebrafish.

    PubMed

    Johansen, Patrick Lie; Fenaroli, Federico; Evensen, Lasse; Griffiths, Gareth; Koster, Gerbrand

    2016-01-01

    Regulation of biological processes is often based on physical interactions between cells and their microenvironment. To unravel how and where interactions occur, micromanipulation methods can be used that offer high-precision control over the duration, position and magnitude of interactions. However, lacking an in vivo system, micromanipulation has generally been done with cells in vitro, which may not reflect the complex in vivo situation inside multicellular organisms. Here using optical tweezers we demonstrate micromanipulation throughout the transparent zebrafish embryo. We show that different cells, as well as injected nanoparticles and bacteria can be trapped and that adhesion properties and membrane deformation of endothelium and macrophages can be analysed. This non-invasive micromanipulation inside a whole-organism gives direct insights into cell interactions that are not accessible using existing approaches. Potential applications include screening of nanoparticle-cell interactions for cancer therapy or tissue invasion studies in cancer and infection biology. PMID:26996121

  18. Optical micromanipulation of nanoparticles and cells inside living zebrafish

    PubMed Central

    Johansen, Patrick Lie; Fenaroli, Federico; Evensen, Lasse; Griffiths, Gareth; Koster, Gerbrand

    2016-01-01

    Regulation of biological processes is often based on physical interactions between cells and their microenvironment. To unravel how and where interactions occur, micromanipulation methods can be used that offer high-precision control over the duration, position and magnitude of interactions. However, lacking an in vivo system, micromanipulation has generally been done with cells in vitro, which may not reflect the complex in vivo situation inside multicellular organisms. Here using optical tweezers we demonstrate micromanipulation throughout the transparent zebrafish embryo. We show that different cells, as well as injected nanoparticles and bacteria can be trapped and that adhesion properties and membrane deformation of endothelium and macrophages can be analysed. This non-invasive micromanipulation inside a whole-organism gives direct insights into cell interactions that are not accessible using existing approaches. Potential applications include screening of nanoparticle-cell interactions for cancer therapy or tissue invasion studies in cancer and infection biology. PMID:26996121

  19. The Dynamics of DNA Topoisomerase IIα in Living Cells

    PubMed Central

    Daum, John R.; Mo, Yin Yuan; Gorbsky, Gary J.

    2010-01-01

    Here we describe methods to prepare a mammalian expression plasmid encoding EGFP fused to the amino terminus of human DNA topoisomerase IIα (TopoIIα). This plasmid is transfected into LLC-Pk cells, a porcine epithelial cell line that remains relatively flat during mitosis. After selection for stable integration, cells are cloned by serial dilution in microwells and used to grow a stable cell line expressing EGFP-TopoIIα. This line is termed LPk-GT2. Using photobleaching methods with conventional and patterned photobleaching LPk-GT2 cells are used demonstrate the rapid dynamics of TopoIIα exchange in both interphase nuclei and mitotic chromosomes. These rapid dynamics are dependent on enzyme activity since ICRF159 a catalytic inhibitor of TopoIIα, slows dynamics significantly. PMID:19763954

  20. Optical micromanipulation of nanoparticles and cells inside living zebrafish

    NASA Astrophysics Data System (ADS)

    Johansen, Patrick Lie; Fenaroli, Federico; Evensen, Lasse; Griffiths, Gareth; Koster, Gerbrand

    2016-03-01

    Regulation of biological processes is often based on physical interactions between cells and their microenvironment. To unravel how and where interactions occur, micromanipulation methods can be used that offer high-precision control over the duration, position and magnitude of interactions. However, lacking an in vivo system, micromanipulation has generally been done with cells in vitro, which may not reflect the complex in vivo situation inside multicellular organisms. Here using optical tweezers we demonstrate micromanipulation throughout the transparent zebrafish embryo. We show that different cells, as well as injected nanoparticles and bacteria can be trapped and that adhesion properties and membrane deformation of endothelium and macrophages can be analysed. This non-invasive micromanipulation inside a whole-organism gives direct insights into cell interactions that are not accessible using existing approaches. Potential applications include screening of nanoparticle-cell interactions for cancer therapy or tissue invasion studies in cancer and infection biology.

  1. Imaging and manipulating the structural machinery of living cells on the micro- and nanoscale

    PubMed Central

    Chown, Matthew G; Kumar, Sanjay

    2007-01-01

    The structure, physiology, and fate of living cells are all highly sensitive to mechanical forces in the cellular microenvironment, including stresses and strains that originate from encounters with the extracellular matrix (ECM), blood and other flowing materials, and neighbouring cells. This relationship between context and physiology bears tremendous implications for the design of cellular micro-or nanotechnologies, since any attempt to control cell behavior in a device must provide the appropriate physical microenvironment for the desired cell behavior. Cells sense, process, and respond to biophysical cues in their environment through a set of integrated, multi-scale structural complexes that span length scales from single molecules to tens of microns, including small clusters of force-sensing molecules at the cell surface, micron-sized cell-ECM focal adhesion complexes, and the cytoskeleton that permeates and defines the entire cell. This review focuses on several key technologies that have recently been developed or adapted for the study of the dynamics of structural micro-and nanosystems in living cells and how these systems contribute to spatially-and temporally-controlled changes in cellular structure and mechanics. We begin by discussing subcellular laser ablation, which permits the precise incision of nanoscale structural elements in living cells in order to discern their mechanical properties and contributions to cell structure. We then discuss fluorescence recovery after photobleaching and fluorescent speckle microscopy, two live-cell fluorescence imaging methods that enable quantitative measurement of the binding and transport properties of specific proteins in the cell. Finally, we discuss methods to manipulate cellular structural networks by engineering the extracellular environment, including microfabrication of ECM distributions of defined geometry and microdevices designed to measure cellular traction forces at micron-scale resolution. Together

  2. Development of exosome surface display technology in living human cells.

    PubMed

    Stickney, Zachary; Losacco, Joseph; McDevitt, Sophie; Zhang, Zhiwen; Lu, Biao

    2016-03-25

    Surface display technology is an emerging key player in presenting functional proteins for targeted drug delivery and therapy. Although a number of technologies exist, a desirable mammalian surface display system is lacking. Exosomes are extracellular vesicles that facilitate cell-cell communication and can be engineered as nano-shuttles for cell-specific delivery. In this study, we report the development of a novel exosome surface display technology by exploiting mammalian cell secreted nano-vesicles and their trans-membrane protein tetraspanins. By constructing a set of fluorescent reporters for both the inner and outer surface display on exosomes at two selected sites of tetraspanins, we demonstrated the successful exosomal display via gene transfection and monitoring fluorescence in vivo. We subsequently validated our system by demonstrating the expected intracellular partitioning of reporter protein into sub-cellular compartments and secretion of exosomes from human HEK293 cells. Lastly, we established the stable engineered cells to harness the ability of this robust system for continuous production, secretion, and uptake of displayed exosomes with minimal impact on human cell biology. In sum, our work paved the way for potential applications of exosome, including exosome tracking and imaging, targeted drug delivery, as well as exosome-mediated vaccine and therapy. PMID:26902116

  3. Chitosan-based nanocoatings for hypothermic storage of living cells.

    PubMed

    Bulwan, Maria; Antosiak-Iwańska, Magdalena; Godlewska, Ewa; Granicka, Ludomira; Zapotoczny, Szczepan; Nowakowska, Maria

    2013-11-01

    The formation of ultrathin chitosan-based nanocoating on HL-60 model cells and their protective function in hypothermic storage are presented. HL-60 cells are encapsulated in ultrathin shells by adsorbing cationic and anionic chitosan derivatives in a stepwise, layer-by-layer, procedure carried out in an aqueous medium under mild conditions. The chitosan-based films are also deposited on model lipid bilayer and the interactions are studied using ellipsometry and atomic force microscopy. The cells covered with the chitosan-based films and stored at 4 °C for 24 h express viability comparable to that of the control sample incubated at 37 °C, while the unprotected cells stored under the same conditions do not show viability. It is shown that the chitosan-based shell protects HL-60 cells against damaging effect of hypothermic storage. Such nanocoatings provide protection, mechanical stability, and support the cell membrane, while ensuring penetration of small molecules such as nutrients/gases what is essential for cell viability. PMID:23966342

  4. Small Molecule-Photoactive Yellow Protein Labeling Technology in Live Cell Imaging.

    PubMed

    Gao, Feng; Gao, Tang; Zhou, Kechao; Zeng, Wenbin

    2016-01-01

    Characterization of the chemical environment, movement, trafficking and interactions of proteins in live cells is essential to understanding their functions. Labeling protein with functional molecules is a widely used approach in protein research to elucidate the protein location and functions both in vitro and in live cells or in vivo. A peptide or a protein tag fused to the protein of interest and provides the opportunities for an attachment of small molecule probes or other fluorophore to image the dynamics of protein localization. Here we reviewed the recent development of no-wash small molecular probes for photoactive yellow protein (PYP-tag), by the means of utilizing a quenching mechanism based on the intramolecular interactions, or an environmental-sensitive fluorophore. Several fluorogenic probes have been developed, with fast labeling kinetics and cell permeability. This technology allows quick live-cell imaging of cell-surface and intracellular proteins without a wash-out procedure. PMID:27589715

  5. Polyamide fluorescent probes for visualization of repeated DNA sequences in living cells.

    PubMed

    Nozeret, Karine; Loll, François; Escudé, Christophe; Boutorine, Alexandre S

    2015-03-01

    DNA imaging in living cells usually requires transgenic approaches that modify the genome. Synthetic pyrrole-imidazole polyamides that bind specifically to the minor groove of double-stranded DNA (dsDNA) represent an attractive approach for in-cell imaging that does not necessitate changes to the genome. Nine hairpin polyamides that target mouse major satellite DNA were synthesized. Their interactions with synthetic target dsDNA fragments were studied by thermal denaturation, gel-shift electrophoresis, circular dichroism, and fluorescence spectroscopy. The polyamides had different affinities for the target DNA, and fluorescent labeling of the polyamides affected their affinity for their targets. We validated the specificity of the probes in fixed cells and provide evidence that two of the probes detect target sequences in mouse living cell lines. This study demonstrates for the first time that synthetic compounds can be used for the visualization of the nuclear substructures formed by repeated DNA sequences in living cells. PMID:25639955

  6. The haemopoietic and immunogenic capacities of living hybrid bone marrow cells tested in tumour allograft rejection.

    PubMed Central

    Kerckhaert, J A; Hofhuis, F M; Willers, J M

    1975-01-01

    In irradiated mice the capacity to reject allogenic tumours can be reconstituted with syngeneic lymphoid cells if the transferred cells are primed with the allogenicantigen. Living semi-allogeneic cells proved to be 30-100-times more acitve as priming antigen than cell membrane fractions. The tumour-suppressive activity of primed lymphoid cells increased in the following order: bone marrow less than Peyer's patches less than thymus less than spleen less than lymph node cells. Even bone marrowcells showed a considerable suppressing activity after priming with live antigen. It was a great advantage that 2 times 10-6 semi-allogeneic bone marrow cells could be used both for the restoration of the haemopoietic system after irradiation and for stimulation of the transferred parental lymphocytes. Priming with large numbers of semi-allogeneic spleen cells abolished the tumour-suppressive activity of the transferred lymphoid cells. This tolerogenic effect disappeared when the priming cells were pretreated with mitomycin. Tolerance could be induced when the cell donors were treated with cyclophosphamide in combination with the living cells. Cell membranes were not effective. PMID:1150311

  7. Biological interaction of living cells with COSAN-based synthetic vesicles

    PubMed Central

    Tarrés, Màrius; Canetta, Elisabetta; Paul, Eleanor; Forbes, Jordan; Azzouni, Karima; Viñas, Clara; Teixidor, Francesc; Harwood, Adrian J.

    2015-01-01

    Cobaltabisdicarbollide (COSAN) [3,3′-Co(1,2-C2B9H11)2]−, is a complex boron-based anion that has the unusual property of self-assembly into membranes and vesicles. These membranes have similar dimensions to biological membranes found in cells, and previously COSAN has been shown to pass through synthetic lipid membranes and those of living cells without causing breakdown of membrane barrier properties. Here, we investigate the interaction of this inorganic membrane system with living cells. We show that COSAN has no immediate effect on cell viability, and cells fully recover when COSAN is removed following exposure for hours to days. COSAN elicits a range of cell biological effects, including altered cell morphology, inhibition of cell growth and, in some cases, apoptosis. These observations reveal a new biology at the interface between inorganic, synthetic COSAN membranes and naturally occurring biological membranes. PMID:25588708

  8. New ways to print living cells promise breakthroughs for engineering complex tissues in vitro

    PubMed Central

    Withers, Ginger S.

    2006-01-01

    The ability to control the placement of cells and the assembly of networks in vitro has tremendous potential for understanding the regulation of development as well as for generating artificial tissues. To date, most engineering tools that can place materials with precision are not compatible with the requirements of living cells, and so approaches to tissue engineering have focused on patterning substrates as a way of controlling cell growth rather than patterning cells directly. In this issue of Biochemical Journal, however, Eagles et al. adapt electrohydrodynamic printing technology to ‘print’ living cells from a neuronal cell line on to a substrate. The importance of this approach is that it has the potential for unprecedented control over the position of cells in culture by directly placing them, thus allowing for the systematic assembly of cell networks. PMID:16479619

  9. Scanning electrochemical microscopy of living cells. 3. Rhodobacter sphaeroides.

    PubMed

    Cai, Chenxin; Liu, Biao; Mirkin, Michael V; Frank, Harry A; Rusling, James F

    2002-01-01

    The scanning electrochemical microscope (SECM) was used to probe the redox activity of individual purple bacteria (Rhodobacter sphaeroides). The approaches developed in our previous studies of mammalian cells were expanded to measure the rates and investigate the pathway of transmembrane charge transfer in bacteria. The two groups of redox mediators (i.e., hydrophilic and hydrophobic redox species) were used to shuttle the electrons between the SECM tip electrode in solution and the redox centers inside the cell. The analysis of the dependencies of the measured rate constant on formal potential and concentration of mediator species in solution yielded information about the permeability of the outer cell membrane to different ionic species and intracellular redox properties. The maps of redox reactivity of the cell surface were obtained with a micrometer or submicrometer spatial resolution. PMID:11795778

  10. Mathematical model of living cells behavior: Case of telocytes

    NASA Astrophysics Data System (ADS)

    Roatesi, Iurie; Roatesi, Simona; Rotaru, Constantin; Cretoiu, Sanda; Cretoiu, Dragos

    2016-06-01

    Telocytes are a novel interstitial cell type characterized by a small cell body and a number of one to five of extremely long and thin prolongations, named telopodes. This article proposes an analytical and numerical modeling for telopodes elongation, based on their appearance and behavior captured from in vitro approaches. Both the analytical and numerical solutions are developed for a viscoelastic model and they are compared and a good agreement is obtained.

  11. Photodegradable macromers and hydrogels for live cell encapsulation and release

    PubMed Central

    Griffin, Donald R.; Kasko, Andrea M.

    2012-01-01

    Hydrogel scaffolds are commonly used as 3D carriers for cells because their properties can be tailored to match natural extra-cellular matrix. Hydrogels may be used in tissue engineering and regenerative medicine to deliver therapeutic cells to injured or diseased tissue through controlled degradation. Hydrolysis and enzymolysis are the two most common mechanisms employed for hydrogel degradation, but neither allows sequential or staged release of cells. In contrast, photodegradation allows external real-time spatial and temporal control over hydrogel degradation, and allows for staged and sequential release of cells. We synthesized and characterized a series of macromers incorporating photodegradbale ortho-nitrobenzyl (o-NB) groups in the macromer backbone. We formed hydrogels from these macromers via redox polymerization and quantified the apparent rate constants of degradation (kapp) of each via photorheology at 370 nm, 10 mW/cm2. Decreasing the number of aryl ethers on the o-NB group increases kapp, and changing the functionality from primary to seconday at the benzylic site dramatically increases kapp. Human mesenchymal stem cells (hMSCs) survive encapsulation in the hydrogels (90% viability post-encapsulation). By exploiting the differences in reactivity of two different o-NB linkers, we quantitatively demonstrate the biased release of one stem cell population (green-fluroescent protein expressing hMSCs) over another (red-fluorescent protein expressing hMSCs). PMID:22765384

  12. Live-cell mass profiling: an emerging approach in quantitative biophysics

    PubMed Central

    Zangle, Thomas A; Teitell, Michael A

    2015-01-01

    Cell mass, volume and growth rate are tightly controlled biophysical parameters in cellular development and homeostasis, and pathological cell growth defines cancer in metazoans. The first measurements of cell mass were made in the 1950s, but only recently have advances in computer science and microfabrication spurred the rapid development of precision mass-quantifying approaches. Here we discuss available techniques for quantifying the mass of single live cells with an emphasis on relative features, capabilities and drawbacks for different applications. PMID:25423019

  13. Long-Lived Binding of Sox2 to DNA Predicts Cell Fate in the Four-Cell Mouse Embryo.

    PubMed

    White, Melanie D; Angiolini, Juan F; Alvarez, Yanina D; Kaur, Gurpreet; Zhao, Ziqing W; Mocskos, Esteban; Bruno, Luciana; Bissiere, Stephanie; Levi, Valeria; Plachta, Nicolas

    2016-03-24

    Transcription factor (TF) binding to DNA is fundamental for gene regulation. However, it remains unknown how the dynamics of TF-DNA interactions change during cell-fate determination in vivo. Here, we use photo-activatable FCS to quantify TF-DNA binding in single cells of developing mouse embryos. In blastocysts, the TFs Oct4 and Sox2, which control pluripotency, bind DNA more stably in pluripotent than in extraembryonic cells. By contrast, in the four-cell embryo, Sox2 engages in more long-lived interactions than does Oct4. Sox2 long-lived binding varies between blastomeres and is regulated by H3R26 methylation. Live-cell tracking demonstrates that those blastomeres with more long-lived binding contribute more pluripotent progeny, and reducing H3R26 methylation decreases long-lived binding, Sox2 target expression, and pluripotent cell numbers. Therefore, Sox2-DNA binding predicts mammalian cell fate as early as the four-cell stage. More generally, we reveal the dynamic repartitioning of TFs between DNA sites driven by physiological epigenetic changes. VIDEO ABSTRACT. PMID:27015308

  14. Apoptosis as a mechanism of cytolysis of tumor cells by a pathogenic free-living amoeba.

    PubMed Central

    Alizadeh, H; Pidherney, M S; McCulley, J P; Niederkorn, J Y

    1994-01-01

    Previous studies have shown that trophozoites of the pathogenic free-living amoeba Acanthamoeba castellanii rapidly lysed a variety of tumor cells in vitro. Tumor cells undergoing parasite-mediated lysis displayed characteristic cell membrane blebbing reminiscent of apoptosis. The present investigation examined the role of apoptosis (programmed cell death) in Acanthamoeba-mediated tumor cell lysis. The results showed that more than 70% of tumor cell DNA was fragmented following exposure to Acanthamoeba cell extracts. By contrast, only 7% of untreated control cells underwent DNA fragmentation. DNA fragmentation increased significantly in a dose-dependent fashion following concentration of the parasite extract. Apoptosis was also confirmed by DNA ladder formation. Characteristic DNA ladders, consisting of multimers of approximately 180 to 200 bp, were produced by tumor cells exposed to Acanthamoeba cell extracts. The morphology of tumor cell lysis was examined by light and scanning electron microscopy. Tumor cells exposed to parasite extract displayed morphological features characteristic of apoptosis including cell shrinkage, cell membrane blebbing, formation of apoptotic bodies, and nuclear condensation. By contrast, similar effects were not found in tumor cells exposed to extract similarly prepared from normal mammalian cells (i.e., human keratocytes). The results suggest that at least one species of pathogenic free-living amoeba is able to lyse tumor cells by a process that culminates in apoptosis. Images PMID:8132336

  15. Optical Detection and Virotherapy of Live Metastatic Tumor Cells in Body Fluids with Vaccinia Strains

    PubMed Central

    Minev, Boris R.; Zimmermann, Martina; Aguilar, Richard J.; Zhang, Qian; Sturm, Julia B.; Fend, Falko; Yu, Yong A.; Cappello, Joseph; Lauer, Ulrich M.; Szalay, Aladar A.

    2013-01-01

    Metastatic tumor cells in body fluids are important targets for treatment, and critical surrogate markers for evaluating cancer prognosis and therapeutic response. Here we report, for the first time, that live metastatic tumor cells in blood samples from mice bearing human tumor xenografts and in blood and cerebrospinal fluid samples from patients with cancer were successfully detected using a tumor cell-specific recombinant vaccinia virus (VACV). In contrast to the FDA-approved CellSearch system, VACV detects circulating tumor cells (CTCs) in a cancer biomarker-independent manner, thus, free of any bias related to the use of antibodies, and can be potentially a universal system for detection of live CTCs of any tumor type, not limited to CTCs of epithelial origin. Furthermore, we demonstrate for the first time that VACV was effective in preventing and reducing circulating tumor cells in mice bearing human tumor xenografts. Importantly, a single intra-peritoneal delivery of VACV resulted in a dramatic decline in the number of tumor cells in the ascitic fluid from a patient with gastric cancer. Taken together, these results suggest VACV to be a useful tool for quantitative detection of live tumor cells in liquid biopsies as well as a potentially effective treatment for reducing or eliminating live tumor cells in body fluids of patients with metastatic disease. PMID:24019862

  16. Morphological Measurement of Living Cells in Methanol with Digital Holographic Microscopy

    PubMed Central

    Wang, Yunxin; Yang, Yishu; Wang, Dayong; Ouyang, Liting; Zhang, Yizhuo; Zhao, Jie; Wang, Xinlong

    2013-01-01

    Cell morphology is the research foundation in many applications related to the estimation of cell status, drug response, and toxicity screening. In biomedical field, the quantitative phase detection is an inevitable trend for living cells. In this paper, the morphological change of HeLa cells treated with methanol of different concentrations is detected using digital holographic microscopy. The compact image-plane digital holographic system is designed based on fiber elements. The quantitative phase image of living cells is obtained in combination with numerical analysis. The statistical analysis shows that the area and average optical thickness of HeLa cells treated with 12.5% or 25% methanol reduce significantly, which indicates that the methanol with lower concentration could cause cellular shrinkage. The area of HeLa cells treated with 50% methanol is similar to that of normal cells (P > 0.05), which reveals the fixative effect of methanol with higher concentration. The maximum optical thickness of the cells treated with 12.5%, 25%, and 50% methanol is greater than that of untreated cells, which implies the pyknosis of HeLa cells under the effect of methanol. All of the results demonstrate that digital holographic microscopy has supplied a noninvasive imaging alternative to measure the morphological change of label-free living cells. PMID:23424605

  17. Astronomy Popularization via Sci-fi Movies

    NASA Astrophysics Data System (ADS)

    Li, Qingkang

    2015-08-01

    It is astronomers’ duty to let more and more young people know a bit astronomy and be interested in astronomy and appreciate the beauty and great achievements in astronomy. One of the most effective methods to popularize astronomy to young people nowadays might be via enjoying some brilliant sci-fi movies related to astronomy with some guidance from astronomers. Firstly, we will introduce the basic information of our selective course “Appreciation of Sci-fi Movies in Astronomy” for the non-major astronomy students in our University, which is surely unique in China, then we will show its effect on astronomy popularization based on several rounds of teaching.

  18. Live cell calcium imaging of dissociated vomeronasal neurons.

    PubMed

    Kaur, Angeldeep; Dey, Sandeepa; Stowers, Lisa

    2013-01-01

    Sensory neurons in the vomeronasal organ (VNO) are thought to mediate a specialized olfactory response. Currently, very little is known about the identity of stimulating ligands or their cognate receptors that initiate neural activation. Each sensory neuron is thought to express 1 of approximately 250 variants of Vmn1Rs, Vmn2Rs (A, B, or D), or FPRs which enables it to be tuned to a subset of ligands (Touhara and Vosshall, Annu Rev Physiol 71:307-332, 2009). The logic of how different sources of native odors or purified ligands are detected by this complex sensory repertoire remains mostly unknown. Here, we describe a method to compare and analyze the response of VNO sensory neurons to multiple stimuli using conventional calcium imaging. This method differs from other olfactory imaging approaches in that we dissociate the tightly packed sensory epithelium into individual single cells. The advantages of this approach include (1) the use of a relatively simple approach and inexpensive microscopy, (2) comparative analysis of several hundreds of neurons to multiple stimuli with single-cell resolution, and (3) the possibility of isolating single cells of interest to further analyze by molecular biology techniques including in situ RNA hybridization, immunofluorescence, or creating single-cell cDNA libraries (Malnic et al., Cell 96:713-723, 1999). PMID:24014362

  19. Tensile stress stimulates microtubule outgrowth in living cells

    NASA Technical Reports Server (NTRS)

    Kaverina, Irina; Krylyshkina, Olga; Beningo, Karen; Anderson, Kurt; Wang, Yu-Li; Small, J. Victor

    2002-01-01

    Cell motility is driven by the sum of asymmetric traction forces exerted on the substrate through adhesion foci that interface with the actin cytoskeleton. Establishment of this asymmetry involves microtubules, which exert a destabilising effect on adhesion foci via targeting events. Here, we demonstrate the existence of a mechano-sensing mechanism that signals microtubule polymerisation and guidance of the microtubules towards adhesion sites under increased stress. Stress was applied either by manipulating the body of cells moving on glass with a microneedle or by stretching a flexible substrate that cells were migrating on. We propose a model for this mechano-sensing phenomenon whereby microtubule polymerisation is stimulated and guided through the interaction of a microtubule tip complex with actin filaments under tension.

  20. Ratiometric Fluorescent Polymeric Thermometer for Thermogenesis Investigation in Living Cells.

    PubMed

    Qiao, Juan; Hwang, Yoon-Ho; Chen, Chuan-Fang; Qi, Li; Dong, Ping; Mu, Xiao-Yu; Kim, Dong-Pyo

    2015-10-20

    Intracellular temperature has a fundamental effect on cellular events. Herein, a novel fluorescent polymer ratiometric nanothermometer has been developed based on transferrin protein-stabilized gold nanoclusters as the targeting and fluorescent ratiometric unit and the thermosensitve polymer as the temperature sensing unit. The resultant nanothermometer could feature a high and spontaneous uptake into the HeLa cells and the ratiometric temperature sensing over the physiological temperature range. Moreover, the precise temperature sensing for intracellular heat generation in HeLa cells following calcium ions stress has been achieved. This practical intracellular thermometry could eliminate the interference of the intracellular surrounding environment in cancer cells without a microinjection procedure, which is user-friendly. The prepared new nanothermometer can provide tools for unveiling the intrinsic relationship between the intracellular temperature and ion channel function. PMID:26393404

  1. Live Cell in Vitro and in Vivo Imaging Applications: Accelerating Drug Discovery

    PubMed Central

    Isherwood, Beverley; Timpson, Paul; McGhee, Ewan J; Anderson, Kurt I; Canel, Marta; Serrels, Alan; Brunton, Valerie G; Carragher, Neil O

    2011-01-01

    Dynamic regulation of specific molecular processes and cellular phenotypes in live cell systems reveal unique insights into cell fate and drug pharmacology that are not gained from traditional fixed endpoint assays. Recent advances in microscopic imaging platform technology combined with the development of novel optical biosensors and sophisticated image analysis solutions have increased the scope of live cell imaging applications in drug discovery. We highlight recent literature examples where live cell imaging has uncovered novel insight into biological mechanism or drug mode-of-action. We survey distinct types of optical biosensors and associated analytical methods for monitoring molecular dynamics, in vitro and in vivo. We describe the recent expansion of live cell imaging into automated target validation and drug screening activities through the development of dedicated brightfield and fluorescence kinetic imaging platforms. We provide specific examples of how temporal profiling of phenotypic response signatures using such kinetic imaging platforms can increase the value of in vitro high-content screening. Finally, we offer a prospective view of how further application and development of live cell imaging technology and reagents can accelerate preclinical lead optimization cycles and enhance the in vitro to in vivo translation of drug candidates. PMID:24310493

  2. Unmasking Chaotic Attributes in Time Series of Living Cell Populations

    PubMed Central

    Laurent, Michel; Deschatrette, Jean; Wolfrom, Claire M.

    2010-01-01

    Background Long-range oscillations of the mammalian cell proliferation rate are commonly observed both in vivo and in vitro. Such complicated dynamics are generally the result of a combination of stochastic events and deterministic regulation. Assessing the role, if any, of chaotic regulation is difficult. However, unmasking chaotic dynamics is essential for analysis of cellular processes related to proliferation rate, including metabolic activity, telomere homeostasis, gene expression, and tumor growth. Methodology/Principal Findings Using a simple, original, nonlinear method based on return maps, we previously found a geometrical deterministic structure coordinating such fluctuations in populations of various cell types. However, nonlinearity and determinism are only necessary conditions for chaos; they do not by themselves constitute a proof of chaotic dynamics. Therefore, we used the same analytical method to analyze the oscillations of four well-known, low-dimensional, chaotic oscillators, originally designed in diverse settings and all possibly well-adapted to model the fluctuations of cell populations: the Lorenz, Rössler, Verhulst and Duffing oscillators. All four systems also display this geometrical structure, coordinating the oscillations of one or two variables of the oscillator. No such structure could be observed in periodic or stochastic fluctuations. Conclusion/Significance Theoretical models predict various cell population dynamics, from stable through periodically oscillating to a chaotic regime. Periodic and stochastic fluctuations were first described long ago in various mammalian cells, but by contrast, chaotic regulation had not previously been evidenced. The findings with our nonlinear geometrical approach are entirely consistent with the notion that fluctuations of cell populations can be chaotically controlled. PMID:20179755

  3. Engineering Synthetic Gene Circuits in Living Cells with CRISPR Technology.

    PubMed

    Jusiak, Barbara; Cleto, Sara; Perez-Piñera, Pablo; Lu, Timothy K

    2016-07-01

    One of the goals of synthetic biology is to build regulatory circuits that control cell behavior, for both basic research purposes and biomedical applications. The ability to build transcriptional regulatory devices depends on the availability of programmable, sequence-specific, and effective synthetic transcription factors (TFs). The prokaryotic clustered regularly interspaced short palindromic repeat (CRISPR) system, recently harnessed for transcriptional regulation in various heterologous host cells, offers unprecedented ease in designing synthetic TFs. We review how CRISPR can be used to build synthetic gene circuits and discuss recent advances in CRISPR-mediated gene regulation that offer the potential to build increasingly complex, programmable, and efficient gene circuits in the future. PMID:26809780

  4. Migration of connexin in the membranes of living cells

    NASA Astrophysics Data System (ADS)

    Bledsoe, Matthew; Rana, Daharsh; May, Karl; Kreft, Jennifer

    2008-11-01

    Movement of connexins within cell lipid bilayers remains somewhat mysterious. In studying their movement, researchers hoped to shed more light on the mechanisms by which they are influenced. We examined this problem by observing the behavior of the connexins directly. Cancerous human liver cells were cultured and their membrane connexins labeled with green fluorescent protein through transvection. The connexins were then filmed by high speed camera and carefully analyzed. The study served to fine-tune the model used in simulations of connexin migration, enabling further study of connexins and their transmembrane environment.

  5. Bewitched, Bothered, and Bored: Harry Potter, The Movie.

    ERIC Educational Resources Information Center

    Nel, Philip

    2002-01-01

    Explores the Harry Potter phenomenon with college students in a university course. Compares the first book with the first movie. Presents an in-depth discussion of the movie and how it relates to the book. (SG)

  6. Smoking in top-grossing US movies, 2011.

    PubMed

    Glantz, Stanton A; Iaccopucci, Anne; Titus, Kori; Polansky, Jonathan R

    2012-01-01

    We reviewed the number of incidents of tobacco use (almost exclusively smoking) depicted in movies in the United States in 2011 to compare that with previously reported trends. We counted use or implied use of a tobacco product by an actor in all movies whose box office gross ranked in the top 10 for at least 1 week. Total tobacco incidents per movie rose 7% from 2010 to 2011, ending 5 years of decline; incidents rose 34% per movie rated G, PG, or PG-13 and 7% per R-rated movie. The reversal of progress toward less onscreen smoking in youth-rated movies underscores the need to rate movies with tobacco imagery as R, establishing an industry-wide market incentive to keep youth-marketed movies tobacco-free. PMID:23017248

  7. Smoking in Top-Grossing US Movies, 2011

    PubMed Central

    Iaccopucci, Anne; Titus, Kori; Polansky, Jonathan R.

    2012-01-01

    We reviewed the number of incidents of tobacco use (almost exclusively smoking) depicted in movies in the United States in 2011 to compare that with previously reported trends. We counted use or implied use of a tobacco product by an actor in all movies whose box office gross ranked in the top 10 for at least 1 week. Total tobacco incidents per movie rose 7% from 2010 to 2011, ending 5 years of decline; incidents rose 34% per movie rated G, PG, or PG-13 and 7% per R-rated movie. The reversal of progress toward less onscreen smoking in youth-rated movies underscores the need to rate movies with tobacco imagery as R, establishing an industry-wide market incentive to keep youth-marketed movies tobacco-free. PMID:23017248

  8. Alkyne-tag Raman imaging of bio-active small molecules in live cells

    NASA Astrophysics Data System (ADS)

    Ando, Jun; Palonpon, Almar F.; Yamakoshi, Hiroyuki; Dodo, Kosuke; Kawata, Satoshi; Sodeoka, Mikiko; Fujita, Katsumasa

    2015-12-01

    Raman microscopy is useful for molecular imaging and analysis of biological specimens. Here, we used alkyne containing a carbon-carbon triple bond as a Raman tag for observing small molecules in live cells. Alkyne tags can maintain original properties of target molecules with providing high chemical specificity owing to its distinct peak in a Raman-silent window of biomolecules. For demonstrations, alkyne-tagged thymidine and coenzyme Q analogue in live cells were visualized with high-spatial resolution. We extended the application of alkyne-tag imaging to visualize cell organelles and specific lipid components in artificial monolayer membranes.

  9. 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).

  10. Biophysical techniques for detection of cAMP and cGMP in living cells.

    PubMed

    Sprenger, Julia U; Nikolaev, Viacheslav O

    2013-01-01

    Cyclic nucleotides cAMP and cGMP are ubiquitous second messengers which regulate myriads of functions in virtually all eukaryotic cells. Their intracellular effects are often mediated via discrete subcellular signaling microdomains. In this review, we will discuss state-of-the-art techniques to measure cAMP and cGMP in biological samples with a particular focus on live cell imaging approaches, which allow their detection with high temporal and spatial resolution in living cells and tissues. Finally, we will describe how these techniques can be applied to the analysis of second messenger dynamics in subcellular signaling microdomains. PMID:23584022

  11. Live cell imaging based on surface plasmon-enhanced fluorescence microscopy using random nanostructures

    NASA Astrophysics Data System (ADS)

    Oh, Youngjin; Lee, Wonju; Son, Taehwang; Kim, Sook Young; Shin, Jeon-Soo; Kim, Donghyun

    2014-02-01

    Localized surface plasmon enhanced microscopy based on nanoislands of random spatial distribution was demonstrated for imaging live cells and molecular interactions. Nanoislands were produced without lithography by high temperature annealing under various processing conditions. The localization of near-field distribution that is associated with localized surface plasmon on metallic random nanoislands was analyzed theoretically and experimentally in comparison with periodic nanostructures. For experimental validation in live cell imaging, mouse macrophage-like cell line stained with Alexa Fluor 488 was prepared on nanoislands. The results suggest the possibility of attaining the imaging resolution on the order of 80 nm.

  12. Supramolecular metal displacement allows on-fluorescence analysis of manganese(II) in living cells.

    PubMed

    Gruppi, Francesca; Liang, Jian; Bartelle, Benjamin B; Royzen, Maksim; Turnbull, Daniel H; Canary, James W

    2012-11-11

    Due to the importance of Mn(2+) ions in biological processes, it is of growing interest to develop protocols for analysis of Mn(2+) uptake and distribution in cells. A supramolecular metal displacement assay can provide ratiometric fluorescence detection of Mn(2+), allowing for quantitative and longitudinal analysis of Mn(2+) uptake in living cells. PMID:23023093

  13. Use of cell cultures as an indicator of pathogenicity of free-living amoebae.

    PubMed Central

    Cursons, R T; Brown, T J

    1978-01-01

    Results comparing the time needed for the development of cytopathic effects in cell cultures with that needed to cause death in mice using inocula of Naegleria and Acanthamoeba are presented. The significance of the source and concentration of the inocula is demonstrated. The use of cell cultures as an indicator of the pathogenicity of free-living amoebae is discussed. Images PMID:342543

  14. Method of freezing living cells and tissues with improved subsequent survival

    DOEpatents

    Senkan, Selim M.; Hirsch, Gerald P.

    1980-01-01

    This invention relates to an improved method for freezing red blood cells, ther living cells, or tissues with improved subsequent survival, wherein constant-volume freezing is utilized that results in significantly improved survival compared with constant-pressure freezing; optimization is attainable through the use of different vessel geometries, cooling baths and warming baths, and sample concentrations.

  15. Localized electroporation and molecular delivery into single living cells by atomic force microscopy

    NASA Astrophysics Data System (ADS)

    Nawarathna, D.; Unal, K.; Wickramasinghe, H. Kumar

    2008-10-01

    We present an efficient and fast method for selective and localized electroporation of a single living cell from a population of millions to tens of cells using the modified tip of an atomic force microscope. Electroporation was observed in real time using an inverted microscope. This technique is proposed as a tool for efficient and controlled delivery of biomolecules, proteins, drugs, and genes.

  16. Probing protein complexes inside living cells using a silicon nanowire-based pull-down assay

    NASA Astrophysics Data System (ADS)

    Choi, Sojoong; Kim, Hyunju; Kim, So Yeon; Yang, Eun Gyeong

    2016-06-01

    Most proteins perform their functions as interacting complexes. Here we propose a novel method for capturing an intracellular protein and its interacting partner out of living cells by utilizing intracellular access of antibody modified vertical silicon nanowire arrays whose surface is covered with a polyethylene glycol layer to prevent strong cell adhesion. Such a feature facilitates the removal of cells by simple washing, enabling subsequent detection of a pulled-down protein and its interacting partner, and further assessment of a drug-induced change in the interacting complex. Our new SiNW-based tool is thus suitable for authentication of protein networks inside living cells.Most proteins perform their functions as interacting complexes. Here we propose a novel method for capturing an intracellular protein and its interacting partner out of living cells by utilizing intracellular access of antibody modified vertical silicon nanowire arrays whose surface is covered with a polyethylene glycol layer to prevent strong cell adhesion. Such a feature facilitates the removal of cells by simple washing, enabling subsequent detection of a pulled-down protein and its interacting partner, and further assessment of a drug-induced change in the interacting complex. Our new SiNW-based tool is thus suitable for authentication of protein networks inside living cells. Electronic supplementary information (ESI) available: Materials, experimental methods and Fig. S1-S8. See DOI: 10.1039/c6nr00171h

  17. Understanding of Protein Synthesis in a Living Cell

    ERIC Educational Resources Information Center

    Mustapha, Y.; Muhammad, S.

    2006-01-01

    The assembly of proteins takes place in the cytoplasm of a cell. There are three main steps. In initiation, far left, all the necessary parts of the process are brought together by a small molecule called a ribosome. During elongation, amino acids, the building blocks of proteins, are joined to one another in a long chain. The sequence in which…

  18. Visualizing how cancer chromosome abnormalities form in living cells

    Cancer.gov

    For the first time, scientists have directly observed events that lead to the formation of a chromosome abnormality that is often found in cancer cells. The abnormality, called a translocation, occurs when part of a chromosome breaks off and becomes attac

  19. Directly lighting up RNA G-quadruplexes from test tubes to living human cells

    PubMed Central

    Xu, Shujuan; Li, Qian; Xiang, Junfeng; Yang, Qianfan; Sun, Hongxia; Guan, Aijiao; Wang, Lixia; Liu, Yan; Yu, Lijia; Shi, Yunhua; Chen, Hongbo; Tang, Yalin

    2015-01-01

    RNA G-quadruplexes (G4s) are one of the key components of the transcriptome that act as efficient post-transcriptional regulatory elements in living cells. To conduct further studies of the unique biological functions of RNA G4s, techniques need to be developed that can efficiently recognize RNA G4 structures under various conditions, in fixed cells and living cells, as well as in vitro. This paper presents the development of such a method, a new technique using a cyanine dye called CyT, which can detect both canonical and non-canonical RNA G4 structures from test tubes to living human cells. The ability of CyT to distinguish between G4 and nonG4 RNA offers a promising tool for future RNA G4-based biomarker discovery and potential diagnostic applications. PMID:26476445

  20. 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

  1. Aptamer-mediated indirect quantum dot labeling and fluorescent imaging of target proteins in living cells

    NASA Astrophysics Data System (ADS)

    Liu, Jianbo; Zhang, Pengfei; Yang, Xiaohai; Wang, Kemin; Guo, Qiuping; Huang, Jin; Li, Wei

    2014-12-01

    Protein labeling for dynamic living cell imaging plays a significant role in basic biological research, as well as in clinical diagnostics and therapeutics. We have developed a novel strategy in which the dynamic visualization of proteins within living cells is achieved by using aptamers as mediators for indirect protein labeling of quantum dots (QDs). With this strategy, the target protein angiogenin was successfully labeled with fluorescent QDs in a minor intactness model, which was mediated by the aptamer AL6-B. Subsequent living cell imaging analyses indicated that the QDs nanoprobes were selectively bound to human umbilical vein endothelial cells, gradually internalized into the cytoplasm, and mostly localized in the lysosome organelle, indicating that the labeled protein retained high activity. Compared with traditional direct protein labeling methods, the proposed aptamer-mediated strategy is simple, inexpensive, and provides a highly selective, stable, and intact labeling platform that has shown great promise for future biomedical labeling and intracellular protein dynamic analyses.

  2. Internal dynamics of a living cell nucleus investigated by dynamic light scattering

    NASA Astrophysics Data System (ADS)

    Suissa, M.; Place, C.; Goillot, E.; Freyssingeas, E.

    2008-08-01

    Recent progresses in cellular biology have shown that the nucleus of a living cell is a structured integration of many functional domains with a complex spatial organization. This organization, as well as molecular and biochemical processes, is time regulated. In the past years many investigations have been performed using fluorescent microscopy techniques to study the internal dynamics of the nucleus of a living cell. These investigations, however, have never focussed on the global internal dynamics of the nucleus, which is still unknown. In this article we present an original light scattering experimental device that we built to investigate this dynamics during biological processes. By means of this experimental set-up, we investigated the global dynamics of the nucleus of a living cell treated with a DNA replication inhibitor. This dynamics presents different and independent kinds of relaxation well separated in time that vary as a function of the cell cycle phases.

  3. Functional Reconstitution of the Insulin-Secreting Porosome Complex in Live Cells.

    PubMed

    Naik, Akshata R; Kulkarni, Sanjana P; Lewis, Kenneth T; Taatjes, Douglas J; Jena, Bhanu P

    2016-01-01

    Supramolecular cup-shaped lipoprotein structures called porosomes embedded in the cell plasma membrane mediate fractional release of intravesicular contents from cells during secretion. The presence of porosomes, have been documented in many cell types including neurons, acinar cells of the exocrine pancreas, GH-secreting cells of the pituitary, and insulin-secreting pancreatic β-cells. Functional reconstitution of porosomes into artificial lipid membranes, have also been accomplished. Earlier studies on mouse insulin-secreting Min6 cells report 100-nm porosome complexes composed of nearly 30 proteins. In the current study, porosomes have been functionally reconstituted for the first time in live cells. Isolated Min6 porosomes reconstituted into live Min6 cells demonstrate augmented levels of porosome proteins and a consequent increase in the potency and efficacy of glucose-stimulated insulin release. Elevated glucose-stimulated insulin secretion 48 hours after reconstitution, reflects on the remarkable stability and viability of reconstituted porosomes, documenting the functional reconstitution of native porosomes in live cells. These results, establish a new paradigm in porosome-mediated insulin secretion in β-cells. PMID:26523491

  4. Global antibody response to Staphylococcus aureus live-cell vaccination.

    PubMed

    Selle, Martina; Hertlein, Tobias; Oesterreich, Babett; Klemm, Theresa; Kloppot, Peggy; Müller, Elke; Ehricht, Ralf; Stentzel, Sebastian; Bröker, Barbara M; Engelmann, Susanne; Ohlsen, Knut

    2016-01-01

    The pathogen Staphylococcus aureus causes a broad range of severe diseases and is feared for its ability to rapidly develop resistance to antibiotic substances. The increasing number of highly resistant S. aureus infections has accelerated the search for alternative treatment options to close the widening gap in anti-S. aureus therapy. This study analyses the humoral immune response to vaccination of Balb/c mice with sublethal doses of live S. aureus. The elicited antibody pattern in the sera of intravenously and intramuscularly vaccinated mice was determined using of a recently developed protein array. We observed a specific antibody response against a broad set of S. aureus antigens which was stronger following i.v. than i.m. vaccination. Intravenous but not intramuscular vaccination protected mice against an intramuscular challenge infection with a high bacterial dose. Vaccine protection was correlated with the strength of the anti-S. aureus antibody response. This study identified novel vaccine candidates by using protein microarrays as an effective tool and showed that successful vaccination against S. aureus relies on the optimal route of administration. PMID:27103319

  5. Global antibody response to Staphylococcus aureus live-cell vaccination

    PubMed Central

    Selle, Martina; Hertlein, Tobias; Oesterreich, Babett; Klemm, Theresa; Kloppot, Peggy; Müller, Elke; Ehricht, Ralf; Stentzel, Sebastian; Bröker, Barbara M.; Engelmann, Susanne; Ohlsen, Knut

    2016-01-01

    The pathogen Staphylococcus aureus causes a broad range of severe diseases and is feared for its ability to rapidly develop resistance to antibiotic substances. The increasing number of highly resistant S. aureus infections has accelerated the search for alternative treatment options to close the widening gap in anti-S. aureus therapy. This study analyses the humoral immune response to vaccination of Balb/c mice with sublethal doses of live S. aureus. The elicited antibody pattern in the sera of intravenously and intramuscularly vaccinated mice was determined using of a recently developed protein array. We observed a specific antibody response against a broad set of S. aureus antigens which was stronger following i.v. than i.m. vaccination. Intravenous but not intramuscular vaccination protected mice against an intramuscular challenge infection with a high bacterial dose. Vaccine protection was correlated with the strength of the anti-S. aureus antibody response. This study identified novel vaccine candidates by using protein microarrays as an effective tool and showed that successful vaccination against S. aureus relies on the optimal route of administration. PMID:27103319

  6. Time- and polarization-resolved cellular autofluorescence towards quantitative biochemistry on living cells

    NASA Astrophysics Data System (ADS)

    Alfveby, John; TImerman, Randi; Soto Velasquez, Monica P.; Wickramasinghe, Dhanushka W. P. M.; Bartusek, Jillian; Heikal, Ahmed A.

    2014-09-01

    Native coenzymes such as the reduced nicotinamide adenine dinucleotide (NADH) and oxidized flavin adenine dinucleotide play pivotal roles in energy metabolism and a myriad of biochemical reactions in living cells/tissues. These coenzymes are naturally fluorescent and, therefore, have the potential to serve as intrinsic biomarkers for mitochondrial activities, programmed cell death (apoptosis), oxidative stress, aging, and neurodegenerative disease. In this contribution, we employ two-photon fluorescence lifetime imaging microscopy (FLIM) and time-resolved anisotropy imaging of intracellular NADH for quantitative, non-invasive biochemistry on living cells in response to hydrogenperoxide- induced oxidative stress. In contrast with steady-state one-photon, UV-excited autofluorescence, two-photon FLIM is sensitive to both molecular conformation and stimuli-induced changes in the local environment in living cells with minimum photodamage and inherently enhanced spatial resolution. On the other hand, time-resolved, two-photon anisotropy imaging of cellular autofluorescence allows for quantitative assessment of binding state and environmental restrictions on the tumbling mobility of intrinsic NADH. Our measurements reveal that free and enzyme-bound NADH exist at equilibrium, with a dominant autofluorescence contribution of the bound fraction in living cells. Parallel studies on NADH-enzyme binding in controlled environments serve as a point of reference in analyzing autofluorescence in living cells. These autofluorescence-based approaches complement the conventional analytical biochemistry methods that require the destruction of cells/tissues, while serving as an important step towards establishing intracellular NADH as a natural biomarker for monitoring changes in energy metabolism and redox state of living cells in response to environmental hazards.

  7. Evolutionary Forces Shaping the Golgi Glycosylation Machinery: Why Cell Surface Glycans Are Universal to Living Cells

    PubMed Central

    Varki, Ajit

    2011-01-01

    Despite more than 3 billion years since the origin of life on earth, the powerful forces of biological evolution seem to have failed to generate any living cell that is devoid of a dense and complex array of cell surface glycans. Thus, cell surface glycans seem to be as essential for life as having a DNA genetic code, diverse RNAs, structural/functional proteins, lipid-based membranes, and metabolites that mediate energy flux and signaling. The likely reasons for this apparently universal law of biology are considered here, and include the fact that glycans have the greatest potential for generating diversity, and thus evading recognition by pathogens. This may also explain why in striking contrast to the genetic code, glycans show widely divergent patterns between taxa. On the other hand, glycans have also been coopted for myriad intrinsic functions, which can vary in their importance for organismal survival. In keeping with these considerations, a significant percentage of the genes in the typical genome are dedicated to the generation and/or turnover of glycans. Among eukaryotes, the Golgi is the subcellular organelle that serves to generate much of the diversity of cell surface glycans, carrying out various glycan modifications of glycoconjugates that transit through the Golgi, en route to the cell surface or extracellular destinations. Here I present an overview of general considerations regarding the selective forces shaping evolution of the Golgi glycosylation machinery, and then briefly discuss the common types of variations seen in each major class of glycans, finally focusing on sialic acids as an extreme example of evolutionary glycan diversity generated by the Golgi. Future studies need to address both the phylogenetic diversity the Golgi and the molecular mechanisms for its rapid responses to intrinsic and environmental stimuli. PMID:21525513

  8. 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

  9. Magnetically driven spinning nanowires as effective materials for eradicating living cells

    NASA Astrophysics Data System (ADS)

    Choi, Daniel S.; Hopkins, Xiaoping; Kringel, Rosemarie; Park, Jungrae; Jeon, In Tak; Keun Kim, Young

    2012-04-01

    We present a method to inflame cells, in vitro, by applying an alternating current (ac) magnetic field to ferromagnetic nanowires (NWs) internalized by living cells. Nickel (Ni) NWs were internalized by human embryonic kidney cells (HEK-293). The application of ac magnetic field to the cells induced spinning of the cells via the motion of internalized NWs. This resulted in cell death by physically causing damage. A study of the response of cytokine to cells with spinning NWs shows increased interleukin-6 effects when compared with responses from non-spinning cells. The spinning effect of cells caused by the application of magnetic field can be used to target and inflame the cells. Such experiments suggest the possibility of inflaming cells for the treatment of cancer.

  10. Photobleaching Methods to Study Golgi Complex Dynamics in Living Cells

    PubMed Central

    Snapp, Erik Lee

    2014-01-01

    The Golgi complex (GC) is a highly dynamic organelle that constantly receives and exports proteins and lipids from both the endoplasmic reticulum and the plasma membrane. While protein trafficking can be monitored with traditional biochemical methods, these approaches average the behaviors of millions of cells, provide modest temporal information and no spatial information. Photobleaching methods enable investigators to monitor protein trafficking in single cells or even single GC stacks with subsecond precision. Furthermore, photobleaching can be exploited to monitor the behaviors of resident GC proteins to provide insight into mechanisms of retention and trafficking. In this chapter, general photobleaching approaches with laser scanning confocal microscopes are described. Importantly, the problems associated with many fluorescent proteins (FPs) and their uses in the secretory pathway are discussed and appropriate choices are suggested. For example, Enhanced Green Fluorescent Protein (EGFP) and most red FPs are extremely problematic. Finally, options for data analyses are described. PMID:24295308

  11. Speciation of uranium in compartments of living cells.

    PubMed

    Geipel, Gerhard; Viehweger, Katrin

    2015-06-01

    Depleted uranium used as ammunition corrodes in the environment forming mineral phases and then dissolved uranium species like uranium carbonates (Schimmack et al., in Radiat Environ Biophys 46:221-227, 2007) and hydroxides. These hydroxide species were contacted with plant cells (canola). After 24 h contact time the cells were fractionated and the uranium speciation in the fraction was determined by time resolved laser-induced fluorescence spectroscopy at room temperature as well at 150 K. It could be shown that the uranium speciation in the fractions is different to that in the nutrient solution. Comparison of the emission bands with literature data allows assignment of the uranium binding forms. PMID:25724950

  12. Mechanics of Individual Keratin Bundles in Living Cells

    PubMed Central

    Nolting, Jens-Friedrich; Möbius, Wiebke; Köster, Sarah

    2014-01-01

    Along with microtubules and microfilaments, intermediate filaments are a major component of the eukaryotic cytoskeleton and play a key role in cell mechanics. In cells, keratin intermediate filaments form networks of bundles that are sparser in structure and have lower connectivity than, for example, actin networks. Because of this, bending and buckling play an important role in these networks. Buckling events, which occur due to compressive intracellular forces and cross-talk between the keratin network and other cytoskeletal components, are measured here in situ. By applying a mechanical model for the bundled filaments, we can access the mechanical properties of both the keratin bundles themselves and the surrounding cytosol. Bundling is characterized by a coupling parameter that describes the strength of the linkage between the individual filaments within a bundle. Our findings suggest that coupling between the filaments is mostly complete, although it becomes weaker for thicker bundles, with some relative movement allowed. PMID:25468348

  13. Mapping diffusion in a living cell via the phasor approach.

    PubMed

    Ranjit, Suman; Lanzano, Luca; Gratton, Enrico

    2014-12-16

    Diffusion of a fluorescent protein within a cell has been measured using either fluctuation-based techniques (fluorescence correlation spectroscopy (FCS) or raster-scan image correlation spectroscopy) or particle tracking. However, none of these methods enables us to measure the diffusion of the fluorescent particle at each pixel of the image. Measurement using conventional single-point FCS at every individual pixel results in continuous long exposure of the cell to the laser and eventual bleaching of the sample. To overcome this limitation, we have developed what we believe to be a new method of scanning with simultaneous construction of a fluorescent image of the cell. In this believed new method of modified raster scanning, as it acquires the image, the laser scans each individual line multiple times before moving to the next line. This continues until the entire area is scanned. This is different from the original raster-scan image correlation spectroscopy approach, where data are acquired by scanning each frame once and then scanning the image multiple times. The total time of data acquisition needed for this method is much shorter than the time required for traditional FCS analysis at each pixel. However, at a single pixel, the acquired intensity time sequence is short; requiring nonconventional analysis of the correlation function to extract information about the diffusion. These correlation data have been analyzed using the phasor approach, a fit-free method that was originally developed for analysis of FLIM images. Analysis using this method results in an estimation of the average diffusion coefficient of the fluorescent species at each pixel of an image, and thus, a detailed diffusion map of the cell can be created. PMID:25517145

  14. Functional DNA Nanomaterials for Sensing and Imaging in Living Cells

    PubMed Central

    Torabi, Seyed-Fakhreddin; Lu, Yi

    2014-01-01

    Recent developments in integrating high selectivity of functional DNA, such as DNAzyme and aptamers, with efficient DNA delivery into cells by gold nanoparticles or superior near-infrared optical properties of upconversion nanoparticles are reviewed. Their applications in sensing and imaging small organic metabolites, toxins, metal ions, pH, DNA, RNA, proteins, and pathogens are summarized. The advantages and future directions of these functional DNA materials are discussed. PMID:24468446

  15. Real-time imaging and tracking of ultrastable organic dye nanoparticles in living cells.

    PubMed

    Xu, Ruirui; Huang, Liming; Wei, Weijia; Chen, Xianfeng; Zhang, Xiaohong; Zhang, Xiujuan

    2016-07-01

    Semiconductor quantum dots and upconversion nanoparticles have been broadly used for live cell imaging due to their color tunability and photostability etc. However, these inorganic materials often contain heavy metals and potentially have metabolism problems. To overcome these issues, herein, we report a type of organic dye nanoparticles (NPs) with coating of a thin silica layer and folic acid targeting molecules on the surface for live cell imaging. These organic NPs possess superior characteristics of high fluorescence intensity, large Stokes shift, good photostability, emission in the NIR range, and targeted delivery, enabling them to be a powerful fluorescent probe for living cell imaging. In our study, we successfully demonstrate their applications in investigating cell division, exploring the cellular uptake kinetics and pathway of NPs, observing the distribution of NPs, and live-time tracking the trajectory of specific NPs. Considering the excellent properties and unique clathrin- and caveollae-independent intracellular uptake pathway, we expect that this type of organic dye NPs will play an important role in live cell imaging. PMID:27064960

  16. Quantitative Live Imaging of Endogenous DNA Replication in Mammalian Cells

    PubMed Central

    Burgess, Andrew; Lorca, Thierry; Castro, Anna

    2012-01-01

    Historically, the analysis of DNA replication in mammalian tissue culture cells has been limited to static time points, and the use of nucleoside analogues to pulse-label replicating DNA. Here we characterize for the first time a novel Chromobody cell line that specifically labels endogenous PCNA. By combining this with high-resolution confocal time-lapse microscopy, and with a simplified analysis workflow, we were able to produce highly detailed, reproducible, quantitative 4D data on endogenous DNA replication. The increased resolution allowed accurate classification and segregation of S phase into early-, mid-, and late-stages based on the unique subcellular localization of endogenous PCNA. Surprisingly, this localization was slightly but significantly different from previous studies, which utilized over-expressed GFP tagged forms of PCNA. Finally, low dose exposure to Hydroxyurea caused the loss of mid- and late-S phase localization patterns of endogenous PCNA, despite cells eventually completing S phase. Taken together, these results indicate that this simplified method can be used to accurately identify and quantify DNA replication under multiple and various experimental conditions. PMID:23029203

  17. Quantitative Probing of Cu(2+) Ions Naturally Present in Single Living Cells.

    PubMed

    Lee, Junho; Lee, Hwa-Rim; Pyo, Jaeyeon; Jung, Youngseob; Seo, Ji-Young; Ryu, Hye Guk; Kim, Kyong-Tai; Je, Jung Ho

    2016-06-01

    Quantitative probing of Cu(2+) ions naturally present in single living cells is realized by developing a quantum-dot-embedded nanowire-waveguide probe. The intracellular Cu(2+) ion concentration is quantified by direct monitoring of photoluminescence quenching during the insertion of the nanowire in a living neuron. The measured intracellular Cu(2+) ion concentration is 3.34 ± 1.04 × 10(-6) m (mean ± s.e.m.) in single hippocampal neurons. PMID:27027298

  18. Put Some Movie Wow! in Your Chemistry Teaching

    ERIC Educational Resources Information Center

    Frey, Christopher A.; Mikasen, Marjorie L.; Griep, Mark A.

    2012-01-01

    Movies and movie clips have been used by many instructors to teach chemistry. Entire movies based on true chemical stories are used because they provide students with a common experience after which instructors can launch writing lessons about the chemistry, the scientists, or engineers, or even postscripts to the story presented in the film. In…

  19. Physics in Movies: Awareness Levels of Teacher Candidates

    ERIC Educational Resources Information Center

    Kizilcik, Hasan Sahin; Damli, Volkan; Unsal, Yasin

    2014-01-01

    The aim of this study is to draw attention to the informal education aspect of the movies shown and to determine the awareness levels about physics in movies of a small group composed of university students. That is an evaluation had been made among the films dealing explicitly with the basic content of physics, except for science fiction movies,…

  20. Multivariate analysis of Raman spectra for in vitro non-invasive studies of living cells

    NASA Astrophysics Data System (ADS)

    Notingher, Ioan; Jell, Gavin; Notingher, Petronela L.; Bisson, Isabelle; Tsigkou, Olga; Polak, Julia M.; Stevens, Molly M.; Hench, Larry L.

    2005-06-01

    Understanding the biochemical and biophysical properties of live cells is fundamental for unravelling the secrets of many diseases and developing new therapies. Raman micro-spectroscopy is a powerful non-invasive technique that allows in vitro studies of individual living cells or groups of cells without the use of any labels or contrast enhancing chemicals. We describe the use of various multivariate statistical methods, such as Principal Component Analysis (PCA), Linear Discriminant Analysis (LDA) and Classical Least Square (CLS) fitting, to extract biochemical information related to various cellular events. Such methods are required because of the high complexity of the Raman spectra obtained from living cells. PCA and LDA are used to discriminate between healthy and tumor cells. A leave-one-out cross-validation method indicated high prediction accuracy (95%) in identification of tumorogenic bone cells. The CLS fitting method using commercially available biopolymers makes it possible to monitor biochemical changes during the differentiation of embryonic stem cells and foetal bone cells. The results suggest that in both cases differentiated cells are characterised by lower concentrations of RNA compared to undifferentiated cells. These studies suggest that Raman micro-spectroscopy could become an invaluable tool for in vitro cellular biochemistry studies.

  1. Ultrafast nanolaser device for detecting cancer in a single live cell.

    SciTech Connect

    Gourley, Paul Lee; McDonald, Anthony Eugene

    2007-11-01

    Emerging BioMicroNanotechnologies have the potential to provide accurate, realtime, high throughput screening of live tumor cells without invasive chemical reagents when coupled with ultrafast laser methods. These optically based methods are critical to advancing early detection, diagnosis, and treatment of disease. The first year goals of this project are to develop a laser-based imaging system integrated with an in- vitro, live-cell, micro-culture to study mammalian cells under controlled conditions. In the second year, the system will be used to elucidate the morphology and distribution of mitochondria in the normal cell respiration state and in the disease state for normal and disease states of the cell. In this work we designed and built an in-vitro, live-cell culture microsystem to study mammalian cells under controlled conditions of pH, temp, CO2, Ox, humidity, on engineered material surfaces. We demonstrated viability of cell culture in the microsystem by showing that cells retain healthy growth rates, exhibit normal morphology, and grow to confluence without blebbing or other adverse influences of the material surfaces. We also demonstrated the feasibility of integrating the culture microsystem with laser-imaging and performed nanolaser flow spectrocytometry to carry out analysis of the cells isolated mitochondria.

  2. Probing mechanical properties of living cells by atomic force microscopy with blunted pyramidal cantilever tips.

    PubMed

    Rico, Félix; Roca-Cusachs, Pere; Gavara, Núria; Farré, Ramon; Rotger, Mar; Navajas, Daniel

    2005-08-01

    Atomic force microscopy (AFM) allows the acquisition of high-resolution images and the measurement of mechanical properties of living cells under physiological conditions. AFM cantilevers with blunted pyramidal tips are commonly used to obtain images of living cells. Measurement of mechanical properties with these tips requires a contact model that takes into account their blunted geometry. The aim of this work was to develop a contact model of a blunted pyramidal tip and to assess the suitability of pyramidal tips for probing mechanical properties of soft gels and living cells. We developed a contact model of a blunted pyramidal tip indenting an elastic half-space. We measured Young's modulus (E) and the complex shear modulus (G*= G' +i G" ) of agarose gels and A549 alveolar epithelial cells with pyramidal tips and compared them with those obtained with spherical tips. The gels exhibited an elastic behavior with almost coincident loading and unloading force curves and negligible values of G". E fell sharply with indentation up to approximately 300 nm , showing a linear regime for deeper indentations. A similar indentation dependence of E with twofold lower values at the linear regime was obtained with the spherical tip fitted with Hertz's model. The dependence of E on indentation in cells paralleled that found in gels. Cells exhibited viscoelastic behavior with G"/G' approximately 1/4 . Pyramidal tips commonly used for AFM imaging are suitable for probing mechanical properties of soft gels and living cells. PMID:16196611

  3. Film as Film; Understanding and Judging Movies.

    ERIC Educational Resources Information Center

    Perkins, V. F.

    The criteria for judging movies which are presented here are based on the belief that film criticism becomes rational, if not "objective", when it displays and inspects the nature of its evidence and the bases of its arguments. The author dissents from the view of early film theorists that montage is the essence of cinema, and that cinema is to be…

  4. The Elements Go to the Movies

    ERIC Educational Resources Information Center

    Taarea, Dina; Thomas, Nicholas C.

    2010-01-01

    The names of many common elements have found their way into the titles of feature films: gold, silver, iron, copper, and lead, for example, appear in hundreds of movie titles. Surprisingly, perhaps, more than two dozen other elements, including iodine, cadmium, zinc, calcium, argon, chlorine, and others, have also been used in film titles. In this…

  5. Portrayed emotions in the movie "Forrest Gump".

    PubMed

    Labs, Annika; Reich, Theresa; Schulenburg, Helene; Boennen, Manuel; Mareike, Gehrke; Golz, Madleen; Hartigs, Benita; Hoffmann, Nico; Keil, Sebastian; Perlow, Malú; Peukmann, Anne Katrin; Rabe, Lea Noell; von Sobbe, Franca-Rosa; Hanke, Michael

    2015-01-01

    Here we present a dataset with a description of portrayed emotions in the movie "Forrest Gump". A total of 12 observers independently annotated emotional episodes regarding their temporal location and duration. The nature of an emotion was characterized with basic attributes, such as arousal and valence, as well as explicit emotion category labels. In addition, annotations include a record of the perceptual evidence for the presence of an emotion. Two variants of the movie were annotated separately: 1) an audio-movie version of Forrest Gump that has been used as a stimulus for the acquisition of a large public functional brain imaging dataset, and 2) the original audio-visual movie. We present reliability and consistency estimates that suggest that both stimuli can be used to study visual and auditory emotion cue processing in real-life like situations. Raw annotations from all observers are publicly released in full in order to maximize their utility for a wide range of applications and possible future extensions. In addition, aggregate time series of inter-observer agreement with respect to particular attributes of portrayed emotions are provided to facilitate adoption of these data. PMID:25977755

  6. Portrayed emotions in the movie "Forrest Gump"

    PubMed Central

    Boennen, Manuel; Mareike, Gehrke; Golz, Madleen; Hartigs, Benita; Hoffmann, Nico; Keil, Sebastian; Perlow, Malú; Peukmann, Anne Katrin; Rabe, Lea Noell; von Sobbe, Franca-Rosa; Hanke, Michael

    2015-01-01

    Here we present a dataset with a description of portrayed emotions in the movie ”Forrest Gump”. A total of 12 observers independently annotated emotional episodes regarding their temporal location and duration. The nature of an emotion was characterized with basic attributes, such as arousal and valence, as well as explicit emotion category labels. In addition, annotations include a record of the perceptual evidence for the presence of an emotion. Two variants of the movie were annotated separately: 1) an audio-movie version of Forrest Gump that has been used as a stimulus for the acquisition of a large public functional brain imaging dataset, and 2) the original audio-visual movie. We present reliability and consistency estimates that suggest that both stimuli can be used to study visual and auditory emotion cue processing in real-life like situations. Raw annotations from all observers are publicly released in full in order to maximize their utility for a wide range of applications and possible future extensions. In addition, aggregate time series of inter-observer agreement with respect to particular attributes of portrayed emotions are provided to facilitate adoption of these data. PMID:25977755

  7. Application and enhancements of MOVIE.BYU

    NASA Technical Reports Server (NTRS)

    Gates, R. L.; Vonofenheim, W. H.

    1984-01-01

    MOVIE.BYU (MOVIE.BRIGHAM YOUNG UNIVERSITY) is a system of programs for the display and manipulation of data representing mathematical, architectural, and topological models in which the geometry may be described in terms of panel (n-sided polygons) and solid elements or contour lines. The MOVIE.BYU system has been used in a series of applications of LaRC. One application has been the display, creation, and manipulation of finite element models in aeronautic/aerospace research. These models have been displayed on both vector and color raster devices, and the user has the option to modify color and shading parameters on these color raster devices. Another application involves the display of scalar functions (temperature, pressure, etc.) over the surface of a given model. This capability gives the researcher added flexibility in the analysis of the model and its accompanying data. Limited animation (frame-by-frame creation) has been another application of MOVIE.BYU in the modeling of kinematic processes in antenna structures.

  8. All-in-One Movie Book.

    ERIC Educational Resources Information Center

    Petzold, Paul

    The amateur movie camera differs from a still camera on several important points. The author explores these differences and discusses the various ways they may be used to advantage. He describes in detail the workings of basic equipment--cameras, exposure meters, lenses, films, and lights--and demonstrates the proper use of each. Techniques such…

  9. Focus on Film: Learning through the Movies

    ERIC Educational Resources Information Center

    Considine, David M.; Baker, Frank

    2006-01-01

    This article explores the use of movies as valuable instructional tools. When students are engaged with the content through a medium they love, they learn better and retain more. Incorporating motion pictures as part of classroom instruction has been spurred by the endorsement of both of the major reading and language arts organizations in the…

  10. Aspects of Video Movie English Teaching.

    ERIC Educational Resources Information Center

    Wood, David John

    1999-01-01

    A discussion of the use of movies on videotape in the English-as-a-Second-Language (ESL) classroom begins with a brief review of the history and emergence of videotape recordings as a popular technology. The advantages of video as a language teaching aid are then examined, including its instructional flexibility, exposure to paralinguistic…

  11. 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

  12. Optical trapping and surgery of living yeast cells using a single laser

    NASA Astrophysics Data System (ADS)

    Ando, Jun; Bautista, Godofredo; Smith, Nicholas; Fujita, Katsumasa; Daria, Vincent Ricardo

    2008-10-01

    We present optical trapping and surgery of living yeast cells using two operational modes of a single laser. We used a focused laser beam operating in continuous-wave mode for noninvasive optical trapping and manipulation of single yeast cell. We verified that such operational mode of the laser does not cause any destructive effect on yeast cell wall. By changing the operation of the laser to femtosecond-pulsed mode, we show that a tightly focused beam dissects the yeast cell walls via nonlinear absorption. Lastly, using the combined technique of optical microsurgery and trapping, we demonstrate intracellular organelle extraction and manipulation from a yeast cell. The technique established here will be useful as an efficient method for both surgery and manipulation of living cells using a single laser beam.

  13. Simultaneous FRAP, FLIM and FAIM for measurements of protein mobility and interaction in living cells

    PubMed Central

    Levitt, James A.; Morton, Penny E.; Fruhwirth, Gilbert O.; Santis, George; Chung, Pei-Hua; Parsons, Maddy; Suhling, Klaus

    2015-01-01

    We present a novel integrated multimodal fluorescence microscopy technique for simultaneous fluorescence recovery after photobleaching (FRAP), fluorescence lifetime imaging (FLIM) and fluorescence anisotropy imaging (FAIM). This approach captures a series of polarization-resolved fluorescence lifetime images during a FRAP recovery, maximizing the information available from a limited photon budget. We have applied this method to analyse the behaviour of GFP-labelled coxsackievirus and adenovirus receptor (CAR) in living human epithelial cells. Our data reveal that CAR exists in oligomeric states throughout the cell, and that these complexes occur in conjunction with high immobile fractions of the receptor at cell-cell junctions. These findings shed light on previously unknown molecular associations between CAR receptors in intact cells and demonstrate the power of combined FRAP, FLIM and FAIM microscopy as a robust method to analyse complex multi-component dynamics in living cells. PMID:26504635

  14. Nanogel-quantum dot hybrid nanoparticles for live cell imaging

    SciTech Connect

    Hasegawa, Urara; Nomura, Shin-ichiro M.; Kaul, Sunil C.; Hirano, Takashi; Akiyoshi, Kazunari; E-mail: akiyoshi.org@tmd.ac.jp

    2005-06-17

    We report here a novel carrier of quantum dots (QDs) for intracellular labeling. Monodisperse hybrid nanoparticles (38 nm in diameter) of QDs were prepared by simple mixing with nanogels of cholesterol-bearing pullulan (CHP) modified with amino groups (CHPNH{sub 2}). The CHPNH{sub 2}-QD nanoparticles were effectively internalized into the various human cells examined. The efficiency of cellular uptake was much higher than that of a conventional carrier, cationic liposome. These hybrid nanoparticles could be a promising fluorescent probe for bioimaging.

  15. Imaging single cells in a beam of live cyanobacteria with an X-ray laser

    DOE Data Explorer

    Schot, Gijs, vander

    2015-02-10

    This entry contains ten diffraction patterns, and reconstructions images, of individual living Cyanobium gracile cells, imaged using 517 eV X-rays from the LCLS XFEL. The Hawk software package was used for phasing. The Uppsala aerosol injector was used for sample injection, assuring very low noise levels. The cells come from various stages of the cell cycle, and were imaged in random orientations.

  16. Quantitative measurement of permeabilization of living cells by terahertz attenuated total reflection

    NASA Astrophysics Data System (ADS)

    Grognot, Marianne; Gallot, Guilhem

    2015-09-01

    Using Attenuated Total Reflection imaging technique in the terahertz domain, we demonstrate non-invasive, non-staining real time measurements of cytoplasm leakage during permeabilization of epithelial cells by saponin. The terahertz signal is mostly sensitive to the intracellular protein concentration in the cells, in a very good agreement with standard bicinchoninic acid protein measurements. It opens the way to in situ real time dynamics of protein content and permeabilization in live cells.

  17. Revealing the Dynamics of Thylakoid Membranes in Living Cyanobacterial Cells

    NASA Astrophysics Data System (ADS)

    Stingaciu, Laura-Roxana; O'Neill, Hugh; Liberton, Michelle; Urban, Volker S.; Pakrasi, Himadri B.; Ohl, Michael

    2016-01-01

    Cyanobacteria are photosynthetic prokaryotes that make major contributions to the production of the oxygen in the Earth atmosphere. The photosynthetic machinery in cyanobacterial cells is housed in flattened membrane structures called thylakoids. The structural organization of cyanobacterial cells and the arrangement of the thylakoid membranes in response to environmental conditions have been widely investigated. However, there is limited knowledge about the internal dynamics of these membranes in terms of their flexibility and motion during the photosynthetic process. We present a direct observation of thylakoid membrane undulatory motion in vivo and show a connection between membrane mobility and photosynthetic activity. High-resolution inelastic neutron scattering experiments on the cyanobacterium Synechocystis sp. PCC 6803 assessed the flexibility of cyanobacterial thylakoid membrane sheets and the dependence of the membranes on illumination conditions. We observed softer thylakoid membranes in the dark that have three-to four fold excess mobility compared to membranes under high light conditions. Our analysis indicates that electron transfer between photosynthetic reaction centers and the associated electrochemical proton gradient across the thylakoid membrane result in a significant driving force for excess membrane dynamics. These observations provide a deeper understanding of the relationship between photosynthesis and cellular architecture.

  18. Entangled active matter: from ants to living cells

    NASA Astrophysics Data System (ADS)

    Brochard-Wyart, Francoise

    2015-03-01

    We introduce the field of ``Entangled Active Matter'' where the building blocks are transiently bound. We will point out strong similarities between aggregates of ants and cells! We will use multicellular aggregates, a model system for tissues. We characterize the tissue mechanical properties using pipette aspiration technique. The aggregate exhibits a viscoelastic response. We observe aggregate reinforcement with pressure, which may results in pulsed contractions or ``shivering.'' We interpret this reinforcement as a mechano-sensitive active response of the acto-myosin cortex. We describe the spreading of aggregates on rigid and soft substrates, varying both intercellular and substrate adhesion. We find both partial and complete wetting, with a precursor film forming a cellular monolayer in a liquid or gas phase. We model the dynamics of spreading from a balance between active cellular driving forces and permeation of cells to enter into the film. Finally we study the motility of aggregates induced by chemical or rigidity gradients, or spontaneous: on soft substrate, the precursor film is unstable, leading to a symmetry breaking and a global motion of the aggregate. We describe the shapes of migrating aggregates, the flow and the force field responsible of the motion. We monitored the center of mass motion and we characterize the stick-slip motions. LABEX CelTisPhyBio, Institut Curie, France

  19. Active invasion of bacteria into living fungal cells

    PubMed Central

    Moebius, Nadine; Üzüm, Zerrin; Dijksterhuis, Jan; Lackner, Gerald; Hertweck, Christian

    2014-01-01

    The rice seedling blight fungus Rhizopus microsporus and its endosymbiont Burkholderia rhizoxinica form an unusual, highly specific alliance to produce the highly potent antimitotic phytotoxin rhizoxin. Yet, it has remained a riddle how bacteria invade the fungal cells. Genome mining for potential symbiosis factors and functional analyses revealed that a type 2 secretion system (T2SS) of the bacterial endosymbiont is required for the formation of the endosymbiosis. Comparative proteome analyses show that the T2SS releases chitinolytic enzymes (chitinase, chitosanase) and chitin-binding proteins. The genes responsible for chitinolytic proteins and T2SS components are highly expressed during infection. Through targeted gene knock-outs, sporulation assays and microscopic investigations we found that chitinase is essential for bacteria to enter hyphae. Unprecedented snapshots of the traceless bacterial intrusion were obtained using cryo-electron microscopy. Beyond unveiling the pivotal role of chitinolytic enzymes in the active invasion of a fungus by bacteria, these findings grant unprecedented insight into the fungal cell wall penetration and symbiosis formation. DOI: http://dx.doi.org/10.7554/eLife.03007.001 PMID:25182414

  20. Safety of Living Donation of Hematopoietic Stem Cells.

    PubMed

    Szer, Jeff; Elmoazzen, Heidi; Fechter, Mirjam; Hwang, William; Korhonen, Matti; Miller, John; Mengling, Thilo; Shaw, Bronwen; Stein, Jerry

    2016-06-01

    More than 12 000 volunteer unrelated hematopoietic stem cell donations are undertaken annually, and the World Marrow Donor Association established an expert committee to examine all reports of adverse events affecting donors globally, eventually making such reporting a necessary part of World Marrow Donor Association accreditation. The committee evaluates and responds to reported events in a nonpunitive confidential process designed to alert the community of rare events which might be missed by local follow-up. Each report is evaluated by the committee for imputability (causal link between the donation and the adverse event) and compared with that submitted by the reporting registry. In 2014, there were 50 reports received from 16 different registries in 15 countries. There were 16 reports of malignancies arising in donors including 3 hematologic malignancies. All but 2 of the 16 occurred more than a year after donation. There were 4 reports of autoimmune phenomena in donors all occurring more than a year postdonation. Of the 30 remaining events, 6 were allergic, 4 cardiac, 3 gastrointestinal, 2 infections, 2 pulmonary, and 13 miscellaneous. Causation was assessed differently to the reporting registry in 17 events with 6 thought to be less likely causally linked to the donation and 10 more likely with 1 requiring more information. Volunteer unrelated hematopoietic stem cell donation is a safe and effective altruistic contribution to the treatment of patients with life-threatening hematologic disorders. A decade of detailed examination of adverse donor events has contributed to the safety of these donations. PMID:27136264

  1. Revealing the Dynamics of Thylakoid Membranes in Living Cyanobacterial Cells

    DOE PAGESBeta

    Stingaciu, Laura-Roxana; O’Neill, Hugh; Liberton, Michelle; Urban, Volker S.; Pakrasi, Himadri B.; Ohl, Michael

    2016-01-21

    Cyanobacteria are photosynthetic prokaryotes that make major contributions to the production of the oxygen in the Earth atmosphere. The photosynthetic machinery in cyanobacterial cells is housed in flattened membrane structures called thylakoids. The structural organization of cyanobacterial cells and the arrangement of the thylakoid membranes in response to environmental conditions have been widely investigated. However, there is limited knowledge about the internal dynamics of these membranes in terms of their flexibility and motion during the photosynthetic process. We present a direct observation of thylakoid membrane undulatory motion in vivo and show a connection between membrane mobility and photosynthetic activity. High-resolutionmore » inelastic neutron scattering experiments on the cyanobacterium Synechocystis sp. PCC 6803 assessed the flexibility of cyanobacterial thylakoid membrane sheets and the dependence of the membranes on illumination conditions. We observed softer thylakoid membranes in the dark that have three-to four fold excess mobility compared to membranes under high light conditions. We find our analysis indicates that electron transfer between photosynthetic reaction centers and the associated electrochemical proton gradient across the thylakoid membrane result in a significant driving force for excess membrane dynamics. Lastly, these observations provide a deeper understanding of the relationship between photosynthesis and cellular architecture.« less

  2. Revealing the Dynamics of Thylakoid Membranes in Living Cyanobacterial Cells

    PubMed Central

    Stingaciu, Laura-Roxana; O’Neill, Hugh; Liberton, Michelle; Urban, Volker S.; Pakrasi, Himadri B.; Ohl, Michael

    2016-01-01

    Cyanobacteria are photosynthetic prokaryotes that make major contributions to the production of the oxygen in the Earth atmosphere. The photosynthetic machinery in cyanobacterial cells is housed in flattened membrane structures called thylakoids. The structural organization of cyanobacterial cells and the arrangement of the thylakoid membranes in response to environmental conditions have been widely investigated. However, there is limited knowledge about the internal dynamics of these membranes in terms of their flexibility and motion during the photosynthetic process. We present a direct observation of thylakoid membrane undulatory motion in vivo and show a connection between membrane mobility and photosynthetic activity. High-resolution inelastic neutron scattering experiments on the cyanobacterium Synechocystis sp. PCC 6803 assessed the flexibility of cyanobacterial thylakoid membrane sheets and the dependence of the membranes on illumination conditions. We observed softer thylakoid membranes in the dark that have three-to four fold excess mobility compared to membranes under high light conditions. Our analysis indicates that electron transfer between photosynthetic reaction centers and the associated electrochemical proton gradient across the thylakoid membrane result in a significant driving force for excess membrane dynamics. These observations provide a deeper understanding of the relationship between photosynthesis and cellular architecture. PMID:26790980

  3. Fluorescent labelling of intestinal epithelial cells reveals independent long-lived intestinal stem cells in a crypt

    SciTech Connect

    Horita, Nobukatsu; Tsuchiya, Kiichiro; Hayashi, Ryohei; Fukushima, Keita; Hibiya, Shuji; Fukuda, Masayoshi; Kano, Yoshihito; Mizutani, Tomohiro; Nemoto, Yasuhiro; Yui, Shiro; Okamoto, Ryuichi; Nakamura, Tetsuya; Watanabe, Mamoru

    2014-11-28

    Highlights: • Lentivirus mixed with Matrigel enables direct infection of intestinal organoids. • Our original approach allows the marking of a single stem cell in a crypt. • Time-lapse imaging shows the dynamics of a single stem cell. • Our lentivirus transgene system demonstrates plural long-lived stem cells in a crypt. - Abstract: Background and aims: The dynamics of intestinal stem cells are crucial for regulation of intestinal function and maintenance. Although crypt stem cells have been identified in the intestine by genetic marking methods, identification of plural crypt stem cells has not yet been achieved as they are visualised in the same colour. Methods: Intestinal organoids were transferred into Matrigel® mixed with lentivirus encoding mCherry. The dynamics of mCherry-positive cells was analysed using time-lapse imaging, and the localisation of mCherry-positive cells was analysed using 3D immunofluorescence. Results: We established an original method for the introduction of a transgene into an organoid generated from mouse small intestine that resulted in continuous fluorescence of the mCherry protein in a portion of organoid cells. Three-dimensional analysis using confocal microscopy showed a single mCherry-positive cell in an organoid crypt that had been cultured for >1 year, which suggested the presence of long-lived mCherry-positive and -negative stem cells in the same crypt. Moreover, a single mCherry-positive stem cell in a crypt gave rise to both crypt base columnar cells and transit amplifying cells. Each mCherry-positive and -negative cell contributed to the generation of organoids. Conclusions: The use of our original lentiviral transgene system to mark individual organoid crypt stem cells showed that long-lived plural crypt stem cells might independently serve as intestinal epithelial cells, resulting in the formation of a completely functional villus.

  4. Comparison of Atomic Force Microscopy and Scanning Ion Conductance Microscopy for Live Cell Imaging.

    PubMed

    Seifert, Jan; Rheinlaender, Johannes; Novak, Pavel; Korchev, Yuri E; Schäffer, Tilman E

    2015-06-23

    Atomic force microscopy (AFM) and scanning ion conductance microscopy (SICM) are excellent and commonly used techniques for imaging the topography of living cells with high resolution. We present a direct comparison of AFM and SICM for imaging microvilli, which are small features on the surface of living cells, and for imaging the shape of whole cells. The imaging quality on microvilli increased significantly after cell fixation for AFM, whereas for SICM it remained constant. The apparent shape of whole cells in the case of AFM depended on the imaging force, which deformed the cell. In the case of SICM, cell deformations were avoided, owing to the contact-free imaging mechanism. We estimated that the lateral resolution on living cells is limited by the cell's elastic modulus for AFM, while it is not for SICM. By long-term, time-lapse imaging of microvilli dynamics, we showed that the imaging quality decreased with time for AFM, while it remained constant for SICM. PMID:26011471

  5. Imaging the nanoscale organization of peptidoglycan in living Lactococcus lactis cells

    PubMed Central

    Andre, Guillaume; Kulakauskas, Saulius; Chapot-Chartier, Marie-Pierre; Navet, Benjamine; Deghorain, Marie; Bernard, Elvis; Hols, Pascal; Dufrêne, Yves F.

    2010-01-01

    The spatial organization of peptidoglycan, the major constituent of bacterial cell-walls, is an important, yet still unsolved issue in microbiology. In this paper, we show that the combined use of atomic force microscopy and cell wall mutants is a powerful platform for probing the nanoscale architecture of cell wall peptidoglycan in living Gram-positive bacteria. Using topographic imaging, we found that Lactococcus lactis wild-type cells display a smooth, featureless surface morphology, whereas mutant strains lacking cell wall exopolysaccharides feature 25-nm-wide periodic bands running parallel to the short axis of the cell. In addition, we used single-molecule recognition imaging to show that parallel bands are made of peptidoglycan. Our data, obtained for the first time on living ovococci, argue for an architectural feature of the cell wall in the plane perpendicular to the long axis of the cell. The non-invasive live cell experiments presented here open new avenues for understanding the architecture and assembly of peptidoglycan in Gram-positive bacteria. PMID:20975688

  6. Digital Holographic Microscopy: Quantitative Phase Imaging and Applications in Live Cell Analysis

    NASA Astrophysics Data System (ADS)

    Kemper, Björn; Langehanenberg, Patrik; Kosmeier, Sebastian; Schlichthaber, Frank; Remmersmann, Christian; von Bally, Gert; Rommel, Christina; Dierker, Christian; Schnekenburger, Jürgen

    The analysis of complex processes in living cells creates a high demand for fast and label-free methods for online monitoring. Widely used fluorescence methods require specific labeling and are often restricted to chemically fixated samples. Thus, methods that offer label-free and minimally invasive detection of live cell processes and cell state alterations are of particular interest. In combination with light microscopy, digital holography provides label-free, multi-focus quantitative phase imaging of living cells. In overview, several methods for digital holographic microscopy (DHM) are presented. First, different experimental setups for the recording of digital holograms and the modular integration of DHM into common microscopes are described. Then the numerical processing of digitally captured holograms is explained. This includes the description of spatial and temporal phase shifting techniques, spatial filtering based reconstruction, holographic autofocusing, and the evaluation of self-interference holograms. Furthermore, the usage of partial coherent light and multi-wavelength approaches is discussed. Finally, potentials of digital holographic microscopy for quantitative cell imaging are illustrated by results from selected applications. It is shown that DHM can be used for automated tracking of migrating cells and cell thickness monitoring as well as for refractive index determination of cells and particles. Moreover, the use of DHM for label-free analysis in fluidics and micro-injection monitoring is demonstrated. The results show that DHM is a highly relevant method that allows novel insights in dynamic cell biology, with applications in cancer research and for drugs and toxicity testing.

  7. An integrated optical platform for micromanipulation of cells and tissue in live animals

    NASA Astrophysics Data System (ADS)

    Turcotte, Raphael

    The hematopoietic stem cell niche is a specialized bone marrow (BM) microenvironment where blood-forming cells reside. Interactions between these rare cells and their niche need to be studied at the single-cell level. While live animal cell tracking with optical microscopy has proven useful for this purpose, a more thorough characterization requires novel approaches. This can be accomplished by using an integrated optical platform for cell and tissue manipulations (cell transplantation and extraction) in the skull bone of live mice. The platform integrates a non-damaging laser ablation microbeam for bone removal and tissue cutting, optical tweezers for single cell trapping, and a video-rate scanning microscope. For single cell delivery, a narrow channel is ablated through bone under imaging guidance. Cells are then transferred from a micropipette into an optical trap, which brings cells into the BM through the channel. The survival and proliferation of implanted cells can be tracked in vivo by imaging. For cell extraction after laser bone thinning, different approaches can be implemented and three of them are presented.

  8. DESIGN, SYNTHESIS, AND APPLICATION OF THE TRIMETHOPRIM-BASED CHEMICAL TAG FOR LIVE CELL IMAGING

    PubMed Central

    Jing, Chaoran; Cornish, Virginia W.

    2013-01-01

    Over the past decade chemical tags have been developed to complement the use of fluorescent proteins in live cell imaging. Chemical tags retain the specificity of protein labeling achieved with fluorescent proteins through genetic encoding, but provide smaller, more robust tags and modular use of organic fluorophores with high photon-output and tailored functionalities. The trimethoprim-based chemical tag (TMP-tag) was initially developed based on the high affinity interaction between E.coli dihydrofolatereductase and the antibiotic trimethoprim and subsequently rendered covalent and fluorogenic via proximity-induced protein labeling reactions. To date, the TMP-tag is one of the few chemical tags that enable intracellular protein labeling and high-resolution live cell imaging. Here we describe the general design, chemical synthesis, and application of TMP-tag for live cell imaging. Alternative protocols for synthesizing and using the covalent and the fluorogenic TMP-tags are also included. PMID:23839994

  9. Holographic optical manipulation of motor-driven membranous structures in living NG-108 cells

    NASA Astrophysics Data System (ADS)

    Farré, Arnau; López-Quesada, Carol; Andilla, Jordi; Martín-Badosa, Estela; Montes-Usategui, Mario

    2010-08-01

    Optical tweezer experiments have partially unveiled the mechanical properties of processive motor proteins while driving polystyrene or silica microbeads in vitro. However, the set of forces underlying the more complex transport mechanisms in living samples remains poorly understood. Several studies have shown that optical tweezers are capable of trapping vesicles and organelles in the cytoplasm of living cells, which can be used as handles to mechanically interact with engaged (active) motors, or other components regulating transport. This may ultimately enable the exploration of the mechanics of this trafficking mechanism in vivo. These cell manipulation experiments have been carried out using different strategies to achieve dynamic beam steering capable of trapping these subcellular structures. We report here the first trapping and manipulation, to our knowledge, of such small motor-propelled cargos in living cells using holographic technology.

  10. 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

  11. Translation dynamics of single mRNAs in live cells and neurons

    PubMed Central

    Wu, Bin; Eliscovich, Carolina; Yoon, Young J.; Singer, Robert H.

    2016-01-01

    Translation is the fundamental biological process converting mRNA information into proteins. Single-molecule imaging in live cells has illuminated the dynamics of RNA transcription; however, it is not yet applicable to translation. Here, we report single-molecule imaging of nascent peptides (SINAPS) to assess translation in live cells. The approach provides direct readout of initiation, elongation, and location of translation. We show that mRNAs coding for endoplasmic reticulum (ER) proteins are translated when they encounter the ER membrane. Single-molecule fluorescence recovery after photobleaching provides direct measurement of elongation speed (5 amino acids per second). In primary neurons, mRNAs are translated in proximal dendrites but repressed in distal dendrites and display “bursting” translation. This technology provides a tool with which to address the spatiotemporal translation mechanism of single mRNAs in living cells. PMID:27313041

  12. Recent advancements in structured-illumination microscopy toward live-cell imaging.

    PubMed

    Hirano, Yasuhiro; Matsuda, Atsushi; Hiraoka, Yasushi

    2015-08-01

    Fluorescence microscopy allows us to observe fluorescently labeled molecules in diverse biological processes and organelle structures within living cells. However, the diffraction limit restricts its spatial resolution to about half of its wavelength, limiting the capability of biological observation at the molecular level. Structured-illumination microscopy (SIM), a type of super-resolution microscopy, doubles the spatial resolution in all three dimensions by illuminating the sample with a patterned excitation light, followed by computer reconstruction. SIM uses a relatively low illumination power compared with other methods of super-resolution microscopy and is easily available for multicolor imaging. SIM has great potential for meeting the requirements of live-cell imaging. Recent developments in diverse types of SIM have achieved higher spatial (∼50 nm lateral) and temporal (∼100 Hz) resolutions. Here, we review recent advancements in SIM and discuss its application in noninvasive live-cell imaging. PMID:26133185

  13. Translation dynamics of single mRNAs in live cells and neurons.

    PubMed

    Wu, Bin; Eliscovich, Carolina; Yoon, Young J; Singer, Robert H

    2016-06-17

    Translation is the fundamental biological process converting mRNA information into proteins. Single-molecule imaging in live cells has illuminated the dynamics of RNA transcription; however, it is not yet applicable to translation. Here, we report single-molecule imaging of nascent peptides (SINAPS) to assess translation in live cells. The approach provides direct readout of initiation, elongation, and location of translation. We show that mRNAs coding for endoplasmic reticulum (ER) proteins are translated when they encounter the ER membrane. Single-molecule fluorescence recovery after photobleaching provides direct measurement of elongation speed (5 amino acids per second). In primary neurons, mRNAs are translated in proximal dendrites but repressed in distal dendrites and display "bursting" translation. This technology provides a tool with which to address the spatiotemporal translation mechanism of single mRNAs in living cells. PMID:27313041

  14. Super-resolution fluorescence imaging of organelles in live cells with photoswitchable membrane probes

    PubMed Central

    Shim, Sang-Hee; Xia, Chenglong; Zhong, Guisheng; Babcock, Hazen P.; Vaughan, Joshua C.; Huang, Bo; Wang, Xun; Xu, Cheng; Bi, Guo-Qiang; Zhuang, Xiaowei

    2012-01-01

    Imaging membranes in live cells with nanometer-scale resolution promises to reveal ultrastructural dynamics of organelles that are essential for cellular functions. In this work, we identified photoswitchable membrane probes and obtained super-resolution fluorescence images of cellular membranes. We demonstrated the photoswitching capabilities of eight commonly used membrane probes, each specific to the plasma membrane, mitochondria, the endoplasmic recticulum (ER) or lysosomes. These small-molecule probes readily label live cells with high probe densities. Using these probes, we achieved dynamic imaging of specific membrane structures in living cells with 30–60 nm spatial resolution at temporal resolutions down to 1–2 s. Moreover, by using spectrally distinguishable probes, we obtained two-color super-resolution images of mitochondria and the ER. We observed previously obscured details of morphological dynamics of mitochondrial fusion/fission and ER remodeling, as well as heterogeneous membrane diffusivity on neuronal processes. PMID:22891300

  15. Live embryo imaging to follow cell cycle and chromosomes stability after nuclear transfer.

    PubMed

    Balbach, Sebastian T; Boiani, Michele

    2015-01-01

    Nuclear transfer (NT) into mouse oocytes yields a transcriptionally and functionally heterogeneous population of cloned embryos. Most studies of NT embryos consider only embryos at predefined key stages (e.g., morula or blastocyst), that is, after the bulk of reprogramming has taken place. These retrospective approaches are of limited use to elucidate mechanisms of reprogramming and to predict developmental success. Observing cloned embryo development using live embryo cinematography has the potential to reveal otherwise undetectable embryo features. However, light exposure necessary for live cell cinematography is highly toxic to cloned embryos. Here we describe a protocol for combined bright-field and fluorescence live-cell imaging of histone H2b-GFP expressing mouse embryos, to record cell divisions up to the blastocyst stage. This protocol, which can be adapted to observe other reporters such as Oct4-GFP or Nanog-GFP, allowed us to quantitatively analyze cleavage kinetics of cloned embryos. PMID:25287344

  16. Cannibalism of live lymphocytes by human metastatic but not primary melanoma cells.

    PubMed

    Lugini, Luana; Matarrese, Paola; Tinari, Antonella; Lozupone, Francesco; Federici, Cristina; Iessi, Elisabetta; Gentile, Massimo; Luciani, Francesca; Parmiani, Giorgio; Rivoltini, Licia; Malorni, Walter; Fais, Stefano

    2006-04-01

    The phenomenon of cell cannibalism, which generally refers to the engulfment of cells within other cells, was described in malignant tumors, but its biological significance is still largely unknown. In the present study, we investigated the occurrence, the in vivo relevance, and the underlying mechanisms of cannibalism in human melanoma. As first evidence, we observed that tumor cannibalism was clearly detectable in vivo in metastatic lesions of melanoma and often involved T cells, which could be found in a degraded state within tumor cells. Then, in vitro experiments confirmed that cannibalism of T cells was a property of metastatic melanoma cells but not of primary melanoma cells. In particular, morphologic analyses, including time-lapse cinematography and electron microscopy, revealed a sequence of events, in which metastatic melanoma cells were able to engulf and digest live autologous melanoma-specific CD8(+) T cells. Importantly, this cannibalistic activity significantly increased metastatic melanoma cell survival, particularly under starvation condition, supporting the evidence that tumor cells may use the eating of live lymphocytes as a way to "feed" in condition of low nutrient supply. The mechanism underlying cannibalism involved a complex framework, including lysosomal protease cathepsin B activity, caveolae formation, and ezrin cytoskeleton integrity and function. In conclusion, our study shows that human metastatic melanoma cells may eat live T cells, which are instead programmed to kill them, suggesting a novel mechanism of tumor immune escape. Moreover, our data suggest that cannibalism may represent a sort of "feeding" activity aimed at sustaining survival and progression of malignant tumor cells in an unfavorable microenvironment. PMID:16585188

  17. Technique of laser confocal and Raman spectroscopy for living cell analysis

    NASA Astrophysics Data System (ADS)

    Meng, Xiaochen; Zhu, Lianqing

    2013-10-01

    Because of the shortcomings of the main methods used to analysis single cell, the need of single living cell analysis with no damage, unmarked and in situ dynamic multi-parameter measurement is urgent in the life sciences and biomedical advanced research field. And the method of for living cells analysis is proposed. The spectral pretreatment technology of living cell is the key work of laser confocal Raman spectroscopy. To study the spectrum processing methods for Raman spectrum on single living cell and develop the pre-process techniques to enhance the signal-to-noise ratio, sensitivity, and decrease the influence of fluorescence, elimination the cosmic rays was used to improve the spectrum. The classification, average and filtration of spectrum were applied to enhance signal-to-noise ratio. The fluorescence was depressed for quantity analysis or utilized for analysis by comparing the background and the spectrum. The results show that the proposed technique for laser confocal Raman spectrum of single cell can perform the sensitive and weak intensity peaks and reflect the information of molecules structures very well.

  18. Bulk electroporation of retinal ganglion cells in live Xenopus tadpoles.

    PubMed

    Ruthazer, Edward S; Schohl, Anne; Schwartz, Neil; Tavakoli, Aydin; Tremblay, Marc; Cline, Hollis T

    2013-08-01

    Individual neurons in the developing nervous system of Xenopus laevis can be visualized by the targeted delivery of a fluorophore. The fluorophore can be delivered as a fluorescent dye or DNA that encodes a fluorescent protein. Local iontophoresis is a method that works well for transfer of fluorescent dye to retinal ganglion cells (RGCs) in the eye, but it does not give a high yield for delivery of DNA. This is largely because the degree of pigmentation of the eyes, even in albino strains, makes it difficult to visualize RGC somata during pipette positioning. Bulk retinal electroporation is a better approach for delivery of plasmid DNA to RGC. The method described here works best in tadpoles older than stage 42. PMID:23906915

  19. High speed microscopy techniques for signaling detection in live cells

    NASA Astrophysics Data System (ADS)

    de Mauro, C.; Cecchetti, C. A.; Alfieri, D.; Borile, Giulia; Urbani, A.; Mongillo, M.; Pavone, F. S.

    2014-05-01

    Alterations in intracellular cardiomyocyte calcium handling have a key role in initiating and sustaining arrhythmias. Arrhythmogenic calcium leak from sarcoplasmic reticulum (SR) can be attributed to all means by which calcium exits the SR store in an abnormal fashion. Abnormal SR calcium exit maymanifest as intracellular Ca2+ sparks and/or Ca2+ waves. Ca2+ signaling in arrhythmogenesis has been mainly studied in isolated cardiomyocytes and given that the extracellular matrix influences both Ca2+ and membrane potential dynamics in the intact heart and underlies environmentally mediated changes, understanding how Ca2+ and voltage are regulated in the intact heart will represent a tremendous advancement in the understanding of arrhythmogenic mechanisms. Using novel high-speed multiphoton microscopy techinques, such as multispot and random access, we investigated animal models with inherited and acquired arrhythmias to assess the role of Ca2+ and voltage signals as arrhythmia triggers in cell and subcellular components of the intact heart and correlate these with electrophysiology.

  20. What does calorimetry and thermodynamics of living cells tell us?

    PubMed

    Maskow, Thomas; Paufler, Sven

    2015-04-01

    This article presents and compares several thermodynamic methods for the quantitative interpretation of data from calorimetric measurements. Heat generation and absorption are universal features of microbial growth and product formation as well as of cell cultures from animals, plants and insects. The heat production rate reflects metabolic changes in real time and is measurable on-line. The detection limit of commercially available calorimetric instruments can be low enough to measure the heat of 100,000 aerobically growing bacteria or of 100 myocardial cells. Heat can be monitored in reaction vessels ranging from a few nanoliters up to many cubic meters. Most important the heat flux measurement does not interfere with the biological process under investigation. The practical advantages of calorimetry include the waiver of labeling and reactants. It is further possible to assemble the thermal transducer in a protected way that reduces aging and thereby signal drifts. Calorimetry works with optically opaque solutions. All of these advantages make calorimetry an interesting method for many applications in medicine, environmental sciences, ecology, biochemistry and biotechnology, just to mention a few. However, in many cases the heat signal is merely used to monitor biological processes but only rarely to quantitatively interpret the data. Therefore, a significant proportion of the information potential of calorimetry remains unutilized. To fill this information gap and to motivate the reader using the full information potential of calorimetry, various methods for quantitative data interpretations are presented, evaluated and compared with each other. Possible errors of interpretation and limitations of quantitative data analysis are also discussed. PMID:25461814