WorldWideScience

Sample records for living cells demonstrated

  1. Förster resonance energy transfer demonstrates a flavonoid metabolon in living plant cells that displays competitive interactions between enzymes

    NARCIS (Netherlands)

    Crosby, K.C.; Pietraszewska-Bogiel, A.; Gadella (jr.), T.W.J.; Winkel, B.S.J.

    2011-01-01

    We have used Förster resonance energy transfer detected by fluorescence lifetime imaging microscopy (FLIM-FRET) to provide the first evidence from living plants cells for the existence of a flavonoid metabolon. The distribution of flux within this system may be regulated by the direct competition of

  2. Fuel Cell Demonstration Program

    Energy Technology Data Exchange (ETDEWEB)

    Gerald Brun

    2006-09-15

    In an effort to promote clean energy projects and aid in the commercialization of new fuel cell technologies the Long Island Power Authority (LIPA) initiated a Fuel Cell Demonstration Program in 1999 with six month deployments of Proton Exchange Membrane (PEM) non-commercial Beta model systems at partnering sites throughout Long Island. These projects facilitated significant developments in the technology, providing operating experience that allowed the manufacturer to produce fuel cells that were half the size of the Beta units and suitable for outdoor installations. In 2001, LIPA embarked on a large-scale effort to identify and develop measures that could improve the reliability and performance of future fuel cell technologies for electric utility applications and the concept to establish a fuel cell farm (Farm) of 75 units was developed. By the end of October of 2001, 75 Lorax 2.0 fuel cells had been installed at the West Babylon substation on Long Island, making it the first fuel cell demonstration of its kind and size anywhere in the world at the time. Designed to help LIPA study the feasibility of using fuel cells to operate in parallel with LIPA's electric grid system, the Farm operated 120 fuel cells over its lifetime of over 3 years including 3 generations of Plug Power fuel cells (Lorax 2.0, Lorax 3.0, Lorax 4.5). Of these 120 fuel cells, 20 Lorax 3.0 units operated under this Award from June 2002 to September 2004. In parallel with the operation of the Farm, LIPA recruited government and commercial/industrial customers to demonstrate fuel cells as on-site distributed generation. From December 2002 to February 2005, 17 fuel cells were tested and monitored at various customer sites throughout Long Island. The 37 fuel cells operated under this Award produced a total of 712,635 kWh. As fuel cell technology became more mature, performance improvements included a 1% increase in system efficiency. Including equipment, design, fuel, maintenance

  3. Live-cell imaging.

    Science.gov (United States)

    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.

  4. Microencapsulation Of Living Cells

    Science.gov (United States)

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

    1989-01-01

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

  5. Navy fuel cell demonstration project.

    Energy Technology Data Exchange (ETDEWEB)

    Black, Billy D.; Akhil, Abbas Ali

    2008-08-01

    This is the final report on a field evaluation by the Department of the Navy of twenty 5-kW PEM fuel cells carried out during 2004 and 2005 at five Navy sites located in New York, California, and Hawaii. The key objective of the effort was to obtain an engineering assessment of their military applications. Particular issues of interest were fuel cell cost, performance, reliability, and the readiness of commercial fuel cells for use as a standalone (grid-independent) power option. Two corollary objectives of the demonstration were to promote technological advances and to improve fuel performance and reliability. From a cost perspective, the capital cost of PEM fuel cells at this stage of their development is high compared to other power generation technologies. Sandia National Laboratories technical recommendation to the Navy is to remain involved in evaluating successive generations of this technology, particularly in locations with greater environmental extremes, and it encourages their increased use by the Navy.

  6. Automatic fatigue monitoring based on real loads. Live demonstration

    International Nuclear Information System (INIS)

    Bergholz, Steffen; Rudolph, Juergen; Bruckmueller, Florian; Heinz, Benedikt; Jouan, Benoit

    2012-01-01

    The fatigue assessment of power plant components based on local fatigue monitoring approaches is an essential part of the integrity concept and modern lifetime management. An integral approach like the AREVA Fatigue Concept (AFC) basically consists of two essential modules: realistic determination of occurring operational thermal loads by means of a high end fatigue monitoring system and related highly qualified fatigue assessment methods and tools. The fatigue monitoring system delivers continuously realistic load data at the fatigue relevant locations. Consequently, realistic operational load sequences are available as input data for all ensuing fatigue analyses. This way, realistic load data are available and qualified fatigue usage factors can be determined. The mode of operation of the fatigue monitoring system will be explained in the framework of a live demonstration by means of the FAMOSi (i = integrated) demonstration wall. The workflow starts with the continuous online measurement of outer wall temperatures transients on a pipe. Visualization is implemented within the FAMOSi viewer software. In a second step, inner wall temperatures are directly calculated. In a third step, the resulting linearly elastic stress history will be calculated as the basis for subsequent code conforming fatigue assessment. Subsequently, the related advanced fatigue assessment methods of the three staged AFC-approach are addressed.

  7. Fast automatic quantitative cell replication with fluorescent live cell imaging

    Directory of Open Access Journals (Sweden)

    Wang Ching-Wei

    2012-01-01

    Full Text Available Abstract Background live cell imaging is a useful tool to monitor cellular activities in living systems. It is often necessary in cancer research or experimental research to quantify the dividing capabilities of cells or the cell proliferation level when investigating manipulations of the cells or their environment. Manual quantification of fluorescence microscopic image is difficult because human is neither sensitive to fine differences in color intensity nor effective to count and average fluorescence level among cells. However, auto-quantification is not a straightforward problem to solve. As the sampling location of the microscopy changes, the amount of cells in individual microscopic images varies, which makes simple measurement methods such as the sum of stain intensity values or the total number of positive stain within each image inapplicable. Thus, automated quantification with robust cell segmentation techniques is required. Results An automated quantification system with robust cell segmentation technique are presented. The experimental results in application to monitor cellular replication activities show that the quantitative score is promising to represent the cell replication level, and scores for images from different cell replication groups are demonstrated to be statistically significantly different using ANOVA, LSD and Tukey HSD tests (p-value Conclusion A robust automated quantification method of live cell imaging is built to measure the cell replication level, providing a robust quantitative analysis system in fluorescent live cell imaging. In addition, the presented unsupervised entropy based cell segmentation for live cell images is demonstrated to be also applicable for nuclear segmentation of IHC tissue images.

  8. Kinase Activity Studied in Living Cells Using an Immunoassay

    Science.gov (United States)

    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…

  9. Biomimetic silica encapsultation of living cells

    Science.gov (United States)

    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. Classroom as Reality: Demonstrating Campaign Effects through Live Simulation

    Science.gov (United States)

    Coffey, Daniel J.; Miller, William J.; Feuerstein, Derek

    2011-01-01

    Scholastic research has demonstrated that when conducted properly, active learning exercises are successful at increasing student awareness, student interest, and knowledge retention. Face-to-face simulations, in particular, have been demonstrated to add positively to classrooms focusing on comparative politics, international relations, public…

  11. Synthetic analog computation in living cells.

    Science.gov (United States)

    Daniel, Ramiz; Rubens, Jacob R; Sarpeshkar, Rahul; Lu, Timothy K

    2013-05-30

    A central goal of synthetic biology is to achieve multi-signal integration and processing in living cells for diagnostic, therapeutic and biotechnology applications. Digital logic has been used to build small-scale circuits, but other frameworks may be needed for efficient computation in the resource-limited environments of cells. Here we demonstrate that synthetic analog gene circuits can be engineered to execute sophisticated computational functions in living cells using just three transcription factors. Such synthetic analog gene circuits exploit feedback to implement logarithmically linear sensing, addition, ratiometric and power-law computations. The circuits exhibit Weber's law behaviour as in natural biological systems, operate over a wide dynamic range of up to four orders of magnitude and can be designed to have tunable transfer functions. Our circuits can be composed to implement higher-order functions that are well described by both intricate biochemical models and simple mathematical functions. By exploiting analog building-block functions that are already naturally present in cells, this approach efficiently implements arithmetic operations and complex functions in the logarithmic domain. Such circuits may lead to new applications for synthetic biology and biotechnology that require complex computations with limited parts, need wide-dynamic-range biosensing or would benefit from the fine control of gene expression.

  12. Interactions between semiconductor nanowires and living cells.

    Science.gov (United States)

    Prinz, Christelle N

    2015-06-17

    Semiconductor nanowires are increasingly used for biological applications and their small dimensions make them a promising tool for sensing and manipulating cells with minimal perturbation. In order to interface cells with nanowires in a controlled fashion, it is essential to understand the interactions between nanowires and living cells. The present paper reviews current progress in the understanding of these interactions, with knowledge gathered from studies where living cells were interfaced with vertical nanowire arrays. The effect of nanowires on cells is reported in terms of viability, cell-nanowire interface morphology, cell behavior, changes in gene expression as well as cellular stress markers. Unexplored issues and unanswered questions are discussed.

  13. Diffusion inside living human cells

    DEFF Research Database (Denmark)

    Leijnse, N.; Jeon, J. -H.; Loft, Steffen

    2012-01-01

    of the cell or within the nucleus. Also, granules in cells which are stressed by intense laser illumination or which have attached to a surface for a long period of time move in a more restricted fashion than those within healthy cells. For granules diffusing in healthy cells, in regions away from the cell...... cells. For these cells the exact diffusional pattern of a particular granule depends on the physiological state of the cell and on the localization of the granule within the cytoplasm. Granules located close to the actin rich periphery of the cell move less than those located towards to the center...

  14. A Good Neighborhood for Cells: Bioreactor Demonstration System (BDS-05)

    Science.gov (United States)

    Chung, Leland W. K.; Goodwin, Thomas J. (Technical Monitor)

    2002-01-01

    Good neighborhoods help you grow. As with a city, the lives of a cell are governed by its neighborhood connections Connections that do not work are implicated in a range of diseases. One of those connections - between prostate cancer and bone cells - will be studied on STS-107 using the Bioreactor Demonstration System (BDS-05). To improve the prospects for finding novel therapies, and to identify biomarkers that predict disease progression, scientists need tissue models that behave the same as metastatic or spreading cancer. This is one of several NASA-sponsored lines of cell science research that use the microgravity environment of orbit in an attempt to grow lifelike tissue models for health research. As cells replicate, they "self associate" to form a complex matrix of collagens, proteins, fibers, and other structures. This highly evolved microenvironment tells each cell who is next door, how it should grow arid into what shapes, and how to respond to bacteria, wounds, and other stimuli. Studying these mechanisms outside the body is difficult because cells do not easily self-associate outside a natural environment. Most cell cultures produce thin, flat specimens that offer limited insight into how cells work together. Ironically, growing cell cultures in the microgravity of space produces cell assemblies that more closely resemble what is found in bodies on Earth. NASA's Bioreactor comprises a miniature life support system and a rotating vessel containing cell specimens in a nutrient medium. Orbital BDS experiments that cultured colon and prostate cancers have been highly promising.

  15. Live cell refractometry using microfluidic devices.

    Science.gov (United States)

    Lue, Niyom; Popescu, Gabriel; Ikeda, Takahiro; Dasari, Ramachandra R; Badizadegan, Kamran; Feld, Michael S

    2006-09-15

    Using Hilbert phase microscopy for extracting quantitative phase images, we measured the average refractive index associated with live cells in culture. To decouple the contributions to the phase signal from the cell refractive index and thickness, we confined the cells in microchannels. The results are confirmed by comparison with measurements of spherical cells in suspension.

  16. Live Cell Characterization of DNA Aggregation Delivered through Lipofection.

    Science.gov (United States)

    Mieruszynski, Stephen; Briggs, Candida; Digman, Michelle A; Gratton, Enrico; Jones, Mark R

    2015-05-27

    DNA trafficking phenomena, such as information on where and to what extent DNA aggregation occurs, have yet to be fully characterised in the live cell. Here we characterise the aggregation of DNA when delivered through lipofection by applying the Number and Brightness (N&B) approach. The N&B analysis demonstrates extensive aggregation throughout the live cell with DNA clusters in the extremity of the cell and peri-nuclear areas. Once within the nucleus aggregation had decreased 3-fold. In addition, we show that increasing serum concentration of cell media results in greater cytoplasmic aggregation. Further, the effects of the DNA fragment size on aggregation was explored, where larger DNA constructs exhibited less aggregation. This study demonstrates the first quantification of DNA aggregation when delivered through lipofection in live cells. In addition, this study has presents a model for alternative uses of this imaging approach, which was originally developed to study protein oligomerization and aggregation.

  17. Quantification of nanowire uptake by live cells

    KAUST Repository

    Margineanu, Michael B.

    2015-01-01

    attempts have been made at tagging and investigating their interaction with living cells. In this study, magnetic iron nanowires with an iron oxide layer are coated with (3-Aminopropyl)triethoxysilane (APTES), and subsequently labeled with a fluorogenic p

  18. RON kinase isoforms demonstrate variable cell motility in normal cells.

    Science.gov (United States)

    Greenbaum, Alissa; Rajput, Ashwani; Wan, Guanghua

    2016-09-01

    Aberrant RON (Recepteur d'Origine Nantais) tyrosine kinase activation causes the epithelial cell to evade normal growth pathways, resulting in unregulated cell proliferation, increased cell motility and decreased apoptosis. Wildtype (wt) RON has been shown to play a role in metastasis of epithelial malignancies. It presents an important potential therapeutic target for colorectal, breast, gastric and pancreatic cancer. Little is known about functional differences amongst RON isoforms RON155, RON160 and RON165. The purpose of this study was to determine the effect of various RON kinase isoforms on cell motility. Cell lines with stable expression of wtRON were generated by inserting the coding region of RON in pTagRFP (tagged red fluorescence protein plasmid). The expression constructs of RON variants (RON155, RON160 and RON165) were generated by creating a mutagenesis-based wtRON-pTag RFP plasmid and stably transfected into HEK 293 cells. The wound closure scratch assay was used to investigate the effect on cell migratory capacity of wild type RON and its variants. RON transfected cells demonstrated increased cell motility compared to HEK293 control cells. RON165 cell motility was significantly increased compared to RON160 (mean percentage of wound covered 37.37% vs. 32.40%; p = 0.03). RON tyrosine kinase isoforms have variable cell motility. This may reflect a difference in the behavior of malignant epithelial cells and their capacity for metastasis.

  19. Polyvalent Display of Biomolecules on Live Cells.

    Science.gov (United States)

    Shi, Peng; Zhao, Nan; Lai, Jinping; Coyne, James; Gaddes, Erin R; Wang, Yong

    2018-06-04

    Surface display of biomolecules on live cells offers new opportunities to treat human diseases and perform basic studies. Existing methods are primarily focused on monovalent functionalization, that is, the display of single biomolecules across the cell surface. Here we show that the surface of live cells can be functionalized to display polyvalent biomolecular structures through two-step reactions under physiological conditions. This polyvalent functionalization enables the cell surface to recognize the microenvironment one order of magnitude more effectively than with monovalent functionalization. Thus, polyvalent display of biomolecules on live cells holds great potential for various biological and biomedical applications. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. Quantification of nanowire uptake by live cells

    KAUST Repository

    Margineanu, Michael B.

    2015-05-01

    Nanostructures fabricated by different methods have become increasingly important for various applications at the cellular level. In order to understand how these nanostructures “behave” and for studying their internalization kinetics, several attempts have been made at tagging and investigating their interaction with living cells. In this study, magnetic iron nanowires with an iron oxide layer are coated with (3-Aminopropyl)triethoxysilane (APTES), and subsequently labeled with a fluorogenic pH-dependent dye pHrodo™ Red, covalently bound to the aminosilane surface. Time-lapse live imaging of human colon carcinoma HCT 116 cells interacting with the labeled iron nanowires is performed for 24 hours. As the pHrodo™ Red conjugated nanowires are non-fluorescent outside the cells but fluoresce brightly inside, internalized nanowires are distinguished from non-internalized ones and their behavior inside the cells can be tracked for the respective time length. A machine learning-based computational framework dedicated to automatic analysis of live cell imaging data, Cell Cognition, is adapted and used to classify cells with internalized and non-internalized nanowires and subsequently determine the uptake percentage by cells at different time points. An uptake of 85 % by HCT 116 cells is observed after 24 hours incubation at NW-to-cell ratios of 200. While the approach of using pHrodo™ Red for internalization studies is not novel in the literature, this study reports for the first time the utilization of a machine-learning based time-resolved automatic analysis pipeline for quantification of nanowire uptake by cells. This pipeline has also been used for comparison studies with nickel nanowires coated with APTES and labeled with pHrodo™ Red, and another cell line derived from the cervix carcinoma, HeLa. It has thus the potential to be used for studying the interaction of different types of nanostructures with potentially any live cell types.

  1. Nanometer scale thermometry in a living cell

    Science.gov (United States)

    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

  2. Laser-Raman spectroscopy of living cells

    International Nuclear Information System (INIS)

    Webb, S.J.

    1980-01-01

    Investigations into the laser-Raman shift spectra of bacterial and mammalian cells have revealed that many Raman lines observed at 4-6 K, do not appear in the spectra of cells held at 300 K. At 300 K, Raman activity, at set frequencies, is observed only when the cells are metabolically active; however, the actual live cell spectrum, between 0 and 3400 cm -1 , has been found to alter in a specific way with time as the cells' progress through their life cycles. Lines above 300 cm -1 , from in vivo Raman active states, appear to shift to higher wave numbers whereas those below 300 cm -1 seem to shift to lower ones. The transient nature of many shift lines observed and the intensity of them when present in the spectrum indicates that, in, vivo, a metabolically induced condensation of closely related states occurs at a set time in the life of a living cell. In addition, the calculated ratio between the intensities of Stokes and anti-Stokes lines observed suggests that the metabolically induced 'collective' Raman active states are produced, in vivo, by non thermal means. It appears, therefore, that the energetics of the well established cell 'time clock' may be studied by laser-Raman spectroscopy; moreover, Raman spectroscopy may yield a new type of information regarding the physics of such biological phenomena as nutrition, virus infection and oncogenesis. (orig.)

  3. Social learning in nest-building birds watching live-streaming video demonstrators.

    Science.gov (United States)

    Guillette, Lauren M; Healy, Susan D

    2018-02-13

    Determining the role that social learning plays in construction behaviours, such as nest building or tool manufacture, could be improved if more experimental control could be gained over the exact public information that is provided by the demonstrator, to the observing individual. Using video playback allows the experimenter to choose what information is provided, but will only be useful in determining the role of social learning if observers attend to, and learn from, videos in a manner that is similar to live demonstration. The goal of the current experiment was to test whether live-streamed video presentations of nest building by zebra finches Taeniopygia guttata would lead observers to copy the material choice demonstrated to them. Here, males that had not previously built a nest were given an initial preference test between materials of two colours. Those observers then watched live-stream footage of a familiar demonstrator building a nest with material of the colour that the observer did not prefer. After this experience, observers were given the chance to build a nest with materials of the two colours. Although two-thirds of the observer males preferred material of the demonstrated colour after viewing the demonstrator build a nest with material of that colour more than they had previously, their preference for the demonstrated material was not as strong as that of observers that had viewed live demonstrator builders in a previous experiment. Our results suggest researchers should proceed with caution before using video demonstration in tests of social learning. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  4. The radiation effects on the living cell

    International Nuclear Information System (INIS)

    Sage, E.; Dutrillaux, B.; Soussi, Th.; Boiteux, S.; Lopez, B.; Feunteun, J.

    1999-06-01

    This publication is a presentation of particular points discussed during the colloquium of the 15-18 june 1999, for which scientific researches are performed at the CEA/CNRS. They deal with the radiobiology, for the radiation effects on living matter; with the DNA, for the knowledge and repair mechanisms on cells submitted to ionizing radiations; with the exposition to UV in correlation with neoplasms; with the P53 gene which is a tumour suppressor. (A.L.B.)

  5. Living labeling techniques of mesenchymal stem cells

    International Nuclear Information System (INIS)

    Dong Qingyu; Chen Li

    2007-01-01

    Mesenchymal stem cells (MSCs) are well known for their self-renew and multi- differentiation potentiality. With the transplantation of the MSCs which can promote the regeneration and repair of the injured tissue, a new route for the treatment of dieases is hopeful to be effective. To trace the distribution, migration, proliferation and differentiation of the implanted MSCs, there need effective labeling techniques, especially living labeling techniques. (authors)

  6. Outcome from 5-year live surgical demonstrations in urinary stone treatment: are outcomes compromised?

    NARCIS (Netherlands)

    Legemate, Jaap D.; Zanetti, Stefano P.; Baard, Joyce; Kamphuis, Guido M.; Montanari, Emanuele; Traxer, Olivier; de la Rosette, Jean Jmch

    2017-01-01

    To compare intra- and post-operative outcomes of endourological live surgical demonstrations (LSDs) and routine surgical practice (RSP) for urinary stones. Consecutive ureterorenoscopic (URS) and percutaneous (PNL) urinary stone procedures over a 5-year period were reviewed. Procedures were divided

  7. PeakForce Tapping resolves individual microvilli on living cells.

    Science.gov (United States)

    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. © 2015 The Authors Journal of Molecular Recognition Published by John Wiley & Sons Ltd.

  8. Circumventing photodamage in live-cell microscopy

    Science.gov (United States)

    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

  9. Thermodynamics of protein destabilization in live cells.

    Science.gov (United States)

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

    2015-10-06

    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.

  10. Raman microscopy of individual living human embryonic stem cells

    DEFF Research Database (Denmark)

    Novikov, Sergey M.; Beermann, Jonas; Bozhevolnyi, Sergey I.

    2010-01-01

    We demonstrate the possibility of mapping the distribution of different biomolecules in living human embryonic stem cells grown on glass substrates, without the need for fluorescent markers. In our work we improve the quality of measurements by finding a buffer that gives low fluorescence, growing...... cells on glass substrates (whose Raman signals are relatively weak compared to that of the cells) and having the backside covered with gold to improve the image contrast under direct white light illumination. The experimental setup used for Raman microscopy is the commercially available confocal...

  11. Direct and dynamic detection of HIV-1 in living cells.

    Directory of Open Access Journals (Sweden)

    Jonas Helma

    Full Text Available In basic and applied HIV research, reliable detection of viral components is crucial to monitor progression of infection. While it is routine to detect structural viral proteins in vitro for diagnostic purposes, it previously remained impossible to directly and dynamically visualize HIV in living cells without genetic modification of the virus. Here, we describe a novel fluorescent biosensor to dynamically trace HIV-1 morphogenesis in living cells. We generated a camelid single domain antibody that specifically binds the HIV-1 capsid protein (CA at subnanomolar affinity and fused it to fluorescent proteins. The resulting fluorescent chromobody specifically recognizes the CA-harbouring HIV-1 Gag precursor protein in living cells and is applicable in various advanced light microscopy systems. Confocal live cell microscopy and super-resolution microscopy allowed detection and dynamic tracing of individual virion assemblies at the plasma membrane. The analysis of subcellular binding kinetics showed cytoplasmic antigen recognition and incorporation into virion assembly sites. Finally, we demonstrate the use of this new reporter in automated image analysis, providing a robust tool for cell-based HIV research.

  12. 2011 ESTCP Live Site Demonstrations, Vallejo CA, ESTCP MR-1165, Demonstration Data Report, Former Mare Island Naval Shipyard, MTADS Discrimination Array, (TEMTADS) Survey

    Science.gov (United States)

    2012-04-05

    Naval Research Laboratory Washington, DC 20375-5320 NRL/MR/6110--12-9397 2011 ESTCP Live Site Demonstrations Vallejo , CA ESTCP MR-1165 Demonstration...b. ABSTRACT c. THIS PAGE 18. NUMBER OF PAGES 17. LIMITATION OF ABSTRACT 2011 ESTCP Live Site Demonstrations Vallejo , CA ESTCP MR-1165 Demonstration...Hertz IVS Instrument Verification Strip (f)MINSY (former) Mare Island Naval Shipyard, located in Vallejo , CA MR Munitions Response MTADS Multi-sensor

  13. Data Analysis for ARRA Early Fuel Cell Market Demonstrations (Presentation)

    Energy Technology Data Exchange (ETDEWEB)

    Kurtz, J.; Wipke, K.; Sprik, S.; Ramsden, T.

    2010-05-01

    Presentation about ARRA Early Fuel Cell Market Demonstrations, including an overview of the ARRE Fuel Cell Project, the National Renewable Energy Laboratory's data analysis objectives, deployment composite data products, and planned analyses.

  14. Axial tomography in live cell laser microscopy

    Science.gov (United States)

    Richter, Verena; Bruns, Sarah; Bruns, Thomas; Weber, Petra; Wagner, Michael; Cremer, Christoph; Schneckenburger, Herbert

    2017-09-01

    Single cell microscopy in a three-dimensional (3-D) environment is reported. Cells are grown in an agarose culture gel, located within microcapillaries and observed from different sides after adaptation of an innovative device for sample rotation. Thus, z-stacks can be recorded by confocal microscopy in different directions and used for illustration in 3-D. This gives additional information, since cells or organelles that appear superimposed in one direction, may be well resolved in another one. The method is tested and validated with single cells expressing a membrane or a mitochondrially associated green fluorescent protein, or cells accumulating fluorescent quantum dots. In addition, axial tomography supports measurements of cellular uptake and distribution of the anticancer drug doxorubicin in the nucleus (2 to 6 h after incubation) or the cytoplasm (24 h). This paper discusses that upon cell rotation an enhanced optical resolution in lateral direction compared to axial direction can be utilized to obtain an improved effective 3-D resolution, which represents an important step toward super-resolution microscopy of living cells.

  15. On strain and stress in living cells

    Science.gov (United States)

    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

  16. Cyborg cells: functionalisation of living cells with polymers and nanomaterials.

    Science.gov (United States)

    Fakhrullin, Rawil F; Zamaleeva, Alsu I; Minullina, Renata T; Konnova, Svetlana A; Paunov, Vesselin N

    2012-06-07

    Living cells interfaced with a range of polyelectrolyte coatings, magnetic and noble metal nanoparticles, hard mineral shells and other complex nanomaterials can perform functions often completely different from their original specialisation. Such "cyborg cells" are already finding a range of novel applications in areas like whole cell biosensors, bioelectronics, toxicity microscreening, tissue engineering, cell implant protection and bioanalytical chemistry. In this tutorial review, we describe the development of novel methods for functionalisation of cells with polymers and nanoparticles and comment on future advances in this technology in the light of other literature approaches. We review recent studies on the cell viability and function upon direct deposition of nanoparticles, coating with polyelectrolytes, polymer assisted assembly of nanomaterials and hard shells on the cell surface. The cell toxicity issues are considered for many practical applications in terms of possible adverse effects of the deposited polymers, polyelectrolytes and nanoparticles on the cell surface.

  17. Microencapsulating and Banking Living Cells for Cell-Based Medicine

    Directory of Open Access Journals (Sweden)

    Wujie Zhang

    2011-01-01

    Full Text Available A major challenge to the eventual success of the emerging cell-based medicine such as tissue engineering, regenerative medicine, and cell transplantation is the limited availability of the desired cell sources. This challenge can be addressed by cell microencapsulation to overcome the undesired immune response (i.e., to achieve immunoisolation so that non-autologous cells can be used to treat human diseases, and by cell/tissue preservation to bank living cells for wide distribution to end users so that they are readily available when needed in the future. This review summarizes the status quo of research in both cell microencapsulation and banking the microencapsulated cells. It is concluded with a brief outlook of future research directions in this important field.

  18. Recent advances in live cell imaging of hepatoma cells

    Science.gov (United States)

    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

  19. Raman spectroscopy for grading of live osteosarcoma cells.

    Science.gov (United States)

    Chiang, Yi-Hung; Wu, Stewart H; Kuo, Yi-Chun; Chen, How-Foo; Chiou, Arthur; Lee, Oscar K

    2015-04-18

    Osteosarcoma is the most common primary malignant bone tumor, and the grading of osteosarcoma cells relies on traditional histopathology and molecular biology methods, which require RNA extraction, protein isolation and immunohistological staining. All these methods require cell isolation, lysis or fixation, which is time-consuming and requires certain amount of tumor specimen. In this study, we report the use of Raman spectroscopy for grading of malignant osteosarcoma cells. We demonstrate that, based on the detection of differential production of mineral species, Raman spectroscopy can be used as a live cell analyzer to accurately assess the grades of osteosarcoma cells by evaluating their mineralization levels. Mineralization level was assessed by measuring amount of hydroxyapatite (HA), which is highly expressed in mature osteoblasts, but not in poorly differentiated osteosarcoma cell or mesenchymal stem cells, the putative cell-of-origin of osteosarcoma. We found that under Raman spectroscopy, the level of HA production was high in MG-63 cells, which are low-grade. Moreover, hydroxyapatite production was low in high-grade osteosarcoma cells such as 143B and SaOS2 cells (p Raman spectroscopy for the measurement of HA production by the protocol reported in this study may serve as a useful tool to rapidly and accurately assess the degree of malignancy in osteosarcoma cells in a label-free manner. Such application may shorten the period of pathological diagnosis and may benefit patients who are inflicted with osteosarcoma.

  20. Deep Learning Automates the Quantitative Analysis of Individual Cells in Live-Cell Imaging Experiments.

    Science.gov (United States)

    Van Valen, David A; Kudo, Takamasa; Lane, Keara M; Macklin, Derek N; Quach, Nicolas T; DeFelice, Mialy M; Maayan, Inbal; Tanouchi, Yu; Ashley, Euan A; Covert, Markus W

    2016-11-01

    Live-cell imaging has opened an exciting window into the role cellular heterogeneity plays in dynamic, living systems. A major critical challenge for this class of experiments is the problem of image segmentation, or determining which parts of a microscope image correspond to which individual cells. Current approaches require many hours of manual curation and depend on approaches that are difficult to share between labs. They are also unable to robustly segment the cytoplasms of mammalian cells. Here, we show that deep convolutional neural networks, a supervised machine learning method, can solve this challenge for multiple cell types across the domains of life. We demonstrate that this approach can robustly segment fluorescent images of cell nuclei as well as phase images of the cytoplasms of individual bacterial and mammalian cells from phase contrast images without the need for a fluorescent cytoplasmic marker. These networks also enable the simultaneous segmentation and identification of different mammalian cell types grown in co-culture. A quantitative comparison with prior methods demonstrates that convolutional neural networks have improved accuracy and lead to a significant reduction in curation time. We relay our experience in designing and optimizing deep convolutional neural networks for this task and outline several design rules that we found led to robust performance. We conclude that deep convolutional neural networks are an accurate method that require less curation time, are generalizable to a multiplicity of cell types, from bacteria to mammalian cells, and expand live-cell imaging capabilities to include multi-cell type systems.

  1. Modulation of protein properties in living cells using nanobodies.

    Science.gov (United States)

    Kirchhofer, Axel; Helma, Jonas; Schmidthals, Katrin; Frauer, Carina; Cui, Sheng; Karcher, Annette; Pellis, Mireille; Muyldermans, Serge; Casas-Delucchi, Corella S; Cardoso, M Cristina; Leonhardt, Heinrich; Hopfner, Karl-Peter; Rothbauer, Ulrich

    2010-01-01

    Protein conformation is critically linked to function and often controlled by interactions with regulatory factors. Here we report the selection of camelid-derived single-domain antibodies (nanobodies) that modulate the conformation and spectral properties of the green fluorescent protein (GFP). One nanobody could reversibly reduce GFP fluorescence by a factor of 5, whereas its displacement by a second nanobody caused an increase by a factor of 10. Structural analysis of GFP-nanobody complexes revealed that the two nanobodies induce subtle opposing changes in the chromophore environment, leading to altered absorption properties. Unlike conventional antibodies, the small, stable nanobodies are functional in living cells. Nanobody-induced changes were detected by ratio imaging and used to monitor protein expression and subcellular localization as well as translocation events such as the tamoxifen-induced nuclear localization of estrogen receptor. This work demonstrates that protein conformations can be manipulated and studied with nanobodies in living cells.

  2. Demonstration of Passive Fuel Cell Thermal Management Technology

    Science.gov (United States)

    Burke, Kenneth A.; Jakupca, Ian; Colozza, Anthony; Wynne, Robert; Miller, Michael; Meyer, Al; Smith, William

    2012-01-01

    The NASA Glenn Research Center is developing advanced passive thermal management technology to reduce the mass and improve the reliability of space fuel cell systems for the NASA Exploration program. The passive thermal management system relies on heat conduction within highly thermally conductive cooling plates to move the heat from the central portion of the cell stack out to the edges of the fuel cell stack. Using the passive approach eliminates the need for a coolant pump and other cooling loop components within the fuel cell system which reduces mass and improves overall system reliability. Previous development demonstrated the performance of suitable highly thermally conductive cooling plates and integrated heat exchanger technology to collect the heat from the cooling plates (Ref. 1). The next step in the development of this passive thermal approach was the demonstration of the control of the heat removal process and the demonstration of the passive thermal control technology in actual fuel cell stacks. Tests were run with a simulated fuel cell stack passive thermal management system outfitted with passive cooling plates, an integrated heat exchanger and two types of cooling flow control valves. The tests were run to demonstrate the controllability of the passive thermal control approach. Finally, successful demonstrations of passive thermal control technology were conducted with fuel cell stacks from two fuel cell stack vendors.

  3. Cooperation between epithelial cells demonstrated by potassium transfer

    International Nuclear Information System (INIS)

    Ledbetter, M.L.; Young, G.J.; Wright, E.R.

    1986-01-01

    Junction-mediated communication can be measured in fibroblast cultures by determining the ability of mixed cultures of cells sensitive and resistant to ouabain to concentrate K+ in the presence of ouabain. We now report the extension of this assay procedure to cultured epithelial cells. Hamster kidney (HaK) cells maintain their ability to concentrate K+ in ouabain at levels inhibitory to dog kidney (MDCK) cells. When HaK and MDCK cells were cultured together in ouabain-containing medium, the K+ (measured as 86Rb+) in the mixed population was greater than expected if the cells were not interacting. The degree of enhancement, expressed as index of cooperation, depended on the numbers of cells in the cultures, their opportunity for cell-to-cell contact, and (above a certain permissive level) the concentration of ouabain. As with other cell types, protein synthesis in MDCK cells depends on maintenance of cell K+. Autoradiography of cells incubated with [3H]leucine demonstrated that MDCK cells in ouabain-treated mixed cultures were able to synthesize proteins only when physically adjacent to HaK cells. The transmission of labeled nucleosides among the cells provides independent evidence of the phenomenon of cooperation, probably mediated by gap junctions. This system offers promise for investigation of stimuli modulating junctional communication

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

    Science.gov (United States)

    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.

  5. Osmotic Pressure, Bacterial Cell Walls, and Penicillin: A Demonstration.

    Science.gov (United States)

    Lennox, John E.

    1984-01-01

    An easily constructed apparatus that models the effect of penicillin on the structure of bacterial cells is described. Background information and procedures for using the apparatus during a classroom demonstration are included. (JN)

  6. An early Oligocene fossil demonstrates treeshrews are slowly evolving "living fossils".

    Science.gov (United States)

    Li, Qiang; Ni, Xijun

    2016-01-14

    Treeshrews are widely considered a "living model" of an ancestral primate, and have long been called "living fossils". Actual fossils of treeshrews, however, are extremely rare. We report a new fossil species of Ptilocercus treeshrew recovered from the early Oligocene (~34 Ma) of China that represents the oldest definitive fossil record of the crown group of treeshrews and nearly doubles the temporal length of their fossil record. The fossil species is strikingly similar to the living Ptilocercus lowii, a species generally recognized as the most plesiomorphic extant treeshrew. It demonstrates that Ptilocercus treeshrews have undergone little evolutionary change in their morphology since the early Oligocene. Morphological comparisons and phylogenetic analysis support the long-standing idea that Ptilocercus treeshrews are morphologically conservative and have probably retained many characters present in the common stock that gave rise to archontans, which include primates, flying lemurs, plesiadapiforms and treeshrews. This discovery provides an exceptional example of slow morphological evolution in a mammalian group over a period of 34 million years. The persistent and stable tropical environment in Southeast Asia through the Cenozoic likely played a critical role in the survival of such a morphologically conservative lineage.

  7. Live 3D TEE demonstrates and guides the management of prosthetic mitral valve obstruction.

    Science.gov (United States)

    Chahal, Mangeet; Pandya, Utpal; Adlakha, Satjit; Khouri, Samer J

    2011-08-01

    A 43-year-old woman, with a remote history of rheumatic mitral stenosis and a St. Jude prosthetic mitral valve replacement, presented with shortness of breath and palpitations, shortly after a long flight. On admission, atrial fibrillation with a rapid ventricular response was noted in the setting of a long history of noncompliance with her anticoagulation. Transesophageal echocardiography (TEE) demonstrated multiple laminated thrombi in the left atrial appendage. Live three-dimensional (3D) TEE confirmed this diagnosis and demonstrated an immobile posterior leaflet of the mitral prosthesis, which had direct implications in her management. She successfully underwent surgery for mitral valve replacement, left atrial appendage ligation, and a Maze procedure on the following day. The multiple thrombi within the atrial appendage were confirmed intraoperatively and pannus formation was determined to be the etiology of the leaflet immobility. © 2011, Wiley Periodicals, Inc.

  8. Nanomembrane-Based, Thermal-Transport Biosensor for Living Cells

    KAUST Repository

    Elafandy, Rami T.; AbuElela, Ayman; Mishra, Pawan; Janjua, Bilal; Oubei, Hassan M.; Buttner, Ulrich; Majid, Mohammed Abdul; Ng, Tien Khee; Merzaban, Jasmeen; Ooi, Boon S.

    2016-01-01

    Knowledge of materials' thermal-transport properties, conductivity and diffusivity, is crucial for several applications within areas of biology, material science and engineering. Specifically, a microsized, flexible, biologically integrated thermal transport sensor is beneficial to a plethora of applications, ranging across plants physiological ecology and thermal imaging and treatment of cancerous cells, to thermal dissipation in flexible semiconductors and thermoelectrics. Living cells pose extra challenges, due to their small volumes and irregular curvilinear shapes. Here a novel approach of simultaneously measuring thermal conductivity and diffusivity of different materials and its applicability to single cells is demonstrated. This technique is based on increasing phonon-boundary-scattering rate in nanomembranes, having extremely low flexural rigidities, to induce a considerable spectral dependence of the bandgap-emission over excitation-laser intensity. It is demonstrated that once in contact with organic or inorganic materials, the nanomembranes' emission spectrally shift based on the material's thermal diffusivity and conductivity. This NM-based technique is further applied to differentiate between different types and subtypes of cancer cells, based on their thermal-transport properties. It is anticipated that this novel technique to enable an efficient single-cell thermal targeting, allow better modeling of cellular thermal distribution and enable novel diagnostic techniques based on variations of single-cell thermal-transport properties.

  9. Nanomembrane-Based, Thermal-Transport Biosensor for Living Cells

    KAUST Repository

    Elafandy, Rami T.

    2016-11-23

    Knowledge of materials\\' thermal-transport properties, conductivity and diffusivity, is crucial for several applications within areas of biology, material science and engineering. Specifically, a microsized, flexible, biologically integrated thermal transport sensor is beneficial to a plethora of applications, ranging across plants physiological ecology and thermal imaging and treatment of cancerous cells, to thermal dissipation in flexible semiconductors and thermoelectrics. Living cells pose extra challenges, due to their small volumes and irregular curvilinear shapes. Here a novel approach of simultaneously measuring thermal conductivity and diffusivity of different materials and its applicability to single cells is demonstrated. This technique is based on increasing phonon-boundary-scattering rate in nanomembranes, having extremely low flexural rigidities, to induce a considerable spectral dependence of the bandgap-emission over excitation-laser intensity. It is demonstrated that once in contact with organic or inorganic materials, the nanomembranes\\' emission spectrally shift based on the material\\'s thermal diffusivity and conductivity. This NM-based technique is further applied to differentiate between different types and subtypes of cancer cells, based on their thermal-transport properties. It is anticipated that this novel technique to enable an efficient single-cell thermal targeting, allow better modeling of cellular thermal distribution and enable novel diagnostic techniques based on variations of single-cell thermal-transport properties.

  10. Raman microscopy of individual living human embryonic stem cells

    Science.gov (United States)

    Novikov, S. M.; Beermann, J.; Bozhevolnyi, S. I.; Harkness, L. M.; Kassem, M.

    2010-04-01

    We demonstrate the possibility of mapping the distribution of different biomolecules in living human embryonic stem cells grown on glass substrates, without the need for fluorescent markers. In our work we improve the quality of measurements by finding a buffer that gives low fluorescence, growing cells on glass substrates (whose Raman signals are relatively weak compared to that of the cells) and having the backside covered with gold to improve the image contrast under direct white light illumination. The experimental setup used for Raman microscopy is the commercially available confocal scanning Raman microscope (Alpha300R) from Witec and sub-μm spatially resolved Raman images were obtained using a 532 nm excitation wavelength.

  11. Local Nucleosome Dynamics Facilitate Chromatin Accessibility in Living Mammalian Cells

    Directory of Open Access Journals (Sweden)

    Saera Hihara

    2012-12-01

    Full Text Available Genome information, which is three-dimensionally organized within cells as chromatin, is searched and read by various proteins for diverse cell functions. Although how the protein factors find their targets remains unclear, the dynamic and flexible nature of chromatin is likely crucial. Using a combined approach of fluorescence correlation spectroscopy, single-nucleosome imaging, and Monte Carlo computer simulations, we demonstrate local chromatin dynamics in living mammalian cells. We show that similar to interphase chromatin, dense mitotic chromosomes also have considerable chromatin accessibility. For both interphase and mitotic chromatin, we observed local fluctuation of individual nucleosomes (∼50 nm movement/30 ms, which is caused by confined Brownian motion. Inhibition of these local dynamics by crosslinking impaired accessibility in the dense chromatin regions. Our findings show that local nucleosome dynamics drive chromatin accessibility. We propose that this local nucleosome fluctuation is the basis for scanning genome information.

  12. Simultaneous detection of mRNA and protein stem cell markers in live cells

    Directory of Open Access Journals (Sweden)

    Bao Gang

    2009-04-01

    Full Text Available Abstract Background Biological studies and medical application of stem cells often require the isolation of stem cells from a mixed cell population, including the detection of cancer stem cells in tumor tissue, and isolation of induced pluripotent stem cells after eliciting the expression of specific genes in adult cells. Here we report the detection of Oct-4 mRNA and SSEA-1 protein in live carcinoma stem cells using respectively molecular beacon and dye-labeled antibody, aiming to establish a new method for stem cells detection and isolation. Results Quantification of Oct-4 mRNA and protein in P19 mouse carcinoma stem cells using respectively RT-PCR and immunocytochemistry confirmed that their levels drastically decreased after differentiation. To visualize Oct-4 mRNA in live stem cells, molecular beacons were designed, synthesized and validated, and the detection specificity was confirmed using control studies. We found that the fluorescence signal from Oct-4-targeting molecular beacons provides a clear discrimination between undifferentiated and retinoic acid-induced differentiated cells. Using deconvolution fluorescence microscopy, Oct-4 mRNAs were found to reside on one side of the cytosol. We demonstrated that, using a combination of Oct-4 mRNA-targeting molecular beacon with SSEA-1 antibody in flow cytometric analysis, undifferentiated stem cells can be clearly distinguished from differentiated cells. We revealed that Oct-4 targeting molecular beacons do not seem to affect stem cell biology. Conclusion Molecular beacons have the potential to provide a powerful tool for highly specific detection and isolation of stem cells, including cancer stem cells and induced pluripotent stem (iPS cells without disturbing cell physiology. It is advantageous to perform simultaneous detection of intracellular (mRNA and cell-surface (protein stem cell markers in flow cytometric analysis, which may lead to high detection sensitivity and efficiency.

  13. How we live and why we die the secret lives of cells

    CERN Document Server

    Wolpert, Lewis

    2009-01-01

    Cells are the basis of all life in the universe. Our bodies are made up of billions of them: an incredibly complex society that governs everything, from movement to memory and imagination. When we age, it is because our cells slow down; when we get ill, it is because our cells mutate or stop working. In "How We Live and Why we Die", Wolpert provides a clear explanation of the science that underpins our lives. He explains how our bodies function and how we derived from a single cell - the embryo. He examines the science behind the topics that are much discussed but rarely understood - stem-cell research, cloning, DNA - and explains how all life evolved from just one cell. Lively and passionate, "How We Live and Why we Die" is an accessible guide to understanding the human body and, essentially, life itself.

  14. A cell transportation solution that preserves live circulating tumor cells in patient blood samples.

    Science.gov (United States)

    Stefansson, Steingrimur; Adams, Daniel L; Ershler, William B; Le, Huyen; Ho, David H

    2016-05-06

    Circulating tumor cells (CTCs) are typically collected into CellSave fixative tubes, which kills the cells, but preserves their morphology. Currently, the clinical utility of CTCs is mostly limited to their enumeration. More detailed investigation of CTC biology can be performed on live cells, but obtaining live CTCs is technically challenging, requiring blood collection into biocompatible solutions and rapid isolation which limits transportation options. To overcome the instability of CTCs, we formulated a sugar based cell transportation solution (SBTS) that stabilizes cell viability at ambient temperature. In this study we examined the long term viability of human cancer cell lines, primary cells and CTCs in human blood samples in the SBTS for transportation purposes. Four cell lines, 5 primary human cells and purified human PBMCs were tested to determine the viability of cells stored in the transportation solution at ambient temperature for up to 7 days. We then demonstrated viability of MCF-7 cells spiked into normal blood with SBTS and stored for up to 7 days. A pilot study was then run on blood samples from 3 patients with metastatic malignancies stored with or without SBTS for 6 days. CTCs were then purified by Ficoll separation/microfilter isolation and identified using CTC markers. Cell viability was assessed using trypan blue or CellTracker™ live cell stain. Our results suggest that primary/immortalized cell lines stored in SBTS remain ~90% viable for > 72 h. Further, MCF-7 cells spiked into whole blood remain viable when stored with SBTS for up to 7 days. Finally, live CTCs were isolated from cancer patient blood samples kept in SBTS at ambient temperature for 6 days. No CTCs were isolated from blood samples stored without SBTS. In this proof of principle pilot study we show that viability of cell lines is preserved for days using SBTS. Further, this solution can be used to store patient derived blood samples for eventual isolation of viable CTCs after

  15. A cell transportation solution that preserves live circulating tumor cells in patient blood samples

    International Nuclear Information System (INIS)

    Stefansson, Steingrimur; Adams, Daniel L.; Ershler, William B.; Le, Huyen; Ho, David H.

    2016-01-01

    Circulating tumor cells (CTCs) are typically collected into CellSave fixative tubes, which kills the cells, but preserves their morphology. Currently, the clinical utility of CTCs is mostly limited to their enumeration. More detailed investigation of CTC biology can be performed on live cells, but obtaining live CTCs is technically challenging, requiring blood collection into biocompatible solutions and rapid isolation which limits transportation options. To overcome the instability of CTCs, we formulated a sugar based cell transportation solution (SBTS) that stabilizes cell viability at ambient temperature. In this study we examined the long term viability of human cancer cell lines, primary cells and CTCs in human blood samples in the SBTS for transportation purposes. Four cell lines, 5 primary human cells and purified human PBMCs were tested to determine the viability of cells stored in the transportation solution at ambient temperature for up to 7 days. We then demonstrated viability of MCF-7 cells spiked into normal blood with SBTS and stored for up to 7 days. A pilot study was then run on blood samples from 3 patients with metastatic malignancies stored with or without SBTS for 6 days. CTCs were then purified by Ficoll separation/microfilter isolation and identified using CTC markers. Cell viability was assessed using trypan blue or CellTracker™ live cell stain. Our results suggest that primary/immortalized cell lines stored in SBTS remain ~90 % viable for > 72 h. Further, MCF-7 cells spiked into whole blood remain viable when stored with SBTS for up to 7 days. Finally, live CTCs were isolated from cancer patient blood samples kept in SBTS at ambient temperature for 6 days. No CTCs were isolated from blood samples stored without SBTS. In this proof of principle pilot study we show that viability of cell lines is preserved for days using SBTS. Further, this solution can be used to store patient derived blood samples for eventual isolation of viable CTCs

  16. Automated analysis of invadopodia dynamics in live cells

    Directory of Open Access Journals (Sweden)

    Matthew E. Berginski

    2014-07-01

    Full Text Available 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.

  17. Functional living biointerfaces to direct cell-material interaction

    OpenAIRE

    Rodrigo Navarro, Aleixandre

    2016-01-01

    [EN] This thesis deals with the development of a living biointerface between synthetic substrates and living cells to engineer cell-material interactions for tissue engineering purposes. This living biointerface is made of Lactococcus lactis, a non-pathogenic lactic bacteria widely used as starter in the dairy industry and, recently, in the expression of heterologous proteins in applications such as oral vaccine delivery or membrane-bound expression of proteins. L. lactis has been engine...

  18. Demonstration of a PC 25 Fuel Cell in Russia

    Energy Technology Data Exchange (ETDEWEB)

    John C. Trocciola; Thomas N. Pompa; Linda S. Boyd

    2004-09-01

    This project involved the installation of a 200kW PC25C{trademark} phosphoric-acid fuel cell power plant at Orgenergogaz, a Gazprom industrial site in Russia. In April 1997, a PC25C{trademark} was sold by ONSI Corporation to Orgenergogaz, a subsidiary of the Russian company ''Gazprom''. Due to instabilities in the Russian financial markets, at that time, the unit was never installed and started by Orgenergogaz. In October of 2001 International Fuel Cells (IFC), now known as UTC Fuel Cells (UTCFC), received a financial assistance award from the United States Department of Energy (DOE) entitled ''Demonstration of PC 25 Fuel Cell in Russia''. Three major tasks were part of this award: the inspection of the proposed site and system, start-up assistance, and installation and operation of the powerplant.

  19. Applications of chitosan-based thermo-sensitive copolymers for harvesting living cell sheet

    International Nuclear Information System (INIS)

    Chen, J.-P.; Yang, T.-F.

    2008-01-01

    A thermo-sensitive chitosan-based copolymer hydrogel was used for harvesting living cell sheets. The hydrogel was tested for harvesting 3T3 cells after carrying out cell culture at 37 deg. C and incubating the confluent cells at 20 deg. C for spontaneous detachment of cell sheets from hydrogel surface without enzyme treatment. Results from cell viability assay and microscopy observations demonstrated that cells could attach to the hydrogel surface and maintain high viability and proliferation ability. Cell detachment efficiency from the hydrogel was about 80%. The detached cell sheet retained high viability and could proliferate again after transferred to a new culture surface

  20. Molecular beacon nanosensors for live cell detection and tracking differentiation and reprogramming

    DEFF Research Database (Denmark)

    Ilieva, Mirolyuba

    2013-01-01

    open to closed state within living cells. Using MBs targeting pluripotent stem cell markers we demonstrated reverse into a more immature state of LUHMES induced by neurosphere-like growth conditions. Moreover, we have been able to trace localisation of this particular population during differentiation...... in separation of fluorophore from quencher and thereby emission of a fluorescent signal that can be detected. In this project the usability and applicability of MBs for live cell detection and tracing of gene expression was demonstrated. MBs library targeting gene markers for pluripotent stem cells as well...... and thus demonstrate the usability of MBs for monitoring cell behaviour within 3D clusters. Finally, MBs detection of expression of human pluripotent markers after reprograming of adult somatic cells with plasmid codding for mouse transcription factors was demonstrated. In conclusion, the method of using...

  1. Photovoltaic Test and Demonstration Project. [for solar cell power systems

    Science.gov (United States)

    Forestieri, A. F.; Brandhorst, H. W., Jr.; Deyo, J. N.

    1976-01-01

    The Photovoltaic Test and Demonstration Project was initiated by NASA in June, 1975, to develop economically feasible photovoltaic power systems suitable for a variety of terrestrial applications. Objectives include the determination of operating characteristic and lifetimes of a variety of solar cell systems and components and development of methodology and techniques for accurate measurements of solar cell and array performance and diagnostic measurements for solar power systems. Initial work will be concerned with residential applications, with testing of the first prototype system scheduled for June, 1976. An outdoor 10 kW array for testing solar power systems is under construction.

  2. Instant live-cell super-resolution imaging of cellular structures by nanoinjection of fluorescent probes.

    Science.gov (United States)

    Hennig, Simon; van de Linde, Sebastian; Lummer, Martina; Simonis, Matthias; Huser, Thomas; Sauer, Markus

    2015-02-11

    Labeling internal structures within living cells with standard fluorescent probes is a challenging problem. Here, we introduce a novel intracellular staining method that enables us to carefully control the labeling process and provides instant access to the inner structures of living cells. Using a hollow glass capillary with a diameter of <100 nm, we deliver functionalized fluorescent probes directly into the cells by (di)electrophoretic forces. The label density can be adjusted and traced directly during the staining process by fluorescence microscopy. We demonstrate the potential of this technique by delivering and imaging a range of commercially available cell-permeable and nonpermeable fluorescent probes to cells.

  3. Colonic metastasis from renal cell carcinoma: helical-CT demonstration

    International Nuclear Information System (INIS)

    Diaz-Candamio, M.J.; Pombo, S.; Pombo, F.

    2000-01-01

    Clinically evident colonic metastasis from renal cell carcinoma (RCC) is rare. In the present study a hypervascular sigmoid mass was demonstrated on arterial-phase helical CT using a water enema in a patient who had suffered left nephrectomy 8 years previously for RCC. The intense and early enhancement of the lesion suggested the possibility of a solitary colonic metastasis from RCC, a diagnosis which was pathologically confirmed. (orig.)

  4. Performing in the surgical amphitheater of today: perception of urologists conducting live case demonstrations.

    Science.gov (United States)

    Salami, Simpa S; Elsamra, Sammy E; Motato, Hector; Leavitt, David A; Friedlander, Justin I; Paoli, Michele A; Duty, Brian; Okeke, Zeph; Smith, Arthur D

    2014-09-01

    To evaluate the perception of urologists who have participated in live case demonstrations (LCDs) regarding safety, educational value/benefits, and ethics. A 19-question anonymous survey was sent to urologists who performed and/or moderated LCDs at the World Congress of Endourology meetings from 2008 to 2012. E-survey was distributed via e-mail, and automatic reminders were sent 2 weeks after original distribution if no response was obtained. Eighty-one percent (92/113) of the urologists responded to the survey of whom 75% practice full time in an academic setting. Only 48.0% were fellowship trained, however. More than 60% had performed more than five LCDs at their home and/or away institutions. Performing LCD at an away institution was associated with a higher level of anxiety when compared with performing LCDs at a home institution (79.8% vs 34.6%; Pfilm equipment and crew; audience and moderator discussions; unfamiliar team, instruments and tools; language barrier; and having to narrate the procedure, as distractors. Seventy-nine percent considered LCD as an ethical practice, and 90.5% regarded LCDs as a "beneficial" and a "great way" to educate. Similarly, considering the various factors and conditions associated with LCDs, 95% would agree to participate in another LCD. LCDs are perceived to be an effective mode of education by performers and moderators of LCDs. Standard guidelines and policies are needed, however, for the selection of patient, surgeon and team, equipment, and facility. Studies are needed to evaluate the impact of this education process.

  5. Live cell imaging reveals at novel view of DNA

    International Nuclear Information System (INIS)

    Moritomi-Yano, Keiko; Yano, Ken-ichi

    2010-01-01

    Non-homologous end-joining (NHEJ) is the major repair pathway for DNA double-strand breaks (DSBs) that are the most severe form of DNA damages. Recently, live cell imaging techniques coupled with laser micro-irradiation were used to analyze the spatio-temporal behavior of the NHEJ core factors upon DSB induction in living cells. Based on the live cell imaging studies, we proposed a novel two-phase model for DSB sensing and protein assembly in the NHEJ pathway. This new model provides a novel view of the dynamic protein behavior on DSBs and broad implications for the molecular mechanism of NHEJ. (author)

  6. Detecting and Tracking Nonfluorescent Nanoparticles Probes in Live Cells

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Gufeng; Fang, Ning

    2012-01-17

    Precisely imaging and tracking dynamic biological processes in live cells are crucial for both fundamental research in life sciences and biomedical applications. Nonfluorescent nanoparticles are emerging as important optical probes in live-cell imaging because of their excellent photostability, large optical cross sections, and low cytotoxicity. Here, we provide a review of recent development in optical imaging of nonfluorescent nanoparticle probes and their applications in dynamic tracking and biosensing in live cells. A brief discussion on cytotoxicity of nanoparticle probes is also provided.

  7. Long term imaging of living brain cancer cells

    Science.gov (United States)

    Farias, Patricia M. A.; Galembeck, André; Milani, Raquel; Andrade, Arnaldo C. D. S.; Stingl, Andreas

    2018-02-01

    QDs synthesized in aqueous medium and functionalized with polyethylene glycol were used as fluorescent probes. They label and monitor living healthy and cancer brain glial cells in culture. Physical-chemical characterization was performed. Toxicological studies were performed by in vivo short and long-term inhalation in animal models. Healthy and cancer glial living cells were incubated in culture media with highly controlled QDs. Specific features of glial cancer cells were enhanced by QD labelling. Cytoplasmic labelling pattern was clearly distinct for healthy and cancer cells. Labelled cells kept their normal activity for same period as non-labelled control samples.

  8. Nanochannel Electroporation as a Platform for Living Cell Interrogation in Acute Myeloid Leukemia.

    Science.gov (United States)

    Zhao, Xi; Huang, Xiaomeng; Wang, Xinmei; Wu, Yun; Eisfeld, Ann-Kathrin; Schwind, Sebastian; Gallego-Perez, Daniel; Boukany, Pouyan E; Marcucci, Guido I; Lee, Ly James

    2015-12-01

    A living cell interrogation platform based on nanochannel electroporation is demonstrated with analysis of RNAs in single cells. This minimally invasive process is based on individual cells and allows both multi-target analysis and stimulus-response analysis by sequential deliveries. The unique platform possesses a great potential to the comprehensive and lysis-free nucleic acid analysis on rare or hard-to-transfect cells.

  9. Imaging in living cells using νB-H Raman spectroscopy: monitoring COSAN uptake.

    Science.gov (United States)

    Tarrés, Màrius; Canetta, Elisabetta; Viñas, Clara; Teixidor, Francesc; Harwood, Adrian J

    2014-03-28

    The boron-rich cobaltabisdicarbollide (COSAN) and its 8,8'-I2 derivative (I2-COSAN), both of purely inorganic nature, are shown to accumulate within living cells, where they can be detected using νB-H Raman microspectroscopy. This demonstrates an alternative method for cell labelling and detection.

  10. Axial tomography in 3D live cell microscopy

    Science.gov (United States)

    Richter, Verena; Bruns, Sarah; Bruns, Thomas; Piper, Mathis; Weber, Petra; Wagner, Michael; Cremer, Christoph; Schneckenburger, Herbert

    2017-07-01

    A miniaturized setup for sample rotation on a microscope stage has been developed, combined with light sheet, confocal or structured illumination microscopy and applied to living cells as well as to small organisms. This setup permits axial tomography with improved visualization of single cells or small cell clusters as well as an enhanced effective 3D resolution upon sample rotation.

  11. Castration-Resistant Lgr5+ Cells Are Long-Lived Stem Cells Required for Prostatic Regeneration

    Directory of Open Access Journals (Sweden)

    Bu-er Wang

    2015-05-01

    Full Text Available The adult prostate possesses a significant regenerative capacity that is of great interest for understanding adult stem cell biology. We demonstrate that leucine-rich repeat-containing G protein-coupled receptor 5 (Lgr5 is expressed in a rare population of prostate epithelial progenitor cells, and a castration-resistant Lgr5+ population exists in regressed prostate tissue. Genetic lineage tracing revealed that Lgr5+ cells and their progeny are primarily luminal. Lgr5+ castration-resistant cells are long lived and upon regeneration, both luminal Lgr5+ cells and basal Lgr5+ cells expand. Moreover, single Lgr5+ cells can generate multilineage prostatic structures in renal transplantation assays. Additionally, Lgr5+ cell depletion revealed that the regenerative potential of the castrated adult prostate depends on Lgr5+ cells. Together, these data reveal insights into the cellular hierarchy of castration-resistant Lgr5+ cells, indicate a requirement for Lgr5+ cells during prostatic regeneration, and identify an Lgr5+ adult stem cell population in the prostate.

  12. Diesel fueled ship propulsion fuel cell demonstration project

    Energy Technology Data Exchange (ETDEWEB)

    Kumm, W.H. [Arctic Energies Ltd., Severna Park, MD (United States)

    1996-12-31

    The paper describes the work underway to adapt a former US Navy diesel electric drive ship as a 2.4 Megawatt fuel cell powered, US Coast Guard operated, demonstrator. The Project will design the new configuration, and then remove the four 600 kW diesel electric generators and auxiliaries. It will design, build and install fourteen or more nominal 180 kW diesel fueled molten carbonate internal reforming direct fuel cells (DFCs). The USCG cutter VINDICATOR has been chosen. The adaptation will be carried out at the USCG shipyard at Curtis Bay, MD. A multi-agency (state and federal) cooperative project is now underway. The USCG prime contractor, AEL, is performing the work under a Phase III Small Business Innovation Research (SBIR) award. This follows their successful completion of Phases I and II under contract to the US Naval Sea Systems (NAVSEA) from 1989 through 1993 which successfully demonstrated the feasibility of diesel fueled DFCs. The demonstrated marine propulsion of a USCG cutter will lead to commercial, naval ship and submarine applications as well as on-land applications such as diesel fueled locomotives.

  13. Live Cell Imaging of Alphaherpes Virus Anterograde Transport and Spread

    Science.gov (United States)

    Taylor, Matthew P.; Kratchmarov, Radomir; Enquist, Lynn W.

    2013-01-01

    Advances in live cell fluorescence microscopy techniques, as well as the construction of recombinant viral strains that express fluorescent fusion proteins have enabled real-time visualization of transport and spread of alphaherpes virus infection of neurons. The utility of novel fluorescent fusion proteins to viral membrane, tegument, and capsids, in conjunction with live cell imaging, identified viral particle assemblies undergoing transport within axons. Similar tools have been successfully employed for analyses of cell-cell spread of viral particles to quantify the number and diversity of virions transmitted between cells. Importantly, the techniques of live cell imaging of anterograde transport and spread produce a wealth of information including particle transport velocities, distributions of particles, and temporal analyses of protein localization. Alongside classical viral genetic techniques, these methodologies have provided critical insights into important mechanistic questions. In this article we describe in detail the imaging methods that were developed to answer basic questions of alphaherpes virus transport and spread. PMID:23978901

  14. Live cell imaging of Arabidopsis root hairs

    NARCIS (Netherlands)

    Ketelaar, T.

    2014-01-01

    Root hairs are tubular extensions from the root surface that expand by tip growth. This highly focused type of cell expansion, combined with position of root hairs on the surface of the root, makes them ideal cells for microscopic observation. This chapter describes the method that is routinely used

  15. Texas LPG fuel cell development and demonstration project

    Energy Technology Data Exchange (ETDEWEB)

    None, None

    2004-07-26

    The State Energy Conservation Office has executed its first Fuel Cell Project which was awarded under a Department of Energy competitive grant process. The Texas LPG Fuel Processor Development and Fuel Cell Demonstration Program is a broad-based public/private partnership led by the Texas State Energy Conservation Office (SECO). Partners include the Alternative Fuels Research and Education Division (AFRED) of the Railroad Commission of Texas; Plug Power, Inc., Latham, NY, UOP/HyRadix, Des Plaines, IL; Southwest Research Institute (SwRI), San Antonio, TX; the Texas Natural Resource Conservation Commission (TNRCC), and the Texas Department of Transportation (TxDOT). The team proposes to mount a development and demonstration program to field-test and evaluate markets for HyRadix's LPG fuel processor system integrated into Plug Power's residential-scale GenSys(TM) 5C (5 kW) PEM fuel cell system in a variety of building types and conditions of service. The program's primary goal is to develop, test, and install a prototype propane-fueled residential fuel cell power system supplied by Plug Power and HyRadix in Texas. The propane industry is currently funding development of an optimized propane fuel processor by project partner UOP/HyRadix through its national checkoff program, the Propane Education and Research Council (PERC). Following integration and independent verification of performance by Southwest Research Institute, Plug Power and HyRadix will produce a production-ready prototype unit for use in a field demonstration. The demonstration unit produced during this task will be delivered and installed at the Texas Department of Transportation's TransGuide headquarters in San Antonio, Texas. Simultaneously, the team will undertake a market study aimed at identifying and quantifying early-entry customers, technical and regulatory requirements, and other challenges and opportunities that need to be addressed in planning commercialization of the units

  16. Colorimetric detection of endogenous hydrogen sulfide production in living cells

    Science.gov (United States)

    Ahn, Yong Jin; Lee, Young Ju; Lee, Jaemyeon; Lee, Doyeon; Park, Hun-Kuk; Lee, Gi-Ja

    2017-04-01

    Hydrogen sulfide (H2S) has received great attention as a third gaseous signal transmitter, following nitric oxide and carbon monoxide. In particular, H2S plays an important role in the regulation of cancer cell biology. Therefore, the detection of endogenous H2S concentrations within biological systems can be helpful to understand the role of gasotransmitters in pathophysiology. Although a simple and inexpensive method for the detection of H2S has been developed, its direct and precise measurement in living cells remains a challenge. In this study, we introduced a simple, facile, and inexpensive colorimetric system for selective H2S detection in living cells using a silver-embedded Nafion/polyvinylpyrrolidone (PVP) membrane. This membrane could be easily applied onto a polystyrene microplate cover. First, we optimized the composition of the coating membrane, such as the PVP/Nafion mixing ratio and AgNO3 concentration, as well as the pH of the Na2S (H2S donor) solution and the reaction time. Next, the in vitro performance of a colorimetric detection assay utilizing the silver/Nafion/PVP membrane was evaluated utilizing a known concentration of Na2S standard solution both at room temperature and at 37 °C in a 5% CO2 incubator. As a result, the sensitivity of the colorimetric assay for H2S at 37 °C in the incubator (0.0056 Abs./μM Na2S, R2 = 0.9948) was similar to that at room temperature (0.0055 Abs./μM Na2S, R2 = 0.9967). Moreover, these assays were less sensitive to interference from compounds such as glutathione, L-cysteine (Cys), and dithiothreitol than to the H2S from Na2S. This assay based on the silver/Nafion/PVP membrane also showed excellent reproducibility (2.8% RSD). Finally, we successfully measured the endogenous H2S concentrations in live C6 glioma cells by s-(5‧-adenosyl)-L-methionine stimulation with and without Cys and L-homocysteine, utilizing the silver/Nafion/PVP membrane. In summary, colorimetric assays using silver

  17. National fuel cell bus program : proterra fuel cell hybrid bus report, Columbia demonstration.

    Science.gov (United States)

    2011-10-01

    This report summarizes the experience and early results from a fuel cell bus demonstration funded by the Federal Transit Administration (FTA) under the National Fuel Cell Bus Program. A team led by the Center for Transportation and the Environment an...

  18. Aberration-free FTIR spectroscopic imaging of live cells in microfluidic devices.

    Science.gov (United States)

    Chan, K L Andrew; Kazarian, Sergei G

    2013-07-21

    The label-free, non-destructive chemical analysis offered by FTIR spectroscopic imaging is a very attractive and potentially powerful tool for studies of live biological cells. FTIR imaging of live cells is a challenging task, due to the fact that cells are cultured in an aqueous environment. While the synchrotron facility has proven to be a valuable tool for FTIR microspectroscopic studies of single live cells, we have demonstrated that high quality infrared spectra of single live cells using an ordinary Globar source can also be obtained by adding a pair of lenses to a common transmission liquid cell. The lenses, when placed on the transmission cell window, form pseudo hemispheres which removes the refraction of light and hence improve the imaging and spectral quality of the obtained data. This study demonstrates that infrared spectra of single live cells can be obtained without the focus shifting effect at different wavenumbers, caused by the chromatic aberration. Spectra of the single cells have confirmed that the measured spectral region remains in focus across the whole range, while spectra of the single cells measured without the lenses have shown some erroneous features as a result of the shift of focus. It has also been demonstrated that the addition of lenses can be applied to the imaging of cells in microfabricated devices. We have shown that it was not possible to obtain a focused image of an isolated cell in a droplet of DPBS in oil unless the lenses are applied. The use of the approach described herein allows for well focused images of single cells in DPBS droplets to be obtained.

  19. Probing the bioelectrochemistry of living cells

    Energy Technology Data Exchange (ETDEWEB)

    Cheran, Larisa-Emilia [Department of Chemistry, University of Toronto, 80 St. George Street, Toronto, Ontario (Canada); Maple Biosciences Lt., 80 St. George Street, Toronto, Ontario (Canada); Cheung, Shilin; Wang, Xiaomang; Thompson, Michael [Department of Chemistry, University of Toronto, 80 St. George Street, Toronto, Ontario (Canada)

    2008-10-01

    Recent times have seen a rapidly expanding interest in the study of both single cell behaviour and populations of cells. This paper presents a concise review of current techniques employed for the transduction and processing of cellular signals. Among these, electrochemical methodology in the form of transistor and impedance methods has figured prominently. Indirectly connected to this approach has been the optical, light addressable potentiometric technique. In our research we are developing vibrational methods which are capable of examining populations of neurons, smooth muscle and human red blood cells on a substrate in a label-free fashion. These are based on transverse acoustic wave methodology and Kelvin nanoprobe physics. With respect to the former, synchronous oscillations of frequency are detected for neurons which are altered by the introduction of certain drugs. The same technique can be used to monitor chemical perturbation of the structure of smooth muscle cells from rat aorta. The Kelvin nanoprobe possesses sub-micron resolution and has been successfully employed in the characterization of both isolated, single neuron and red blood cells. Alterations in cell behaviour are reflected in apparent changes in work function, which in turn is associated with changes in cellular potential and dielectric properties. (author)

  20. Live cell imaging of in vitro human trophoblast syncytialization.

    Science.gov (United States)

    Wang, Rui; Dang, Yan-Li; Zheng, Ru; Li, Yue; Li, Weiwei; Lu, Xiaoyin; Wang, Li-Juan; Zhu, Cheng; Lin, Hai-Yan; Wang, Hongmei

    2014-06-01

    Human trophoblast syncytialization, a process of cell-cell fusion, is one of the most important yet least understood events during placental development. Investigating the fusion process in a placenta in vivo is very challenging given the complexity of this process. Application of primary cultured cytotrophoblast cells isolated from term placentas and BeWo cells derived from human choriocarcinoma formulates a biphasic strategy to achieve the mechanism of trophoblast cell fusion, as the former can spontaneously fuse to form the multinucleated syncytium and the latter is capable of fusing under the treatment of forskolin (FSK). Live-cell imaging is a powerful tool that is widely used to investigate many physiological or pathological processes in various animal models or humans; however, to our knowledge, the mechanism of trophoblast cell fusion has not been reported using a live- cell imaging manner. In this study, a live-cell imaging system was used to delineate the fusion process of primary term cytotrophoblast cells and BeWo cells. By using live staining with Hoechst 33342 or cytoplasmic dyes or by stably transfecting enhanced green fluorescent protein (EGFP) and DsRed2-Nuc reporter plasmids, we observed finger-like protrusions on the cell membranes of fusion partners before fusion and the exchange of cytoplasmic contents during fusion. In summary, this study provides the first video recording of the process of trophoblast syncytialization. Furthermore, the various live-cell imaging systems used in this study will help to yield molecular insights into the syncytialization process during placental development. © 2014 by the Society for the Study of Reproduction, Inc.

  1. Analysis of live cell images: Methods, tools and opportunities.

    Science.gov (United States)

    Nketia, Thomas A; Sailem, Heba; Rohde, Gustavo; Machiraju, Raghu; Rittscher, Jens

    2017-02-15

    Advances in optical microscopy, biosensors and cell culturing technologies have transformed live cell imaging. Thanks to these advances live cell imaging plays an increasingly important role in basic biology research as well as at all stages of drug development. Image analysis methods are needed to extract quantitative information from these vast and complex data sets. The aim of this review is to provide an overview of available image analysis methods for live cell imaging, in particular required preprocessing image segmentation, cell tracking and data visualisation methods. The potential opportunities recent advances in machine learning, especially deep learning, and computer vision provide are being discussed. This review includes overview of the different available software packages and toolkits. Copyright © 2017. Published by Elsevier Inc.

  2. 4Pi-confocal microscopy of live cells

    Science.gov (United States)

    Bahlmann, Karsten; Jakobs, Stefan; Hell, Stefan W.

    2002-06-01

    By coherently adding the spherical wavefronts of two opposing lenses, two-photon excitation 4Pi-confocal fluorescence microscopy has achieved three-dimensional imaging with an axial resolution 3-7 times better than confocal microscopy. So far this improvement was possible only in glycerol-mounted, fixed cells. Here we report 4Pi-confocal microscopy of watery objects and its application to the imaging of live cells. Water immersion 4Pi-confocal microscopy of membrane stained live Escherichia coli bacteria attains a 4.3 fold better axial resolution as compared to the best water immersion confocal microscope. The resolution enhancement results into a vastly improved three-dimensional representation of the bacteria. The first images of live biological samples with an all-directional resolution in the 190-280 nm range are presented here, thus establishing a new resolution benchmark in live cell microscopy.

  3. Functional memory B cells and long-lived plasma cells are generated after a single Plasmodium chabaudi infection in mice.

    Directory of Open Access Journals (Sweden)

    Francis Maina Ndungu

    2009-12-01

    Full Text Available Antibodies have long been shown to play a critical role in naturally acquired immunity to malaria, but it has been suggested that Plasmodium-specific antibodies in humans may not be long lived. The cellular mechanisms underlying B cell and antibody responses are difficult to study in human infections; therefore, we have investigated the kinetics, duration and characteristics of the Plasmodium-specific memory B cell response in an infection of P. chabaudi in mice. Memory B cells and plasma cells specific for the C-terminal region of Merozoite Surface Protein 1 were detectable for more than eight months following primary infection. Furthermore, a classical memory response comprised predominantly of the T-cell dependent isotypes IgG2c, IgG2b and IgG1 was elicited upon rechallenge with the homologous parasite, confirming the generation of functional memory B cells. Using cyclophosphamide treatment to discriminate between long-lived and short-lived plasma cells, we demonstrated long-lived cells secreting Plasmodium-specific IgG in both bone marrow and in spleens of infected mice. The presence of these long-lived cells was independent of the presence of chronic infection, as removal of parasites with anti-malarial drugs had no impact on their numbers. Thus, in this model of malaria, both functional Plasmodium-specific memory B cells and long-lived plasma cells can be generated, suggesting that defects in generating these cell populations may not be the reason for generating short-lived antibody responses.

  4. High-frequency microrheology reveals cytoskeleton dynamics in living cells

    Science.gov (United States)

    Rigato, Annafrancesca; Miyagi, Atsushi; Scheuring, Simon; Rico, Felix

    2017-08-01

    Living cells are viscoelastic materials, dominated by an elastic response on timescales longer than a millisecond. On shorter timescales, the dynamics of individual cytoskeleton filaments are expected to emerge, but active microrheology measurements on cells accessing this regime are scarce. Here, we develop high-frequency microrheology experiments to probe the viscoelastic response of living cells from 1 Hz to 100 kHz. We report the viscoelasticity of different cell types under cytoskeletal drug treatments. On previously inaccessible short timescales, cells exhibit rich viscoelastic responses that depend on the state of the cytoskeleton. Benign and malignant cancer cells revealed remarkably different scaling laws at high frequencies, providing a unique mechanical fingerprint. Microrheology over a wide dynamic range--up to the frequency characterizing the molecular components--provides a mechanistic understanding of cell mechanics.

  5. Quantification of cytoskeletal deformation in living cells

    NARCIS (Netherlands)

    van Engeland, S.; Kuijpers, N.H.L.

    In order to get a better insight in the mechanisms causing tissue damage there is an interest from within the biology community to quantify cellular deformations upon external loading. The cytoskeleton plays an important role in the transmission of forces throughout the cell. This study aims to

  6. Living Well with Sickle Cell Disease

    Science.gov (United States)

    ... Healthy Habits People with sickle cell disease should drink 8 to 10 glasses of water every day and eat healthy food. Try not to get too hot, too cold, or too tired. Children can, and should, participate in ... tired, and drink plenty of water. Look for clinical studies New ...

  7. Vital Autofluorescence: Application to the Study of Plant Living Cells

    Directory of Open Access Journals (Sweden)

    Victoria V. Roshchina

    2012-01-01

    approach to study the autofluorescence of plant living cells—from cell diagnostics up to modelling the cell-cell contacts and cell interactions with fluorescent biologically active substances. It bases on the direct observations of secretions released from allelopathic and medicinal species and the cell-donor interactions with cell-acceptors as biosensors (unicellular plant generative and vegetative microspores. Special attention was paid to the interactions with pigmented and fluorescing components of the secretions released by the cells-donors from plant species. Colored components of secretions are considered as histochemical dyes for the analysis of cellular mechanisms at the cell-cell contacts and modelling of cell-cell interactions. The fluorescence of plant biosensors was also recommended for the testing of natural plant excretions as medical drugs.

  8. Noninvasive imaging of protein-protein interactions from live cells and living subjects using bioluminescence resonance energy transfer.

    Science.gov (United States)

    De, Abhijit; Gambhir, Sanjiv Sam

    2005-12-01

    This study demonstrates a significant advancement of imaging of a distance-dependent physical process, known as the bioluminescent resonance energy transfer (BRET2) signal in living subjects, by using a cooled charge-coupled device (CCD) camera. A CCD camera-based spectral imaging strategy enables simultaneous visualization and quantitation of BRET signal from live cells and cells implanted in living mice. We used the BRET2 system, which utilizes Renilla luciferase (hRluc) protein and its substrate DeepBlueC (DBC) as an energy donor and a mutant green fluorescent protein (GFP2) as the acceptor. To accomplish this objective in this proof-of-principle study, the donor and acceptor proteins were fused to FKBP12 and FRB, respectively, which are known to interact only in the presence of the small molecule mediator rapamycin. Mammalian cells expressing these fusion constructs were imaged using a cooled-CCD camera either directly from culture dishes or by implanting them into mice. By comparing the emission photon yields in the presence and absence of rapamycin, the specific BRET signal was determined. The CCD imaging approach of BRET signal is particularly appealing due to its capacity to seamlessly bridge the gap between in vitro and in vivo studies. This work validates BRET as a powerful tool for interrogating and observing protein-protein interactions directly at limited depths in living mice.

  9. Assessing resolution in live cell structured illumination microscopy

    Science.gov (United States)

    Pospíšil, Jakub; Fliegel, Karel; Klíma, Miloš

    2017-12-01

    Structured Illumination Microscopy (SIM) is a powerful super-resolution technique, which is able to enhance the resolution of optical microscope beyond the Abbe diffraction limit. In the last decade, numerous SIM methods that achieve the resolution of 100 nm in the lateral dimension have been developed. The SIM setups with new high-speed cameras and illumination pattern generators allow rapid acquisition of the live specimen. Therefore, SIM is widely used for investigation of the live structures in molecular and live cell biology. Quantitative evaluation of resolution enhancement in a real sample is essential to describe the efficiency of super-resolution microscopy technique. However, measuring the resolution of a live cell sample is a challenging task. Based on our experimental findings, the widely used Fourier ring correlation (FRC) method does not seem to be well suited for measuring the resolution of SIM live cell video sequences. Therefore, the resolution assessing methods based on Fourier spectrum analysis are often used. We introduce a measure based on circular average power spectral density (PSDca) estimated from a single SIM image (one video frame). PSDca describes the distribution of the power of a signal with respect to its spatial frequency. Spatial resolution corresponds to the cut-off frequency in Fourier space. In order to estimate the cut-off frequency from a noisy signal, we use a spectral subtraction method for noise suppression. In the future, this resolution assessment approach might prove useful also for single-molecule localization microscopy (SMLM) live cell imaging.

  10. Live-cell visualization of gasdermin D-driven pyroptotic cell death.

    Science.gov (United States)

    Rathkey, Joseph K; Benson, Bryan L; Chirieleison, Steven M; Yang, Jie; Xiao, Tsan S; Dubyak, George R; Huang, Alex Y; Abbott, Derek W

    2017-09-01

    Pyroptosis is a form of cell death important in defenses against pathogens that can also result in a potent and sometimes pathological inflammatory response. During pyroptosis, GSDMD (gasdermin D), the pore-forming effector protein, is cleaved, forms oligomers, and inserts into the membranes of the cell, resulting in rapid cell death. However, the potent cell death induction caused by GSDMD has complicated our ability to understand the biology of this protein. Studies aimed at visualizing GSDMD have relied on expression of GSDMD fragments in epithelial cell lines that naturally lack GSDMD expression and also lack the proteases necessary to cleave GSDMD. In this work, we performed mutagenesis and molecular modeling to strategically place tags and fluorescent proteins within GSDMD that support native pyroptosis and facilitate live-cell imaging of pyroptotic cell death. Here, we demonstrate that these fusion proteins are cleaved by caspases-1 and -11 at Asp-276. Mutations that disrupted the predicted p30-p20 autoinhibitory interface resulted in GSDMD aggregation, supporting the oligomerizing activity of these mutations. Furthermore, we show that these novel GSDMD fusions execute inflammasome-dependent pyroptotic cell death in response to multiple stimuli and allow for visualization of the morphological changes associated with pyroptotic cell death in real time. This work therefore provides new tools that not only expand the molecular understanding of pyroptosis but also enable its direct visualization. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  11. Semi-automated quantification of living cells with internalized nanostructures

    KAUST Repository

    Margineanu, Michael B.

    2016-01-15

    Background Nanostructures fabricated by different methods have become increasingly important for various applications in biology and medicine, such as agents for medical imaging or cancer therapy. In order to understand their interaction with living cells and their internalization kinetics, several attempts have been made in tagging them. Although methods have been developed to measure the number of nanostructures internalized by the cells, there are only few approaches aimed to measure the number of cells that internalize the nanostructures, and they are usually limited to fixed-cell studies. Flow cytometry can be used for live-cell assays on large populations of cells, however it is a single time point measurement, and does not include any information about cell morphology. To date many of the observations made on internalization events are limited to few time points and cells. Results In this study, we present a method for quantifying cells with internalized magnetic nanowires (NWs). A machine learning-based computational framework, CellCognition, is adapted and used to classify cells with internalized and no internalized NWs, labeled with the fluorogenic pH-dependent dye pHrodo™ Red, and subsequently to determine the percentage of cells with internalized NWs at different time points. In a “proof-of-concept”, we performed a study on human colon carcinoma HCT 116 cells and human epithelial cervical cancer HeLa cells interacting with iron (Fe) and nickel (Ni) NWs. Conclusions This study reports a novel method for the quantification of cells that internalize a specific type of nanostructures. This approach is suitable for high-throughput and real-time data analysis and has the potential to be used to study the interaction of different types of nanostructures in live-cell assays.

  12. The effects of atomic force microscopy upon nominated living cells

    Energy Technology Data Exchange (ETDEWEB)

    O' Hagan, Barry Michael Gerard [School of Biomedical Sciences, University of Ulster, Cromore Road, Coleraine, County Londonderry, BT52 1SA (United Kingdom)]. E-mail: bmg.ohagan@ulstser.ac.uk; Doyle, Peter [Unilever Research, Port Sunlight, The Wirral, Merseyside (United Kingdom); Allen, James M. [School of Biomedical Sciences, University of Ulster, Cromore Road, Coleraine, County Londonderry, BT52 1SA (United Kingdom); Sutton, Kerry [School of Biomedical Sciences, University of Ulster, Cromore Road, Coleraine, County Londonderry, BT52 1SA (United Kingdom); McKerr, George [School of Biomedical Sciences, University of Ulster, Cromore Road, Coleraine, County Londonderry, BT52 1SA (United Kingdom)

    2004-12-15

    This work describes a system for precise re-location of cells within a monolayer after atomic force imaging. As we know little about probe interaction with soft biological surfaces any corroborative evidence is of great importance. For example, it is of paramount importance in living cell force microscopy that interrogated cells can be re-located and imaged by other corroborative technologies. Methodologies expressed here have shown that non-invasive force parameters can be established for specific cell types. Additionally, we show that the same sample can be transferred reliably to an SEM. Results here indicate that further work with live cells should initially establish appropriate prevailing force parameters and that cell damage should be checked for before and after an imaging experiment.

  13. The effects of atomic force microscopy upon nominated living cells

    International Nuclear Information System (INIS)

    O'Hagan, Barry Michael Gerard; Doyle, Peter; Allen, James M.; Sutton, Kerry; McKerr, George

    2004-01-01

    This work describes a system for precise re-location of cells within a monolayer after atomic force imaging. As we know little about probe interaction with soft biological surfaces any corroborative evidence is of great importance. For example, it is of paramount importance in living cell force microscopy that interrogated cells can be re-located and imaged by other corroborative technologies. Methodologies expressed here have shown that non-invasive force parameters can be established for specific cell types. Additionally, we show that the same sample can be transferred reliably to an SEM. Results here indicate that further work with live cells should initially establish appropriate prevailing force parameters and that cell damage should be checked for before and after an imaging experiment

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  15. A hybrid bio-jetting approach for directly engineering living cells

    International Nuclear Information System (INIS)

    Kwok, Albert; Irvine, Scott; Arumuganathar, Sumathy; Jayasinghe, Suwan N; McEwan, Jean R

    2008-01-01

    This paper reports developments on a hybrid cell-engineering protocol coupling both bio-electrosprays and aerodynamically assisted bio-jets for process-handling living cells. The current work demonstrates the ability to couple these two cell-jetting protocols for handling a wide range of cells for deposition. The post-treated cells are assessed for their viability by way of flow cytometry, which illustrates a significant population of viable cells post-treatment in comparison to those controls. This work is the first example of coupling these two protocols for the process handling of living cells. The hybrid protocol demonstrates the achievement of stable cone jetting of a cellular suspension in the single-needle configuration which was previously unachieved with single-needle bio-electrosprays. Furthermore the living cells explored in these investigations expressed GFP, thus demonstrating the ability to couple gene therapy with this hybrid protocol. Hence, this approach could one day be explored for building biologically viable tissues incorporating a therapeutic payload for combating a range of cellular/tissue-based pathologies

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

    Directory of Open Access Journals (Sweden)

    Andrea Brown Forrester

    2015-09-01

    Full Text Available 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.

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

    Science.gov (United States)

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

    2015-01-01

    To explore the lived experiences of adolescents with sickle cell disease, in Kingston, Jamaica. 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. 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. 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.

  18. Optical detection and virotherapy of live metastatic tumor cells in body fluids with vaccinia strains.

    Directory of Open Access Journals (Sweden)

    Huiqiang Wang

    Full Text Available 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.

  19. Demonstration of Advanced EMI Models for Live-Site UXO Discrimination at Waikoloa, Hawaii

    Science.gov (United States)

    2015-12-01

    SITE UXO DISCRIMINATION AT WAIKOLOA, HAWAII 5b. GRANT NUMBER 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) Dr. Fridon Shubitidze Thayer...UXO demonstration study at the former Waikoloa Maneuver Area (WMA) in Waikoloa, Hawaii , under ESTCP Munitions Response Project MR-201227. 15

  20. A near-infrared fluorescent sensor for H+ in aqueous solution and living cells

    OpenAIRE

    WU, Aibin; DUAN, Liping

    2014-01-01

    A heptamethine cyanine-based sensor (1) was designed and synthesized by incorporating heptamethine cyanine fluorophore and methylpiperazine. Sensor 1 exhibited good response to the change of pH levels, and a large Stokes shift (>100 nm) was obtained. Fluorescent image experiments in living cells further demonstrated its potential applications in biological systems.

  1. Design of microdevices for long-term live cell imaging

    International Nuclear Information System (INIS)

    Chen, Huaying; Nordon, Robert E; Rosengarten, Gary; Li, Musen

    2012-01-01

    Advances in fluorescent live cell imaging provide high-content information that relates a cell's life events to its ancestors. An important requirement to track clonal growth and development is the retention of motile cells derived from an ancestor within the same microscopic field of view for days to weeks, while recording fluorescence images and controlling the mechanical and biochemical microenvironments that regulate cell growth and differentiation. The aim of this study was to design a microwell device for long-term, time-lapse imaging of motile cells with the specific requirements of (a) inoculating devices with an average of one cell per well and (b) retaining progeny of cells within a single microscopic field of view for extended growth periods. A two-layer PDMS microwell culture device consisting of a parallel-plate flow cell bonded on top of a microwell array was developed for cell capture and clonal culture. Cell deposition statistics were related to microwell geometry (plate separation and well depth) and the Reynolds number. Computational fluid dynamics was used to simulate flow in the microdevices as well as cell–fluid interactions. Analysis of the forces acting upon a cell was used to predict cell docking zones, which were confirmed by experimental observations. Cell–fluid dynamic interactions are important considerations for design of microdevices for long-term, live cell imaging. The analysis of force and torque balance provides a reasonable approximation for cell displacement forces. It is computationally less intensive compared to simulation of cell trajectories, and can be applied to a wide range of microdevice geometries to predict the cell docking behavior. (paper)

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

    Directory of Open Access Journals (Sweden)

    Jun Wang

    2012-04-01

    Full Text Available 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.

  3. [Development of a Fluorescence Probe for Live Cell Imaging].

    Science.gov (United States)

    Shibata, Aya

    2017-01-01

     Probes that detect specific biological materials are indispensable tools for deepening our understanding of various cellular phenomena. In live cell imaging, the probe must emit fluorescence only when a specific substance is detected. In this paper, we introduce a new probe we developed for live cell imaging. Glutathione S-transferase (GST) activity is higher in tumor cells than in normal cells and is involved in the development of resistance to various anticancer drugs. We previously reported the development of a general strategy for the synthesis of probes for detection of GST enzymes, including fluorogenic, bioluminogenic, and 19 F-NMR probes. Arylsulfonyl groups were used as caging groups during probe design. The fluorogenic probes were successfully used to quantitate very low levels of GST activity in cell extracts and were also successfully applied to the imaging of microsomal MGST1 activity in living cells. The bioluminogenic and 19 F-NMR probes were able to detect GST activity in Escherichia coli cells. Oligonucleotide-templated reactions are powerful tools for nucleic acid sensing. This strategy exploits the target strand as a template for two functionalized probes and provides a simple molecular mechanism for multiple turnover reactions. We developed a nucleophilic aromatic substitution reaction-triggered fluorescent probe. The probe completed its reaction within 30 s of initiation and amplified the fluorescence signal from 0.5 pM target oligonucleotide by 1500 fold under isothermal conditions. Additionally, we applied the oligonucleotide-templated reaction for molecular releasing and peptide detection.

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

    Science.gov (United States)

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

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

  5. Information management for high content live cell imaging

    Directory of Open Access Journals (Sweden)

    White Michael RH

    2009-07-01

    Full Text Available Abstract Background High content live cell imaging experiments are able to track the cellular localisation of labelled proteins in multiple live cells over a time course. Experiments using high content live cell imaging will generate multiple large datasets that are often stored in an ad-hoc manner. This hinders identification of previously gathered data that may be relevant to current analyses. Whilst solutions exist for managing image data, they are primarily concerned with storage and retrieval of the images themselves and not the data derived from the images. There is therefore a requirement for an information management solution that facilitates the indexing of experimental metadata and results of high content live cell imaging experiments. Results We have designed and implemented a data model and information management solution for the data gathered through high content live cell imaging experiments. Many of the experiments to be stored measure the translocation of fluorescently labelled proteins from cytoplasm to nucleus in individual cells. The functionality of this database has been enhanced by the addition of an algorithm that automatically annotates results of these experiments with the timings of translocations and periods of any oscillatory translocations as they are uploaded to the repository. Testing has shown the algorithm to perform well with a variety of previously unseen data. Conclusion Our repository is a fully functional example of how high throughput imaging data may be effectively indexed and managed to address the requirements of end users. By implementing the automated analysis of experimental results, we have provided a clear impetus for individuals to ensure that their data forms part of that which is stored in the repository. Although focused on imaging, the solution provided is sufficiently generic to be applied to other functional proteomics and genomics experiments. The software is available from: fhttp://code.google.com/p/livecellim/

  6. The live cell irradiation and observation setup at SNAKE

    Energy Technology Data Exchange (ETDEWEB)

    Hable, V. [Angewandte Physik und Messtechnik LRT2, UniBw-Muenchen, 85577 Neubiberg (Germany)], E-mail: volker.hable@unibw.de; Greubel, C.; Bergmaier, A.; Reichart, P. [Angewandte Physik und Messtechnik LRT2, UniBw-Muenchen, 85577 Neubiberg (Germany); Hauptner, A.; Kruecken, R. [Physik Department E12, TU-Muenchen, 85748 Garching (Germany); Strickfaden, H.; Dietzel, S.; Cremer, T. [Department Biologie II, LMU-Muenchen, 82152 Martinsried (Germany); Drexler, G.A.; Friedl, A.A. [Strahlenbiologisches Institut, LMU-Muenchen, 80336 Muenchen (Germany); Dollinger, G. [Angewandte Physik und Messtechnik LRT2, UniBw-Muenchen, 85577 Neubiberg (Germany)

    2009-06-15

    We describe a new setup at the ion microprobe SNAKE (Superconducting Nanoscope for Applied nuclear (Kern-) physics Experiments) at the Munich 14 MV Tandem accelerator that facilitates both living cell irradiation with sub micrometer resolution and online optical imaging of the cells before and after irradiation by state of the art phase contrast and fluorescence microscopy. The cells are kept at standard cell growth conditions at 37 {sup o}C in cell culture medium. After irradiation it is possible to switch from single ion irradiation conditions to cell observation within 0.5 s. First experiments were performed targeting substructures of a cell nucleus that were tagged by TexasRed labeled nucleotides incorporated in the cellular DNA by 55 MeV single carbon ion irradiation. In addition we show first online sequences of short time kinetics of Mdc1 protein accumulation in the vicinity of double strand breaks after carbon ion irradiation.

  7. Label-free evanescent microscopy for membrane nano-tomography in living cells.

    Science.gov (United States)

    Bon, Pierre; Barroca, Thomas; Lévèque-Fort, Sandrine; Fort, Emmanuel

    2014-11-01

    We show that through-the-objective evanescent microscopy (epi-EM) is a powerful technique to image membranes in living cells. Readily implementable on a standard inverted microscope, this technique enables full-field and real-time tracking of membrane processes without labeling and thus signal fading. In addition, we demonstrate that the membrane/interface distance can be retrieved with 10 nm precision using a multilayer Fresnel model. We apply this nano-axial tomography of living cell membranes to retrieve quantitative information on membrane invagination dynamics. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

  8. Following the Dynamics of pH in Endosomes of Live Cells with SERS Nanosensors

    DEFF Research Database (Denmark)

    Kneipp, J.; Kneipp, Harald; Wittig, B.

    2010-01-01

    The surface enhanced Raman scattering (SERS) spectrum of a reporter molecule attached to gold or silver nanostructures, which is pH-sensitive, can deliver information on the local pH in the environment of the nanostructure. Here, we demonstrate the use of a mobile SERS nanosensor made from gold...... nanaoaggregates and 4-mercaptobenzoic acid (pMBA) attached as a reporter for monitoring changes in local pH of the cellular compartments of living NIH/3T3 cells. We show that SERS nanosensors enable the dynamics of local pH in individual live cells to be followed at subendosomal resolution in a timeline...

  9. Live-cell imaging: new avenues to investigate retinal regeneration

    Directory of Open Access Journals (Sweden)

    Manuela Lahne

    2017-01-01

    Full Text Available Sensing and responding to our environment requires functional neurons that act in concert. Neuronal cell loss resulting from degenerative diseases cannot be replaced in humans, causing a functional impairment to integrate and/or respond to sensory cues. In contrast, zebrafish (Danio rerio possess an endogenous capacity to regenerate lost neurons. Here, we will focus on the processes that lead to neuronal regeneration in the zebrafish retina. Dying retinal neurons release a damage signal, tumor necrosis factor α, which induces the resident radial glia, the Müller glia, to reprogram and re-enter the cell cycle. The Müller glia divide asymmetrically to produce a Müller glia that exits the cell cycle and a neuronal progenitor cell. The arising neuronal progenitor cells undergo several rounds of cell divisions before they migrate to the site of damage to differentiate into the neuronal cell types that were lost. Molecular and immunohistochemical studies have predominantly provided insight into the mechanisms that regulate retinal regeneration. However, many processes during retinal regeneration are dynamic and require live-cell imaging to fully discern the underlying mechanisms. Recently, a multiphoton imaging approach of adult zebrafish retinal cultures was developed. We will discuss the use of live-cell imaging, the currently available tools and those that need to be developed to advance our knowledge on major open questions in the field of retinal regeneration.

  10. Light microscopical demonstration and zonal distribution of parasinusoidal cells (Ito cells) in normal human liver

    DEFF Research Database (Denmark)

    Horn, T; Junge, Jette; Nielsen, O

    1988-01-01

    The parasinusoidal cells of the liver (Ito cells) were demonstrated light microscopically in autopsy specimens fixed in formalin and stained with Oil red O after dichromate treatment. The method allows examination of large samples containing numerous acini. Quantitative assessment showed a zonal...

  11. Light microscopical demonstration and zonal distribution of parasinusoidal cells (Ito cells) in normal human liver

    DEFF Research Database (Denmark)

    Horn, T; Junge, Jette; Nielsen, O

    1988-01-01

    The parasinusoidal cells of the liver (Ito cells) were demonstrated light microscopically in autopsy specimens fixed in formalin and stained with Oil red O after dichromate treatment. The method allows examination of large samples containing numerous acini. Quantitative assessment showed a zonal ...

  12. Imaging Proteolysis by Living Human Breast Cancer Cells

    Directory of Open Access Journals (Sweden)

    Mansoureh Sameni

    2000-01-01

    Full Text Available Malignant progression is accompanied by degradation of extracellular matrix proteins. Here we describe a novel confocal assay in which we can observe proteolysis by living human breast cancer cells (BT20 and BT549 through the use of quenchedfluorescent protein substrates. Degradation thus was imaged, by confocal optical sectioning, as an accumulation of fluorescent products. With the BT20 cells, fluorescence was localized to pericellular focal areas that coincide with pits in the underlying matrix. In contrast, fluorescence was localized to intracellular vesicles in the BT549 cells, vesicles that also label for lysosomal markers. Neither intracellular nor pericellular fluorescence was observed in the BT549 cells in the presence of cytochalasin B, suggesting that degradation occurred intracellularly and was dependent on endocytic uptake of substrate. In the presence of a cathepsin 13-selective cysteine protease inhibitor, intracellular fluorescence was decreased ~90% and pericellular fluorescence decreased 67% to 96%, depending on the protein substrate. Matrix metallo protease inhibitors reduced pericellular fluorescence ~50%, i.e., comparably to a serine and a broad spectrum cysteine protease inhibitor. Our results suggest that: 1 a proteolytic cascade participates in pericellular digestion of matrix proteins by living human breast cancer cells, and 2 the cysteine protease cathepsin B participates in both pericellular and intracellular digestion of matrix proteins by living human breast cancer cells.

  13. A nucleic acid dependent chemical photocatalysis in live human cells

    DEFF Research Database (Denmark)

    Arian, Dumitru; Cló, Emiliano; Gothelf, Kurt V

    2010-01-01

    Only two nucleic acid directed chemical reactions that are compatible with live cells have been reported to date. Neither of these processes generate toxic species from nontoxic starting materials. Reactions of the latter type could be applied as gene-specific drugs, for example, in the treatment...

  14. Energy, control and DNA structure in the living cell

    DEFF Research Database (Denmark)

    Wijker, J.E.; Jensen, Peter Ruhdal; Gomes, A. Vaz

    1995-01-01

    Maintenance (let alone growth) of the highly ordered living cell is only possible through the continuous input of free energy. Coupling of energetically downhill processes (such as catabolic reactions) to uphill processes is essential to provide this free energy and is catalyzed by enzymes either...

  15. Lives of a Cell: 40 Years Later, A Third Interpretation

    Centers for Disease Control (CDC) Podcasts

    2015-06-16

    Reginald Tucker reads an abridged version of the article Lives of a Cell: 40 Years Later, A Third Interpretation.  Created: 6/16/2015 by National Center for Emerging and Zoonotic Infectious Diseases (NCEZID).   Date Released: 6/18/2015.

  16. THE NISSL SUBSTANCE OF LIVING AND FIXED SPINAL GANGLION CELLS

    Science.gov (United States)

    Deitch, Arline D.; Moses, Montrose J.

    1957-01-01

    Living chick spinal ganglion neurons grown for 19 to 25 days in vitro were photographed with a color-translating ultraviolet microscope (UV-91) at 265, 287, and 310 mµ. This instrument was unique in permitting rapid accumulation of ultraviolet information with minimal damage to the cell. In the photographs taken at 265 mµ of the living neurons, discrete ultraviolet-absorbing cytoplasmic masses were observed which were found to be virtually unchanged in appearance after formalin fixation. These were identical with the Nissl bodies of the same cells seen after staining with basic dyes. The correlation of ultraviolet absorption, ribonuclease extraction, and staining experiments with acid and basic dyes confirmed the ribonucleoprotein nature of these Nissl bodies in the living and fixed cells. No change in distribution or concentration of ultraviolet-absorbing substance was observed in the first 12 ultraviolet photographs of a neuron, and it is concluded that the cells had not been subjected to significant ultraviolet damage during the period of photography. On the basis of these observations, as well as previous findings with phase contrast microscopy, it is concluded that Nissl bodies preexist in the living neuron as discrete aggregates containing high concentrations of nucleoprotein. PMID:13438929

  17. AFM review study on pox viruses and living cells.

    Science.gov (United States)

    Ohnesorge, F M; Hörber, J K; Häberle, W; Czerny, C P; Smith, D P; Binnig, G

    1997-10-01

    Single living cells were studied in growth medium by atomic force microscopy at a high--down to one image frame per second--imaging rate over time periods of many hours, stably producing hundreds of consecutive scans with a lateral resolution of approximately 30-40 nm. The cell was held by a micropipette mounted onto the scanner-piezo as shown in Häberle, W., J. K. H. Hörber, and G. Binnig. 1991. Force microscopy on living cells. J. Vac. Sci. Technol. B9:1210-0000. To initiate specific processes on the cell surface the cells had been infected with pox viruses as reported earlier and, most likely, the liberation of a progeny virion by the still-living cell was observed, hence confirming and supporting earlier results (Häberle, W., J. K. H. Hörber, F. Ohnesorge, D. P. E. Smith, and G. Binnig. 1992. In situ investigations of single living cells infected by viruses. Ultramicroscopy. 42-44:1161-0000; Hörber, J. K. H., W. Häberle, F. Ohnesorge, G. Binnig, H. G. Liebich, C. P. Czerny, H. Mahnel, and A. Mayr. 1992. Investigation of living cells in the nanometer regime with the atomic force microscope. Scanning Microscopy. 6:919-930). Furthermore, the pox viruses used were characterized separately by AFM in an aqueous environment down to the molecular level. Quasi-ordered structural details were resolved on a scale of a few nm where, however, image distortions and artifacts due to multiple tip effects are probably involved--just as in very high resolution (small dark spots in the light microscope, that we believed to be the regions in the cell plasma where viruses are assembled; this is known from the literature on electron microscopy on pox-infected cells and referred to there as "virus factories" (e.g., Moss, B. 1986. Replication of pox viruses. In Fundamental Virology, B. N. Fields and D. M. Knape, editors. Raven Press, New York. 637-655). Therefore, we assume that the cells stay alive during imaging, in our experience for approximately 30-45 h p.i.).

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

    International Nuclear Information System (INIS)

    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

    2014-01-01

    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

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

    Energy Technology Data Exchange (ETDEWEB)

    Horita, Nobukatsu [Department of Gastroenterology and Hepatology, Graduate School, Tokyo Medical and Dental University (Japan); Tsuchiya, Kiichiro, E-mail: kii.gast@tmd.ac.jp [Department of Advanced Therapeutics for Gastrointestinal Diseases, Graduate School, Tokyo Medical and Dental University (Japan); Hayashi, Ryohei [Department of Gastroenterology and Hepatology, Graduate School, Tokyo Medical and Dental University (Japan); Department of Gastroenterology and Metabolism, Hiroshima University (Japan); Fukushima, Keita; Hibiya, Shuji; Fukuda, Masayoshi; Kano, Yoshihito; Mizutani, Tomohiro; Nemoto, Yasuhiro; Yui, Shiro [Department of Gastroenterology and Hepatology, Graduate School, Tokyo Medical and Dental University (Japan); Okamoto, Ryuichi; Nakamura, Tetsuya [Department of Advanced Therapeutics for Gastrointestinal Diseases, Graduate School, Tokyo Medical and Dental University (Japan); Watanabe, Mamoru [Department of Gastroenterology and Hepatology, Graduate School, Tokyo Medical and Dental University (Japan)

    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.

  20. Living with a diagnosis of non-small cell lung cancer: patients' lived experiences.

    LENUS (Irish Health Repository)

    McCarthy, Ita

    2012-01-31

    The aim of this study was to explore patients\\' experience of living with non-small cell lung cancer (NSCLC). Patients diagnosed with NSCLC know that their treatment is not with curative intent and can expect distressing symptoms. In this phenomenological study, six adults with a diagnosis of NSCLC were interviewed. Data was analysed guided by van Manen\\'s six-step process. Four main themes were interpreted: \\'Maintaining my life\\'; \\'The enemy within\\'; \\'Staying on the train\\

  1. Combining bio-electrospraying with gene therapy: a novel biotechnique for the delivery of genetic material via living cells.

    Science.gov (United States)

    Ward, Eliot; Chan, Emma; Gustafsson, Kenth; Jayasinghe, Suwan N

    2010-05-01

    The investigations reported in this article demonstrate the ability of bio-electrosprays and cell electrospinning to deliver a genetic construct in association with living cells. Previous studies on both bio-electrosprays and cell electrospinning demonstrated great promise for tissue engineering and regenerative biology/medicine. The investigations described herein widen the applicability of these biotechniques by combining gene therapy protocols, resulting in a novel drug delivery methodology previously unexplored. In these studies a human cell line was transduced with recombinant self-inactivating lentiviral particles. These particles incorporated a green fluorescent protein fused to an endosomal targeting construct. This construct encodes a peptide, which can subsequently be detected on the surface of cells by specific T-cells. The transduced cell line was subsequently manipulated in association with either bio-electrospraying or cell electrospinning. Hence this demonstrates (i) the ability to safely handle genetically modified living cells and (ii) the ability to directly form pre-determined architectures bearing living therapeutic cells. This merged technology demonstrates a unique approach for directly forming living therapeutic architectures for controlled and targeted release of experimental cells/genes, as well as medical cell/gene therapeutics for a plethora of biological and medical applications. Hence, such developments could be applied to personalised medicine.

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

    Science.gov (United States)

    Li, Gene-Wei; Xie, X Sunney

    2011-07-20

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

  3. Self-adhesive microculture system for extended live cell imaging.

    Science.gov (United States)

    Skommer, J; McGuinness, D; Wlodkowic, D

    2011-06-01

    Gas permeable and biocompatible soft polymers are convenient for biological applications. Using the soft polymer poly(dimethylsiloxane) (PDMS), we established a straightforward technique for in-house production of self-adhesive and optical grade microculture devices. A gas permeable PDMS layer effectively protects against medium evaporation, changes in osmolarity, contamination and drug diffusion. These chip-based devices can be used effectively for long term mammalian cell culture and support a range of bioassays used in pharmacological profiling of anti-cancer drugs. Results obtained on a panel of hematopoietic and solid tumor cell lines during screening of investigative anti-cancer agents corresponded well to those obtained in a conventional cell culture on polystyrene plates. The cumulative correlation analysis of multiple cell lines and anti-cancer drugs showed no adverse effects on cell viability or cell growth retardation during microscale static cell culture. PDMS devices also can be custom modified for many bio-analytical purposes and are interfaced easily with both inverted and upright cell imaging platforms. Moreover, PDMS microculture devices are suitable for extended real time cell imaging. Data from the multicolor, real time analysis of apoptosis on human breast cancer MCF-7 cells provided further evidence that elimination of redundant centrifugation/washing achieved during microscale real time analysis facilitates preservation of fragile apoptotic cells and provides dynamic cellular information at high resolution. Because only small reaction volumes are required, such devices offer reduced use of consumables as well as simplified manipulations during all stages of live cell imaging.

  4. A Microfluidic Platform for Correlative Live-Cell and Super-Resolution Microscopy

    Science.gov (United States)

    Tam, Johnny; Cordier, Guillaume Alan; Bálint, Štefan; Sandoval Álvarez, Ángel; Borbely, Joseph Steven; Lakadamyali, Melike

    2014-01-01

    Recently, super-resolution microscopy methods such as stochastic optical reconstruction microscopy (STORM) have enabled visualization of subcellular structures below the optical resolution limit. Due to the poor temporal resolution, however, these methods have mostly been used to image fixed cells or dynamic processes that evolve on slow time-scales. In particular, fast dynamic processes and their relationship to the underlying ultrastructure or nanoscale protein organization cannot be discerned. To overcome this limitation, we have recently developed a correlative and sequential imaging method that combines live-cell and super-resolution microscopy. This approach adds dynamic background to ultrastructural images providing a new dimension to the interpretation of super-resolution data. However, currently, it suffers from the need to carry out tedious steps of sample preparation manually. To alleviate this problem, we implemented a simple and versatile microfluidic platform that streamlines the sample preparation steps in between live-cell and super-resolution imaging. The platform is based on a microfluidic chip with parallel, miniaturized imaging chambers and an automated fluid-injection device, which delivers a precise amount of a specified reagent to the selected imaging chamber at a specific time within the experiment. We demonstrate that this system can be used for live-cell imaging, automated fixation, and immunostaining of adherent mammalian cells in situ followed by STORM imaging. We further demonstrate an application by correlating mitochondrial dynamics, morphology, and nanoscale mitochondrial protein distribution in live and super-resolution images. PMID:25545548

  5. Quantification of plant cell coupling with live-cell microscopy

    DEFF Research Database (Denmark)

    Liesche, Johannes; Schulz, Alexander

    2015-01-01

    by confocal microscopy, loaded tracer is activated by UV illumination in a target cell and its spread to neighboring cells monitored. When combined with high-speed acquisition by resonant scanning or spinning disc confocal microscopy, the high signal-to-noise ratio of photoactivation allows collection...

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

    Science.gov (United States)

    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. Temperature-dependent imaging of living cells by AFM

    International Nuclear Information System (INIS)

    Espenel, Cedric; Giocondi, Marie-Cecile; Seantier, Bastien; Dosset, Patrice; Milhiet, Pierre-Emmanuel; Le Grimellec, Christian

    2008-01-01

    Characterization of lateral organization of plasma membranes is a prerequisite to the understanding of membrane structure-function relationships in living cells. Lipid-lipid and lipid-protein interactions are responsible for the existence of various membrane microdomains involved in cell signalization and in numerous pathologies. Developing approaches for characterizing microdomains associate identification tools like recognition imaging with high-resolution topographical imaging. Membrane properties are markedly dependent on temperature. However, mesoscopic scale topographical information of cell surface in a temperature range covering most of cell biology experimentation is still lacking. In this work we have examined the possibility of imaging the temperature-dependent behavior of eukaryotic cells by atomic force microscopy (AFM). Our results establish that the surface of living CV1 kidney cells can be imaged by AFM, between 5 and 37 deg. C, both in contact and tapping modes. These first temperature-dependent data show that large cell structures appeared essentially stable at a microscopic scale. On the other hand, as shown by contact mode AFM, the surface was highly dynamic at a mesoscopic scale, with marked changes in apparent topography, friction, and deflection signals. When keeping the scanning conditions constant, a progressive loss in the image contrast was however observed, using tapping mode, on decreasing the temperature

  8. Automotive Fuel Processor Development and Demonstration with Fuel Cell Systems

    Energy Technology Data Exchange (ETDEWEB)

    Nuvera Fuel Cells

    2005-04-15

    The potential for fuel cell systems to improve energy efficiency and reduce emissions over conventional power systems has generated significant interest in fuel cell technologies. While fuel cells are being investigated for use in many applications such as stationary power generation and small portable devices, transportation applications present some unique challenges for fuel cell technology. Due to their lower operating temperature and non-brittle materials, most transportation work is focusing on fuel cells using proton exchange membrane (PEM) technology. Since PEM fuel cells are fueled by hydrogen, major obstacles to their widespread use are the lack of an available hydrogen fueling infrastructure and hydrogen's relatively low energy storage density, which leads to a much lower driving range than conventional vehicles. One potential solution to the hydrogen infrastructure and storage density issues is to convert a conventional fuel such as gasoline into hydrogen onboard the vehicle using a fuel processor. Figure 2 shows that gasoline stores roughly 7 times more energy per volume than pressurized hydrogen gas at 700 bar and 4 times more than liquid hydrogen. If integrated properly, the fuel processor/fuel cell system would also be more efficient than traditional engines and would give a fuel economy benefit while hydrogen storage and distribution issues are being investigated. Widespread implementation of fuel processor/fuel cell systems requires improvements in several aspects of the technology, including size, startup time, transient response time, and cost. In addition, the ability to operate on a number of hydrocarbon fuels that are available through the existing infrastructure is a key enabler for commercializing these systems. In this program, Nuvera Fuel Cells collaborated with the Department of Energy (DOE) to develop efficient, low-emission, multi-fuel processors for transportation applications. Nuvera's focus was on (1) developing fuel

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

    Science.gov (United States)

    Gadalla, Atef; Dehoux, Thomas; Audoin, Bertrand

    2014-05-01

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

  10. Live cell imaging reveals marked variability in myoblast proliferation and fate

    Science.gov (United States)

    2013-01-01

    Background During the process of muscle regeneration, activated stem cells termed satellite cells proliferate, and then differentiate to form new myofibers that restore the injured area. Yet not all satellite cells contribute to muscle repair. Some continue to proliferate, others die, and others become quiescent and are available for regeneration following subsequent injury. The mechanisms that regulate the adoption of different cell fates in a muscle cell precursor population remain unclear. Methods We have used live cell imaging and lineage tracing to study cell fate in the C2 myoblast line. Results Analyzing the behavior of individual myoblasts revealed marked variability in both cell cycle duration and viability, but similarities between cells derived from the same parental lineage. As a consequence, lineage sizes and outcomes differed dramatically, and individual lineages made uneven contributions toward the terminally differentiated population. Thus, the cohort of myoblasts undergoing differentiation at the end of an experiment differed dramatically from the lineages present at the beginning. Treatment with IGF-I increased myoblast number by maintaining viability and by stimulating a fraction of cells to complete one additional cell cycle in differentiation medium, and as a consequence reduced the variability of the terminal population compared with controls. Conclusion Our results reveal that heterogeneity of responses to external cues is an intrinsic property of cultured myoblasts that may be explained in part by parental lineage, and demonstrate the power of live cell imaging for understanding how muscle differentiation is regulated. PMID:23638706

  11. Cell-permeable nanobodies for targeted immunolabelling and antigen manipulation in living cells.

    Science.gov (United States)

    Herce, Henry D; Schumacher, Dominik; Schneider, Anselm F L; Ludwig, Anne K; Mann, Florian A; Fillies, Marion; Kasper, Marc-André; Reinke, Stefan; Krause, Eberhard; Leonhardt, Heinrich; Cardoso, M Cristina; Hackenberger, Christian P R

    2017-08-01

    Functional antibody delivery in living cells would enable the labelling and manipulation of intracellular antigens, which constitutes a long-thought goal in cell biology and medicine. Here we present a modular strategy to create functional cell-permeable nanobodies capable of targeted labelling and manipulation of intracellular antigens in living cells. The cell-permeable nanobodies are formed by the site-specific attachment of intracellularly stable (or cleavable) cyclic arginine-rich cell-penetrating peptides to camelid-derived single-chain VHH antibody fragments. We used this strategy for the non-endocytic delivery of two recombinant nanobodies into living cells, which enabled the relocalization of the polymerase clamp PCNA (proliferating cell nuclear antigen) and tumour suppressor p53 to the nucleolus, and thereby allowed the detection of protein-protein interactions that involve these two proteins in living cells. Furthermore, cell-permeable nanobodies permitted the co-transport of therapeutically relevant proteins, such as Mecp2, into the cells. This technology constitutes a major step in the labelling, delivery and targeted manipulation of intracellular antigens. Ultimately, this approach opens the door towards immunostaining in living cells and the expansion of immunotherapies to intracellular antigen targets.

  12. Cell-permeable nanobodies for targeted immunolabelling and antigen manipulation in living cells

    Science.gov (United States)

    Herce, Henry D.; Schumacher, Dominik; Schneider, Anselm F. L.; Ludwig, Anne K.; Mann, Florian A.; Fillies, Marion; Kasper, Marc-André; Reinke, Stefan; Krause, Eberhard; Leonhardt, Heinrich; Cardoso, M. Cristina; Hackenberger, Christian P. R.

    2017-08-01

    Functional antibody delivery in living cells would enable the labelling and manipulation of intracellular antigens, which constitutes a long-thought goal in cell biology and medicine. Here we present a modular strategy to create functional cell-permeable nanobodies capable of targeted labelling and manipulation of intracellular antigens in living cells. The cell-permeable nanobodies are formed by the site-specific attachment of intracellularly stable (or cleavable) cyclic arginine-rich cell-penetrating peptides to camelid-derived single-chain VHH antibody fragments. We used this strategy for the non-endocytic delivery of two recombinant nanobodies into living cells, which enabled the relocalization of the polymerase clamp PCNA (proliferating cell nuclear antigen) and tumour suppressor p53 to the nucleolus, and thereby allowed the detection of protein-protein interactions that involve these two proteins in living cells. Furthermore, cell-permeable nanobodies permitted the co-transport of therapeutically relevant proteins, such as Mecp2, into the cells. This technology constitutes a major step in the labelling, delivery and targeted manipulation of intracellular antigens. Ultimately, this approach opens the door towards immunostaining in living cells and the expansion of immunotherapies to intracellular antigen targets.

  13. Enhanced fluorescence imaging of live cells by effective cytosolic delivery of probes.

    Directory of Open Access Journals (Sweden)

    Marzia Massignani

    Full Text Available BACKGROUND: Microscopic techniques enable real-space imaging of complex biological events and processes. They have become an essential tool to confirm and complement hypotheses made by biomedical scientists and also allow the re-examination of existing models, hence influencing future investigations. Particularly imaging live cells is crucial for an improved understanding of dynamic biological processes, however hitherto live cell imaging has been limited by the necessity to introduce probes within a cell without altering its physiological and structural integrity. We demonstrate herein that this hurdle can be overcome by effective cytosolic delivery. PRINCIPAL FINDINGS: We show the delivery within several types of mammalian cells using nanometre-sized biomimetic polymer vesicles (a.k.a. polymersomes that offer both highly efficient cellular uptake and endolysomal escape capability without any effect on the cellular metabolic activity. Such biocompatible polymersomes can encapsulate various types of probes including cell membrane probes and nucleic acid probes as well as labelled nucleic acids, antibodies and quantum dots. SIGNIFICANCE: We show the delivery of sufficient quantities of probes to the cytosol, allowing sustained functional imaging of live cells over time periods of days to weeks. Finally the combination of such effective staining with three-dimensional imaging by confocal laser scanning microscopy allows cell imaging in complex three-dimensional environments under both mono-culture and co-culture conditions. Thus cell migration and proliferation can be studied in models that are much closer to the in vivo situation.

  14. Live Cells Decreased Methane Production in Intestinal Content of Pigs

    Directory of Open Access Journals (Sweden)

    Y. L. Gong

    2013-06-01

    Full Text Available An in vitro gas production technique was used in this study to elucidate the effect of two strains of active live yeast on methane (CH4 production in the large intestinal content of pigs to provide an insight to whether active live yeast could suppress CH4 production in the hindgut of pigs. Treatments used in this study include blank (no substrate and no live yeast cells, control (no live yeast cells and yeast (YST supplementation groups (supplemented with live yeast cells, YST1 or YST2. The yeast cultures contained 1.8×1010 cells per g, which were added at the rates of 0.2 mg and 0.4 mg per ml of the fermented inoculum. Large intestinal contents were collected from 2 Duroc×Landrace×Yorkshire pigs, mixed with a phosphate buffer (1:2, and incubated anaerobically at 39°C for 24 h using 500 mg substrate (dry matter (DM basis. Total gas and CH4 production decreased (p<0.05 with supplementation of yeast. The methane production reduction potential (MRP was calculated by assuming net methane concentration for the control as 100%. The MRP of yeast 2 was more than 25%. Compared with the control group, in vitro DM digestibility (IVDMD and total volatile fatty acids (VFA concentration increased (p<0.05 in 0.4 mg/ml YST1 and 0.2 mg/ml YST2 supplementation groups. Proportion of propionate, butyrate and valerate increased (p<0.05, but that of acetate decreased (p<0.05, which led to a decreased (p<0.05 acetate: propionate (A: P ratio in the both YST2 treatments and the 0.4 mg/ml YST 1 supplementation groups. Hydrogen recovery decreased (p<0.05 with yeast supplementation. Quantity of methanogenic archaea per milliliter of inoculum decreased (p<0.05 with yeast supplementation after 24 h of incubation. Our results suggest that live yeast cells suppressed in vitro CH4 production when inoculated into the large intestinal contents of pigs and shifted the fermentation pattern to favor propionate production together with an increased population of acetogenic

  15. DESIGN OF SMALL AUTOMATION WORK CELL SYSTEM DEMONSTRATIONS

    International Nuclear Information System (INIS)

    TURNER, C.; PEHL, J.

    2000-01-01

    The introduction of automation systems into many of the facilities dealing with the production, use and disposition of nuclear materials has been an ongoing objective. Many previous attempts have been made, using a variety of monolithic and, in some cases, modular technologies. Many of these attempts were less than successful, owing to the difficulty of the problem, the lack of maturity of the technology, and over optimism about the capabilities of a particular system. Consequently, it is not surprising that suggestions that automation can reduce worker Occupational Radiation Exposure (ORE) levels are often met with skepticism and caution. The development of effective demonstrations of these technologies is of vital importance if automation is to become an acceptable option for nuclear material processing environments. The University of Texas Robotics Research Group (UTRRG) has been pursuing the development of technologies to support modular small automation systems (each of less than 5 degrees-of-freedom) and the design of those systems for more than two decades. Properly designed and implemented, these technologies have a potential to reduce the worker ORE associated with work in nuclear materials processing facilities. Successful development of systems for these applications requires the development of technologies that meet the requirements of the applications. These application requirements form a general set of rules that applicable technologies and approaches need to adhere to, but in and of themselves are generally insufficient for the design of a specific automation system. For the design of an appropriate system, the associated task specifications and relationships need to be defined. These task specifications also provide a means by which appropriate technology demonstrations can be defined. Based on the requirements and specifications of the operations of the Advanced Recovery and Integrated Extraction System (ARIES) pilot line at Los Alamos National

  16. Secondary Metabolite Localization by Autofluorescence in Living Plant Cells

    Directory of Open Access Journals (Sweden)

    Pascale Talamond

    2015-03-01

    Full Text Available 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.

  17. Autofluorescence-Free Live-Cell Imaging Using Terbium Nanoparticles.

    Science.gov (United States)

    Cardoso Dos Santos, M; Goetz, J; Bartenlian, H; Wong, K-L; Charbonnière, L J; Hildebrandt, N

    2018-04-18

    Fluorescent nanoparticles (NPs) have become irreplaceable tools for advanced cellular and subcellular imaging. While very bright NPs require excitation with UV or visible light, which can create strong autofluorescence of biological components, NIR-excitable NPs without autofluorescence issues exhibit much lower brightness. Here, we show the application of a new type of surface-photosensitized terbium NPs (Tb-NPs) for autofluorescence-free intracellular imaging in live HeLa cells. The combination of exceptionally high brightness, high photostability, and long photoluminecence (PL) lifetimes for highly efficient suppression of the short-lived autofluorescence allowed for time-gated PL imaging of intracellular vesicles over 72 h without toxicity and at extremely low Tb-NP concentrations down to 12 pM. Detection of highly resolved long-lifetime (ms) PL decay curves from small (∼10 μm 2 ) areas within single cells within a few seconds emphasized the unprecedented photophysical properties of Tb-NPs for live-cell imaging that extend well beyond currently available nanometric imaging agents.

  18. Collective Dynamics of Intracellular Water in Living Cells

    International Nuclear Information System (INIS)

    Orecchini, A; Sebastiani, F; Paciaroni, A; Petrillo, C; Sacchetti, F; Jasnin, M; Francesco, A De; Zaccai, G; Moulin, M; Haertlein, M

    2012-01-01

    Water dynamics plays a fundamental role for the fulfillment of biological functions in living organisms. Decades of hydrated protein powder studies have revealed the peculiar dynamical properties of hydration water with respect to pure water, due to close coupling interactions with the macromolecule. In such a framework, we have studied coherent collective dynamics in protein and DNA hydration water. State-of-the-art neutron instrumentation has allowed us to observe the propagation of coherent density fluctuations within the hydration shell of the biomolecules. The corresponding dispersion curves resulted to be only slightly affected by the coupling with the macromolecules. Nevertheless, the effects of the interaction appeared as a marked increase of the mode damping factors, which suggested a destructuring of the water hydrogen-bond network. Such results were interpreted as the signature of a 'glassy' dynamical character of macromolecule hydration water, in agreement with indications from measurements of the density of vibrational states. Extending the investigations to living organisms at physiological conditions, we present here an in-vivo study of collective dynamics of intracellular water in Escherichia coli cells. The cells and water were fully deuterated to minimise the incoherent neutron scattering background. The water dynamics observed in the living cells is discussed in terms of the dynamics of pure bulk water and that of hydration water measured in powder samples.

  19. Simulations of living cell origins using a cellular automata model.

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  1. Picosecond orientational dynamics of water in living cells.

    Science.gov (United States)

    Tros, Martijn; Zheng, Linli; Hunger, Johannes; Bonn, Mischa; Bonn, Daniel; Smits, Gertien J; Woutersen, Sander

    2017-10-12

    Cells are extremely crowded, and a central question in biology is how this affects the intracellular water. Here, we use ultrafast vibrational spectroscopy and dielectric-relaxation spectroscopy to observe the random orientational motion of water molecules inside living cells of three prototypical organisms: Escherichia coli, Saccharomyces cerevisiae (yeast), and spores of Bacillus subtilis. In all three organisms, most of the intracellular water exhibits the same random orientational motion as neat water (characteristic time constants ~9 and ~2 ps for the first-order and second-order orientational correlation functions), whereas a smaller fraction exhibits slower orientational dynamics. The fraction of slow intracellular water varies between organisms, ranging from ~20% in E. coli to ~45% in B. subtilis spores. Comparison with the water dynamics observed in solutions mimicking the chemical composition of (parts of) the cytosol shows that the slow water is bound mostly to proteins, and to a lesser extent to other biomolecules and ions.The cytoplasm's crowdedness leads one to expect that cell water is different from bulk water. By measuring the rotational motion of water molecules in living cells, Tros et al. find that apart from a small fraction of water solvating biomolecules, cell water has the same dynamics as bulk water.

  2. Bio-electrospraying and droplet-based microfluidics: control of cell numbers within living residues

    Energy Technology Data Exchange (ETDEWEB)

    Hong Jongin; DeMello, Andrew J [Nanostructured Materials and Devices Group, Department of Chemistry, Imperial College London, Exhibition Road, London SW7 2AZ (United Kingdom); Jayasinghe, Suwan N, E-mail: a.demello@imperial.ac.u, E-mail: s.jayasinghe@ucl.ac.u [BioPhysics Group, Department of Mechanical Engineering, University College London, Torrington Place, London WC1E 7JE (United Kingdom)

    2010-04-15

    Bio-electrospraying (BES) has demonstrated great promise as a rapidly evolving strategy for tissue engineering and regenerative biology/medicine. Since its discovery in 2005, many studies have confirmed that cells (immortalized, primary and stem cells) and whole organisms (Danio rerio, Xenopus tropicalis, Caenorhabditis elegans to Drosophila) remain viable post-bio-electrospraying. Although this bio-protocol has achieved much, it suffers from one crucial problem, namely the ability to precisely control the number of cells within droplets and or encapsulations. If overcome, BES has the potential to become a high-efficiency biotechnique for controlled cell encapsulation, a technique most useful for a wide range of applications in biology and medicine ranging from the forming of three-dimensional cultures to an approach for treating diseases such as type I diabetes. In this communication, we address this issue by demonstrating the coupling of BES with droplet-based microfluidics for controlling live cell numbers within droplets and residues. (communication)

  3. Bio-electrospraying and droplet-based microfluidics: control of cell numbers within living residues

    International Nuclear Information System (INIS)

    Hong Jongin; DeMello, Andrew J; Jayasinghe, Suwan N

    2010-01-01

    Bio-electrospraying (BES) has demonstrated great promise as a rapidly evolving strategy for tissue engineering and regenerative biology/medicine. Since its discovery in 2005, many studies have confirmed that cells (immortalized, primary and stem cells) and whole organisms (Danio rerio, Xenopus tropicalis, Caenorhabditis elegans to Drosophila) remain viable post-bio-electrospraying. Although this bio-protocol has achieved much, it suffers from one crucial problem, namely the ability to precisely control the number of cells within droplets and or encapsulations. If overcome, BES has the potential to become a high-efficiency biotechnique for controlled cell encapsulation, a technique most useful for a wide range of applications in biology and medicine ranging from the forming of three-dimensional cultures to an approach for treating diseases such as type I diabetes. In this communication, we address this issue by demonstrating the coupling of BES with droplet-based microfluidics for controlling live cell numbers within droplets and residues. (communication)

  4. Cocompartmentation of proteins and K+ within the living cell

    International Nuclear Information System (INIS)

    Kellermayer, M.; Ludany, A.; Jobst, K.; Szucs, G.; Trombitas, K.; Hazlewood, C.F.

    1986-01-01

    Monolayer H-50 tissue culture cells were treated with Triton X-100 and Brij 58 nonionic detergents, and their electron microscopic morphology along with the release of the intracellular proteins [ 35 S]methionine-labelled and 42 K-labelled K + were studied. Although Triton X-100 was more effective, both detergents removed the lipoid membranes within 5 min. The mobilization and solubilization of the cytoplasmic and nuclear proteins occurred much faster with Triton X-100 than with Brij 58. In Triton X-100-treated cells, the loss of K + was complete within 2 min. The loss of K + from the Brij 58-treated cells was complete only after 10 min and the mobilization of K + showed sigmoid-type release kinetics. These results support the view that most of K + and diffusible proteins are not freely dissolved in the cellular water, but they are cocompartmentalized inside the living cell

  5. Digital photocontrol of the network of live excitable cells

    Science.gov (United States)

    Erofeev, I. S.; Magome, N.; Agladze, K. I.

    2011-11-01

    Recent development of tissue engineering techniques allows creating and maintaining almost indefinitely networks of excitable cells with desired architecture. We coupled the network of live excitable cardiac cells with a common computer by sensitizing them to light, projecting a light pattern on the layer of cells, and monitoring excitation with the aid of fluorescent probes (optical mapping). As a sensitizing substance we used azobenzene trimethylammonium bromide (AzoTAB). This substance undergoes cis-trans-photoisomerization and trans-isomer of AzoTAB inhibits excitation in the cardiac cells, while cis-isomer does not. AzoTAB-mediated sensitization allows, thus, reversible and dynamic control of the excitation waves through the entire cardiomyocyte network either uniformly, or in a preferred spatial pattern. Technically, it was achieved by coupling a common digital projector with a macroview microscope and using computer graphic software for creating the projected pattern of conducting pathways. This approach allows real time interactive photocontrol of the heart tissue.

  6. Arginine-rich intracellular delivery peptides noncovalently transport protein into living cells

    International Nuclear Information System (INIS)

    Wang, Y.-H.; Chen, C.-P.; Chan, M.-H.; Chang, M.; Hou, Y.-W.; Chen, H.-H.; Hsu, H.-R.; Liu, Kevin; Lee, H.-J.

    2006-01-01

    Plasma membranes of plant or animal cells are generally impermeable to peptides or proteins. Many basic peptides have previously been investigated and covalently cross-linked with cargoes for cellular internalization. In the current study, we demonstrate that arginine-rich intracellular delivery (AID) peptides are able to deliver fluorescent proteins or β-galactosidase enzyme into animal and plant cells, as well as animal tissue. Cellular internalization and transdermal delivery of protein could be mediated by effective and nontoxic AID peptides in a neither fusion protein nor conjugation fashion. Therefore, noncovalent AID peptides may provide a useful strategy to have active proteins function in living cells and tissues in vivo

  7. Fully synthetic phage-like system for screening mixtures of small molecules in live cells.

    Science.gov (United States)

    Byk, Gerardo; Partouche, Shirly; Weiss, Aryeh; Margel, Shlomo; Khandadash, Raz

    2010-05-10

    A synthetic "phage-like" system was designed for screening mixtures of small molecules in live cells. The core of the system consists of 2 mum diameter cross-linked monodispersed microspheres bearing a panel of fluorescent tags and peptides or small molecules either directly synthesized or covalently conjugated to the microspheres. The microsphere mixtures were screened for affinity to cell line PC-3 (prostate cancer model) by incubation with live cells, and as was with phage-display peptide methods, unbound microspheres were removed by repeated washings followed by total lysis of cells and analysis of the bound microspheres by flow-cytometry. Similar to phage-display peptide screening, this method can be applied even in the absence of prior information about the cellular targets of the candidate ligands, which makes the system especially interesting for selection of molecules with high affinity for desired cells, tissues, or tumors. The advantage of the proposed system is the possibility of screening synthetic non-natural peptides or small molecules that cannot be expressed and screened using phage display libraries. A library composed of small molecules synthesized by the Ugi reaction was screened, and a small molecule, Rak-2, which strongly binds to PC-3 cells was found. Rak-2 was then individually synthesized and validated in a complementary whole cell-based binding assay, as well as by live cell microscopy. This new system demonstrates that a mixture of molecules bound to subcellular sized microspheres can be screened on plated cells. Together with other methods using subcellular sized particles for cellular multiplexing, this method represents an important milestone toward high throughput screening of mixtures of small molecules in live cells and in vivo with potential applications in the fields of drug delivery and diagnostic imaging.

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

    Science.gov (United States)

    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

  9. Intergovernmental Advanced Stationary PEM Fuel Cell System Demonstration Final Report

    Energy Technology Data Exchange (ETDEWEB)

    Rich Chartrand

    2011-08-31

    A program to complete the design, construction and demonstration of a PEMFC system fuelled by Ethanol, LPG or NG for telecom applications was initiated in October 2007. Early in the program the economics for Ethanol were shown to be unfeasible and permission was given by DOE to focus on LPG only. The design and construction of a prototype unit was completed in Jun 2009 using commercially available PEM FC stack from Ballard Power Systems. During the course of testing, the high pressure drop of the stack was shown to be problematic in terms of control and stability of the reformer. Also, due to the power requirements for air compression the overall efficiency of the system was shown to be lower than a similar system using internally developed low pressure drop FC stack. In Q3 2009, the decision was made to change to the Plug power stack and a second prototype was built and tested. Overall net efficiency was shown to be 31.5% at 3 kW output. Total output of the system is 6 kW. Using the new stack hardware, material cost reduction of 63% was achieved over the previous Alpha design. During a November 2009 review meeting Plug Power proposed and was granted permission, to demonstrate the new, commercial version of Plug Power's telecom system at CERL. As this product was also being tested as part of a DOE Topic 7A program, this part of the program was transferred to the Topic 7A program. In Q32008, the scope of work of this program was expanded to include a National Grid demonstration project of a micro-CHP system using hightemperature PEM technology. The Gensys Blue system was cleared for unattended operation, grid connection, and power generation in Aug 2009 at Union College in NY state. The system continues to operate providing power and heat to Beuth House. The system is being continually evaluated and improvements to hardware and controls will be implemented as more is learned about the system's operation. The program is instrumental in improving the

  10. Skin vaccination with live virus vectored microneedle arrays induce long lived CD8(+) T cell memory.

    Science.gov (United States)

    Becker, Pablo D; Hervouet, Catherine; Mason, Gavin M; Kwon, Sung-Yun; Klavinskis, Linda S

    2015-09-08

    A simple dissolvable microneedle array (MA) platform has emerged as a promising technology for vaccine delivery, due to needle-free injection with a formulation that preserves the immunogenicity of live viral vectored vaccines dried in the MA matrix. While recent studies have focused largely on design parameters optimized to induce primary CD8(+) T cell responses, the hallmark of a vaccine is synonymous with engendering long-lasting memory. Here, we address the capacity of dried MA vaccination to programme phenotypic markers indicative of effector/memory CD8(+) T cell subsets and also responsiveness to recall antigen benchmarked against conventional intradermal (ID) injection. We show that despite a slightly lower frequency of dividing T cell receptor transgenic CD8(+) T cells in secondary lymphoid tissue at an early time point, the absolute number of CD8(+) T cells expressing an effector memory (CD62L(-)CD127(+)) and central memory (CD62L(+)CD127(+)) phenotype during peak expansion were comparable after MA and ID vaccination with a recombinant human adenovirus type 5 vector (AdHu5) encoding HIV-1 gag. Similarly, both vaccination routes generated CD8(+) memory T cell subsets detected in draining LNs for at least two years post-vaccination capable of responding to secondary antigen. These data suggest that CD8(+) T cell effector/memory generation and long-term memory is largely unaffected by physical differences in vaccine delivery to the skin via dried MA or ID suspension. Copyright © 2015 Elsevier Ltd. All rights reserved.

  11. Labeling proteins inside living cells using external fluorophores for microscopy.

    Science.gov (United States)

    Teng, Kai Wen; Ishitsuka, Yuji; Ren, Pin; Youn, Yeoan; Deng, Xiang; Ge, Pinghua; Lee, Sang Hak; Belmont, Andrew S; Selvin, Paul R

    2016-12-09

    Site-specific fluorescent labeling of proteins inside live mammalian cells has been achieved by employing Streptolysin O, a bacterial enzyme which forms temporary pores in the membrane and allows delivery of virtually any fluorescent probes, ranging from labeled IgG's to small ligands, with high efficiency (>85% of cells). The whole process, including recovery, takes 30 min, and the cell is ready to be imaged immediately. A variety of cell viability tests were performed after treatment with SLO to ensure that the cells have intact membranes, are able to divide, respond normally to signaling molecules, and maintains healthy organelle morphology. When combined with Oxyrase, a cell-friendly photostabilizer, a ~20x improvement in fluorescence photostability is achieved. By adding in glutathione, fluorophores are made to blink, enabling super-resolution fluorescence with 20-30 nm resolution over a long time (~30 min) under continuous illumination. Example applications in conventional and super-resolution imaging of native and transfected cells include p65 signal transduction activation, single molecule tracking of kinesin, and specific labeling of a series of nuclear and cytoplasmic protein complexes.

  12. Sorting live stem cells based on Sox2 mRNA expression.

    Directory of Open Access Journals (Sweden)

    Hans M Larsson

    Full Text Available While cell sorting usually relies on cell-surface protein markers, molecular beacons (MBs offer the potential to sort cells based on the presence of any expressed mRNA and in principle could be extremely useful to sort rare cell populations from primary isolates. We show here how stem cells can be purified from mixed cell populations by sorting based on MBs. Specifically, we designed molecular beacons targeting Sox2, a well-known stem cell marker for murine embryonic (mES and neural stem cells (NSC. One of our designed molecular beacons displayed an increase in fluorescence compared to a nonspecific molecular beacon both in vitro and in vivo when tested in mES and NSCs. We sorted Sox2-MB(+SSEA1(+ cells from a mixed population of 4-day retinoic acid-treated mES cells and effectively isolated live undifferentiated stem cells. Additionally, Sox2-MB(+ cells isolated from primary mouse brains were sorted and generated neurospheres with higher efficiency than Sox2-MB(- cells. These results demonstrate the utility of MBs for stem cell sorting in an mRNA-specific manner.

  13. Selective turn-on fluorescent probes for imaging hydrogen sulfide in living cells.

    Science.gov (United States)

    Montoya, Leticia A; Pluth, Michael D

    2012-05-16

    Hydrogen sulfide (H(2)S) is an important biological messenger but few biologically-compatible methods are available for its detection. Here we report two bright fluorescent probes that are selective for H(2)S over cysteine, glutathione and other reactive sulfur, nitrogen, and oxygen species. Both probes are demonstrated to detect H(2)S in live cells. This journal is © The Royal Society of Chemistry 2012

  14. Detecting protein-protein interactions in living cells

    DEFF Research Database (Denmark)

    Gottschalk, Marie; Bach, Anders; Hansen, Jakob Lerche

    2009-01-01

    to the endogenous C-terminal peptide of the NMDA receptor, as evaluated by a cell-free protein-protein interaction assay. However, it is important to address both membrane permeability and effect in living cells. Therefore a bioluminescence resonance energy transfer (BRET) assay was established, where the C......-terminal of the NMDA receptor and PDZ2 of PSD-95 were fused to green fluorescent protein (GFP) and Renilla luciferase (Rluc) and expressed in COS7 cells. A robust and specific BRET signal was obtained by expression of the appropriate partner proteins and subsequently, the assay was used to evaluate a Tat......The PDZ domain mediated interaction between the NMDA receptor and its intracellular scaffolding protein, PSD-95, is a potential target for treatment of ischemic brain diseases. We have recently developed a number of peptide analogues with improved affinity for the PDZ domains of PSD-95 compared...

  15. A new fluorescent pH probe for imaging lysosomes in living cells.

    Science.gov (United States)

    Lv, Hong-Shui; Huang, Shu-Ya; Xu, Yu; Dai, Xi; Miao, Jun-Ying; Zhao, Bao-Xiang

    2014-01-15

    A new rhodamine B-based pH fluorescent probe has been synthesized and characterized. The probe responds to acidic pH with short response time, high selectivity and sensitivity, and exhibits a more than 20-fold increase in fluorescence intensity within the pH range of 7.5-4.1 with the pKa value of 5.72, which is valuable to study acidic organelles in living cells. Also, it has been successfully applied to HeLa cells, for its low cytotoxicity, brilliant photostability, good membrane permeability and no 'alkalizing effect' on lysosomes. The results demonstrate that this probe is a lysosome-specific probe, which can selectively stain lysosomes and monitor lysosomal pH changes in living cells. Copyright © 2013 Elsevier Ltd. All rights reserved.

  16. Tracking chemical changes in a live cell: Biomedical applications of SR-FTIR spectromicroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Holman, Hoi-Ying N.; Martin, Michael C.; McKinney, Wayne R.

    2002-07-25

    Synchrotron radiation-based Fourier transform infrared (SR-FTIR) spectromicroscopy is a newly emerging bioanalytical and imaging tool. This unique technique provides mid-infrared (IR) spectra, hence chemical information, with high signal-to-noise at spatial resolutions as fine as 3 to 10 microns. Thus it enables researchers to locate, identify, and track specific chemical events within an individual living mammalian cell. Mid-IR photons are too low in energy (0.05 - 0.5 eV) to either break bonds or to cause ionization. In this review, we show that the synchrotron IR beam has no detectable effects on the short- and long-term viability, reproductive integrity, cell-cycle progression, and mitochondrial metabolism in living human cells, and produces only minimal sample heating (< 0.5 degrees C). We will then present several examples demonstrating the application potentials of SR-FTIR spectromicroscopy in biomedical research. These will include monitoring living cells progressing through the cell cycle, including death, and cells reacting to dilute concentrations of toxins.

  17. Tracking chemical changes in a live cell: Biomedical applications of SR-FTIR spectromicroscopy

    International Nuclear Information System (INIS)

    Holman, Hoi-Ying N.; Martin, Michael C.; McKinney, Wayne R.

    2002-01-01

    Synchrotron radiation-based Fourier transform infrared (SR-FTIR) spectromicroscopy is a newly emerging bioanalytical and imaging tool. This unique technique provides mid-infrared (IR) spectra, hence chemical information, with high signal-to-noise at spatial resolutions as fine as 3 to 10 microns. Thus it enables researchers to locate, identify, and track specific chemical events within an individual living mammalian cell. Mid-IR photons are too low in energy (0.05 - 0.5 eV) to either break bonds or to cause ionization. In this review, we show that the synchrotron IR beam has no detectable effects on the short- and long-term viability, reproductive integrity, cell-cycle progression, and mitochondrial metabolism in living human cells, and produces only minimal sample heating (< 0.5 degrees C). We will then present several examples demonstrating the application potentials of SR-FTIR spectromicroscopy in biomedical research. These will include monitoring living cells progressing through the cell cycle, including death, and cells reacting to dilute concentrations of toxins

  18. Labeling RNAs in Live Cells Using Malachite Green Aptamer Scaffolds as Fluorescent Probes.

    Science.gov (United States)

    Yerramilli, V Siddartha; Kim, Kyung Hyuk

    2018-03-16

    RNAs mediate many different processes that are central to cellular function. The ability to quantify or image RNAs in live cells is very useful in elucidating such functions of RNA. RNA aptamer-fluorogen systems have been increasingly used in labeling RNAs in live cells. Here, we use the malachite green aptamer (MGA), an RNA aptamer that can specifically bind to malachite green (MG) dye and induces it to emit far-red fluorescence signals. Previous studies on MGA showed a potential for the use of MGA for genetically tagging other RNA molecules in live cells. However, these studies also exhibited low fluorescence signals and high background noise. Here we constructed and tested RNA scaffolds containing multiple tandem repeats of MGA as a strategy to increase the brightness of the MGA aptamer-fluorogen system as well as to make the system fluoresce when tagging various RNA molecules, in live cells. We demonstrate that our MGA scaffolds can induce fluorescence signals by up to ∼20-fold compared to the basal level as a genetic tag for other RNA molecules. We also show that our scaffolds function reliably as genetically encoded fluorescent tags for mRNAs of fluorescent proteins and other RNA aptamers.

  19. Fluorescent tags of protein function in living cells.

    Science.gov (United States)

    Whitaker, M

    2000-02-01

    A cell's biochemistry is now known to be the biochemistry of molecular machines, that is, protein complexes that are assembled and dismantled in particular locations within the cell as needed. One important element in our understanding has been the ability to begin to see where proteins are in cells and what they are doing as they go about their business. Accordingly, there is now a strong impetus to discover new ways of looking at the workings of proteins in living cells. Although the use of fluorescent tags to track individual proteins in cells has a long history, the availability of laser-based confocal microscopes and the imaginative exploitation of the green fluorescent protein from jellyfish have provided new tools of great diversity and utility. It is now possible to watch a protein bind its substrate or its partners in real time and with submicron resolution within a single cell. The importance of processes of self-organisation represented by protein folding on the one hand and subcellular organelles on the other are well recognised. Self-organisation at the intermediate level of multimeric protein complexes is now open to inspection. BioEssays 22:180-187, 2000. Copyright 2000 John Wiley & Sons, Inc.

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

    International Nuclear Information System (INIS)

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

    2010-01-01

    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.

  1. Opportunities for PEM fuel cell commercialization : fuel cell electric vehicle demonstration in Shanghai

    Energy Technology Data Exchange (ETDEWEB)

    Ma, Z.F. [Shanghai Jiao Tong Univ., Shanghai (China). Dept. of Chemical Engineering

    2006-07-01

    The research and development activities devoted to the development of the proton exchange membrane fuel cell (PEMFC) were discussed with reference to its application in the fuel cell electric vehicle (FCEV). In the past decade, PEMFC technology has been successfully applied in both the automobile and residential sector worldwide. In China, more than one billion RMB yuan has been granted by the Chinese government to develop PEM fuel cell technology over the past 5 years, particularly for commercialization of the fuel cell electric vehicle (FCEV). The City of Shanghai has played a significant role in the FCEV demonstration with involvement by Shanghai Auto Industrial Company (SAIC), Tongji University, Shanghai Jiaotong University, and Shanghai Shenli High Tech Co. Ltd. These participants were involved in the development and integration of the following components into the FCEV: fuel cell engines, batteries, FCEV electric control systems, and primary materials for the fuel cell stack. During the course of the next five year-plan (2006-2010), Shanghai will promote the commercialization of FCEV. More than one thousand FCEVs will be manufactured and an FCEV fleet will be in operation throughout Shanghai City by 2010.

  2. Imaging proteolytic activity in live cells and animal models.

    Directory of Open Access Journals (Sweden)

    Stefanie Galbán

    Full Text Available In addition to their degradative role in protein turnover, proteases play a key role as positive or negative regulators of signal transduction pathways and therefore their dysregulation contributes to many disease states. Regulatory roles of proteases include their hormone-like role in triggering G protein-coupled signaling (Protease-Activated-Receptors; their role in shedding of ligands such as EGF, Notch and Fas; and their role in signaling events that lead to apoptotic cell death. Dysregulated activation of apoptosis by the caspase family of proteases has been linked to diseases such as cancer, autoimmunity and inflammation. In an effort to better understand the role of proteases in health and disease, a luciferase biosensor is described which can quantitatively report proteolytic activity in live cells and mouse models. The biosensor, hereafter referred to as GloSensor Caspase 3/7 has a robust signal to noise (50-100 fold and dynamic range such that it can be used to screen for pharmacologically active compounds in high throughput campaigns as well as to study cell signaling in rare cell populations such as isolated cancer stem cells. The biosensor can also be used in the context of genetically engineered mouse models of human disease wherein conditional expression using the Cre/loxP technology can be implemented to investigate the role of a specific protease in living subjects. While the regulation of apoptosis by caspase's was used as an example in these studies, biosensors to study additional proteases involved in the regulation of normal and pathological cellular processes can be designed using the concepts presented herein.

  3. Localization of mitochondria in living cells with rhodamine 123.

    Science.gov (United States)

    Johnson, L V; Walsh, M L; Chen, L B

    1980-01-01

    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. Images PMID:6965798

  4. Live cell microscopy of DNA damage response in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Pinela da Silva, Sonia Cristina; Gallina, Irene; Eckert-Boulet, Nadine Valerie

    2012-01-01

    live cell imaging allows for multiple cellular markers to be monitored over several hours. This chapter reviews useful fluorescent markers and genotoxic agents for studying the DNA damage response in living cells and provides protocols for live cell imaging, time-lapse microscopy, and for induction...

  5. 78 FR 49528 - Consolidation of Wound Care Products Containing Live Cells

    Science.gov (United States)

    2013-08-14

    ...] Consolidation of Wound Care Products Containing Live Cells AGENCY: Food and Drug Administration, HHS. ACTION... certain wound care products containing live cells from the Center for Devices and Radiological Health... CDRH and CBER. FDA believes that as more wound care products containing live cells are developed such...

  6. An integrated on-line irradiation and in situ live cell imaging system

    Energy Technology Data Exchange (ETDEWEB)

    Liang, Ying; Fu, Qibin; Wang, Weikang; Liu, Yu; Liu, Feng; Yang, Gen, E-mail: gen.yang@pku.edu.cn; Wang, Yugang

    2015-09-01

    Ionizing radiation poses a threat to genome integrity by introducing DNA damages, particularly DNA double-strand breaks (DSB) in cells. Understanding how cells react to DSB and maintain genome integrity is of major importance, since increasing evidences indicate the links of DSB with genome instability and cancer predispositions. However, tracking the dynamics of DNA damages and repair response to ionizing radiation in individual cell is difficult. Here we describe the development of an on-line irradiation and in situ live cell imaging system based on isotopic sources at Institute of Heavy Ion Physics, Peking University. The system was designed to irradiate cells and in situ observe the cellular responses to ionizing radiation in real time. On-line irradiation was achieved by mounting a metal framework that hold an isotopic γ source above the cell culture dish for γ irradiation; or by integrating an isotopic α source to an objective lens under the specialized cell culture dish for α irradiation. Live cell imaging was performed on a confocal microscope with an environmental chamber installed on the microscope stage. Culture conditions in the environment chamber such as CO{sub 2}, O{sub 2} concentration as well as temperature are adjustable, which further extends the capacity of the system and allows more flexible experimental design. We demonstrate the use of this system by tracking the DSB foci formation and disappearance in individual cells after exposure to irradiation. On-line irradiation together with in situ live cell imaging in adjustable culture conditions, the system overall provides a powerful tool for investigation of cellular and subcellular response to ionizing radiation under different physiological conditions such as hyperthermia or hypoxia.

  7. An integrated on-line irradiation and in situ live cell imaging system

    International Nuclear Information System (INIS)

    Liang, Ying; Fu, Qibin; Wang, Weikang; Liu, Yu; Liu, Feng; Yang, Gen; Wang, Yugang

    2015-01-01

    Ionizing radiation poses a threat to genome integrity by introducing DNA damages, particularly DNA double-strand breaks (DSB) in cells. Understanding how cells react to DSB and maintain genome integrity is of major importance, since increasing evidences indicate the links of DSB with genome instability and cancer predispositions. However, tracking the dynamics of DNA damages and repair response to ionizing radiation in individual cell is difficult. Here we describe the development of an on-line irradiation and in situ live cell imaging system based on isotopic sources at Institute of Heavy Ion Physics, Peking University. The system was designed to irradiate cells and in situ observe the cellular responses to ionizing radiation in real time. On-line irradiation was achieved by mounting a metal framework that hold an isotopic γ source above the cell culture dish for γ irradiation; or by integrating an isotopic α source to an objective lens under the specialized cell culture dish for α irradiation. Live cell imaging was performed on a confocal microscope with an environmental chamber installed on the microscope stage. Culture conditions in the environment chamber such as CO 2 , O 2 concentration as well as temperature are adjustable, which further extends the capacity of the system and allows more flexible experimental design. We demonstrate the use of this system by tracking the DSB foci formation and disappearance in individual cells after exposure to irradiation. On-line irradiation together with in situ live cell imaging in adjustable culture conditions, the system overall provides a powerful tool for investigation of cellular and subcellular response to ionizing radiation under different physiological conditions such as hyperthermia or hypoxia

  8. An integrated on-line irradiation and in situ live cell imaging system

    Science.gov (United States)

    Liang, Ying; Fu, Qibin; Wang, Weikang; Liu, Yu; Liu, Feng; Yang, Gen; Wang, Yugang

    2015-09-01

    Ionizing radiation poses a threat to genome integrity by introducing DNA damages, particularly DNA double-strand breaks (DSB) in cells. Understanding how cells react to DSB and maintain genome integrity is of major importance, since increasing evidences indicate the links of DSB with genome instability and cancer predispositions. However, tracking the dynamics of DNA damages and repair response to ionizing radiation in individual cell is difficult. Here we describe the development of an on-line irradiation and in situ live cell imaging system based on isotopic sources at Institute of Heavy Ion Physics, Peking University. The system was designed to irradiate cells and in situ observe the cellular responses to ionizing radiation in real time. On-line irradiation was achieved by mounting a metal framework that hold an isotopic γ source above the cell culture dish for γ irradiation; or by integrating an isotopic α source to an objective lens under the specialized cell culture dish for α irradiation. Live cell imaging was performed on a confocal microscope with an environmental chamber installed on the microscope stage. Culture conditions in the environment chamber such as CO2, O2 concentration as well as temperature are adjustable, which further extends the capacity of the system and allows more flexible experimental design. We demonstrate the use of this system by tracking the DSB foci formation and disappearance in individual cells after exposure to irradiation. On-line irradiation together with in situ live cell imaging in adjustable culture conditions, the system overall provides a powerful tool for investigation of cellular and subcellular response to ionizing radiation under different physiological conditions such as hyperthermia or hypoxia.

  9. Live cell imaging of actin dynamics in dexamethasone-treated porcine trabecular meshwork cells.

    Science.gov (United States)

    Fujimoto, Tomokazu; Inoue, Toshihiro; Inoue-Mochita, Miyuki; Tanihara, Hidenobu

    2016-04-01

    The regulation of the actin cytoskeleton in trabecular meshwork (TM) cells is important for controlling outflow of the aqueous humor. In some reports, dexamethasone (DEX) increased the aqueous humor outflow resistance and induced unusual actin structures, such as cross-linked actin networks (CLAN), in TM cells. However, the functions and dynamics of CLAN in TM cells are not completely known, partly because actin stress fibers have been observed only in fixed cells. We conducted live-cell imaging of the actin dynamics in TM cells with or without DEX treatment. An actin-green fluorescent protein (GFP) fusion construct with a modified insect virus was transfected into porcine TM cells. Time-lapse imaging of live TM cells treated with 25 μM Y-27632 and 100 nM DEX was performed using an inverted fluorescence microscope. Fluorescent images were recorded every 15 s for 30 min after Y-27632 treatment or every 30 min for 72 h after DEX treatment. The GFP-actin was expressed in 22.7 ± 10.9% of the transfected TM cells. In live TM cells, many actin stress fibers were observed before the Y-27632 treatment. Y-27632 changed the cell shape and decreased stress fibers in a time-dependent manner. In fixed cells, CLAN-like structures were seen in 26.5 ± 1.7% of the actin-GFP expressed PTM cells treated with DEX for 72 h. In live imaging, there was 28% CLAN-like structure formation at 72 h after DEX treatment, and the lifetime of CLAN-like structures increased after DEX treatment. The DEX-treated cells with CLAN-like structures showed less migration than DEX-treated cells without CLAN-like structures. Furthermore, the control cells (without DEX treatment) with CLAN-like structures also showed less migration than the control cells without CLAN-like structures. These results suggested that CLAN-like structure formation was correlated with cell migration in TM cells. Live cell imaging of the actin cytoskeleton provides valuable information on the actin dynamics in TM

  10. Demonstration of a visual cell-based assay for screening glucose ...

    Indian Academy of Sciences (India)

    GLUT4 translocation is visualized by live cell imaging based on GFP fluorescence by employing a cooled charge-coupled device camera attached to a fluorescent microscope. This video imaging method and further quantitative analysis of GLUT4 on the cell membrane provide rapid and foolproof visual evidence that this ...

  11. Sub-15 fs multiphoton lithography of three-dimensional structures for live cell applications

    International Nuclear Information System (INIS)

    Licht, Martin; Uchugonova, Aisada; König, Karsten; Straub, Martin

    2012-01-01

    Development, morphology and intratissue location of cells are influenced by the 3D nano- and microenvironment. In this paper we demonstrate multiphoton photopolymerization to generate three-dimensional structures for cell culture applications with micro- and nanotopographic features using SU-8 photoresist and mr-NIL 6000 nanoimprint resist. Moving the focal spot of high-repetition rate near-infrared sub-15 fs pulsed laser light by a galvanometric beam scanner in combination with a piezoelectric vertical stage, nearly arbitrary trajectories of polymerized photoresist were generated. This technique can be used to generate cage structures with submicron interior features for live cell applications. Preliminary experiments with PC-3 and HT-1080 cells indicate the influence of the structures on cell behavior. (paper)

  12. g-force induced giant efficiency of nanoparticles internalization into living cells

    Science.gov (United States)

    Ocampo, Sandra M.; Rodriguez, Vanessa; de La Cueva, Leonor; Salas, Gorka; Carrascosa, Jose. L.; Josefa Rodríguez, María; García-Romero, Noemí; Luis, Jose; Cuñado, F.; Camarero, Julio; Miranda, Rodolfo; Belda-Iniesta, Cristobal; Ayuso-Sacido, Angel

    2015-10-01

    Nanotechnology plays an increasingly important role in the biomedical arena. Iron oxide nanoparticles (IONPs)-labelled cells is one of the most promising approaches for a fast and reliable evaluation of grafted cells in both preclinical studies and clinical trials. Current procedures to label living cells with IONPs are based on direct incubation or physical approaches based on magnetic or electrical fields, which always display very low cellular uptake efficiencies. Here we show that centrifugation-mediated internalization (CMI) promotes a high uptake of IONPs in glioblastoma tumour cells, just in a few minutes, and via clathrin-independent endocytosis pathway. CMI results in controllable cellular uptake efficiencies at least three orders of magnitude larger than current procedures. Similar trends are found in human mesenchymal stem cells, thereby demonstrating the general feasibility of the methodology, which is easily transferable to any laboratory with great potential for the development of improved biomedical applications.

  13. A benzothiazole-based fluorescent probe for hypochlorous acid detection and imaging in living cells

    Science.gov (United States)

    Nguyen, Khac Hong; Hao, Yuanqiang; Zeng, Ke; Fan, Shengnan; Li, Fen; Yuan, Suke; Ding, Xuejing; Xu, Maotian; Liu, You-Nian

    2018-06-01

    A benzothiazole-based turn-on fluorescent probe with a large Stokes shift (190 nm) has been developed for hypochlorous acid detection. The probe displays prompt fluorescence response for HClO with excellent selectivity over other reactive oxygen species as well as a low detection limit of 0.08 μM. The sensing mechanism involves the HClO-induced specific oxidation of oxime moiety of the probe to nitrile oxide, which was confirmed by HPLC-MS technique. Furthermore, imaging studies demonstrated that the probe is cell permeable and can be applied to detect HClO in living cells.

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

    Science.gov (United States)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Stickney, Zachary, E-mail: zstickney@scu.edu; Losacco, Joseph, E-mail: jlosacco@scu.edu; McDevitt, Sophie, E-mail: smmcdevitt@scu.edu; Zhang, Zhiwen, E-mail: zzhang@scu.edu; Lu, Biao, E-mail: blu2@scu.edu

    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.

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

    International Nuclear Information System (INIS)

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

    2016-01-01

    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.

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

    Science.gov (United States)

    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.

  18. Direct imaging of APP proteolysis in living cells.

    Science.gov (United States)

    Parenti, Niccoló; Del Grosso, Ambra; Antoni, Claudia; Cecchini, Marco; Corradetti, Renato; Pavone, Francesco S; Calamai, Martino

    2017-01-01

    Alzheimer's disease is a multifactorial disorder caused by the interaction of genetic, epigenetic and environmental factors. The formation of cytotoxic oligomers consisting of A β peptide is widely accepted as being one of the main key events triggering the development of Alzheimer's disease. A β peptide production results from the specific proteolytic processing of the amyloid precursor protein (APP). Deciphering the factors governing the activity of the secretases responsible for the cleavage of APP is still a critical issue. Kits available commercially measure the enzymatic activity of the secretases from cells lysates, in vitro . By contrast, we have developed a prototypal rapid bioassay that provides visible information on the proteolytic processing of APP directly in living cells. APP was fused to a monomeric variant of the green fluorescent protein and a monomeric variant of the red fluorescent protein at the C-terminal and N-terminal (mChAPPmGFP), respectively. Changes in the proteolytic processing rate in transfected human neuroblastoma and rat neuronal cells were imaged with confocal microscopy as changes in the red/green fluorescence intensity ratio. The significant decrease in the mean red/green ratio observed in cells over-expressing the β -secretase BACE1, or the α -secretase ADAM10, fused to a monomeric blue fluorescent protein confirms that the proteolytic site is still accessible. Specific siRNA was used to evaluate the contribution of endogenous BACE1. Interestingly, we found that the degree of proteolytic processing of APP is not completely homogeneous within the same single cell, and that there is a high degree of variability between cells of the same type. We were also able to follow with a fluorescence spectrometer the changes in the red emission intensity of the extracellular medium when BACE1 was overexpressed. This represents a complementary approach to fluorescence microscopy for rapidly detecting changes in the proteolytic processing

  19. Gold nanoparticles delivery in mammalian live cells: a critical review

    Directory of Open Access Journals (Sweden)

    Raphaël Lévy

    2010-02-01

    the University of Liverpool as a Post-doctoral Marie Curie Research Fellow. In 2006, he obtained a prestigious David Phillips Fellowship, to develop single particle-based imaging in living cells (photothermal microscopy. His research interests include the design and characterization of nanomaterials and their interactions with living cells. Umbreen Shaheen completed her Master in Zoology and then lectured at the University of Balochistan. She studied biotechnology at the National Institute of Biotechnology and Genetic Engineering (NIBGE, Pakistan and is currently doing her PhD at the University of Liverpool, on intracellular delivery of peptide-capped gold nanoparticles. Yann Cesbron is a PhD student at the University of Liverpool, developing photothermal microscopy for biological imaging. He graduated at the University Louis Pasteur (Strasbourg, France with a Master of Science in Condensed Matter Physics and a second Master of Science in Polymer Materials. He moved to Liverpool in 2006 to start his PhD. Violaine Sée is a BBSRC David Phillips Research Fellow at the University of Liverpool. She graduated in Chemistry and Molecular and Cellular Biology at the University Louis Pasteur in Strasbourg (France. After a Master in Pharmacology, in 2001 she obtained her PhD in Pharmacology and Neurobiology at the University Louis Pasteur. She was then assistant lecturer and subsequently moved to the University of Liverpool as a Post-doctoral Research Fellow. In 2005, she obtained a prestigious David Phillips Fellowship, to develop her work on intracellular signaling dynamics. She is focusing on the imaging of single living cells in order to understand regulation of gene transcription and cell fate. She has recently been interested in using new techniques for single molecule imaging in live cells based on the use of gold nanoparticles.

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

    Science.gov (United States)

    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.

  1. Comprehensive studies on the interactions between chitosan nanoparticles and some live cells

    International Nuclear Information System (INIS)

    Zheng Aiping; Liu Huixue; Yuan Lan; Meng Meng; Wang Jiancheng; Zhang Xuan; Zhang Qiang

    2011-01-01

    As more and more oral formulations of nanoparticles are used in clinical contexts, a comprehensive study on the mechanisms of interaction between polymer nanoparticles and live cells seems merited. Such a study was conducted and the results were compared to the polymer itself in order to demonstrate different kinds of effects that are brought into the cell by polymer and its nanoparticles, especially the effects on the biomembrane. Several techniques, including surface plasmon resonance (SPR), Fourier transformed infrared spectroscopy (FTIR), Raman spectroscopy, fluorescence polarization spectroscopy (FP), flow cytometry (FCM) with quantitative analysis, and confocal images with antibody staining were employed toward this end. The cytotoxicity in vitro was also evaluated. Chitosan (CS), a polycationic polymer, was used to prepare the nanoparticles. We demonstrate that chitosan nanoparticles (CS-NP) induce strong alterations in the distribution of membrane proteins, fluidity of membrane lipids, and general membrane structure. Furthermore, the uptake of CS-NP into Caco-2 cells was found to have a similar mechanism to that of CS molecules, but the differences in degree were noted. These results indicate that positive charge and nanoscale size were the factors that most significantly affected the interactions between the nanoparticles of polycationic polymers and live cells. However, no difference in cytotoxicity toward the Caco-2 cells was found between CS and CS-NP. This supports the idea that CS-NP is an effective and safe carrier for oral drug delivery.

  2. A combined optical and atomic force microscope for live cell investigations

    Energy Technology Data Exchange (ETDEWEB)

    Madl, Josef [Institute for Biophysics, Johannes Kepler University Linz, Altenbergerstr. 69, 4040 Linz (Austria); Rhode, Sebastian [Institute for Biophysics, Johannes Kepler University Linz, Altenbergerstr. 69, 4040 Linz (Austria); Stangl, Herbert [Institute for Medical Chemistry, Medical University Vienna, Waehringerstr. 10, 1090 Vienna (Austria); Stockinger, Hannes [Department of Molecular Immunology, Center for Biomolecular Medicine and Pharmacology, Medical University Vienna, Lazarettgasse 19, 1090 Vienna (Austria); Hinterdorfer, Peter [Institute for Biophysics, Johannes Kepler University Linz, Altenbergerstr. 69, 4040 Linz (Austria); Schuetz, Gerhard J. [Institute for Biophysics, Johannes Kepler University Linz, Altenbergerstr. 69, 4040 Linz (Austria); Kada, Gerald [Scientec, Mitterbauerweg 4, 4020 Linz (Austria)]. E-mail: gerald_kada@agilent.com

    2006-06-15

    We present an easy-to-use combination of an atomic force microscope (AFM) and an epi-fluorescence microscope, which allows live cell imaging under physiological conditions. High-resolution AFM images were acquired while simultaneously monitoring either the fluorescence image of labeled membrane components, or a high-contrast optical image (DIC, differential interference contrast). By applying two complementary techniques at the same time, additional information and correlations between structure and function of living organisms were obtained. The synergy effects between fluorescence imaging and AFM were further demonstrated by probing fluorescence-labeled receptor clusters in the cell membrane via force spectroscopy using antibody-functionalized tips. The binding probability on receptor-containing areas identified with fluorescence microscopy ('receptor-positive sites') was significantly higher than that on sites lacking receptors.

  3. A combined optical and atomic force microscope for live cell investigations

    International Nuclear Information System (INIS)

    Madl, Josef; Rhode, Sebastian; Stangl, Herbert; Stockinger, Hannes; Hinterdorfer, Peter; Schuetz, Gerhard J.; Kada, Gerald

    2006-01-01

    We present an easy-to-use combination of an atomic force microscope (AFM) and an epi-fluorescence microscope, which allows live cell imaging under physiological conditions. High-resolution AFM images were acquired while simultaneously monitoring either the fluorescence image of labeled membrane components, or a high-contrast optical image (DIC, differential interference contrast). By applying two complementary techniques at the same time, additional information and correlations between structure and function of living organisms were obtained. The synergy effects between fluorescence imaging and AFM were further demonstrated by probing fluorescence-labeled receptor clusters in the cell membrane via force spectroscopy using antibody-functionalized tips. The binding probability on receptor-containing areas identified with fluorescence microscopy ('receptor-positive sites') was significantly higher than that on sites lacking receptors

  4. Quantitative determination of optical trapping strength and viscoelastic moduli inside living cells

    DEFF Research Database (Denmark)

    Mas, Josep; Richardson, Andrew Callum; Reihani, S. Nader S.

    2013-01-01

    is viscoelastic, it would be incorrect to use a calibration procedure relying on a viscous environment. Here we demonstrate a method to perform a correct force calibration inside a living cell. This method (theoretically proposed in Fischer and Berg-Sørensen (2007 J. Opt. A: Pure Appl. Opt. 9 S239)) takes......With the success of in vitro single-molecule force measurements obtained in recent years, the next step is to perform quantitative force measurements inside a living cell. Optical traps have proven excellent tools for manipulation, also in vivo, where they can be essentially non-invasive under...... correct wavelength and exposure conditions. It is a pre-requisite for in vivo quantitative force measurements that a precise and reliable force calibration of the tweezers is performed. There are well-established calibration protocols in purely viscous environments; however, as the cellular cytoplasm...

  5. Wavelength-Dependent Differential Interference Contrast Microscopy: Selectively Imaging Nanoparticle Probes in Live Cells

    Energy Technology Data Exchange (ETDEWEB)

    Sun, Wei; Wang, Gufeng; Fang, Ning; and Yeung, Edward S.

    2009-11-15

    Gold and silver nanoparticles display extraordinarily large apparent refractive indices near their plasmon resonance (PR) wavelengths. These nanoparticles show good contrast in a narrow spectral band but are poorly resolved at other wavelengths in differential interference contrast (DIC) microscopy. The wavelength dependence of DIC contrast of gold/silver nanoparticles is interpreted in terms of Mie's theory and DIC working principles. We further exploit this wavelength dependence by modifying a DIC microscope to enable simultaneous imaging at two wavelengths. We demonstrate that gold/silver nanoparticles immobilized on the same glass slides through hybridization can be differentiated and imaged separately. High-contrast, video-rate images of living cells can be recorded both with and without illuminating the gold nanoparticle probes, providing definitive probe identification. Dual-wavelength DIC microscopy thus presents a new approach to the simultaneous detection of multiple probes of interest for high-speed live-cell imaging.

  6. Turn-on fluorescence chemosensor for fluoride ions and its applicability in imaging of living cells

    Energy Technology Data Exchange (ETDEWEB)

    Ponnuvel, Kandasamy; Padmini, Vediappen, E-mail: padimini_tamilenthi@yahoo.co.in

    2016-01-15

    The study was easy to prepare fluorescent chemosensor, the urea based triphenylamine conjugated ligand and structurally simple anion probes displayed great selectivity for the fluoride anion over other anions in an aqueous tetrahydrofuran solution. The probe was characterized using NMR spectroscopy, UV–visible, emission spectroscopy and mass spectrometry. The sensor showed spectral shifts and intensity changes in the presence of fluoride anions. The Job’s plot analysis indicates that the binding stoichiometry to be 1:1. Furthermore, by means of confocal fluorescent microscopy experiments, it has been demonstrated that it can be used as a fluorescent probe for monitoring fluoride ions in the living cells. - Highlights: • A novel fluorescent chemosensor for the detection of F{sup −} anions. • Detection of F{sup −} anions can be performed in water at pH=7.4. • The chemosensor could be efficiently delivered to live cells for bioimaging of F{sup −}.

  7. Vibrational imaging of newly synthesized proteins in live cells by stimulated Raman scattering microscopy

    Science.gov (United States)

    Wei, Lu; Yu, Yong; Shen, Yihui; Wang, Meng C.; Min, Wei

    2013-01-01

    Synthesis of new proteins, a key step in the central dogma of molecular biology, has been a major biological process by which cells respond rapidly to environmental cues in both physiological and pathological conditions. However, the selective visualization of a newly synthesized proteome in living systems with subcellular resolution has proven to be rather challenging, despite the extensive efforts along the lines of fluorescence staining, autoradiography, and mass spectrometry. Herein, we report an imaging technique to visualize nascent proteins by harnessing the emerging stimulated Raman scattering (SRS) microscopy coupled with metabolic incorporation of deuterium-labeled amino acids. As a first demonstration, we imaged newly synthesized proteins in live mammalian cells with high spatial–temporal resolution without fixation or staining. Subcellular compartments with fast protein turnover in HeLa and HEK293T cells, and newly grown neurites in differentiating neuron-like N2A cells, are clearly identified via this imaging technique. Technically, incorporation of deuterium-labeled amino acids is minimally perturbative to live cells, whereas SRS imaging of exogenous carbon–deuterium bonds (C–D) in the cell-silent Raman region is highly sensitive, specific, and compatible with living systems. Moreover, coupled with label-free SRS imaging of the total proteome, our method can readily generate spatial maps of the quantitative ratio between new and total proteomes. Thus, this technique of nonlinear vibrational imaging of stable isotope incorporation will be a valuable tool to advance our understanding of the complex spatial and temporal dynamics of newly synthesized proteome in vivo. PMID:23798434

  8. Zero Emission Bay Area (ZEBA) Fuel Cell Bus Demonstration Results: Sixth Report

    Science.gov (United States)

    2017-09-01

    This report presents results of a demonstration of fuel cell electric buses (FCEBs) operating in Oakland, California. Alameda-Contra Costa Transit District (AC Transit) leads the Zero Emission Bay Area (ZEBA) demonstration that includes 13 advanced-d...

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

    Science.gov (United States)

    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.

  10. Nanomechanical and topographical imaging of living cells by atomic force microscopy with colloidal probes

    Energy Technology Data Exchange (ETDEWEB)

    Puricelli, Luca; Galluzzi, Massimiliano; Schulte, Carsten; Podestà, Alessandro, E-mail: alessandro.podesta@mi.infn.it; Milani, Paolo [CIMaINa and Department of Physics, Università degli Studi di Milano, Via Celoria 16, 20133 Milano (Italy)

    2015-03-15

    Atomic Force Microscopy (AFM) has a great potential as a tool to characterize mechanical and morphological properties of living cells; these properties have been shown to correlate with cells’ fate and patho-physiological state in view of the development of novel early-diagnostic strategies. Although several reports have described experimental and technical approaches for the characterization of cellular elasticity by means of AFM, a robust and commonly accepted methodology is still lacking. Here, we show that micrometric spherical probes (also known as colloidal probes) are well suited for performing a combined topographic and mechanical analysis of living cells, with spatial resolution suitable for a complete and accurate mapping of cell morphological and elastic properties, and superior reliability and accuracy in the mechanical measurements with respect to conventional and widely used sharp AFM tips. We address a number of issues concerning the nanomechanical analysis, including the applicability of contact mechanical models and the impact of a constrained contact geometry on the measured Young’s modulus (the finite-thickness effect). We have tested our protocol by imaging living PC12 and MDA-MB-231 cells, in order to demonstrate the importance of the correction of the finite-thickness effect and the change in Young’s modulus induced by the action of a cytoskeleton-targeting drug.

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

    Science.gov (United States)

    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...

  12. Characterizing the interactions between prolyl isomerase pin1 and phosphatase inhibitor-2 in living cells with FRET and FCS

    Science.gov (United States)

    Sun, Yuansheng; Wang, Lifu; Jyothikumar, Vinod; Brautigan, David L.; Periasamy, Ammasi

    2012-03-01

    Phosphatase inhibitor-2 (I2) was discovered as a regulator of protein Ser/Thr phosphatase-1 and is conserved from yeast to human. Binding between purified recombinant I2 from different species and the prolyl isomerase Pin1 has been demonstrated with pull-down assays, size exclusion chromatography and nuclear magnetic resonance spectroscopy. Despite this, questions persist as to whether these proteins associate together in living cells. In this study, we prepared fluorescent protein (FP) fusions of I2 and Pin1 and employed both Förster Resonance Energy Transfer (FRET) and Fluorescence Correlation Spectroscopy (FCS) imaging techniques to characterize their interactions in living cells. In both intensity-based and time-resolved FRET studies, we observed FRET uniformly across whole cells co-expressing I2-Cerulean and Pin1-Venus that was significantly higher than in negative controls expressing Cerulean FP (without fusing to I2) as the FRET donor and Pin1-Venus, showing a specific interaction between I2-Cerulean and Pin1-Venus in living cells. We also observed the co-diffusion of I2-Cerulean and Pin1-mCherry in Fluorescence Cross Correlation Spectroscopy (FCCS) measurements. We further showed that I2 itself as well as I2-Pin1 formed complexes in living cells (predicted from in vitro studies) via a quantitative FRET assay, and demonstrated from FCS measurements that both I2 and Pin1 (fused to Cerulean) are highly mobile in living cells.

  13. Biofilm growth program and architecture revealed by single-cell live imaging

    Science.gov (United States)

    Yan, Jing; Sabass, Benedikt; Stone, Howard; Wingreen, Ned; Bassler, Bonnie

    Biofilms are surface-associated bacterial communities. Little is known about biofilm structure at the level of individual cells. We image living, growing Vibrio cholerae biofilms from founder cells to ten thousand cells at single-cell resolution, and discover the forces underpinning the architectural evolution of the biofilm. Mutagenesis, matrix labeling, and simulations demonstrate that surface-adhesion-mediated compression causes V. cholerae biofilms to transition from a two-dimensional branched morphology to a dense, ordered three-dimensional cluster. We discover that directional proliferation of rod-shaped bacteria plays a dominant role in shaping the biofilm architecture, and this growth pattern is controlled by a single gene. Competition analyses reveal the advantages of the dense growth mode in providing the biofilm with superior mechanical properties. We will further present continuum theory to model the three-dimensional growth of biofilms at the solid-liquid interface as well as solid-air interface.

  14. Live-cell imaging of invasion and intravasation in an artificial microvessel platform.

    Science.gov (United States)

    Wong, Andrew D; Searson, Peter C

    2014-09-01

    Methods to visualize metastasis exist, but additional tools to better define the biologic and physical processes underlying invasion and intravasation are still needed. One difficulty in studying metastasis stems from the complexity of the interface between the tumor microenvironment and the vascular system. Here, we report the development of an investigational platform that positions tumor cells next to an artificial vessel embedded in an extracellular matrix. On this platform, we used live-cell fluorescence microscopy to analyze the complex interplay between metastatic cancer cells and a functional artificial microvessel that was lined with endothelial cells. The platform recapitulated known interactions, and its use demonstrated the capabilities for a systematic study of novel physical and biologic parameters involved in invasion and intravasation. In summary, our work offers an important new tool to advance knowledge about metastasis and candidate antimetastatic therapies. ©2014 American Association for Cancer Research.

  15. The Next Frontier: Quantitative Biochemistry in Living Cells.

    Science.gov (United States)

    Honigmann, Alf; Nadler, André

    2018-01-09

    Researchers striving to convert biology into an exact science foremost rely on structural biology and biochemical reconstitution approaches to obtain quantitative data. However, cell biological research is moving at an ever-accelerating speed into areas where these approaches lose much of their edge. Intrinsically unstructured proteins and biochemical interaction networks composed of interchangeable, multivalent, and unspecific interactions pose unique challenges to quantitative biology, as do processes that occur in discrete cellular microenvironments. Here we argue that a conceptual change in our way of conducting biochemical experiments is required to take on these new challenges. We propose that reconstitution of cellular processes in vitro should be much more focused on mimicking the cellular environment in vivo, an approach that requires detailed knowledge of the material properties of cellular compartments, essentially requiring a material science of the cell. In a similar vein, we suggest that quantitative biochemical experiments in vitro should be accompanied by corresponding experiments in vivo, as many newly relevant cellular processes are highly context-dependent. In essence, this constitutes a call for chemical biologists to convert their discipline from a proof-of-principle science to an area that could rightfully be called quantitative biochemistry in living cells. In this essay, we discuss novel techniques and experimental strategies with regard to their potential to fulfill such ambitious aims.

  16. Structural model of radiation effects in living cells

    International Nuclear Information System (INIS)

    Neyman, J.; Puri, P.S.

    1976-01-01

    The chance mechanism of cell damage and of repair in the course of irradiation involves two details familiar to biologists that thus far seem to have been overlooked in mathematical treatment. One of these details is that, generally, the passage of a single ''primary'' radiation particle generates a ''cluster'' of secondaries which can produce ''hits'' that damage the living cell. With high linear energy transfer, each cluster contains very many secondary particles. With low linear energy transfer, the number of secondaries per cluster is generally small. The second overlooked detail of the chance mechanism is concerned with what may be called the time scales of radiation damage and of the subsequent repair. The generation of a cluster of secondary particles and the possible hits occur so rapidly that, for all practical purposes, they may be considered as occurring instantly. On the other hand, the subsequent changes in the damaged cells appear to require measurable amounts of time. The constructed stochastic model embodies these details, the clustering of secondary particles and the time scale difference. The results explain certain details of observed phenomena

  17. High-throughput screening of hybridoma supernatants using multiplexed fluorescent cell barcoding on live cells.

    Science.gov (United States)

    Lu, Mei; Chan, Brian M; Schow, Peter W; Chang, Wesley S; King, Chadwick T

    2017-12-01

    With current available assay formats using either immobilized protein (ELISA, enzyme-linked immunosorbent assay) or immunostaining of fixed cells for primary monoclonal antibody (mAb) screening, researchers often fail to identify and characterize antibodies that recognize the native conformation of cell-surface antigens. Therefore, screening using live cells has become an integral and important step contributing to the successful identification of therapeutic antibody candidates. Thus the need for developing high-throughput screening (HTS) technologies using live cells has become a major priority for therapeutic mAb discovery and development. We have developed a novel technique called Multiplexed Fluorescent Cell Barcoding (MFCB), a flow cytometry-based method based upon the Fluorescent Cell Barcoding (FCB) technique and the Luminex fluorescent bead array system, but is applicable to high-through mAb screens on live cells. Using this technique in our system, we can simultaneously identify or characterize the antibody-antigen binding of up to nine unique fluorescent labeled cell populations in the time that it would normally take to process a single population. This has significantly reduced the amount of time needed for the identification of potential lead candidates. This new technology enables investigators to conduct large-scale primary hybridoma screens using flow cytometry. This in turn has allowed us to screen antibodies more efficiently than before and streamline identification and characterization of lead molecules. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. Direct measurement of catalase activity in living cells and tissue biopsies.

    Science.gov (United States)

    Scaglione, Christine N; Xu, Qijin; Ramanujan, V Krishnan

    2016-01-29

    Spatiotemporal regulation of enzyme-substrate interactions governs the decision-making steps in biological systems. Enzymes, being functional units of every living cell, contribute to the macromolecular stability of cell survival, proliferation and hence are vital windows to unraveling the biological complexity. Experimental measurements capturing this dynamics of enzyme-substrate interactions in real time add value to this understanding. Furthermore these measurements, upon validation in realistic biological specimens such as clinical biopsies - can further improve our capability in disease diagnostics and treatment monitoring. Towards this direction, we describe here a novel, high-sensitive measurement system for measuring diffusion-limited enzyme-substrate kinetics in real time. Using catalase (enzyme) and hydrogen peroxide (substrate) as the example pair, we demonstrate that this system is capable of direct measurement of catalase activity in vitro and the measured kinetics follows the classical Michaelis-Menten reaction kinetics. We further demonstrate the system performance by measuring catalase activity in living cells and in very small amounts of liver biopsies (down to 1 μg total protein). Catalase-specific enzyme activity is demonstrated by genetic and pharmacological tools. Finally we show the clinically-relevant diagnostic capability of our system by comparing the catalase activities in liver biopsies from young and old mouse (liver and serum) samples. We discuss the potential applicability of this system in clinical diagnostics as well as in intraoperative surgical settings. Copyright © 2016 Elsevier Inc. All rights reserved.

  19. In Situ Live-Cell Nucleus Fluorescence Labeling with Bioinspired Fluorescent Probes.

    Science.gov (United States)

    Ding, Pan; Wang, Houyu; Song, Bin; Ji, Xiaoyuan; Su, Yuanyuan; He, Yao

    2017-08-01

    Fluorescent imaging techniques for visualization of nuclear structure and function in live cells are fundamentally important for exploring major cellular events. The ideal cellular labeling method is capable of realizing label-free, in situ, real-time, and long-term nucleus labeling in live cells, which can fully obtain the nucleus-relative information and effectively alleviate negative effects of alien probes on cellular metabolism. However, current established fluorescent probes-based strategies (e.g., fluorescent proteins-, organic dyes-, fluorescent organic/inorganic nanoparticles-based imaging techniques) are unable to simultaneously realize label-free, in situ, long-term, and real-time nucleus labeling, resulting in inevitable difficulties in fully visualizing nuclear structure and function in live cells. To this end, we present a type of bioinspired fluorescent probes, which are highly efficacious for in situ and label-free tracking of nucleus in long-term and real-time manners. Typically, the bioinspired polydopamine (PDA) nanoparticles, served as fluorescent probes, can be readily synthesized in situ within live cell nucleus without any further modifications under physiological conditions (37 °C, pH ∼7.4). Compared with other conventional nuclear dyes (e.g., propidium iodide (PI), Hoechst), superior spectroscopic properties (e.g., quantum yield of ∼35.8% and high photostability) and low cytotoxicity of PDA-based probes enable long-term (e.g., 3 h) fluorescence tracking of nucleus. We also demonstrate the generality of this type of bioinspired fluorescent probes in different cell lines and complex biological samples.

  20. Quantitative determination of optical trapping strength and viscoelastic moduli inside living cells

    International Nuclear Information System (INIS)

    Mas, Josep; Berg-Sørensen, Kirstine; Richardson, Andrew C; Reihani, S Nader S; Oddershede, Lene B

    2013-01-01

    With the success of in vitro single-molecule force measurements obtained in recent years, the next step is to perform quantitative force measurements inside a living cell. Optical traps have proven excellent tools for manipulation, also in vivo, where they can be essentially non-invasive under correct wavelength and exposure conditions. It is a pre-requisite for in vivo quantitative force measurements that a precise and reliable force calibration of the tweezers is performed. There are well-established calibration protocols in purely viscous environments; however, as the cellular cytoplasm is viscoelastic, it would be incorrect to use a calibration procedure relying on a viscous environment. Here we demonstrate a method to perform a correct force calibration inside a living cell. This method (theoretically proposed in Fischer and Berg-Sørensen (2007 J. Opt. A: Pure Appl. Opt. 9 S239)) takes into account the viscoelastic properties of the cytoplasm and relies on a combination of active and passive recordings of the motion of the cytoplasmic object of interest. The calibration procedure allows us to extract absolute values for the viscoelastic moduli of the living cell cytoplasm as well as the force constant describing the optical trap, thus paving the way for quantitative force measurements inside the living cell. Here, we determine both the spring constant of the optical trap and the elastic contribution from the cytoplasm, influencing the motion of naturally occurring tracer particles. The viscoelastic moduli that we find are of the same order of magnitude as moduli found in other cell types by alternative methods. (paper)

  1. Direct measurement of catalase activity in living cells and tissue biopsies

    Energy Technology Data Exchange (ETDEWEB)

    Scaglione, Christine N.; Xu, Qijin; Ramanujan, V. Krishnan, E-mail: Ramanujanv@csmc.edu

    2016-01-29

    Spatiotemporal regulation of enzyme-substrate interactions governs the decision-making steps in biological systems. Enzymes, being functional units of every living cell, contribute to the macromolecular stability of cell survival, proliferation and hence are vital windows to unraveling the biological complexity. Experimental measurements capturing this dynamics of enzyme-substrate interactions in real time add value to this understanding. Furthermore these measurements, upon validation in realistic biological specimens such as clinical biopsies – can further improve our capability in disease diagnostics and treatment monitoring. Towards this direction, we describe here a novel, high-sensitive measurement system for measuring diffusion-limited enzyme-substrate kinetics in real time. Using catalase (enzyme) and hydrogen peroxide (substrate) as the example pair, we demonstrate that this system is capable of direct measurement of catalase activity in vitro and the measured kinetics follows the classical Michaelis-Menten reaction kinetics. We further demonstrate the system performance by measuring catalase activity in living cells and in very small amounts of liver biopsies (down to 1 μg total protein). Catalase-specific enzyme activity is demonstrated by genetic and pharmacological tools. Finally we show the clinically-relevant diagnostic capability of our system by comparing the catalase activities in liver biopsies from young and old mouse (liver and serum) samples. We discuss the potential applicability of this system in clinical diagnostics as well as in intraoperative surgical settings. - Highlights: • A novel, direct measurement of Catalase enzyme activity via, oxygen sensing method. • Steady-stateprofiles of Catalase activity follow the Michaelis-Menten Kinetics. • Catalase-specific activity demonstrated using genetic and pharmacological tools. • Overcomes limitations of spectroscopic methods and indirect calorimetric approaches. • Clear

  2. Direct measurement of catalase activity in living cells and tissue biopsies

    International Nuclear Information System (INIS)

    Scaglione, Christine N.; Xu, Qijin; Ramanujan, V. Krishnan

    2016-01-01

    Spatiotemporal regulation of enzyme-substrate interactions governs the decision-making steps in biological systems. Enzymes, being functional units of every living cell, contribute to the macromolecular stability of cell survival, proliferation and hence are vital windows to unraveling the biological complexity. Experimental measurements capturing this dynamics of enzyme-substrate interactions in real time add value to this understanding. Furthermore these measurements, upon validation in realistic biological specimens such as clinical biopsies – can further improve our capability in disease diagnostics and treatment monitoring. Towards this direction, we describe here a novel, high-sensitive measurement system for measuring diffusion-limited enzyme-substrate kinetics in real time. Using catalase (enzyme) and hydrogen peroxide (substrate) as the example pair, we demonstrate that this system is capable of direct measurement of catalase activity in vitro and the measured kinetics follows the classical Michaelis-Menten reaction kinetics. We further demonstrate the system performance by measuring catalase activity in living cells and in very small amounts of liver biopsies (down to 1 μg total protein). Catalase-specific enzyme activity is demonstrated by genetic and pharmacological tools. Finally we show the clinically-relevant diagnostic capability of our system by comparing the catalase activities in liver biopsies from young and old mouse (liver and serum) samples. We discuss the potential applicability of this system in clinical diagnostics as well as in intraoperative surgical settings. - Highlights: • A novel, direct measurement of Catalase enzyme activity via, oxygen sensing method. • Steady-stateprofiles of Catalase activity follow the Michaelis-Menten Kinetics. • Catalase-specific activity demonstrated using genetic and pharmacological tools. • Overcomes limitations of spectroscopic methods and indirect calorimetric approaches. • Clear

  3. Live-cell imaging study of mitochondrial morphology in mammalian cells exposed to X-rays

    International Nuclear Information System (INIS)

    Noguchi, M.; Yokoya, A.; Narita, A.; Fujii, K.; Kanari, Y.

    2015-01-01

    Morphological changes in mitochondria induced by X-irradiation in normal murine mammary gland cells were studied with a live-cell microscopic imaging technique. Mitochondria were visualised by staining with a specific fluorescent probe in the cells, which express fluorescent ubiquitination-based cell-cycle indicator 2 (Fucci2) probes to visualise cell cycle. In unirradiated cells, the number of cells with fragmented mitochondria was about 20 % of the total cells through observation period (96 h). In irradiated cells, the population with fragmented mitochondria significantly increased depending on the absorbed dose. Particularly, for 8 Gy irradiation, the accumulation of fragmentation persists even in the cells whose cell cycle came to a stand (80 % in G1 (G0-like) phase). The fraction reached to a maximum at 96 h after irradiation. The kinetics of the fraction with fragmented mitochondria was similar to that for cells in S/G2/M phase (20 %) through the observation period (120 h). The evidences show that, in irradiated cells, some signals are continually released from a nucleus or cytoplasm even in the G0-like cells to operate some sort of protein machineries involved in mitochondrial fission. It is inferred that this delayed mitochondrial fragmentation is strongly related to their dysfunction, and hence might modulate radiobiological effects such as mutation or cell death. (authors)

  4. Non-Flow-Through Fuel Cell System Test Results and Demonstration on the SCARAB Rover

    Science.gov (United States)

    Scheidegger, Brianne, T.; Burke, Kenneth A.; Jakupca, Ian J.

    2012-01-01

    This paper describes the results of the demonstration of a non-flow-through PEM fuel cell as part of a power system on the SCARAB rover. A 16-cell non-flow-through fuel cell stack from Infinity Fuel Cell and Hydrogen, Inc. was incorporated into a power system designed to act as a range extender by providing power to the rover s hotel loads. This work represents the first attempt at a ground demonstration of this new technology aboard a mobile test platform. Development and demonstration were supported by the Office of the Chief Technologist s Space Power Systems Project and the Advanced Exploration System Modular Power Systems Project.

  5. Merkel cells are long-lived cells whose production is stimulated by skin injury✰

    Science.gov (United States)

    Wright, Margaret C.; Logan, Gregory J.; Bolock, Alexa M.; Kubicki, Adam C.; Hemphill, Julie A.; Sanders, Timothy A.; Maricich, Stephen M.

    2017-01-01

    Mechanosensitive Merkel cells are thought to have finite lifespans, but controversy surrounds the frequency of their replacement and which precursor cells maintain the population. We found by embryonic EdU administration that Merkel cells undergo terminal cell division in late embryogenesis and survive long into adulthood. We also found that new Merkel cells are produced infrequently during normal skin homeostasis and that their numbers do not change during natural or induced hair cycles. In contrast, live imaging and EdU experiments showed that mild mechanical injury produced by skin shaving dramatically increases Merkel cell production. We confirmed with genetic cell ablation and fate-mapping experiments that new touch dome Merkel cells in adult mice arise from touch dome keratinocytes. Together, these independent lines of evidence show that Merkel cells in adult mice are long-lived, are replaced rarely during normal adult skin homeostasis, and that their production can be induced by repeated shaving. These results have profound implications for understanding sensory neurobiology and human diseases such as Merkel cell carcinoma. PMID:27998808

  6. Merkel cells are long-lived cells whose production is stimulated by skin injury.

    Science.gov (United States)

    Wright, Margaret C; Logan, Gregory J; Bolock, Alexa M; Kubicki, Adam C; Hemphill, Julie A; Sanders, Timothy A; Maricich, Stephen M

    2017-02-01

    Mechanosensitive Merkel cells are thought to have finite lifespans, but controversy surrounds the frequency of their replacement and which precursor cells maintain the population. We found by embryonic EdU administration that Merkel cells undergo terminal cell division in late embryogenesis and survive long into adulthood. We also found that new Merkel cells are produced infrequently during normal skin homeostasis and that their numbers do not change during natural or induced hair cycles. In contrast, live imaging and EdU experiments showed that mild mechanical injury produced by skin shaving dramatically increases Merkel cell production. We confirmed with genetic cell ablation and fate-mapping experiments that new touch dome Merkel cells in adult mice arise from touch dome keratinocytes. Together, these independent lines of evidence show that Merkel cells in adult mice are long-lived, are replaced rarely during normal adult skin homeostasis, and that their production can be induced by repeated shaving. These results have profound implications for understanding sensory neurobiology and human diseases such as Merkel cell carcinoma. Copyright © 2016 Elsevier Inc. All rights reserved.

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

    Science.gov (United States)

    Gambhir, Sanjiv [Portola Valley, CA; Pritha, Ray [Mountain View, CA

    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.

  8. Direct imaging of APP proteolysis in living cells

    Directory of Open Access Journals (Sweden)

    Niccoló Parenti

    2017-04-01

    Full Text Available Alzheimer’s disease is a multifactorial disorder caused by the interaction of genetic, epigenetic and environmental factors. The formation of cytotoxic oligomers consisting of Aβ peptide is widely accepted as being one of the main key events triggering the development of Alzheimer’s disease. Aβ peptide production results from the specific proteolytic processing of the amyloid precursor protein (APP. Deciphering the factors governing the activity of the secretases responsible for the cleavage of APP is still a critical issue. Kits available commercially measure the enzymatic activity of the secretases from cells lysates, in vitro. By contrast, we have developed a prototypal rapid bioassay that provides visible information on the proteolytic processing of APP directly in living cells. APP was fused to a monomeric variant of the green fluorescent protein and a monomeric variant of the red fluorescent protein at the C-terminal and N-terminal (mChAPPmGFP, respectively. Changes in the proteolytic processing rate in transfected human neuroblastoma and rat neuronal cells were imaged with confocal microscopy as changes in the red/green fluorescence intensity ratio. The significant decrease in the mean red/green ratio observed in cells over-expressing the β-secretase BACE1, or the α-secretase ADAM10, fused to a monomeric blue fluorescent protein confirms that the proteolytic site is still accessible. Specific siRNA was used to evaluate the contribution of endogenous BACE1. Interestingly, we found that the degree of proteolytic processing of APP is not completely homogeneous within the same single cell, and that there is a high degree of variability between cells of the same type. We were also able to follow with a fluorescence spectrometer the changes in the red emission intensity of the extracellular medium when BACE1 was overexpressed. This represents a complementary approach to fluorescence microscopy for rapidly detecting changes in the

  9. N-way FRET microscopy of multiple protein-protein interactions in live cells.

    Directory of Open Access Journals (Sweden)

    Adam D Hoppe

    Full Text Available Fluorescence Resonance Energy Transfer (FRET microscopy has emerged as a powerful tool to visualize nanoscale protein-protein interactions while capturing their microscale organization and millisecond dynamics. Recently, FRET microscopy was extended to imaging of multiple donor-acceptor pairs, thereby enabling visualization of multiple biochemical events within a single living cell. These methods require numerous equations that must be defined on a case-by-case basis. Here, we present a universal multispectral microscopy method (N-Way FRET to enable quantitative imaging for any number of interacting and non-interacting FRET pairs. This approach redefines linear unmixing to incorporate the excitation and emission couplings created by FRET, which cannot be accounted for in conventional linear unmixing. Experiments on a three-fluorophore system using blue, yellow and red fluorescent proteins validate the method in living cells. In addition, we propose a simple linear algebra scheme for error propagation from input data to estimate the uncertainty in the computed FRET images. We demonstrate the strength of this approach by monitoring the oligomerization of three FP-tagged HIV Gag proteins whose tight association in the viral capsid is readily observed. Replacement of one FP-Gag molecule with a lipid raft-targeted FP allowed direct observation of Gag oligomerization with no association between FP-Gag and raft-targeted FP. The N-Way FRET method provides a new toolbox for capturing multiple molecular processes with high spatial and temporal resolution in living cells.

  10. Live cell CRISPR-imaging in plants reveals dynamic telomere movements

    KAUST Repository

    Dreissig, Steven

    2017-05-16

    Elucidating the spatio-temporal organization of the genome inside the nucleus is imperative to understand the regulation of genes and non-coding sequences during development and environmental changes. Emerging techniques of chromatin imaging promise to bridge the long-standing gap between sequencing studies which reveal genomic information and imaging studies that provide spatial and temporal information of defined genomic regions. Here, we demonstrate such an imaging technique based on two orthologues of the bacterial CRISPR-Cas9 system. By fusing eGFP/mRuby2 to the catalytically inactive version of Streptococcus pyogenes and Staphylococcus aureus Cas9, we show robust visualization of telomere repeats in live leaf cells of Nicotiana benthamiana. By tracking the dynamics of telomeres visualized by CRISPR-dCas9, we reveal dynamic telomere movements of up to 2 μm within 30 minutes during interphase. Furthermore, we show that CRISPR-dCas9 can be combined with fluorescence-labelled proteins to visualize DNA-protein interactions in vivo. By simultaneously using two dCas9 orthologues, we pave the way for imaging of multiple genomic loci in live plants cells. CRISPR-imaging bears the potential to significantly improve our understanding of the dynamics of chromosomes in live plant cells.

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

    Science.gov (United States)

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

    2018-03-23

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

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

    Science.gov (United States)

    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.

  13. Silicon nanocrystals and nanodiamonds in live cells: photoluminescence characteristics, cytotoxicity and interaction with cell cytoskeleton

    Czech Academy of Sciences Publication Activity Database

    Fučíková, A.; Valenta, J.; Pelant, Ivan; Hubálek Kalbáčová, M.; Brož, A.; Rezek, Bohuslav; Kromka, Alexander; Bakaeva, Zulfiya

    2014-01-01

    Roč. 4, č. 20 (2014), s. 10334-10342 ISSN 2046-2069 R&D Projects: GA ČR(CZ) GBP108/12/G108; GA ČR GA202/09/2078 Institutional support: RVO:68378271 ; RVO:61389013 Keywords : silicon nanocrystals * nanodiamonds * live cells * photoluminescence Subject RIV: BO - Biophysics Impact factor: 3.840, year: 2014

  14. Live Imaging of HIV-1 Transfer across T Cell Virological Synapse to Epithelial Cells that Promotes Stromal Macrophage Infection.

    Science.gov (United States)

    Real, Fernando; Sennepin, Alexis; Ganor, Yonatan; Schmitt, Alain; Bomsel, Morgane

    2018-05-08

    During sexual intercourse, HIV-1 crosses epithelial barriers composing the genital mucosa, a poorly understood feature that requires an HIV-1-infected cell vectoring efficient mucosal HIV-1 entry. Therefore, urethral mucosa comprising a polarized epithelium and a stroma composed of fibroblasts and macrophages were reconstructed in vitro. Using this system, we demonstrate by live imaging that efficient HIV-1 transmission to stromal macrophages depends on cell-mediated transfer of the virus through virological synapses formed between HIV-1-infected CD4 + T cells and the epithelial cell mucosal surface. We visualized HIV-1 translocation through mucosal epithelial cells via transcytosis in regions where virological synapses occurred. In turn, interleukin-13 is secreted and HIV-1 targets macrophages, which develop a latent state of infection reversed by lipopolysaccharide (LPS) activation. The live observation of virological synapse formation reported herein is key in the design of vaccines and antiretroviral therapies aimed at blocking HIV-1 access to cellular reservoirs in genital mucosa. Copyright © 2018. Published by Elsevier Inc.

  15. Uranium and thorium uptake by live and dead cells of Pseudomonas Sp

    International Nuclear Information System (INIS)

    Siva Prasath, C.S.; Manikandan, N.; Prakash, S.

    2010-01-01

    This study presents uptake of uranium (U) and thorium (Th) by live and dead cells of Pseudomonas Sp. Increasing concentration of U and Tb showed decrease in absorption by Pseudomonas Sp. Dead cells of Pseudomonas Sp. exhibited same or more uptake of U and Th than living cells. Increasing temperature promotes uptake of U and Th by Pseudomonas Sp. (author)

  16. New nanocomposites for SERS studies of living cells and mitochondria

    DEFF Research Database (Denmark)

    Sarycheva, A. S.; Brazhe, N. A.; Baizhumanov, A. A.

    2016-01-01

    A great enhancement in Raman scattering (SERS) from heme-containing submembrane biomolecules inside intact erythrocytes and functional mitochondria is demonstrated for the first time using silver–silica beads prepared using a new method involving aerosol pyrolysis with aqueous diamminesilver...... molecules. The SERS spectra of functional mitochondria are sensitive to the activity of the mitochondrial electron transport chain, thus making the method a novel label-free approach to monitor the redox state and conformation of cytochromes in their natural cell environment. The developed nanocomposites...

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

    International Nuclear Information System (INIS)

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

    2010-01-01

    Prion diseases are fatal neurodegenerative disorders resulting from structural conversion of the cellular isoform of PrP C to the infectious scrapie isoform PrP 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 C to the cell membrane and in initiating PrP C endocytosis.

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

    Energy Technology Data Exchange (ETDEWEB)

    Luo, Kan [State Key Laboratory of Virology and Modern Virology Research Centre, College of Life Sciences, Wuhan University, Wuhan 430072 (China); Li, Shu [AIDS Research Centre, Institute of Pathogen Biology, Chinese Academy of Medical Science, Beijing 100730 (China); Xie, Min [College of Chemistry and Molecular Science, Wuhan University, Wuhan 430072 (China); Wu, Di; Wang, WenXi; Chen, Rui; Huang, Liqin; Huang, Tao [State Key Laboratory of Virology and Modern Virology Research Centre, College of Life Sciences, Wuhan University, Wuhan 430072 (China); Pang, Daiwen, E-mail: dwpang@whu.edu.cn [College of Chemistry and Molecular Science, Wuhan University, Wuhan 430072 (China); Xiao, Gengfu, E-mail: xiaogf@wh.iov.cn [State Key Laboratory of Virology and Modern Virology Research Centre, College of Life Sciences, Wuhan University, Wuhan 430072 (China); Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071 (China)

    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.

  19. Application of laser-accelerated protons to the demonstration of DNA double-strand breaks in human cancer cells

    Science.gov (United States)

    Yogo, A.; Sato, K.; Nishikino, M.; Mori, M.; Teshima, T.; Numasaki, H.; Murakami, M.; Demizu, Y.; Akagi, S.; Nagayama, S.; Ogura, K.; Sagisaka, A.; Orimo, S.; Nishiuchi, M.; Pirozhkov, A. S.; Ikegami, M.; Tampo, M.; Sakaki, H.; Suzuki, M.; Daito, I.; Oishi, Y.; Sugiyama, H.; Kiriyama, H.; Okada, H.; Kanazawa, S.; Kondo, S.; Shimomura, T.; Nakai, Y.; Tanoue, M.; Sasao, H.; Wakai, D.; Bolton, P. R.; Daido, H.

    2009-05-01

    We report the demonstrated irradiation effect of laser-accelerated protons on human cancer cells. In vitro (living) A549 cells are irradiated with quasimonoenergetic proton bunches of 0.8-2.4 MeV with a single bunch duration of 15 ns. Irradiation with the proton dose of 20 Gy results in a distinct formation of γ-H2AX foci as an indicator of DNA double-strand breaks generated in the cancer cells. This is a pioneering result that points to future investigations of the radiobiological effects of laser-driven ion beams. Unique high-current and short-bunch features make laser-driven proton bunches an excitation source for time-resolved determination of radical yields.

  20. A single-cell scraper based on an atomic force microscope for detaching a living cell from a substrate

    Energy Technology Data Exchange (ETDEWEB)

    Iwata, Futoshi, E-mail: iwata.futoshi@shizuoka.ac.jp [Department of Mechanical Engineering, Faculty of Engineering, Shizuoka University, Johoku, Naka-ku, Hamamatsu 432-8561 (Japan); Research Institute of Electronics, Shizuoka University, Johoku, Naka-ku, Hamamatsu 432-8011 (Japan); Adachi, Makoto; Hashimoto, Shigetaka [Department of Mechanical Engineering, Faculty of Engineering, Shizuoka University, Johoku, Naka-ku, Hamamatsu 432-8561 (Japan)

    2015-10-07

    We describe an atomic force microscope (AFM) manipulator that can detach a single, living adhesion cell from its substrate without compromising the cell's viability. The micrometer-scale cell scraper designed for this purpose was fabricated from an AFM micro cantilever using focused ion beam milling. The homemade AFM equipped with the scraper was compact and standalone and could be mounted on a sample stage of an inverted optical microscope. It was possible to move the scraper using selectable modes of operation, either a manual mode with a haptic device or a computer-controlled mode. The viability of the scraped single cells was evaluated using a fluorescence dye of calcein-acetoxymethl ester. Single cells detached from the substrate were collected by aspiration into a micropipette capillary glass using an electro-osmotic pump. As a demonstration, single HeLa cells were selectively detached from the substrate and collected by the micropipette. It was possible to recultivate HeLa cells from the single cells collected using the system.

  1. Optical imaging of non-fluorescent nanoparticle probes in live cells

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Gufeng; Stender, Anthony S.; Sun, Wei; and Fang, Ning

    2009-12-17

    Precise imaging of cellular and subcellular structures and dynamic processes in live cells is crucial for fundamental research in life sciences and in medical applications. Non-fluorescent nanoparticles are an important type of optical probe used in live-cell imaging due to their photostability, large optical cross-sections, and low toxicity. Here, we provide an overview of recent developments in the optical imaging of non-fluorescent nanoparticle probes in live cells.

  2. Time-resolved analysis of DNA-protein interactions in living cells by UV laser pulses.

    Science.gov (United States)

    Nebbioso, Angela; Benedetti, Rosaria; Conte, Mariarosaria; Carafa, Vincenzo; De Bellis, Floriana; Shaik, Jani; Matarese, Filomena; Della Ventura, Bartolomeo; Gesuele, Felice; Velotta, Raffaele; Martens, Joost H A; Stunnenberg, Hendrik G; Altucci, Carlo; Altucci, Lucia

    2017-09-15

    Interactions between DNA and proteins are mainly studied through chemical procedures involving bi-functional reagents, mostly formaldehyde. Chromatin immunoprecipitation is used to identify the binding between transcription factors (TFs) and chromatin, and to evaluate the occurrence and impact of histone/DNA modifications. The current bottleneck in probing DNA-protein interactions using these approaches is caused by the fact that chemical crosslinkers do not discriminate direct and indirect bindings or short-lived chromatin occupancy. Here, we describe a novel application of UV laser-induced (L-) crosslinking and demonstrate that a combination of chemical and L-crosslinking is able to distinguish between direct and indirect DNA-protein interactions in a small number of living cells. The spatial and temporal dynamics of TF bindings to chromatin and their role in gene expression regulation may thus be assessed. The combination of chemical and L-crosslinking offers an exciting and unprecedented tool for biomedical applications.

  3. Diffraction-unlimited optical imaging of unstained living cells in liquid by electron beam scanning of luminescent environmental cells.

    Science.gov (United States)

    Miyazaki, Hideki T; Kasaya, Takeshi; Takemura, Taro; Hanagata, Nobutaka; Yasuda, Takeshi; Miyazaki, Hiroshi

    2013-11-18

    An environmental cell with a 50-nm-thick cathodoluminescent window was attached to a scanning electron microscope, and diffraction-unlimited near-field optical imaging of unstained living human lung epithelial cells in liquid was demonstrated. Electrons with energies as low as 0.8 - 1.2 kV are sufficiently blocked by the window without damaging the specimens, and form a sub-wavelength-sized illumination light source. A super-resolved optical image of the specimen adhered to the opposite window surface was acquired by a photomultiplier tube placed below. The cells after the observation were proved to stay alive. The image was formed by enhanced dipole radiation or energy transfer, and features as small as 62 nm were resolved.

  4. Development of controlled release silicone adhesive-based mupirocin patch demonstrates antibacterial activity on live rat skin against Staphylococcus aureus.

    Science.gov (United States)

    David, Sheba R; Malek, Nurafiqah; Mahadi, Abdul Hanif; Chakravarthi, Srikumar; Rajabalaya, Rajan

    2018-01-01

    Peritonitis is the most serious complication of peritoneal dialysis. Staphylococcus aureus infections could lead to peritonitis which causes reversal of peritoneal dialysis treatment back to hemodialysis. The aim of this study was to develop a controlled release silicone adhesive-based mupirocin patch for prophylactic effect and analyze its antibacterial effectiveness against S. aureus . The matrix patches were prepared by using different polymers, with and without silicone adhesive, dibutyl sebacate and mupirocin. The patches were characterized for mechanical properties, drug content, moisture content, water absorption capacity and Fourier transform infrared spectrum. In vitro release studies were performed by using Franz diffusion cell. In vitro disk diffusion assay was performed on the Mueller-Hinton Agar plate to measure the zone of inhibition of the patches. The in vivo study was performed on four groups of rats with bacterial counts at three different time intervals, along with skin irritancy and histopathologic studies. The patches showed appropriate average thickness (0.63-1.12 mm), tensile strength (5.08-10.08 MPa) and modulus of elasticity (21.53-42.19 MPa). The drug content ranged from 94.5% to 97.4%, while the moisture content and water absorption capacities at two relative humidities (75% and 93%) were in the range of 1.082-3.139 and 1.287-4.148 wt%, respectively. Fourier transform infrared spectra showed that there were no significant interactions between the polymer and the drug. The highest percentage of drug release at 8 hours was 47.94%. The highest zone of inhibition obtained was 28.3 mm against S. aureus . The in vivo studies showed that the bacterial colonies were fewer at 1 cm (7×10 1 CFU/mL) than at 2 cm (1.3×10 2 CFU/mL) over a 24-hour period. The patches were nonirritant to the skin, and histopathologic results also showed no toxic or damaging effects to the skin. The in vitro and in vivo studies indicated that controlled release patches

  5. Long-Term Live Cell Imaging of Cell Migration: Effects of Pathogenic Fungi on Human Epithelial Cell Migration.

    Science.gov (United States)

    Wöllert, Torsten; Langford, George M

    2016-01-01

    Long-term live cell imaging was used in this study to determine the responses of human epithelial cells to pathogenic biofilms formed by Candida albicans. Epithelial cells of the skin represent the front line of defense against invasive pathogens such as C. albicans but under certain circumstances, especially when the host's immune system is compromised, the skin barrier is breached. The mechanisms by which the fungal pathogen penetrates the skin and invade the deeper layers are not fully understood. In this study we used keratinocytes grown in culture as an in vitro model system to determine changes in host cell migration and the actin cytoskeleton in response to virulence factors produced by biofilms of pathogenic C. albicans. It is clear that changes in epithelial cell migration are part of the response to virulence factors secreted by biofilms of C. albicans and the actin cytoskeleton is the downstream effector that mediates cell migration. Our goal is to understand the mechanism by which virulence factors hijack the signaling pathways of the actin cytoskeleton to alter cell migration and thereby invade host tissues. To understand the dynamic changes of the actin cytoskeleton during infection, we used long-term live cell imaging to obtain spatial and temporal information of actin filament dynamics and to identify signal transduction pathways that regulate the actin cytoskeleton and its associated proteins. Long-term live cell imaging was achieved using a high resolution, multi-mode epifluorescence microscope equipped with specialized light sources, high-speed cameras with high sensitivity detectors, and specific biocompatible fluorescent markers. In addition to the multi-mode epifluorescence microscope, a spinning disk confocal long-term live cell imaging system (Olympus CV1000) equipped with a stage incubator to create a stable in vitro environment for long-term real-time and time-lapse microscopy was used. Detailed descriptions of these two long-term live

  6. Weak Ergodicity Breaking of Receptor Motion in Living Cells Stemming from Random Diffusivity

    Science.gov (United States)

    Manzo, Carlo; Torreno-Pina, Juan A.; Massignan, Pietro; Lapeyre, Gerald J.; Lewenstein, Maciej; Garcia Parajo, Maria F.

    2015-01-01

    Molecular transport in living systems regulates numerous processes underlying biological function. Although many cellular components exhibit anomalous diffusion, only recently has the subdiffusive motion been associated with nonergodic behavior. These findings have stimulated new questions for their implications in statistical mechanics and cell biology. Is nonergodicity a common strategy shared by living systems? Which physical mechanisms generate it? What are its implications for biological function? Here, we use single-particle tracking to demonstrate that the motion of dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN), a receptor with unique pathogen-recognition capabilities, reveals nonergodic subdiffusion on living-cell membranes In contrast to previous studies, this behavior is incompatible with transient immobilization, and, therefore, it cannot be interpreted according to continuous-time random-walk theory. We show that the receptor undergoes changes of diffusivity, consistent with the current view of the cell membrane as a highly dynamic and diverse environment. Simulations based on a model of an ordinary random walk in complex media quantitatively reproduce all our observations, pointing toward diffusion heterogeneity as the cause of DC-SIGN behavior. By studying different receptor mutants, we further correlate receptor motion to its molecular structure, thus establishing a strong link between nonergodicity and biological function. These results underscore the role of disorder in cell membranes and its connection with function regulation. Because of its generality, our approach offers a framework to interpret anomalous transport in other complex media where dynamic heterogeneity might play a major role, such as those found, e.g., in soft condensed matter, geology, and ecology.

  7. Raman tweezers in microfluidic systems for analysis and sorting of living cells

    Science.gov (United States)

    Pilát, Zdeněk.; Ježek, Jan; Kaňka, Jan; Zemánek, Pavel

    2014-12-01

    We have devised an analytical and sorting system combining optical trapping with Raman spectroscopy in microfluidic environment, dedicated to identification and sorting of biological objects, such as living cells of various unicellular organisms. Our main goal was to create a robust and universal platform for non-destructive and non-contact sorting of micro-objects based on their Raman spectral properties. This approach allowed us to collect spectra containing information about the chemical composition of the objects, such as the presence and composition of pigments, lipids, proteins, or nucleic acids, avoiding artificial chemical probes such as fluorescent markers. The non-destructive nature of this optical analysis and manipulation allowed us to separate individual living cells of our interest in a sterile environment and provided the possibility to cultivate the selected cells for further experiments. We used a mixture of polystyrene micro-particles and algal cells to test and demonstrate the function of our analytical and sorting system. The devised system could find its use in many medical, biotechnological, and biological applications.

  8. Demonstration of S-100 protein in sustentacular cells of phaeochromocytomas and paragangliomas

    DEFF Research Database (Denmark)

    Schroder, H D; Johannsen, L

    1986-01-01

    to the sustentacular cells of normal paraganglia and adrenal medulla were found in all paragangliomas and in the benign and aggressively growing phaeochromocytomas. In the two malignant tumours no positive reaction was demonstrated. In one tumour the sustentacular cells were shown to contain glial fibrillary acidic......Eighteen phaeochromocytomas, including both sporadic and familial cases, four cervical paragangliomas, two jugular paragangliomas, and one abdominal paraganglioma were examined immunohistochemically for the presence of S-100 protein. Positive staining in cells morphologically similar...... protein further supporting their Schwann cell relationship. The number of S-100 positive cells varied considerably. They demonstrated a spindle celled or elongated configuration with long slender processes. The nature of the sustentacular cell proliferation, neoplastic versus reactive, is discussed....

  9. Specific insulin binding in bovine chromaffin cells; demonstration of preferential binding to adrenalin-storing cells

    International Nuclear Information System (INIS)

    Serck-Hanssen, G.; Soevik, O.

    1987-01-01

    Insulin binding was studied in subpopulations of bovine chromaffin cells enriched in adrenalin-producing cells (A-cells) or noradrenalin-producing cells (NA-cells). Binding of 125 I-insulin was carried out at 15 0 C for 3 hrs in the absence or presence of excess unlabeled hormone. Four fractions of cells were obtained by centrifugation on a stepwise bovine serum albumin gradient. The four fractions were all shown to bind insulin in a specific manner and the highest binding was measured in the cell layers of higher densities, containing mainly A-cells. The difference in binding of insulin to the four subpopulations of chromaffin cells seemed to be related to differences in numbers of receptors as opposed to receptor affinities. The authors conclude that bovine chromaffin cells possess high affinity binding sites for insulin and that these binding sites are mainly confined to A-cells. 24 references, 2 figures, 1 table

  10. Mechanics of Cellulose Synthase Complexes in Living Plant Cells

    Science.gov (United States)

    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.

  11. Towards programming languages for genetic engineering of living cells.

    Science.gov (United States)

    Pedersen, Michael; Phillips, Andrew

    2009-08-06

    Synthetic biology aims at producing novel biological systems to carry out some desired and well-defined functions. An ultimate dream is to design these systems at a high level of abstraction using engineering-based tools and programming languages, press a button, and have the design translated to DNA sequences that can be synthesized and put to work in living cells. We introduce such a programming language, which allows logical interactions between potentially undetermined proteins and genes to be expressed in a modular manner. Programs can be translated by a compiler into sequences of standard biological parts, a process that relies on logic programming and prototype databases that contain known biological parts and protein interactions. Programs can also be translated to reactions, allowing simulations to be carried out. While current limitations on available data prevent full use of the language in practical applications, the language can be used to develop formal models of synthetic systems, which are otherwise often presented by informal notations. The language can also serve as a concrete proposal on which future language designs can be discussed, and can help to guide the emerging standard of biological parts which so far has focused on biological, rather than logical, properties of parts.

  12. Long-lived tissue resident HIV-1 specific memory CD8+ T cells are generated by skin immunization with live virus vectored microneedle arrays.

    Science.gov (United States)

    Zaric, Marija; Becker, Pablo Daniel; Hervouet, Catherine; Kalcheva, Petya; Ibarzo Yus, Barbara; Cocita, Clement; O'Neill, Lauren Alexandra; Kwon, Sung-Yun; Klavinskis, Linda Sylvia

    2017-12-28

    The generation of tissue resident memory (T RM ) cells at the body surfaces to provide a front line defence against invading pathogens represents an important goal in vaccine development for a wide variety of pathogens. It has been widely assumed that local vaccine delivery to the mucosae is necessary to achieve that aim. Here we characterise a novel micro-needle array (MA) delivery system fabricated to deliver a live recombinant human adenovirus type 5 vaccine vector (AdHu5) encoding HIV-1 gag. We demonstrate rapid dissolution kinetics of the microneedles in skin. Moreover, a consequence of MA vaccine cargo release was the generation of long-lived antigen-specific CD8 + T cells that accumulate in mucosal tissues, including the female genital and respiratory tract. The memory CD8 + T cell population maintained in the peripheral mucosal tissues was attributable to a MA delivered AdHu5 vaccine instructing CD8 + T cell expression of CXCR3 + , CD103 +, CD49a + , CD69 + , CD127 + homing, retention and survival markers. Furthermore, memory CD8 + T cells generated by MA immunization significantly expanded upon locally administered antigenic challenge and showed a predominant poly-functional profile producing high levels of IFNγ and Granzyme B. These data demonstrate that skin vaccine delivery using microneedle technology induces mobilization of long lived, poly-functional CD8 + T cells to peripheral tissues, phenotypically displaying hallmarks of residency and yields new insights into how to design and deliver effective vaccine candidates with properties to exert local immunosurveillance at the mucosal surfaces. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  13. Computed tomographic demonstration of a spontaneous subcapsular hematoma due to a small renal cell carcinoma

    International Nuclear Information System (INIS)

    Hilton, S.; Bosniak, M.A.; Megibow, A.J.; Ambos, M.A.

    1981-01-01

    Computed tomography (CT) was able to demonstrate a small renal cell carcinoma as the cause of a spontaneous subcapsular hematoma. Angiographic and pathologic correlation were obtained. A review of the causes for nontraumatic renal subcapsular hematoma is included

  14. Chloroquine Analog Interaction with C2- and Iota-Toxin in Vitro and in Living Cells.

    Science.gov (United States)

    Kronhardt, Angelika; Beitzinger, Christoph; Barth, Holger; Benz, Roland

    2016-08-10

    C2-toxin from Clostridium botulinum and Iota-toxin from Clostridium perfringens belong both to the binary A-B-type of toxins consisting of two separately secreted components, an enzymatic subunit A and a binding component B that facilitates the entry of the corresponding enzymatic subunit into the target cells. The enzymatic subunits are in both cases actin ADP-ribosyltransferases that modify R177 of globular actin finally leading to cell death. Following their binding to host cells' receptors and internalization, the two binding components form heptameric channels in endosomal membranes which mediate the translocation of the enzymatic components Iota a and C2I from endosomes into the cytosol of the target cells. The binding components form ion-permeable channels in artificial and biological membranes. Chloroquine and related 4-aminoquinolines were able to block channel formation in vitro and intoxication of living cells. In this study, we extended our previous work to the use of different chloroquine analogs and demonstrate that positively charged aminoquinolinium salts are able to block channels formed in lipid bilayer membranes by the binding components of C2- and Iota-toxin. Similarly, these molecules protect cultured mammalian cells from intoxication with C2- and Iota-toxin. The aminoquinolinium salts did presumably not interfere with actin ADP-ribosylation or receptor binding but blocked the pores formed by C2IIa and Iota b in living cells and in vitro. The blocking efficiency of pores formed by Iota b and C2IIa by the chloroquine analogs showed interesting differences indicating structural variations between the types of protein-conducting nanochannels formed by Iota b and C2IIa.

  15. Experimental demonstration of programmable multi-functional spin logic cell based on spin Hall effect

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, X.; Wan, C.H., E-mail: wancaihua@iphy.ac.cn; Yuan, Z.H.; Fang, C.; Kong, W.J.; Wu, H.; Zhang, Q.T.; Tao, B.S.; Han, X.F., E-mail: xfhan@iphy.ac.cn

    2017-04-15

    Confronting with the gigantic volume of data produced every day, raising integration density by reducing the size of devices becomes harder and harder to meet the ever-increasing demand for high-performance computers. One feasible path is to actualize more logic functions in one cell. In this respect, we experimentally demonstrate a prototype spin-orbit torque based spin logic cell integrated with five frequently used logic functions (AND, OR, NOT, NAND and NOR). The cell can be easily programmed and reprogrammed to perform desired function. Furthermore, the information stored in cells is symmetry-protected, making it possible to expand into logic gate array where the cell can be manipulated one by one without changing the information of other undesired cells. This work provides a prospective example of multi-functional spin logic cell with reprogrammability and nonvolatility, which will advance the application of spin logic devices. - Highlights: • Experimental demonstration of spin logic cell based on spin Hall effect. • Five logic functions are realized in a single logic cell. • The logic cell is reprogrammable. • Information in the cell is symmetry-protected. • The logic cell can be easily expanded to logic gate array.

  16. Induction of Live Cell Phagocytosis by a Specific Combination of Inflammatory Stimuli

    Directory of Open Access Journals (Sweden)

    Takamasa Ishidome

    2017-08-01

    Full Text Available Conditions of severe hyper-inflammation can lead to uncontrolled activation of macrophages, and the ensuing phagocytosis of live cells. However, relationships between inflammatory stimuli and uncontrolled phagocytosis of live cells by macrophages are poorly understood. To identify mediators of this process, we established phagocytosis assays of live cells by stimulating macrophages with CpG DNA, interferon-γ, and anti-interleukin-10 receptor antibody. In this model, various cell surface receptors were upregulated on macrophages, and phagocytosis of live cells was induced in a Rac1-dependent manner. Subsequent inhibition of the ICAM-1, VCAM-1, and both of these receptors abolished in vitro and in vivo phagocytosis of live T cells, myeloid cells, and B cells, respectively. Specifically, the reduction in lymphocyte numbers due to in vivo activation of macrophages was ameliorated in Icam-1-deficient mice. In addition, overexpression of ICAM-1 or VCAM-1 in non-phagocytic NIH3T3 cells led to active phagocytosis of live cells. These data indicate molecular mechanisms underlying live cell phagocytosis induced by hyper-inflammation, and this experimental model will be useful to clarify the pathophysiological mechanisms of hemophagocytosis and to indicate therapeutic targets.

  17. Overcoming the Philosophy/Life, Body/Mind Rift: Demonstrating Yoga as Embodied-Lived-Philosophical-Practice

    Science.gov (United States)

    Ergas, Oren

    2014-01-01

    Philosophy's essence depicted by Socrates lies in its role as pedagogy for living, yet its traditional treatment of "body" as a hindrance to "knowledge" in fact severs it from life, transforming it into "an escape from life" (James, 1978, p. 18). The philosophy/life dichotomy is thus an inherent flaw preventing…

  18. Development of controlled release silicone adhesive–based mupirocin patch demonstrates antibacterial activity on live rat skin against Staphylococcus aureus

    Directory of Open Access Journals (Sweden)

    David SR

    2018-03-01

    Full Text Available Sheba R David,1 Nurafiqah Malek,1 Abdul Hanif Mahadi,2 Srikumar Chakravarthi,3 Rajan Rajabalaya1 1PAPRSB Institute of Health Sciences, Universiti Brunei Darussalam, Bandar Seri Begawan, Brunei Darussalam; 2Centre for Advanced Material and Energy Sciences (CAMES, Universiti Brunei Darussalam, Bandar Seri Begawan, Brunei Darussalam; 3School of Medicine, Perdana University, Jalan MAEPS Perdana, Serdang, Selangor, Malaysia Background: Peritonitis is the most serious complication of peritoneal dialysis. Staphylococcus aureus infections could lead to peritonitis which causes reversal of peritoneal dialysis treatment back to hemodialysis. The aim of this study was to develop a controlled release silicone adhesive-based mupirocin patch for prophylactic effect and analyze its antibacterial effectiveness against S. aureus.Methods: The matrix patches were prepared by using different polymers, with and without silicone adhesive, dibutyl sebacate and mupirocin. The patches were characterized for mechanical properties, drug content, moisture content, water absorption capacity and Fourier transform infrared spectrum. In vitro release studies were performed by using Franz diffusion cell. In vitro disk diffusion assay was performed on the Mueller–Hinton Agar plate to measure the zone of inhibition of the patches. The in vivo study was performed on four groups of rats with bacterial counts at three different time intervals, along with skin irritancy and histopathologic studies. Results: The patches showed appropriate average thickness (0.63–1.12 mm, tensile strength (5.08–10.08 MPa and modulus of elasticity (21.53–42.19 MPa. The drug content ranged from 94.5% to 97.4%, while the moisture content and water absorption capacities at two relative humidities (75% and 93% were in the range of 1.082–3.139 and 1.287–4.148 wt%, respectively. Fourier transform infrared spectra showed that there were no significant interactions between the polymer and the drug

  19. Visualization and measurement of ATP levels in living cells replicating hepatitis C virus genome RNA.

    Directory of Open Access Journals (Sweden)

    Tomomi Ando

    Full Text Available Adenosine 5'-triphosphate (ATP is the primary energy currency of all living organisms and participates in a variety of cellular processes. Although ATP requirements during viral lifecycles have been examined in a number of studies, a method by which ATP production can be monitored in real-time, and by which ATP can be quantified in individual cells and subcellular compartments, is lacking, thereby hindering studies aimed at elucidating the precise mechanisms by which viral replication energized by ATP is controlled. In this study, we investigated the fluctuation and distribution of ATP in cells during RNA replication of the hepatitis C virus (HCV, a member of the Flaviviridae family. We demonstrated that cells involved in viral RNA replication actively consumed ATP, thereby reducing cytoplasmic ATP levels. Subsequently, a method to measure ATP levels at putative subcellular sites of HCV RNA replication in living cells was developed by introducing a recently-established Förster resonance energy transfer (FRET-based ATP indicator, called ATeam, into the NS5A coding region of the HCV replicon. Using this method, we were able to observe the formation of ATP-enriched dot-like structures, which co-localize with non-structural viral proteins, within the cytoplasm of HCV-replicating cells but not in non-replicating cells. The obtained FRET signals allowed us to estimate ATP concentrations within HCV replicating cells as ∼5 mM at possible replicating sites and ∼1 mM at peripheral sites that did not appear to be involved in HCV replication. In contrast, cytoplasmic ATP levels in non-replicating Huh-7 cells were estimated as ∼2 mM. To our knowledge, this is the first study to demonstrate changes in ATP concentration within cells during replication of the HCV genome and increased ATP levels at distinct sites within replicating cells. ATeam may be a powerful tool for the study of energy metabolism during replication of the viral genome.

  20. Explicit memory creation during sleep demonstrates a causal role of place cells in navigation.

    Science.gov (United States)

    de Lavilléon, Gaetan; Lacroix, Marie Masako; Rondi-Reig, Laure; Benchenane, Karim

    2015-04-01

    Hippocampal place cells assemblies are believed to support the cognitive map, and their reactivations during sleep are thought to be involved in spatial memory consolidation. By triggering intracranial rewarding stimulations by place cell spikes during sleep, we induced an explicit memory trace, leading to a goal-directed behavior toward the place field. This demonstrates that place cells' activity during sleep still conveys relevant spatial information and that this activity is functionally significant for navigation.

  1. Canola Oil Fuel Cell Demonstration: Volume 2 - Market Availability of Agricultural Crops for Fuel Cell Applications

    National Research Council Canada - National Science Library

    Adams, John W; Cassarino, Craig; Spangler, Lee; Johnson, Duane; Lindstrom, Joel; Binder, Michael J; Holcomb, Franklin H; Lux, Scott M

    2006-01-01

    .... The reformation of vegetable oil crops for fuel cell uses is not well known; yet vegetable oils such as canola oil represent a viable alternative and complement to traditional fuel cell feedstocks...

  2. Real-Time Gene Expression Profiling of Live Shewanella Oneidensis Cells

    Energy Technology Data Exchange (ETDEWEB)

    Xiaoliang Sunney Xie

    2009-03-30

    The overall objective of this proposal is to make real-time observations of gene expression in live Shewanella oneidensis cells with high sensitivity and high throughput. Gene expression, a central process to all life, is stochastic because most genes often exist in one or two copies per cell. Although the central dogma of molecular biology has been proven beyond doubt, due to insufficient sensitivity, stochastic protein production has not been visualized in real time in an individual cell at the single-molecule level. We report the first direct observation of single protein molecules as they are generated, one at a time in a single live E. coli cell, yielding quantitative information about gene expression [Science 2006; 311: 1600-1603]. We demonstrated a general strategy for live-cell single-molecule measurements: detection by localization. It is difficult to detect single fluorescence protein molecules inside cytoplasm - their fluorescence is spread by fast diffusion to the entire cell and overwhelmed by the strong autofluorescence. We achieved single-molecule sensitivity by immobilizing the fluorescence protein on the cell membrane, where the diffusion is much slowed. We learned that under the repressed condition protein molecules are produced in bursts, with each burst originating from a stochastically-transcribed single messenger RNA molecule, and that protein copy numbers in the bursts follow a geometric distribution. We also simultaneously published a paper reporting a different method using β-glactosidase as a reporter [Nature 440, 358 (2006)]. Many important proteins are expressed at low levels, inaccessible by previous proteomic techniques. Both papers allowed quantification of protein expression with unprecedented sensitivity and received overwhelming acclaim from the scientific community. The Nature paper has been identified as one of the most-cited papers in the past year [http://esi-topics.com/]. We have also an analytical framework describing the

  3. Green biosynthesis of biocompatible CdSe quantum dots in living Escherichia coli cells

    International Nuclear Information System (INIS)

    Yan, Zhengyu; Qian, Jing; Su, Yilong; Ai, Xiaoxia; Wu, Shengmei; Gu, Yueqing

    2014-01-01

    A green and efficient biosynthesis method to prepare fluorescence-tunable biocompatible cadmium selenide quantum dots using Escherichia coli cells as biological matrix was proposed. Decisive factors in biosynthesis of cadmium selenide quantum dots in a designed route in Escherichia coli cells were elaborately investigated, including the influence of the biological matrix growth stage, the working concentration of inorganic reactants, and the co-incubation duration of inorganic metals to biomatrix. Ultraviolet-visible, photoluminescence, and inverted fluorescence microscope analysis confirmed the unique optical properties of the biosynthesized cadmium selenide quantum dots. The size distribution of the nanocrystals extracted from cells and the location of nanocrystals foci in vivo were also detected seriously by transmission electron microscopy. A surface protein capping layer outside the nanocrystals was confirmed by Fourier transform infrared spectroscopy measurements, which were supposed to contribute to reducing cytotoxicity and maintain a high viability of cells when incubating with quantum dots at concentrations as high as 2 μM. Cell morphology observation indicated an effective labeling of living cells by the biosynthesized quantum dots after a 48 h co-incubation. The present work demonstrated an economical and environmentally friendly approach to fabricating highly fluorescent quantum dots which were expected to be an excellent fluorescent dye for broad bio-imaging and labeling. (papers)

  4. Simultaneous live cell imaging using dual FRET sensors with a single excitation light.

    Directory of Open Access Journals (Sweden)

    Yusuke Niino

    Full Text Available Fluorescence resonance energy transfer (FRET between fluorescent proteins is a powerful tool for visualization of signal transduction in living cells, and recently, some strategies for imaging of dual FRET pairs in a single cell have been reported. However, these necessitate alteration of excitation light between two different wavelengths to avoid the spectral overlap, resulting in sequential detection with a lag time. Thus, to follow fast signal dynamics or signal changes in highly motile cells, a single-excitation dual-FRET method should be required. Here we reported this by using four-color imaging with a single excitation light and subsequent linear unmixing to distinguish fluorescent proteins. We constructed new FRET sensors with Sapphire/RFP to combine with CFP/YFP, and accomplished simultaneous imaging of cAMP and cGMP in single cells. We confirmed that signal amplitude of our dual FRET measurement is comparable to of conventional single FRET measurement. Finally, we demonstrated to monitor both intracellular Ca(2+ and cAMP in highly motile cardiac myocytes. To cancel out artifacts caused by the movement of the cell, this method expands the applicability of the combined use of dual FRET sensors for cell samples with high motility.

  5. Engineering amount of cell-cell contact demonstrates biphasic proliferative regulation through RhoA and the actin cytoskeleton

    International Nuclear Information System (INIS)

    Gray, Darren S.; Liu, Wendy F.; Shen, Colette J.; Bhadriraju, Kiran; Nelson, Celeste M.; Chen, Christopher S.

    2008-01-01

    Endothelial cell-cell contact via VE-cadherin plays an important role in regulating numerous cell functions, including proliferation. However, using different experimental approaches to manipulate cell-cell contact, investigators have observed both inhibition and stimulation of proliferation depending on the adhesive context. In this study, we used micropatterned wells combined with active positioning of cells by dielectrophoresis in order to investigate whether the number of contacting neighbors affected the proliferative response. Varying cell-cell contact resulted in a biphasic effect on proliferation; one contacting neighbor increased proliferation, while two or more neighboring cells partially inhibited this increase. We also observed that cell-cell contact increased the formation of actin stress fibers, and that expression of dominant negative RhoA (RhoN19) blocked the contact-mediated increase in stress fibers and proliferation. Furthermore, examination of heterotypic pairs of untreated cells in contact with RhoN19-expressing cells revealed that intracellular, but not intercellular, tension is required for the contact-mediated stimulation of proliferation. Moreover, engagement of VE-cadherin with cadherin-coated beads was sufficient to stimulate proliferation in the absence of actual cell-cell contact. In all, these results demonstrate that cell-cell contact signals through VE-cadherin, RhoA, and intracellular tension in the actin cytoskeleton to regulate proliferation

  6. Zero Emission Bay Area (ZEBA) Fuel Cell Bus Demonstration Results. Fourth Report

    Energy Technology Data Exchange (ETDEWEB)

    Eudy, Leslie [National Renewable Energy Laboratory (NREL), Golden, CO (United States); Post, Matthew [National Renewable Energy Laboratory (NREL), Golden, CO (United States)

    2015-07-02

    This report presents results of a demonstration of fuel cell electric buses (FCEB) operating in Oakland, California. Alameda-Contra Costa Transit District (AC Transit) leads the Zero Emission Bay Area (ZEBA) demonstration, which includes 12 advanced-design fuel cell buses and two hydrogen fueling stations. The FCEBs in service at AC Transit are 40-foot, low-floor buses built by Van Hool with a hybrid electric propulsion system that includes a US Hybrid fuel cell power system and EnerDel lithium-based energy storage system. The buses began revenue service in May 2010.

  7. Developing new optical imaging techniques for single particle and molecule tracking in live cells

    Energy Technology Data Exchange (ETDEWEB)

    Sun, Wei [Iowa State Univ., Ames, IA (United States)

    2010-01-01

    Differential interference contrast (DIC) microscopy is a far-field as well as wide-field optical imaging technique. Since it is non-invasive and requires no sample staining, DIC microscopy is suitable for tracking the motion of target molecules in live cells without interfering their functions. In addition, high numerical aperture objectives and condensers can be used in DIC microscopy. The depth of focus of DIC is shallow, which gives DIC much better optical sectioning ability than those of phase contrast and dark field microscopies. In this work, DIC was utilized to study dynamic biological processes including endocytosis and intracellular transport in live cells. The suitability of DIC microscopy for single particle tracking in live cells was first demonstrated by using DIC to monitor the entire endocytosis process of one mesoporous silica nanoparticle (MSN) into a live mammalian cell. By taking advantage of the optical sectioning ability of DIC, we recorded the depth profile of the MSN during the endocytosis process. The shape change around the nanoparticle due to the formation of a vesicle was also captured. DIC microscopy was further modified that the sample can be illuminated and imaged at two wavelengths simultaneously. By using the new technique, noble metal nanoparticles with different shapes and sizes were selectively imaged. Among all the examined metal nanoparticles, gold nanoparticles in rod shapes were found to be especially useful. Due to their anisotropic optical properties, gold nanorods showed as diffraction-limited spots with disproportionate bright and dark parts that are strongly dependent on their orientation in the 3D space. Gold nanorods were developed as orientation nanoprobes and were successfully used to report the self-rotation of gliding microtubules on kinesin coated substrates. Gold nanorods were further used to study the rotational motions of cargoes during the endocytosis and intracellular transport processes in live mammalian

  8. Intracellular Delivery of Nanobodies for Imaging of Target Proteins in Live Cells.

    Science.gov (United States)

    Röder, Ruth; Helma, Jonas; Preiß, Tobias; Rädler, Joachim O; Leonhardt, Heinrich; Wagner, Ernst

    2017-01-01

    Cytosolic delivery of nanobodies for molecular target binding and fluorescent labeling in living cells. Fluorescently labeled nanobodies were formulated with sixteen different sequence-defined oligoaminoamides. The delivery of formulated anti-GFP nanobodies into different target protein-containing HeLa cell lines was investigated by flow cytometry and fluorescence microscopy. Nanoparticle formation was analyzed by fluorescence correlation spectroscopy. The initial oligomer screen identified two cationizable four-arm structured oligomers (734, 735) which mediate intracellular nanobody delivery in a receptor-independent (734) or folate receptor facilitated (735) process. The presence of disulfide-forming cysteines in the oligomers was found critical for the formation of stable protein nanoparticles of around 20 nm diameter. Delivery of labeled GFP nanobodies or lamin nanobodies to their cellular targets was demonstrated by fluorescence microscopy including time lapse studies. Two sequence-defined oligoaminoamides with or without folate for receptor targeting were identified as effective carriers for intracellular nanobody delivery, as exemplified by GFP or lamin binding in living cells. Due to the conserved nanobody core structure, the methods should be applicable for a broad range of nanobodies directed to different intracellular targets.

  9. Combining PALM and SOFI for quantitative imaging of focal adhesions in living cells

    Science.gov (United States)

    Deschout, Hendrik; Lukes, Tomas; Sharipov, Azat; Feletti, Lely; Lasser, Theo; Radenovic, Aleksandra

    2017-02-01

    Focal adhesions are complicated assemblies of hundreds of proteins that allow cells to sense their extracellular matrix and adhere to it. Although most focal adhesion proteins have been identified, their spatial organization in living cells remains challenging to observe. Photo-activated localization microscopy (PALM) is an interesting technique for this purpose, especially since it allows estimation of molecular parameters such as the number of fluorophores. However, focal adhesions are dynamic entities, requiring a temporal resolution below one minute, which is difficult to achieve with PALM. In order to address this problem, we merged PALM with super-resolution optical fluctuation imaging (SOFI) by applying both techniques to the same data. Since SOFI tolerates an overlap of single molecule images, it can improve the temporal resolution compared to PALM. Moreover, an adaptation called balanced SOFI (bSOFI) allows estimation of molecular parameters, such as the fluorophore density. We therefore performed simulations in order to assess PALM and SOFI for quantitative imaging of dynamic structures. We demonstrated the potential of our PALM-SOFI concept as a quantitative imaging framework by investigating moving focal adhesions in living cells.

  10. Femtosecond UV-laser pulses to unveil protein-protein interactions in living cells.

    Science.gov (United States)

    Itri, Francesco; Monti, Daria M; Della Ventura, Bartolomeo; Vinciguerra, Roberto; Chino, Marco; Gesuele, Felice; Lombardi, Angelina; Velotta, Raffaele; Altucci, Carlo; Birolo, Leila; Piccoli, Renata; Arciello, Angela

    2016-02-01

    A hallmark to decipher bioprocesses is to characterize protein-protein interactions in living cells. To do this, the development of innovative methodologies, which do not alter proteins and their natural environment, is particularly needed. Here, we report a method (LUCK, Laser UV Cross-linKing) to in vivo cross-link proteins by UV-laser irradiation of living cells. Upon irradiation of HeLa cells under controlled conditions, cross-linked products of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were detected, whose yield was found to be a linear function of the total irradiation energy. We demonstrated that stable dimers of GAPDH were formed through intersubunit cross-linking, as also observed when the pure protein was irradiated by UV-laser in vitro. We proposed a defined patch of aromatic residues located at the enzyme subunit interface as the cross-linking sites involved in dimer formation. Hence, by this technique, UV-laser is able to photofix protein surfaces that come in direct contact. Due to the ultra-short time scale of UV-laser-induced cross-linking, this technique could be extended to weld even transient protein interactions in their native context.

  11. Near-IR Two-Photon Fluorescent Sensor for K(+) Imaging in Live Cells.

    Science.gov (United States)

    Sui, Binglin; Yue, Xiling; Kim, Bosung; Belfield, Kevin D

    2015-08-19

    A new two-photon excited fluorescent K(+) sensor is reported. The sensor comprises three moieties, a highly selective K(+) chelator as the K(+) recognition unit, a boron-dipyrromethene (BODIPY) derivative modified with phenylethynyl groups as the fluorophore, and two polyethylene glycol chains to afford water solubility. The sensor displays very high selectivity (>52-fold) in detecting K(+) over other physiological metal cations. Upon binding K(+), the sensor switches from nonfluorescent to highly fluorescent, emitting red to near-IR (NIR) fluorescence. The sensor exhibited a good two-photon absorption cross section, 500 GM at 940 nm. Moreover, it is not sensitive to pH in the physiological pH range. Time-dependent cell imaging studies via both one- and two-photon fluorescence microscopy demonstrate that the sensor is suitable for dynamic K(+) sensing in living cells.

  12. A Simple BODIPY-Based Viscosity Probe for Imaging of Cellular Viscosity in Live Cells

    Directory of Open Access Journals (Sweden)

    Dongdong Su

    2016-08-01

    Full Text Available Intracellular viscosity is a fundamental physical parameter that indicates the functioning of cells. In this work, we developed a simple boron-dipyrromethene (BODIPY-based probe, BTV, for cellular mitochondria viscosity imaging by coupling a simple BODIPY rotor with a mitochondria-targeting unit. The BTV exhibited a significant fluorescence intensity enhancement of more than 100-fold as the solvent viscosity increased. Also, the probe showed a direct linear relationship between the fluorescence lifetime and the media viscosity, which makes it possible to trace the change of the medium viscosity. Furthermore, it was demonstrated that BTV could achieve practical applicability in the monitoring of mitochondrial viscosity changes in live cells through fluorescence lifetime imaging microscopy (FLIM.

  13. Biophysical response of living cells to boron nitride nanoparticles: uptake mechanism and bio-mechanical characterization

    Energy Technology Data Exchange (ETDEWEB)

    Rasel, Md. Alim Iftekhar; Li, Tong; Nguyen, Trung Dung; Singh, Sanjleena [Queensland University of Technology (QUT), School of Chemistry, Physics and Mechanical Engineering (Australia); Zhou, Yinghong; Xiao, Yin [Queensland University of Technology (QUT), Institute of Health and Biomedical Innovation (Australia); Gu, YuanTong, E-mail: yuantong.gu@qut.edu.au [Queensland University of Technology (QUT), School of Chemistry, Physics and Mechanical Engineering (Australia)

    2015-11-15

    Boron nitride nanomaterials have attracted significant interest due to their superior chemical and physical properties. Despite these novel properties, investigation on the interaction between boron nitride nanoparticle (BN NP) and living systems has been limited. In this study, BN NP (100–250 nm) is assessed as a promising biomaterial for medical applications. The toxicity of BN NP is evaluated by assessing the cells behaviours both biologically (MTT assay, ROS detection etc.) and physically (atomic force microscopy). The uptake mechanism of BN NP is studied by analysing the alternations in cellular morphology based on cell imaging techniques. The results demonstrate in vitro cytocompatibility of BN NP with immense potential for use as an effective nanoparticle for various bio-medical applications.

  14. A magnetic trap for living cells suspended in a paramagnetic buffer

    Science.gov (United States)

    Winkleman, Adam; Gudiksen, Katherine L.; Ryan, Declan; Whitesides, George M.; Greenfield, Derek; Prentiss, Mara

    2004-09-01

    This manuscript describes the fabrication and use of a three-dimensional magnetic trap for diamagnetic objects in an aqueous solution of paramagnetic ions; this trap uses permanent magnets. It demonstrates trapping of polystyrene spheres, and of various types of living cells: mouse fibroblast (NIH-3T3), yeast (Saccharomyces cerevisiae), and algae (Chlamydomonas reinhardtii). For a 40mM solution of gadolinium (III) diethylenetriaminepentaacetic acid (Gd .DTPA) in aqueous buffer, the smallest cell (particle) that could be trapped had a radius of ˜2.5μm. The trapped particle and location of the magnetic trap can be translated in three dimensions by independent manipulation of the permanent magnets. This letter a1so characterizes the biocompatibility of the trapping solution.

  15. Red fluorescent chitosan nanoparticles grafted with poly(2-methacryloyloxyethyl phosphorylcholine) for live cell imaging.

    Science.gov (United States)

    Wang, Ke; Fan, Xingliang; Zhang, Xiaoyong; Zhang, Xiqi; Chen, Yi; Wei, Yen

    2016-08-01

    Poly(2-methacryloyloxyethyl phosphorylcholine) conjugated red fluorescent chitosan nanoparticles (GCC-pMPC) were facilely fabricated by "grafting from" method via surface initiated atom transfer radical polymerization (ATRP). Firstly, glutaraldehyde crosslinked red fluorescent chitosan nanoparticles (GCC NPs) with many amino groups and hydroxyl groups on their surface were prepared, which were then reacted with 2-bromoisobutyryl bromide to form GCC-Br; subsequently, poly(MPC) (pMPC) brushes were grafted onto GCC NPs surface using GCC-Br as initiator via ATRP. Compared with PEGylated nanoparticles, zwitterionic polymers modified nanoparticles demonstrated better performance in their cellular uptake. Moreover, the obtained GCC-pMPC demonstrated excellent water-dispersibility, biocompatibility, and photostability, which made them highly potential for long-term tracing applications. Importantly, the successful live cell imaging of GCC-pMPC would remarkably advance the research of their further bioapplications. Copyright © 2016 Elsevier B.V. All rights reserved.

  16. Multiple origins of spontaneously arising micronuclei in HeLa cells: Direct evidence from long-term live cell imaging

    International Nuclear Information System (INIS)

    Rao Xiaotang; Zhang Yingyin; Yi Qiyi; Hou Heli; Xu Bo; Chu Liang; Huang Yun; Zhang Wenrui; Fenech, Michael; Shi Qinghua

    2008-01-01

    Although micronuclei (MNi) are extensively used to evaluate genotoxic effects and chromosome instability, the most basic issue regarding their origins has not been completely addressed due to limitations of traditional methods. Recently, long-term live cell imaging was developed to monitor the dynamics of single cell in a real-time and high-throughput manner. In the present study, this state-of-the-art technique was employed to examine spontaneous micronucleus (MN) formation in untreated HeLa cells. We demonstrate that spontaneous MNi are derived from incorrectly aligned chromosomes in metaphase (displaced chromosomes, DCs), lagging chromosomes (LCs) and broken chromosome bridges (CBs) in later mitotic stages, but not nuclear buds in S phase. However, most of bipolar mitoses with DCs (91.29%), LCs (73.11%) and broken CBs (88.93%) did not give rise to MNi. Our data also show directly, for the first time, that MNi could originate spontaneously from (1) MNi already presented in the mother cells; (2) nuclear fragments that appeared during mitosis with CB; and (3) chromosomes being extruded into a minicell which fused with one of the daughter cells later. Quantitatively, most of MNi originated from LCs (63.66%), DCs (10.97%) and broken CBs (9.25%). Taken together, these direct evidences show that there are multiple origins for spontaneously arising MNi in HeLa cells and each mechanism contributes to overall MN formation to different extents

  17. Multiple origins of spontaneously arising micronuclei in HeLa cells: Direct evidence from long-term live cell imaging

    Energy Technology Data Exchange (ETDEWEB)

    Rao Xiaotang; Zhang Yingyin; Yi Qiyi; Hou Heli; Xu Bo; Chu Liang; Huang Yun; Zhang Wenrui [Laboratory of Molecular and Cell Genetics, Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230027 (China); Fenech, Michael [CSIRO Human Nutrition, PO Box 10041, Adelaide BC, Adelaide, SA 5000 (Australia); Shi Qinghua [Laboratory of Molecular and Cell Genetics, Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230027 (China)], E-mail: qshi@ustc.edu.cn

    2008-11-10

    Although micronuclei (MNi) are extensively used to evaluate genotoxic effects and chromosome instability, the most basic issue regarding their origins has not been completely addressed due to limitations of traditional methods. Recently, long-term live cell imaging was developed to monitor the dynamics of single cell in a real-time and high-throughput manner. In the present study, this state-of-the-art technique was employed to examine spontaneous micronucleus (MN) formation in untreated HeLa cells. We demonstrate that spontaneous MNi are derived from incorrectly aligned chromosomes in metaphase (displaced chromosomes, DCs), lagging chromosomes (LCs) and broken chromosome bridges (CBs) in later mitotic stages, but not nuclear buds in S phase. However, most of bipolar mitoses with DCs (91.29%), LCs (73.11%) and broken CBs (88.93%) did not give rise to MNi. Our data also show directly, for the first time, that MNi could originate spontaneously from (1) MNi already presented in the mother cells; (2) nuclear fragments that appeared during mitosis with CB; and (3) chromosomes being extruded into a minicell which fused with one of the daughter cells later. Quantitatively, most of MNi originated from LCs (63.66%), DCs (10.97%) and broken CBs (9.25%). Taken together, these direct evidences show that there are multiple origins for spontaneously arising MNi in HeLa cells and each mechanism contributes to overall MN formation to different extents.

  18. Demonstration of Hydrogen Energy Network and Fuel Cells in Residential Homes

    International Nuclear Information System (INIS)

    Hirohisa Aki; Tetsuhiko Maeda; Itaru Tamura; Akeshi Kegasa; Yoshiro Ishikawa; Ichiro Sugimoto; Itaru Ishii

    2006-01-01

    The authors proposed the setting up of an energy interchange system by establishing energy networks of electricity, hot water, and hydrogen in residential homes. In such networks, some homes are equipped with fuel cell stacks, fuel processors, hydrogen storage devices, and large storage tanks for hot water. The energy network enables the flexible operation of the fuel cell stacks and fuel processors. A demonstration project has been planned in existing residential homes to evaluate the proposal. The demonstration will be presented in a small apartment building. The building will be renovated and will be equipped with a hydrogen production facility, a hydrogen interchange pipe, and fuel cell stacks with a heat recovery device. The energy flow process from hydrogen production to consumption in the homes will be demonstrated. This paper presents the proposed energy interchange system and demonstration project. (authors)

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

    Science.gov (United States)

    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.

  20. Identification of novel direct protein-protein interactions by irradiating living cells with femtosecond UV laser pulses.

    Science.gov (United States)

    Itri, Francesco; Monti, Daria Maria; Chino, Marco; Vinciguerra, Roberto; Altucci, Carlo; Lombardi, Angela; Piccoli, Renata; Birolo, Leila; Arciello, Angela

    2017-10-07

    The identification of protein-protein interaction networks in living cells is becoming increasingly fundamental to elucidate main biological processes and to understand disease molecular bases on a system-wide level. We recently described a method (LUCK, Laser UV Cross-linKing) to cross-link interacting protein surfaces in living cells by UV laser irradiation. By using this innovative methodology, that does not require any protein modification or cell engineering, here we demonstrate that, upon UV laser irradiation of HeLa cells, a direct interaction between GAPDH and alpha-enolase was "frozen" by a cross-linking event. We validated the occurrence of this direct interaction by co-immunoprecipitation and Immuno-FRET analyses. This represents a proof of principle of the LUCK capability to reveal direct protein interactions in their physiological environment. Copyright © 2017 Elsevier Inc. All rights reserved.

  1. Online quantitative monitoring of live cell engineered cartilage growth using diffuse fiber-optic Raman spectroscopy.

    Science.gov (United States)

    Bergholt, Mads S; Albro, Michael B; Stevens, Molly M

    2017-09-01

    Tissue engineering (TE) has the potential to improve the outcome for patients with osteoarthritis (OA). The successful clinical translation of this technique as part of a therapy requires the ability to measure extracellular matrix (ECM) production of engineered tissues in vitro, in order to ensure quality control and improve the likelihood of tissue survival upon implantation. Conventional techniques for assessing the ECM content of engineered cartilage, such as biochemical assays and histological staining are inherently destructive. Raman spectroscopy, on the other hand, represents a non-invasive technique for in situ biochemical characterization. Here, we outline current roadblocks in translational Raman spectroscopy in TE and introduce a comprehensive workflow designed to non-destructively monitor and quantify ECM biomolecules in large (>3 mm), live cell TE constructs online. Diffuse near-infrared fiber-optic Raman spectra were measured from live cell cartilaginous TE constructs over a 56-day culturing period. We developed a multivariate curve resolution model that enabled quantitative biochemical analysis of the TE constructs. Raman spectroscopy was able to non-invasively quantify the ECM components and showed an excellent correlation with biochemical assays for measurement of collagen (R 2  = 0.84) and glycosaminoglycans (GAGs) (R 2  = 0.86). We further demonstrated the robustness of this technique for online prospective analysis of live cell TE constructs. The fiber-optic Raman spectroscopy strategy developed in this work offers the ability to non-destructively monitor construct growth online and can be adapted to a broad range of TE applications in regenerative medicine toward controlled clinical translation. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

  2. Massachusetts Fuel Cell Bus Project: Demonstrating a Total Transit Solution for Fuel Cell Electric Buses in Boston

    Energy Technology Data Exchange (ETDEWEB)

    2017-05-22

    The Federal Transit Administration's National Fuel Cell Bus Program focuses on developing commercially viable fuel cell bus technologies. Nuvera is leading the Massachusetts Fuel Cell Bus project to demonstrate a complete transit solution for fuel cell electric buses that includes one bus and an on-site hydrogen generation station for the Massachusetts Bay Transportation Authority (MBTA). A team consisting of ElDorado National, BAE Systems, and Ballard Power Systems built the fuel cell electric bus, and Nuvera is providing its PowerTap on-site hydrogen generator to provide fuel for the bus.

  3. In Cell Footprinting Coupled with Mass Spectrometry for the Structural Analysis of Proteins in Live Cells.

    Science.gov (United States)

    Espino, Jessica A; Mali, Vishaal S; Jones, Lisa M

    2015-08-04

    Protein footprinting coupled with mass spectrometry has become a widely used tool for the study of protein-protein and protein-ligand interactions and protein conformational change. These methods provide residue-level analysis on protein interaction sites and have been successful in studying proteins in vitro. The extension of these methods for in cell footprinting would open an avenue to study proteins that are not amenable for in vitro studies and would probe proteins in their native environment. Here we describe the application of an oxidative-based footprinting approach inside cells in which hydroxyl radicals are used to oxidatively modify proteins. Mass spectrometry is used to detect modification sites and to calculate modification levels. The method is probing biologically relevant proteins in live cells, and proteins in various cellular compartments can be oxdiatively modified. Several different amino acid residues are modified making the method a general labeling strategy for the study of a variety of proteins. Further, comparison of the extent of oxidative modification with solvent accessible surface area reveals the method successfully probes solvent accessibility. This marks the first time protein footprinting has been performed in live cells.

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

    NARCIS (Netherlands)

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

    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

  5. Demonstration of clonable alloreactive host T cells in a primate model for bone marrow transplantation

    International Nuclear Information System (INIS)

    Reisner, Y.; Ben-Bassat, I.; Douer, D.; Kaploon, A.; Schwartz, E.; Ramot, B.

    1986-01-01

    The phenomenon of marrow rejection following supralethal radiochemotherapy was explained in the past mainly by non-T-cell mechanisms known to be resistant to high-dose irradiation. In the present study a low but significant number of radiochemoresistant-clonable T cells was found in the peripheral blood and spleen of Rhesus monkeys following the cytoreductive protocol used for treatment of leukemia patients prior to bone marrow transplantation. More than 95% of the clonable cells are concentrated in the spleen 5 days after transplant. The cells possess immune memory as demonstrated by the generation of alloreactive-specific cytotoxicity. The present findings suggest that host-versus-graft activity may be mediated by alloreactive T cells. It is hoped that elimination of such cells prior to bone marrow transplantation will increase the engraftment rate of HLA-nonidentical marrow in leukemia patients

  6. Supramolecular oligothiophene microfibers spontaneously assembled on surfaces or coassembled with proteins inside live cells.

    Science.gov (United States)

    Barbarella, Giovanna; Di Maria, Francesca

    2015-08-18

    -overrich" hexamers and octamers, leads to surface-independent self-assembly of microfibers-helical or rodlike depending on the groups attached to the same identical inner core-that are crystalline, fluorescent, and conductive and display chirality despite the lack of chiral carbon atoms on the building blocks. Supramolecular polymorphic microfibers are also formed, and they are characterized by very different functional properties. The second, based on a rigid oligothiophene-S,S-dioxide, leads to coassembled protein-oligothiophene microfibers that are physiologically formed inside live cells. The oligothiophene-S,S-dioxide can indeed spontaneously cross the membrane of live cells and be directed toward the perinuclear region, where it is recognized and incorporated by specific peptides during the formation of fibrillar proteins without being harmful to the cells. Coassembled oligothiophene-protein microfibers are progressively formed through a cell-mediated physiological process. Thanks to the oligothiophene blocks, the microfibers possess fluorescence and charge-conduction properties. By means of fluorescence imaging, we demonstrated that various types of live cells seeded on these microfibers were able to internalize and degrade them, experiencing in turn different effects on their morphology and viability, suggesting a possible use of the microfibers as multiscale biomaterials to direct cell behavior. On the whole, our results show the great versatility of oligothiophene building blocks and allow us to foresee that their capabilities of spontaneous assembly in the most different environments could be exploited in much more exciting research fields than those explored to date.

  7. Zero Emission Bay Area (ZEBA) Fuel Cell Bus Demonstration Results: Fifth Report

    Energy Technology Data Exchange (ETDEWEB)

    Eudy, Leslie [National Renewable Energy Lab. (NREL), Golden, CO (United States); Post, Matthew [National Renewable Energy Lab. (NREL), Golden, CO (United States); Jeffers, Matthew [National Renewable Energy Lab. (NREL), Golden, CO (United States)

    2016-06-01

    This report presents results of a demonstration of fuel cell electric buses (FCEB) operating in Oakland, California. Alameda-Contra Costa Transit District (AC Transit) leads the Zero Emission Bay Area (ZEBA) demonstration, which includes 13 advanced-design fuel cell buses and two hydrogen fueling stations. The ZEBA partners are collaborating with the U.S. Department of Energy (DOE) and DOE's National Renewable Energy Laboratory (NREL) to evaluate the buses in revenue service. NREL has published four previous reports describing operation of these buses. This report presents new and updated results covering data from January 2015 through December 2015.

  8. Zero Emission Bay Area (ZEBA) Fuel Cell Bus Demonstration Results: Sixth Report

    Energy Technology Data Exchange (ETDEWEB)

    Eudy, Leslie [National Renewable Energy Laboratory (NREL), Golden, CO (United States); Post, Matthew B. [National Renewable Energy Laboratory (NREL), Golden, CO (United States); Jeffers, Matthew A. [National Renewable Energy Laboratory (NREL), Golden, CO (United States)

    2017-09-11

    This report presents results of a demonstration of fuel cell electric buses (FCEB) operating in Oakland, California. Alameda-Contra Costa Transit District (AC Transit) leads the Zero Emission Bay Area (ZEBA) demonstration, which includes 13 advanced-design fuel cell buses and two hydrogen fueling stations. The ZEBA partners are collaborating with the U.S. Department of Energy (DOE) and DOE's National Renewable Energy Laboratory (NREL) to evaluate the buses in revenue service. NREL has published five previous reports describing operation of these buses. This report presents new and updated results covering data from January 2016 through December 2016.

  9. Continued maturing of SOFC cell production technology and development and demonstration of SOFC stacks. Final report

    Energy Technology Data Exchange (ETDEWEB)

    2008-08-15

    The overall objective of the 6385 project was to develop stack materials, components and stack technology including industrial relevant manufacturing methods for cells components and stacks. Furthermore, the project should include testing and demonstration of the stacks under relevant operating conditions. A production of 6.829 cells, twenty 75-cell stacks and a number of small stacks was achieved. Major improvements were also made in the manufacturing methods and in stack design. Two test and demonstration activities were included in the project. The first test unit was established at H.C. OErsted power plant at the Copenhagen waterfront in order to perform test of SOFC stacks. The unit will be used for tests in other projects. The second demonstration unit is the alpha prototype demonstration in a system running on natural gas in Finland. The alpha prototype demonstration system with 24 TOFC (Topsoe Fuel Cell) stacks was established and started running in October 2007 and operational experience was gained in the period from October 2007 to February 2008. (auther)

  10. Biothermodynamics of live cells: a tool for biotechnology and biochemical engineering

    Science.gov (United States)

    von Stockar, Urs

    2010-12-01

    The aim of this contribution is to review the application of thermodynamics to live cultures of microbial and other cells and to explore to what extent this may be put to practical use. A major focus is on energy dissipation effects in industrially relevant cultures, both in terms of heat and Gibbs energy dissipation. The experimental techniques for calorimetric measurements in live cultures are reviewed and their use for monitoring and control is discussed. A detailed analysis of the dissipation of Gibbs energy in chemotrophic growth shows that it reflects the entropy production by metabolic processes in the cells and thus also the driving force for growth and metabolism. By splitting metabolism conceptually up into catabolism and biosynthesis, it can be shown that this driving force decreases as the growth yield increases. This relationship is demonstrated by using experimental measurements on a variety of microbial strains. On the basis of these data, several literature correlations were tested as tools for biomass yield prediction. The prediction of other culture performance characteristics, including product yields for biorefinery planning, energy yields for biofuel manufacture, maximum growth rates, maintenance requirements, and threshold concentrations is also briefly reviewed.

  11. Structure, cell wall elasticity and polysaccharide properties of living yeast cells, as probed by AFM

    International Nuclear Information System (INIS)

    Alsteens, David; Dupres, Vincent; Evoy, Kevin Mc; Dufrene, Yves F; Wildling, Linda; Gruber, Hermann J

    2008-01-01

    Although the chemical composition of yeast cell walls is known, the organization, assembly, and interactions of the various macromolecules remain poorly understood. Here, we used in situ atomic force microscopy (AFM) in three different modes to probe the ultrastructure, cell wall elasticity and polymer properties of two brewing yeast strains, i.e. Saccharomyces carlsbergensis and S. cerevisiae. Topographic images of the two strains revealed smooth and homogeneous cell surfaces, and the presence of circular bud scars on dividing cells. Nanomechanical measurements demonstrated that the cell wall elasticity of S. carlsbergensis is homogeneous. By contrast, the bud scar of S. cerevisiae was found to be stiffer than the cell wall, presumably due to the accumulation of chitin. Notably, single molecule force spectroscopy with lectin-modified tips revealed major differences in polysaccharide properties of the two strains. Polysaccharides were clearly more extended on S. cerevisiae, suggesting that not only oligosaccharides, but also polypeptide chains of the mannoproteins were stretched. Consistent with earlier cell surface analyses, these findings may explain the very different aggregation properties of the two organisms. This study demonstrates the power of using multiple complementary AFM modalities for probing the organization and interactions of the various macromolecules of microbial cell walls

  12. Meaningful interpretation of subdiffusive measurements in living cells (crowded environment) by fluorescence fluctuation microscopy.

    Science.gov (United States)

    Baumann, Gerd; Place, Robert F; Földes-Papp, Zeno

    2010-08-01

    In living cell or its nucleus, the motions of molecules are complicated due to the large crowding and expected heterogeneity of the intracellular environment. Randomness in cellular systems can be either spatial (anomalous) or temporal (heterogeneous). In order to separate both processes, we introduce anomalous random walks on fractals that represented crowded environments. We report the use of numerical simulation and experimental data of single-molecule detection by fluorescence fluctuation microscopy for detecting resolution limits of different mobile fractions in crowded environment of living cells. We simulate the time scale behavior of diffusion times tau(D)(tau) for one component, e.g. the fast mobile fraction, and a second component, e.g. the slow mobile fraction. The less the anomalous exponent alpha the higher the geometric crowding of the underlying structure of motion that is quantified by the ratio of the Hausdorff dimension and the walk exponent d(f)/d(w) and specific for the type of crowding generator used. The simulated diffusion time decreases for smaller values of alpha # 1 but increases for a larger time scale tau at a given value of alpha # 1. The effect of translational anomalous motion is substantially greater if alpha differs much from 1. An alpha value close to 1 contributes little to the time dependence of subdiffusive motions. Thus, quantitative determination of molecular weights from measured diffusion times and apparent diffusion coefficients, respectively, in temporal auto- and crosscorrelation analyses and from time-dependent fluorescence imaging data are difficult to interpret and biased in crowded environments of living cells and their cellular compartments; anomalous dynamics on different time scales tau must be coupled with the quantitative analysis of how experimental parameters change with predictions from simulated subdiffusive dynamics of molecular motions and mechanistic models. We first demonstrate that the crowding exponent

  13. Effective scheme of photolysis of GFP in live cell as revealed with confocal fluorescence microscopy

    Science.gov (United States)

    Glazachev, Yu I.; Orlova, D. Y.; Řezníčková, P.; Bártová, E.

    2018-05-01

    We proposed an effective kinetics scheme of photolysis of green fluorescent protein (GFP) observed in live cells with a commercial confocal fluorescence microscope. We investigated the photolysis of GFP-tagged heterochromatin protein, HP1β-GFP, in live nucleus with the pulse position modulation approach, which has several advantages over the classical pump-and-probe method. At the basis of the proposed scheme lies a process of photoswitching from the native fluorescence state to the intermediate fluorescence state, which has a lower fluorescence yield and recovers back to native state in the dark. This kinetics scheme includes four effective parameters (photoswitching, reverse switching, photodegradation rate constants, and relative brightness of the intermediate state) and covers the time scale from dozens of milliseconds to minutes of the experimental fluorescence kinetics. Additionally, the applicability of the scheme was demonstrated in the cases of continuous irradiation and the classical pump-and-probe approach using numerical calculations and analytical solutions. An interesting finding of experimental data analysis was that the overall photodegradation of GFP proceeds dominantly from the intermediate state, and demonstrated approximately the second-order reaction versus irradiation power. As a practical example, the proposed scheme elucidates the artifacts of fluorescence recovery after the photobleaching method, and allows us to propose some suggestions on how to diminish them.

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

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Yun [Iowa State Univ., Ames, IA (United States)

    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

  15. Cell-Like Entities: On the Boundary Between Non-Living and Living

    National Research Council Canada - National Science Library

    Frazier, John M; Kelley-Loughnane, Nancy; Rodriguez, Mauricio; Viveros, Leamon; Trott, Sandra; Paliy, Oleg; Tomczak, Melanie

    2006-01-01

    ... direct and control cellular processes at the molecular level. Using this knowledge as a foundation, it is theoretically possible to conceive of designing biological constructs, which we refer to as cell-like entities (CLEs...

  16. Tracking single cells in live animals using a photoconvertible near-infrared cell membrane label.

    Science.gov (United States)

    Carlson, Alicia L; Fujisaki, Joji; Wu, Juwell; Runnels, Judith M; Turcotte, Raphaël; Spencer, Joel A; 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.

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

    Science.gov (United States)

    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.

  18. Use of an optical trap for study of host-pathogen interactions for dynamic live cell imaging.

    Science.gov (United States)

    Tam, Jenny M; Castro, Carlos E; Heath, Robert J W; Mansour, Michael K; Cardenas, Michael L; Xavier, Ramnik J; Lang, Matthew J; Vyas, Jatin M

    2011-07-28

    , chemotherapy, and organ transplantation patients), were optically trapped using non-destructive laser intensities and moved adjacent to macrophages, which can phagocytose the pathogen. High resolution, transmitted light and fluorescence-based movies established the ability to observe early events of phagocytosis in living cells. To demonstrate the broad applicability in immunology, primary T-cells were also trapped and manipulated to form synapses with anti-CD3 coated microspheres in vivo, and time-lapse imaging of synapse formation was also obtained. By providing a method to exert fine spatial control of live pathogens with respect to immune cells, cellular interactions can be captured by fluorescence microscopy with minimal perturbation to cells and can yield powerful insight into early responses of innate and adaptive immunity.

  19. A novel colorimetric and off–on fluorescent sensor for Hg2+ and its application in live cell imaging

    International Nuclear Information System (INIS)

    Li, Man; Jiang, Yuhua; Zhang, Di; Ding, Peigang; Wang, Zhenji; Ye, Yong; Zhao, Yufen

    2014-01-01

    A novel rhodamine-based fluorescent probe L2 with two “S” groups was synthesized and characterized. As expected, L2 exhibited high selectivity and sensitivity for Hg 2+ over other commonly coexistent metal ions. Its selectivity is excellent, and the detection limit was measured to be 1 ppm. The significant changes in the fluorescence color could be used for naked-eye detection. Furthermore, fluorescence imaging experiments of Hg 2+ ions in living EC-109 cells demonstrated its value of practical applications in biological systems. -- Highlights: • A new rhodamine-based chemodosimeter (L2) was synthesized and characterized. • L2 exhibited high selectivity and excellent sensitivity detection of Hg 2+ . • The significant changes in color could be used for naked-eye detection. • The fluorescence imaging experiments of Hg 2+ ions in living EC-109 cells demonstrated its value of practical applications in biological systems

  20. Exploring the Leishmania Hydrophilic Acylated Surface Protein B (HASPB) Export Pathway by Live Cell Imaging Methods.

    Science.gov (United States)

    MacLean, Lorna; Price, Helen; O'Toole, Peter

    2016-01-01

    Leishmania major is a human-infective protozoan parasite transmitted by the bite of the female phlebotomine sand fly. The L. major hydrophilic acylated surface protein B (HASPB) is only expressed in infective parasite stages suggesting a role in parasite virulence. HASPB is a "nonclassically" secreted protein that lacks a conventional signal peptide, reaching the cell surface by an alternative route to the classical ER-Golgi pathway. Instead HASPB trafficking to and exposure on the parasite plasma membrane requires dual N-terminal acylation. Here, we use live cell imaging methods to further explore this pathway allowing visualization of key events in real time at the individual cell level. These methods include live cell imaging using fluorescent reporters to determine the subcellular localization of wild type and acylation site mutation HASPB18-GFP fusion proteins, fluorescence recovery after photobleaching (FRAP) to analyze the dynamics of HASPB in live cells, and live antibody staining to detect surface exposure of HASPB by confocal microscopy.

  1. Langerin negative dendritic cells promote potent CD8+ T-cell priming by skin delivery of live adenovirus vaccine microneedle arrays.

    Science.gov (United States)

    Bachy, Veronique; Hervouet, Catherine; Becker, Pablo D; Chorro, Laurent; Carlin, Leo M; Herath, Shanthi; Papagatsias, Timos; Barbaroux, Jean-Baptiste; Oh, Sea-Jin; Benlahrech, Adel; Athanasopoulos, Takis; Dickson, George; Patterson, Steven; Kwon, Sung-Yun; Geissmann, Frederic; Klavinskis, Linda S

    2013-02-19

    Stabilization of virus protein structure and nucleic acid integrity is challenging yet essential to preserve the transcriptional competence of live recombinant viral vaccine vectors in the absence of a cold chain. When coupled with needle-free skin delivery, such a platform would address an unmet need in global vaccine coverage against HIV and other global pathogens. Herein, we show that a simple dissolvable microneedle array (MA) delivery system preserves the immunogenicity of vaccines encoded by live recombinant human adenovirus type 5 (rAdHu5). Specifically, dried rAdHu5 MA immunization induced CD8(+) T-cell expansion and multifunctional cytokine responses equipotent with conventional injectable routes of immunization. Intravital imaging demonstrated MA cargo distributed both in the epidermis and dermis, with acquisition by CD11c(+) dendritic cells (DCs) in the dermis. The MA immunizing properties were attributable to CD11c(+) MHCII(hi) CD8α(neg) epithelial cell adhesion molecule (EpCAM(neg)) CD11b(+) langerin (Lang; CD207)(neg) DCs, but neither Langerhans cells nor Lang(+) DCs were required for CD8(+) T-cell priming. This study demonstrates an important technical advance for viral vaccine vectors progressing to the clinic and provides insights into the mechanism of CD8(+) T-cell priming by live rAdHu5 MAs.

  2. Bayesian approach to MSD-based analysis of particle motion in live cells.

    Science.gov (United States)

    Monnier, Nilah; Guo, Syuan-Ming; Mori, Masashi; He, Jun; Lénárt, Péter; Bathe, Mark

    2012-08-08

    Quantitative tracking of particle motion using live-cell imaging is a powerful approach to understanding the mechanism of transport of biological molecules, organelles, and cells. However, inferring complex stochastic motion models from single-particle trajectories in an objective manner is nontrivial due to noise from sampling limitations and biological heterogeneity. Here, we present a systematic Bayesian approach to multiple-hypothesis testing of a general set of competing motion models based on particle mean-square displacements that automatically classifies particle motion, properly accounting for sampling limitations and correlated noise while appropriately penalizing model complexity according to Occam's Razor to avoid over-fitting. We test the procedure rigorously using simulated trajectories for which the underlying physical process is known, demonstrating that it chooses the simplest physical model that explains the observed data. Further, we show that computed model probabilities provide a reliability test for the downstream biological interpretation of associated parameter values. We subsequently illustrate the broad utility of the approach by applying it to disparate biological systems including experimental particle trajectories from chromosomes, kinetochores, and membrane receptors undergoing a variety of complex motions. This automated and objective Bayesian framework easily scales to large numbers of particle trajectories, making it ideal for classifying the complex motion of large numbers of single molecules and cells from high-throughput screens, as well as single-cell-, tissue-, and organism-level studies. Copyright © 2012 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  3. Caged Molecular Glues as Photoactivatable Tags for Nuclear Translocation of Guests in Living Cells.

    Science.gov (United States)

    Arisaka, Akio; Mogaki, Rina; Okuro, Kou; Aida, Takuzo

    2018-02-21

    We developed dendritic caged molecular glues ( Caged Glue-R) as tags for nucleus-targeted drug delivery, whose multiple guanidinium ion (Gu + ) pendants are protected by an anionic photocleavable unit (butyrate-substituted nitroveratryloxycarbonyl; BA NVOC). Negatively charged Caged Glue-R hardly binds to anionic biomolecules because of their electrostatic repulsion. However, upon exposure of Caged Glue-R to UV light or near-infrared (NIR) light, the BA NVOC groups of Caged Glue-R are rapidly detached to yield an uncaged molecular glue ( Uncaged Glue-R) that carries multiple Gu + pendants. Because Gu + forms a salt bridge with PO 4 - , Uncaged Glue-R tightly adheres to anionic biomolecules such as DNA and phospholipids in cell membranes by a multivalent salt-bridge formation. When tagged with Caged Glue-R, guests can be taken up into living cells via endocytosis and hide in endosomes. However, when the Caged Glue-R tag is photochemically uncaged to form Uncaged Glue-R, the guests escape from the endosome and migrate into the cytoplasm followed by the cell nucleus. We demonstrated that quantum dots (QDs) tagged with Caged Glue-R can be delivered efficiently to cell nuclei eventually by irradiation with light.

  4. Graphene-augmented nanofiber scaffolds demonstrate new features in cells behaviour

    Science.gov (United States)

    Kazantseva, Jekaterina; Ivanov, Roman; Gasik, Michael; Neuman, Toomas; Hussainova, Irina

    2016-07-01

    Three-dimensional (3D) customized scaffolds capable to mimic a native extracellular matrix open new frontiers in cells manipulation and advanced therapy. The major challenge is in a proper substrate for in vitro models on engineered scaffolds, capable to modulate cells differentiation. Here for the first time we demonstrate novel design and functionality of the 3D porous scaffolds of aligned, self-assembled ceramic nanofibers of ultra-high anisotropy ratio (~107), augmented into graphene shells. This unique hybrid nano-network allows an exceptional combination of selective guidance stimuli of stem cells differentiation, immune reactions variations, and local immobilization of cancer cells, which was not available before. The scaffolds were shown to be able to direct human mesenchymal stem cells (important for stimulation of neuronal and muscle cells) preferential orientation, to suppress major inflammatory factors, and to localize cancer cells; all without additions of specific culture media. The selective downregulation of specific cytokines is anticipated as a new tool for understanding of human immune system and ways of treatment of associated diseases. The effects observed are self-regulated by cells only, without side effects, usually arising from use of external factors. New scaffolds may open new horizons for stem cells fate control such as towards axons and neurites regeneration (Alzheimer’s disease) as well as cancer therapy development.

  5. HaloTag protein-mediated specific labeling of living cells with quantum dots

    International Nuclear Information System (INIS)

    So, Min-kyung; Yao Hequan; Rao Jianghong

    2008-01-01

    Quantum dots emerge as an attractive alternative to small molecule fluorophores as fluorescent tags for in vivo cell labeling and imaging. This communication presents a method for specific labeling of live cells using quantum dots. The labeling is mediated by HaloTag protein expressed at the cell surface which forms a stable covalent adduct with its ligand (HaloTag ligand). The labeling can be performed in one single step with quantum dot conjugates that are functionalized with HaloTag ligand, or in two steps with biotinylated HaloTag ligand first and followed by streptavidin coated quantum dots. Live cell fluorescence imaging indicates that the labeling is specific and takes place at the cell surface. This HaloTag protein-mediated cell labeling method should facilitate the application of quantum dots for live cell imaging

  6. Cell volume and geometric parameters determination in living cells using confocal microscopy and 3D reconstruction

    OpenAIRE

    sprotocols

    2015-01-01

    Authors: David Hevia, Aida Rodriguez-Garcia, Marta Alonso-Gervós, Isabel Quirós-González, Henar M Cimadevilla, Carmen Gómez-Cordovés, Rosa M Sainz & Juan C Mayo ### Abstract The protocol reported here describes a simple, easy, fast and reproducible method aimed to know the geometric parameters of living cells based on confocal laser scanning microscopy combined with 3D reconstruction software. Briefly, the method is based on intrinsic fluorescence properties of acridine orange (AO), a...

  7. The Belgian demonstration programme for the disposal of high-level and long-lived radioactive waste

    International Nuclear Information System (INIS)

    Bernier, F.; Demarche, M.

    2006-01-01

    The EIG EURIDICE is responsible for performing large-scale tests, technical demonstrations and experiments so as to assess the feasibility of a final disposal of vitrified radioactive waste in deep clay layers. This programme is part of the Belgian Research and Development programme managed by ONDRAF/NIRAS. The research infrastructure includes the Underground Research Facilities HADES (URF HADES) in the Boom Clay geological formation and surface facilities. The achievements of the demonstration programme are the demonstration of the construction of shafts and galleries at industrial scale, the characterisation of the hydro-mechanical response of the host rock, and the 'OPHELIE mock-up' a large scale hydration test under thermal load of pre-fabricated bentonite blocks. The future works will consist mainly in the realisation of the 'PRACLAY experiments' including a large scale heater test. The results of this test will constitute an important input for the Safety and Feasibility Cases 1 (SFC-1, 2013) and 2 (SFC-2, 2020). (author)

  8. TNX GeoSiphon Cell (TGSC-1) Phase II Single Cell Deployment/Demonstration Final Report

    Energy Technology Data Exchange (ETDEWEB)

    Phifer, M.A.

    1999-04-15

    This Phase II final report documents the Phase II testing conducted from June 18, 1998 through November 13, 1998, and it focuses on the application of the siphon technology as a sub-component of the overall GeoSiphon Cell technology. [Q-TPL-T-00004

  9. Power conversion and quality of the Santa Clara 2 MW direct carbonate fuel cell demonstration plant

    Energy Technology Data Exchange (ETDEWEB)

    Skok, A.J. [Fuel Cell Engineering Corp., Danbury, CT (United States); Abueg, R.Z. [Basic Measuring Instruments, Santa Clara, CA (United States); Schwartz, P. [Fluor Daniel, Inc., Irvine, CA (United States)] [and others

    1996-12-31

    The Santa Clara Demonstration Project (SCDP) is the first application of a commercial-scale carbonate fuel cell power plant on a US electric utility system. It is also the largest fuel cell power plant ever operated in the United States. The 2MW plant, located in Santa Clara, California, utilizes carbonate fuel cell technology developed by Energy Research Corporation (ERC) of Danbury, Connecticut. The ultimate goal of a fuel cell power plant is to deliver usable power into an electrical distribution system. The power conversion sub-system does this for the Santa Clara Demonstration Plant. A description of this sub-system and its capabilities follows. The sub-system has demonstrated the capability to deliver real power, reactive power and to absorb reactive power on a utility grid. The sub-system can be operated in the same manner as a conventional rotating generator except with enhanced capabilities for reactive power. Measurements demonstrated the power quality from the plant in various operating modes was high quality utility grade power.

  10. Live imaging of individual cell divisions in mouse neuroepithelium shows asymmetry in cilium formation and Sonic hedgehog response

    Directory of Open Access Journals (Sweden)

    Piotrowska-Nitsche Karolina

    2012-05-01

    Full Text Available Abstract Background Primary cilia are microtubule-based sensory organelles that play important roles in developmental signaling pathways. Recent work demonstrated that, in cell culture, the daughter cell that inherits the older mother centriole generates a primary cilium and responds to external stimuli prior to its sister cell. This asynchrony in timing of cilia formation could be especially critical during development as cell divisions are required for both differentiation and maintenance of progenitor cell niches. Methods Here we integrate several fluorescent markers and use ex vivo live imaging of a single cell division within the mouse E8.5 neuroepithelium to reveal both the formation of a primary cilium and the transcriptional response to Sonic hedgehog in the daughter cells. Results We show that, upon cell division, cilia formation and the Sonic hedgehog response are asynchronous between the daughter cells. Conclusions Our results demonstrate that we can directly observe single cell divisions within the developing neuroepithelium and concomitantly monitor cilium formation or Sonic hedgehog response. We expect this method to be especially powerful in examining whether cellular behavior can lead to both differentiation and maintenance of cells in a progenitor niche.

  11. Hydrogen fueling stations in Japan hydrogen and fuel cell demonstration project

    International Nuclear Information System (INIS)

    Koseki, K.; Tomuro, J.; Sato, H.; Maruyama, S.

    2004-01-01

    A new national demonstration project of fuel cell vehicles, which is called Japan Hydrogen and Fuel Cell Demonstration Project (JHFC Project), has started in FY2002 on a four-year plan. In this new project, ten hydrogen fueling stations have been constructed in Tokyo and Kanagawa area in FY2002-2003. The ten stations adopt the following different types of fuel and fueling methods: LPG reforming, methanol reforming, naphtha reforming, desulfurized-gasoline reforming, kerosene reforming, natural gas reforming, water electrolysis, liquid hydrogen, by-product hydrogen, and commercially available cylinder hydrogen. Approximately fifty fuel cell passenger cars and a fuel cell bus are running on public roads using these stations. In addition, two hydrogen stations will be constructed in FY2004 in Aichi prefecture where The 2005 World Exposition (EXPO 2005) will be held. The stations will service eight fuel cell buses used as pick-up buses for visitors. We, Engineering Advancement Association of Japan (ENAA), are commissioned to construct and operate a total of twelve stations by Ministry of Economy Trade and Industry (METI). We are executing to demonstrate or identify the energy-saving effect, reduction of the environmental footprint, and issues for facilitating the acceptance of hydrogen stations on the basis of the data obtained from the operation of the stations. (author)

  12. Cationic Organochalcogen with Monomer/Excimer Emissions for Dual-Color Live Cell Imaging and Cell Damage Diagnosis.

    Science.gov (United States)

    Chao, Xi-Juan; Wang, Kang-Nan; Sun, Li-Li; Cao, Qian; Ke, Zhuo-Feng; Cao, Du-Xia; Mao, Zong-Wan

    2018-04-25

    Studies on the development of fluorescent organic molecules with different emission colors for imaging of organelles and their biomedical application are gaining lots of focus recently. Here, we report two cationic organochalcogens 1 and 2, both of which exhibit very weak green emission (Φ 1 = 0.12%; Φ 2 = 0.09%) in dilute solution as monomers, but remarkably enhanced green emission upon interaction with nucleic acids and large red-shifted emission in aggregate state by the formation of excimers at high concentration. More interestingly, the monomer emission and excimer-like emission can be used for dual color imaging of different organelles. Upon passively diffusing into cells, both probes selectively stain nucleoli with strong green emission upon 488 nm excitation, whereas upon 405 nm excitation, a completely different stain pattern by staining lysosomes (for 1) or mitochondria (for 2) with distinct red emission is observed because of the highly concentrated accumulation in these organelles. Studies on the mechanism of the accumulation in lysosomes (for 1) or mitochondria (for 2) found that the accumulations of the probes are dependent on the membrane permeabilization, which make the probes have great potential in diagnosing cell damage by sensing lysosomal or mitochondrial membrane permeabilization. The study is demonstrative, for the first time, of two cationic molecules for dual-color imaging nucleoli and lysosomes (1)/mitochondria (2) simultaneously in live cell based on monomer and excimer-like emission, respectively, and more importantly, for diagnosing cell damage.

  13. What befalls the proteins and water in a living cell when the cell dies?

    Science.gov (United States)

    Ling, Gilbert N; Fu, Ya-zhen

    2005-01-01

    The solvency of solutes of varying molecular size in the intracellular water of freshly-killed Ehrlich carcinoma cells fits the same theoretical curve that describes the solvency of similar solutes in a 36% solution of native bovine hemoglobin--a protein found only in red blood cells and making up 97.3% of the red cell's total intracellular proteins. The merging of the two sets of data confirms the prediction of the AI Hypothesis that key intracellular protein(s) in dying cells undergo(es) a transition from: (1) one in which the polypeptide NHCO groups assume a fully-extended conformation with relatively strong power of polarizing and orienting the bulk-phase water in multilayers; to (2) one in which most of the polypeptide NHCO groups are engaged in alpha-helical and other "introvert" conformations (see below for definition) with much weaker power in polarizing-orienting multilayers of bulk-phase water. This concordance of the two sets of data also shows that what we now call native hemoglobin--supposedly denoting hemoglobin found in its natural state in living red blood cells--, in fact, more closely resembles the water-polarizing, and -orienting intracellular proteins in dead cells. Although in the dead Ehrlich carcinoma cells as well as in the 36% solution of native hemoglobin, much of the protein's polypeptide NHCO groups are engaged in alpha-helical and other "introvert" conformation (Perutz 1969; Weissbluth 1974), both systems produce a weak but nonetheless pervasive and "long-range" water polarization and orientation. It is suggested that in both the dead Ehrlich carcinoma ascites cells and in the 36% native bovine hemoglobin solution, enough polypeptide NHCO groups assume the fully-extended conformation to produce the weak but far-reaching multilayer water polarization and orientation observed.

  14. Determination of cell cycle phases in live B16 melanoma cells using IRMS.

    Science.gov (United States)

    Bedolla, Diana E; Kenig, Saša; Mitri, Elisa; Ferraris, Paolo; Marcello, Alessandro; Grenci, Gianluca; Vaccari, Lisa

    2013-07-21

    The knowledge of cell cycle phase distribution is of paramount importance for understanding cellular behaviour under normal and stressed growth conditions. This task is usually assessed using Flow Cytometry (FC) or immunohistochemistry. Here we report on the use of FTIR microspectroscopy in Microfluidic Devices (MD-IRMS) as an alternative technique for studying cell cycle distribution in live cells. Asynchronous, S- and G0-synchronized B16 mouse melanoma cells were studied by running parallel experiments based on MD-IRMS and FC using Propidium Iodide (PI) staining. MD-IRMS experiments have been done using silicon-modified BaF2 devices, where the thin silicon layer prevents BaF2 dissolution without affecting the transparency of the material and therefore enabling a better assessment of the Phosphate I (PhI) and II (PhII) bands. Hierarchical Cluster Analysis (HCA) of cellular microspectra in the 1300-1000 cm(-1) region pointed out a distribution of cells among clusters, which is in good agreement with FC results among G0/G1, S and G2/M phases. The differentiation is mostly driven by the intensity of PhI and PhII bands. In particular, PhI almost doubles from the G0/G1 to G2/M phase, in agreement with the trend followed by nucleic acids during cellular progression. MD-IRMS is then proposed as a powerful method for the in situ determination of the cell cycle stage of an individual cell, without any labelling or staining, which gives the advantage of possibly monitoring specific cellular responses to several types of stimuli by clearly separating the spectral signatures related to the cellular response from those of cells that are normally progressing.

  15. Live birth potential of good morphology and vitrified blastocysts presenting abnormal cell divisions

    DEFF Research Database (Denmark)

    Azzarello, Antonino; Høst, Thomas; Hay-Schmidt, Anders

    2017-01-01

    a lower live birth rate (17.0%) than blastocyst with solely regular cell divisions (29.3%). ACDs could occur at more than one cell division in the same good morphology blastocyst. Reported as independent events, we observed ACDs occurring more frequently at the later cell cycles (1st: 1.3%; 2nd: 8.0%; 3rd...

  16. Effects of high-gradient magnetic fields on living cell machinery

    Czech Academy of Sciences Publication Activity Database

    Zablotskyy, V.; Lunov, O.; Kubinová, Šárka; Polyakova, T.; Syková, Eva; Dejneka, A.

    2016-01-01

    Roč. 49, č. 2016 (2016), s. 493003 ISSN 0022-3727 R&D Projects: GA MŠk(CZ) LO1309 Institutional support: RVO:68378041 Keywords : living cell * magnetic gradient force * cell mechanics * stem cell * magnetic field Subject RIV: FP - Other Medical Disciplines Impact factor: 2.588, year: 2016

  17. A simple optical fiber device for quantitative fluorescence microscopy of single living cells

    NARCIS (Netherlands)

    van Graft, M.; van Graft, Marja; Oosterhuis, B.; Oosterhuis, Bernard; van der Werf, Kees; de Grooth, B.G.; Greve, Jan

    1993-01-01

    simple and relatively inexpensive system is described for obtaining quantitative fluorescence measurements on single living cells loaded with a fluorescent probe to study cell physiological processes. The light emitted from the fluorescent cells is captured by and transported through an optical

  18. Quantification of GPCR internalization by single-molecule microscopy in living cells.

    NARCIS (Netherlands)

    Serge, A.; Keijzer, S. de; Hemert, F. Van; Hickman, M.R.; Hereld, D.; Spaink, H.P.; Schmidt, T.; Snaar-Jagalska, B.E.

    2011-01-01

    Receptor internalization upon ligand stimulation is a key component of a cell's response and allows a cell to correctly sense its environment. Novel fluorescent methods have enabled the direct visualization of the agonist-stimulated G-protein-coupled receptors (GPCR) trafficking in living cells.

  19. A rhodol-based fluorescent chemosensor for hydrazine and its application in live cell bioimaging

    Science.gov (United States)

    Tiensomjitr, Khomsan; Noorat, Rattha; Wechakorn, Kanokorn; Prabpai, Samran; Suksen, Kanoknetr; Kanjanasirirat, Phongthon; Pewkliang, Yongyut; Borwornpinyo, Suparerk; Kongsaeree, Palangpon

    2017-10-01

    A rhodol cinnamate fluorescent chemosensor (RC) has been developed for selective detection of hydrazine (N2H4). In aqueous medium, the rhodol-based probe exhibited high selectivity for hydrazine among other molecules. The addition of hydrazine triggered a fluorescence emission with 48-fold enhancement based on hydrazinolysis and a subsequent ring-opening process. The chemical probe also displayed a selective colorimetric response toward N2H4 from colorless solution to pink, readily observed by the naked eye. The detection limit of RC for hydrazine was calculated to be 300 nM (9.6 ppb). RC is membrane permeable and was successfully demonstrated to detect hydrazine in live HepG2 cells by confocal fluorescence microscopy.

  20. Safety evaluation report of hot cell facilities for demonstration of advanced spent fuel conditioning process

    International Nuclear Information System (INIS)

    You, Gil Sung; Choung, W. M.; Ku, J. H.; Cho, I. J.; Kook, D. H.; Park, S. W.; Bek, S. Y.; Lee, E. P.

    2004-10-01

    The advanced spent fuel conditioning process(ACP) proposed to reduce the overall volume of the PWR spent fuel and improve safety and economy of the long-term storage of spent fuel. In the next phase(2004∼2006), the hot test will be carried out for verification of the ACP in a laboratory scale. For the hot test, the hot cell facilities of α- type and auxiliary facilities are required essentially for safe handling of high radioactive materials. As the hot cell facilities for demonstration of the ACP, a existing hot cell of β- type will be refurbished to minimize construction expenditures of hot cell facility. Up to now, the detail design of hot cell facilities and process were completed, and the safety analysis was performed to substantiate secure of conservative safety. The design data were submitted for licensing which was necessary for construction and operation of hot cell facilities. The safety investigation of KINS on hot cell facilities was completed, and the license for construction and operation of hot cell facilities was acquired already from MOST. In this report, the safety analysis report submitted to KINS was summarized. And also, the questionnaires issued from KINS and answers of KAERI in process of safety investigation were described in detail

  1. Photoinducible bioorthogonal chemistry: a spatiotemporally controllable tool to visualize and perturb proteins in live cells.

    Science.gov (United States)

    Lim, Reyna K V; Lin, Qing

    2011-09-20

    Visualization in biology has been greatly facilitated by the use of fluorescent proteins as in-cell probes. The genes coding for these wavelength-tunable proteins can be readily fused with the DNA coding for a protein of interest, which enables direct monitoring of natural proteins in real time inside living cells. Despite their success, however, fluorescent proteins have limitations that have only begun to be addressed in the past decade through the development of bioorthogonal chemistry. In this approach, a very small bioorthogonal tag is embedded within the basic building blocks of the cell, and then a variety of external molecules can be selectively conjugated to these pretagged biomolecules. The result is a veritable palette of biophysical probes for the researcher to choose from. In this Account, we review our progress in developing a photoinducible, bioorthogonal tetrazole-alkene cycloaddition reaction ("photoclick chemistry") and applying it to probe protein dynamics and function in live cells. The work described here summarizes the synthesis, structure, and reactivity studies of tetrazoles, including their optimization for applications in biology. Building on key insights from earlier reports, our initial studies of the reaction have revealed full water compatibility, high photoactivation quantum yield, tunable photoactivation wavelength, and broad substrate scope; an added benefit is the formation of fluorescent cycloadducts. Subsequent studies have shown fast reaction kinetics (up to 11.0 M(-1) s(-1)), with the rate depending on the HOMO energy of the nitrile imine dipole as well as the LUMO energy of the alkene dipolarophile. Moreover, through the use of photocrystallography, we have observed that the photogenerated nitrile imine adopts a bent geometry in the solid state. This observation has led to the synthesis of reactive, macrocyclic tetrazoles that contain a short "bridge" between two flanking phenyl rings. This photoclick chemistry has been used

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

    Science.gov (United States)

    Sundaram, S K; Sacksteder, Colette A; Weber, Thomas J; Riley, Brian J; Addleman, R Shane; Harrer, Bruce J; Peterman, John W

    2013-01-01

    A significant challenge to realize the full potential of nanotechnology for therapeutic and diagnostic applications is to understand and evaluate how live cells interact with an external stimulus, such as a nanosized particle, and the toxicity and broad risk associated with these stimuli. It is difficult to capture the complexity and dynamics of these interactions by following omics-based approaches exclusively, which can be expensive and time-consuming. Attenuated total reflectance-Fourier transform infrared spectroscopy is well suited to provide noninvasive live-cell monitoring of cellular responses to potentially toxic nanosized particles or other stimuli. This alternative approach provides the ability to carry out rapid toxicity screenings and nondisruptive monitoring of live-cell cultures. We review the technical basis of the approach, the instrument configuration and interface with the biological media, the various effects that impact the data, subsequent data analysis and toxicity, and present some preliminary results on live-cell monitoring.

  3. Bridging the gap between cell culture and live tissue

    Directory of Open Access Journals (Sweden)

    Stefan Przyborski

    2017-11-01

    Full Text Available Traditional in vitro two-dimensional (2-D culture systems only partly imitate the physiological and biochemical features of cells in their original tissue. In vivo, in organs and tissues, cells are surrounded by a three-dimensional (3-D organization of supporting matrix and neighbouring cells, and a gradient of chemical and mechanical signals. Furthermore, the presence of blood flow and mechanical movement provides a dynamic environment (Jong et al., 2011. In contrast, traditional in vitro culture, carried out on 2-D plastic or glass substrates, typically provides a static environment, which, however is the base of the present understanding of many biological processes, tissue homeostasis as well as disease. It is clear that this is not an exact representation of what is happening in vivo and the microenvironment provided by in vitro cell culture models are significantly different and can cause deviations in cell response and behaviour from those distinctive of in vivo tissues. In order to translate the present basic knowledge in cell control, cell repair and regeneration from the laboratory bench to the clinical application, we need a better understanding of the cell and tissue interactions. This implies a detailed comprehension of the natural tissue environment, with its organization and local signals, in order to more closely mimic what happens in vivo, developing more physiological models for efficient in vitro systems. In particular, it is imperative to understand the role of the environmental cues which can be mainly divided into those of a chemical and mechanical nature.

  4. The Secret Lives of Pluripotent Cells: There and Back Again

    Directory of Open Access Journals (Sweden)

    Paolo Cinelli

    2010-03-01

    Full Text Available Embryonic stem cells (ESCs and induced pluripotent stem cells (IPSCs hold great promise for the therapeutic treatment of human diseases, but their functional similarity, their stability and especially the mechanism underlying their derivation are not yet clearly explained. [...

  5. Imaging protein-protein interactions in living cells

    NARCIS (Netherlands)

    Hink, M.A.; Bisseling, T.; Visser, A.J.W.G.

    2002-01-01

    The complex organization of plant cells makes it likely that the molecular behaviour of proteins in the test tube and the cell is different. For this reason, it is essential though a challenge to study proteins in their natural environment. Several innovative microspectroscopic approaches provide

  6. Cancer cell-oriented migration of mesenchymal stem cells engineered with an anticancer gene (PTEN: an imaging demonstration

    Directory of Open Access Journals (Sweden)

    Yang ZS

    2014-03-01

    Full Text Available Zhuo-Shun Yang,1,* Xiang-Jun Tang,2,* Xing-Rong Guo,1 Dan-Dan Zou,1 Xu-Yong Sun,3 Jing-Bo Feng,1 Jie Luo,1 Long-Jun Dai,1,4 Garth L Warnock4 1Hubei Key Laboratory of Stem Cell Research, Taihe Hospital, Hubei University of Medicine, Shiyan, People’s Republic of China; 2Department of Neurosurgery, Taihe Hospital, Hubei University of Medicine, Shiyan, People’s Republic of China; 3Guangxi Key Laboratory for Transplant Medicine, 303 Hospital of PLA, Nanning, People’s Republic of China; 4Department of Surgery, University of British Columbia, Vancouver, BC, Canada *These authors contributed equally to this work Background: Mesenchymal stem cells (MSCs have been considered to hold great potential as ideal carriers for the delivery of anticancer agents since the discovery of their tumor tropism. This study was performed to demonstrate the effects of phosphatase and tensin homolog (PTEN engineering on MSCs’ capacity for cancer cell-oriented migration. Methods: MSCs were engineered with a PTEN-bearing plasmid and the expression was confirmed with Western blotting. A human glioma cell line (DBTRG was used as the target cell; DBTRG cell-oriented migration of MSCs was monitored with a micro speed photographic system. Results: The expression of transfected PTEN in MSCs was identified by immunoblotting analysis and confirmed with cell viability assessment of target cells. The DBTRG cell-oriented migration of PTEN-engineered MSCs was demonstrated by a real-time dynamic monitoring system, and a phagocytosis-like action of MSCs was also observed. Conclusion: MSCs maintained their capacity for cancer cell-directed migration after they were engineered with anticancer genes. This study provides the first direct evidence of MSCs’ tropism post-anticancer gene engineering. Keywords: gene therapy, mesenchymal stem cells, phosphatase and tensin homolog, cancer

  7. Cell tracking using iron oxide fails to distinguish dead from living transplanted cells in the infarcted heart.

    Science.gov (United States)

    Winter, E M; Hogers, B; van der Graaf, L M; Gittenberger-de Groot, A C; Poelmann, R E; van der Weerd, L

    2010-03-01

    Recently, debate has arisen about the usefulness of cell tracking using iron oxide-labeled cells. Two important issues in determining the usefulness of cell tracking with MRI are generally overlooked; first, the effect of graft rejection in immunocompetent models, and second, the necessity for careful histological confirmation of the fate of the labeled cells in the presence of iron oxide. Therefore, both iron oxide-labeled living as well as dead epicardium-derived cells (EPDCs) were investigated in ischemic myocardium of immunodeficient non-obese diabetic (NOD)/acid: non-obese diabetic severe combined immunodeficient (NOD/scid) mice with 9.4T MRI until 6 weeks after surgery, at which time immunohistochemical analysis was performed. In both groups, voids on MRI scans were observed that did not change in number, size, or localization over time. Based on MRI, no distinction could be made between living and dead injected cells. Prussian blue staining confirmed that the hypointense spots on MRI corresponded to iron-loaded cells. However, in the dead-EPDC recipients, all iron-positive cells appeared to be macrophages, while the living-EPDC recipients also contained engrafted iron-loaded EPDCs. Iron labeling is inadequate for determining the fate of transplanted cells in the immunodeficient host, since dead cells produce an MRI signal indistinguishable from incorporated living cells. (c) 2010 Wiley-Liss, Inc.

  8. RADIOAUTOGRAPHIC DEMONSTRATION OF 5-HYDROXYTRYPTAMINE-3H UPTAKE BY PULMONARY ENDOTHELIAL CELLS

    Science.gov (United States)

    Strum, Judy M.; Junod, Alain F.

    1972-01-01

    The lung is able to rapidly remove 5-hydroxytryptamme (5-HT) from the circulation by a Na+-dependent transport mechanism. In order to identify the sites of uptake, radioautographic studies were done on rat lungs which had been isolated and perfused with 5-HT-3H and 0 5 mM iproniazid, a monoamine oxidase inhibitor. In control experiments 10-4 M imipramine was added to the perfusate to inhibit the membrane transport of 5-HT At the light microscope level, silver grains were seen concentrated near capillaries and in the endothelium of large vessels From electron microscope radioautographs a semiquantitative grain count was made and 90% of the silver grains were observed over capillary endothelial cells. The grains were found over the nucleus and cytoplasm of the cell and shewed no preferential association with any particular cytoplasmic inclusion bodies, organelles, or vesicles Other cell types were unlabeled except for a few mast cells, certain vascular smooth muscle cells, and one nerve ending. This radioautographic demonstration of the cell type responsible for the rapid removal of 5-HT from the lung circulation clearly establishes the existence of a new metabolic role for pulmonary endothelial cells. PMID:5044755

  9. Detection of Mitochondrial Caspase Activity in Real Time In Situ in Live Cells

    Science.gov (United States)

    Zhang, Yingpei; Haskins, Catherine; Lopez-Cruzan, Marisa; Zhang, Jianhua; Centonze, Victoria E.; Herman, Brian

    2004-08-01

    Apoptosis plays an important role in many physiological and pathological processes. The initiation and execution of the cell death program requires activation of multiple caspases in a stringently temporal order. Here we describe a method that allows real-time observation of caspase activation in situ in live cells based on fluorescent resonance energy transfer (FRET) measurement using the prism and reflector imaging spectroscopy system (PARISS). When a fusion protein consisting of CFP connected to YFP via an intervening caspase substrate that has been targeted to a specific subcellular location is excited with a light source whose wavelength matches the cyan fluorescent protein (CFP) excitation peak, the energy absorbed by the CFP fluorophore is not emitted as fluorescence. Instead, the excitation energy is absorbed by the nearby yellow fluorescent protein (YFP) fluorophore that is covalently linked to CFP through a short peptide containing the caspase substrate. Cleavage of the linker peptide by caspases results in loss of FRET due to the separation of CFP and YFP fluorophores. Using a mitochondrially targeted CFP caspase 3 substrate YFP construct (mC3Y), we demonstrate for the first time that there is caspase-3-like activity in the mitochondrial matrix of some cells at very late stage of apoptosis.

  10. Surface plasmon excitation using a Fourier-transform infrared spectrometer: Live cell and bacteria sensing

    Science.gov (United States)

    Lirtsman, Vladislav; Golosovsky, Michael; Davidov, Dan

    2017-10-01

    We report an accessory for beam collimation to be used as a plug-in for a conventional Fourier-Transform Infrared (FTIR) spectrometer. The beam collimator makes use of the built-in focusing mirror of the FTIR spectrometer which focuses the infrared beam onto the pinhole mounted in the place usually reserved for the sample. The beam is collimated by a small parabolic mirror and is redirected to the sample by a pair of plane mirrors. The reflected beam is conveyed by another pair of plane mirrors to the built-in detector of the FTIR spectrometer. This accessory is most useful for the surface plasmon excitation. We demonstrate how it can be employed for label-free and real-time sensing of dynamic processes in bacterial and live cell layers. In particular, by measuring the intensity of the CO2 absorption peak one can assess the cell layer metabolism, while by measuring the position of the surface plasmon resonance one assesses the cell layer morphology.

  11. Low cost label-free live cell imaging for biological samples

    Science.gov (United States)

    Seniya, C.; Towers, C. E.; Towers, D. P.

    2017-02-01

    This paper reports the progress to develop a practical phase measuring microscope offering new capabilities in terms of phase measurement accuracy and quantification of cell:cell interactions over the longer term. A novel, low cost phase interference microscope for imaging live cells (label-free) is described. The method combines the Zernike phase contrast approach with a dual mirror design to enable phase modulation between the scattered and un-scattered optical fields. Two designs are proposed and demonstrated, one of which retains the common path nature of Zernike's original microscopy concept. In both setups the phase shift is simple to control via a piezoelectric driven mirror in the back focal plane of the imaging system. The approach is significantly cheaper to implement than those based on spatial light modulators (SLM) at approximately 20% of the cost. A quantitative assessment of the performance of a set of phase shifting algorithms is also presented, specifically with regard to broad bandwidth illumination in phase contrast microscopy. The simulation results show that the phase measurement accuracy is strongly dependent on the algorithm selected and the optical path difference in the sample.

  12. Targeting and tracing of specific DNA sequences with dTALEs in living cells

    Science.gov (United States)

    Thanisch, Katharina; Schneider, Katrin; Morbitzer, Robert; Solovei, Irina; Lahaye, Thomas; Bultmann, Sebastian; Leonhardt, Heinrich

    2014-01-01

    Epigenetic regulation of gene expression involves, besides DNA and histone modifications, the relative positioning of DNA sequences within the nucleus. To trace specific DNA sequences in living cells, we used programmable sequence-specific DNA binding of designer transcription activator-like effectors (dTALEs). We designed a recombinant dTALE (msTALE) with variable repeat domains to specifically bind a 19-bp target sequence of major satellite DNA. The msTALE was fused with green fluorescent protein (GFP) and stably expressed in mouse embryonic stem cells. Hybridization with a major satellite probe (3D-fluorescent in situ hybridization) and co-staining for known cellular structures confirmed in vivo binding of the GFP-msTALE to major satellite DNA present at nuclear chromocenters. Dual tracing of major satellite DNA and the replication machinery throughout S-phase showed co-localization during mid to late S-phase, directly demonstrating the late replication timing of major satellite DNA. Fluorescence bleaching experiments indicated a relatively stable but still dynamic binding, with mean residence times in the range of minutes. Fluorescently labeled dTALEs open new perspectives to target and trace DNA sequences and to monitor dynamic changes in subnuclear positioning as well as interactions with functional nuclear structures during cell cycle progression and cellular differentiation. PMID:24371265

  13. Targeting and tracing of specific DNA sequences with dTALEs in living cells.

    Science.gov (United States)

    Thanisch, Katharina; Schneider, Katrin; Morbitzer, Robert; Solovei, Irina; Lahaye, Thomas; Bultmann, Sebastian; Leonhardt, Heinrich

    2014-04-01

    Epigenetic regulation of gene expression involves, besides DNA and histone modifications, the relative positioning of DNA sequences within the nucleus. To trace specific DNA sequences in living cells, we used programmable sequence-specific DNA binding of designer transcription activator-like effectors (dTALEs). We designed a recombinant dTALE (msTALE) with variable repeat domains to specifically bind a 19-bp target sequence of major satellite DNA. The msTALE was fused with green fluorescent protein (GFP) and stably expressed in mouse embryonic stem cells. Hybridization with a major satellite probe (3D-fluorescent in situ hybridization) and co-staining for known cellular structures confirmed in vivo binding of the GFP-msTALE to major satellite DNA present at nuclear chromocenters. Dual tracing of major satellite DNA and the replication machinery throughout S-phase showed co-localization during mid to late S-phase, directly demonstrating the late replication timing of major satellite DNA. Fluorescence bleaching experiments indicated a relatively stable but still dynamic binding, with mean residence times in the range of minutes. Fluorescently labeled dTALEs open new perspectives to target and trace DNA sequences and to monitor dynamic changes in subnuclear positioning as well as interactions with functional nuclear structures during cell cycle progression and cellular differentiation.

  14. Laser-induced radiation microbeam technology and simultaneous real-time fluorescence imaging in live cells.

    Science.gov (United States)

    Botchway, Stanley W; Reynolds, Pamela; Parker, Anthony W; O'Neill, Peter

    2012-01-01

    The use of nano- and microbeam techniques to induce and identify subcellular localized energy deposition within a region of a living cell provides a means to investigate the effects of low radiation doses. Particularly within the nucleus where the propagation and processing of deoxyribonucleic acid (DNA) damage (and repair) in both targeted and nontargeted cells, the latter being able to study cell-cell (bystander) effects. We have pioneered a near infrared (NIR) femtosecond laser microbeam to mimic ionizing radiation through multiphoton absorption within a 3D femtoliter volume of a highly focused Gaussian laser beam. The novel optical microbeam mimics both complex ionizing and UV-radiation-type cell damage including double strand breaks (DSBs). Using the microbeam technology, we have been able to investigate the formation of DNA DSB and subsequent recruitment of repair proteins to the submicrometer size site of damage introduced in viable cells. The use of a phosphorylated H2AX (γ-H2AX a marker for DSBs, visualized by immunofluorescent staining) and real-time imaging of fluorescently labeling proteins, the dynamics of recruitment of repair proteins in viable mammalian cells can be observed. Here we show the recruitment of ATM, p53 binding protein 1 (53BP1), and RAD51, an integral protein of the homologous recombination process in the DNA repair pathway and Ku-80-GFP involved in the nonhomologous end joining (NHEJ) pathway as exemplar repair process to show differences in the repair kinetics of DNA DSBs. The laser NIR multiphoton microbeam technology shows persistent DSBs at later times post laser irradiation which are indicative of DSBs arising at replication presumably from UV photoproducts or clustered damage containing single strand breaks (SSBs) that are also observed. Effects of the cell cycle may also be investigated in real time. Postirradiation and fixed cells studies show that in G1 cells a fraction of multiphoton laser-induced DSBs is persistent for >6h

  15. Solar cells. Proposal for a national strategy for research, development and demonstration; Solceller. Oplaeg til en national strategi for forskning, udvikling og demonstration

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    2004-07-01

    The Danish Energy Authority, Elkraft System and Eltra have initiated collaboration on the development of national R and D strategies for a number of energy technologies including solar cells. The aim is to ensure a coordinated national effort as regards research, development and demonstration within societal and energy political frames, and, furthermore, to ensure coordination with similar international initiatives, especially within the European Union. The overall aim is for the Danish solar cell strategy to contribute to support Danish national energy policy and to ensure and improve Danish competence, which can manifest itself internationally. The efforts within solar cell technology must aim at increasing solar cell systems' efficiency and service life, and furthermore, aim at reducing production costs. Hereby the efforts can contribute to an improvement of solar cell systems' competitive power in relation to other power production technologies with a view to make installation of solar cell systems attractive, both in Denmark and internationally. (BA)

  16. Site-Specific Bioorthogonal Labeling for Fluorescence Imaging of Intracellular Proteins in Living Cells.

    Science.gov (United States)

    Peng, Tao; Hang, Howard C

    2016-11-02

    Over the past years, fluorescent proteins (e.g., green fluorescent proteins) have been widely utilized to visualize recombinant protein expression and localization in live cells. Although powerful, fluorescent protein tags are limited by their relatively large sizes and potential perturbation to protein function. Alternatively, site-specific labeling of proteins with small-molecule organic fluorophores using bioorthogonal chemistry may provide a more precise and less perturbing method. This approach involves site-specific incorporation of unnatural amino acids (UAAs) into proteins via genetic code expansion, followed by bioorthogonal chemical labeling with small organic fluorophores in living cells. While this approach has been used to label extracellular proteins for live cell imaging studies, site-specific bioorthogonal labeling and fluorescence imaging of intracellular proteins in live cells is still challenging. Herein, we systematically evaluate site-specific incorporation of diastereomerically pure bioorthogonal UAAs bearing stained alkynes or alkenes into intracellular proteins for inverse-electron-demand Diels-Alder cycloaddition reactions with tetrazine-functionalized fluorophores for live cell labeling and imaging in mammalian cells. Our studies show that site-specific incorporation of axial diastereomer of trans-cyclooct-2-ene-lysine robustly affords highly efficient and specific bioorthogonal labeling with monosubstituted tetrazine fluorophores in live mammalian cells, which enabled us to image the intracellular localization and real-time dynamic trafficking of IFITM3, a small membrane-associated protein with only 137 amino acids, for the first time. Our optimized UAA incorporation and bioorthogonal labeling conditions also enabled efficient site-specific fluorescence labeling of other intracellular proteins for live cell imaging studies in mammalian cells.

  17. Zero Emission Bay Area (ZEBA) Fuel Cell Bus Demonstration: First Results Report

    Energy Technology Data Exchange (ETDEWEB)

    Chandler, K.; Eudy, L.

    2011-08-01

    This report documents the early implementation experience for the Zero Emission Bay Area (ZEBA) Demonstration, the largest fleet of fuel cell buses in the United States. The ZEBA Demonstration group includes five participating transit agencies: AC Transit (lead transit agency), Santa Clara Valley Transportation Authority (VTA), Golden Gate Transit (GGT), San Mateo County Transit District (SamTrans), and San Francisco Municipal Railway (Muni). The ZEBA partners are collaborating with the U.S. Department of Energy (DOE) and DOE's National Renewable Energy Laboratory (NREL) to evaluate the buses in revenue service.

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

    Science.gov (United States)

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

    2015-01-15

    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.

  19. Live microbial cells adsorb Mg2+ more effectively than lifeless organic matter

    Science.gov (United States)

    Qiu, Xuan; Yao, Yanchen; Wang, Hongmei; Duan, Yong

    2018-03-01

    The Mg2+ content is essential in determining different Mg-CaCO3 minerals. It has been demonstrated that both microbes and the organic matter secreted by microbes are capable of allocating Mg2+ and Ca2+ during the formation of Mg-CaCO3, yet detailed scenarios remain unclear. To investigate the mechanism that microbes and microbial organic matter potentially use to mediate the allocation of Mg2+ and Ca2+ in inoculating systems, microbial mats and four marine bacterial strains ( Synechococcus elongatus, Staphylococcus sp., Bacillus sp., and Desulfovibrio vulgaris) were incubated in artificial seawater media with Mg/Ca ratios ranging from 0.5 to 10.0. At the end of the incubation, the morphology of the microbial mats and the elements adsorbed on them were analyzed using scanning electronic microscopy (SEM) and energy diffraction spectra (EDS), respectively. The content of Mg2+ and Ca2+ adsorbed by the extracellular polysaccharide substances (EPS) and cells of the bacterial strains were analyzed with atomic adsorption spectroscopy (AAS). The functional groups on the surface of the cells and EPS of S. elongatus were estimated using automatic potentiometric titration combined with a chemical equilibrium model. The results show that live microbial mats generally adsorb larger amounts of Mg2+ than Ca2+, while this rarely is the case for autoclaved microbial mats. A similar phenomenon was also observed for the bacterial strains. The living cells adsorb more Mg2+ than Ca2+, yet a reversed trend was observed for EPS. The functional group analysis indicates that the cell surface of S. elongatus contains more basic functional groups (87.24%), while the EPS has more acidic and neutral functional groups (83.08%). These features may be responsible for the different adsorption behavior of Mg2+ and Ca2+ by microbial cells and EPS. Our work confirms the differential Mg2+ and Ca2+ mediation by microbial cells and EPS, which may provide insight into the processes that microbes use to

  20. Exploring hope and healing in patients living with advanced non-small cell lung cancer.

    Science.gov (United States)

    Eustache, Chloe; Jibb, Emily; Grossman, Mary

    2014-09-01

    To explore the experience and meaning of hope in relation to the healing process of patients living with stage IIIb or IV non-small cell lung cancer. Interpretative qualitative study design. Peter Brojde Lung Cancer Centre in the Jewish General Hospital in Montreal, Quebec, Canada. 12 English- and French-speaking patients, aged 36-78 years. One 60-90-minute semistructured interview per participant was conducted. An inductive approach to data analysis was taken, involving immersion in the data, coding, classifying, and creating linkages. Four main themes emerged: (a) the morass of shattered hope, (b) tentative steps toward a new hope paradigm, (c) reframing hope within the context of a life-threatening illness, and (d) strengthening the link between hope and wellness. Patients described a process where hope was diminished or lost entirely, regained, and reshaped as they learned to live and grow following their diagnosis. This study adds to the literature by describing the dynamic nature of hope as well as factors facilitating or hindering the hope process. It demonstrates how finding meaning, a structural component of healing, can be used to envision a new hopeful future. This study suggests hope and healing cannot exist in isolation, and highlights the importance of understanding the fluctuating nature of hope in patients with advanced lung cancer to foster it, therefore promoting healing.

  1. Electroporation and microinjection successfully deliver single-stranded and duplex DNA into live cells as detected by FRET measurements.

    Directory of Open Access Journals (Sweden)

    Rosemary A Bamford

    Full Text Available Förster resonance energy transfer (FRET technology relies on the close proximity of two compatible fluorophores for energy transfer. Tagged (Cy3 and Cy5 complementary DNA strands forming a stable duplex and a doubly-tagged single strand were shown to demonstrate FRET outside of a cellular environment. FRET was also observed after transfecting these DNA strands into fixed and live cells using methods such as microinjection and electroporation, but not when using lipid based transfection reagents, unless in the presence of the endosomal acidification inhibitor bafilomycin. Avoiding the endocytosis pathway is essential for efficient delivery of intact DNA probes into cells.

  2. Characterization of nanostructures in the live cell plasma membrane utilizing advanced single molecule fluorescence techniques

    International Nuclear Information System (INIS)

    Brameshuber, M.

    2009-01-01

    Unrevealing the detailed structure of the cellular plasma membrane at a nanoscopic length scale is the key for understanding the regulation of various signaling pathways or interaction mechanism. Hypotheses postulate the existence of nanoscopic lipid platforms in the cell membrane which are termed lipid- or membrane rafts. Based on biochemical studies, rafts are believed to play a crucial role in many signaling processes. However, there is currently not much information on their size, shape, stability, surface density, composition and heterogeneity. In this thesis I present an ultra-sensitive fluorescence based method which allows for the first time the direct imaging of single mobile rafts in the live cell plasma membrane. The method senses rafts by their property to assemble a characteristic set of fluorescent marker-proteins or lipids on a time-scale of seconds. A special photobleaching protocol was developed and used to reduce the surface density of labeled mobile rafts down to the level of well-isolated diffraction-limited spots, without altering the single spot brightness. The statistical distribution of probe molecules per raft was determined by single molecule brightness analysis. For demonstration, I used the consensus markers Bodipy-GM1, a fluorescent lipid analogue, and glycosylphosphatidyl-inositol-anchored monomeric GFP. For both markers I found cholesterol-dependent association in the plasma membrane of living CHO and Jurkat T cells in the resting state, indicating the presence of mobile, stable rafts hosting these probes. I further characterized these structures by taking cell-to-cell variations under consideration. By comparing Bodipy-GM1 with mGFP-GPI homo-association upon temperature variation, two different states - a non-equilibrated and an equilibrated state - could be identified. I conclude that rafts are loaded non-randomly; the characteristic load is maintained during its lifetime in the plasma membrane of a non-activated cell. Beside these

  3. Tracking neuronal marker expression inside living differentiating cells using molecular beacons

    DEFF Research Database (Denmark)

    Ilieva, Mirolyuba; Della Vedova, Paolo; Hansen, Ole

    2013-01-01

    and tyrosine hydroxylase mRNAs were expressed 2 and 3 days post induction of differentiation, respectively. Oct 4 was not detected with MB in these cells and signal was not increased over time suggesting that MB are generally stable inside the cells. The gene expression changes measured using MBs were...... confirmed using qRT-PCR. These results suggest that MBs are simple to use sensors inside living cell, and particularly useful for studying dynamic gene expression in heterogeneous cell populations....

  4. Safe sorting of GFP-transduced live cells for subsequent culture using a modified FACS vantage

    DEFF Research Database (Denmark)

    Sørensen, T U; Gram, G J; Nielsen, S D

    1999-01-01

    BACKGROUND: A stream-in-air cell sorter enables rapid sorting to a high purity, but it is not well suited for sorting of infectious material due to the risk of airborne spread to the surroundings. METHODS: A FACS Vantage cell sorter was modified for safe use with potentially HIV infected cells...... culture. CONCLUSIONS: Sorting of live infected cells can be performed safely and with no deleterious effects on vector expression using the modified FACS Vantage instrument....

  5. Proteorhodopsin in living color: diversity of spectral properties within living bacterial cells.

    Science.gov (United States)

    Kelemen, Bradley R; Du, Mai; Jensen, Rasmus B

    2003-12-03

    Proteorhodopsin is a family of over 50 proteins that provide phototrophic capability to marine bacteria by acting as light-powered proton pumps. The potential importance of proteorhodopsin to global ocean ecosystems and the possible applications of proteorhodopsin in optical data storage and optical signal processing have spurred diverse research in this new family of proteins. We show that proteorhodopsin expressed in Escherichia coli is functional and properly inserted in the membrane. At high expression levels, it appears to self-associate. We present a method for determining spectral properties of proteorhodopsin in intact E. coli cells that matches results obtained with detergent-solubilized, purified proteins. Using this method, we observe distinctly different spectra for protonated and deprotonated forms of 21 natural proteorhodopsin proteins in intact E. coli cells. Upon protonation, the wavelength maxima red shifts between 13 and 53 nm. We find that pKa values between 7.1 and 8.5 describe the pH-dependent spectral shift for all of the 21 natural variants of proteorhodopsin. The wavelength maxima of the deprotonated forms of the 21 natural proteorhodopsins cluster in two sequence-related groups: blue proteorhodopsins (B-PR) and green proteorhodopsins (G-PR). The site-directed substitution Leu105Gln in Bac31A8 proteorhodopsin shifts this G-PR's wavelength maximum to a wavelength maximum the same as that of the B-PR Hot75m1 proteorhodopsin. The site-directed substitution Gln107Leu in Hot75m1 proteorhodopsin shifts this B-PR's wavelength maximum to a wavelength maximum as that of Bac31A8 proteorhodopsin.

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

    Directory of Open Access Journals (Sweden)

    Feng Gao

    2016-08-01

    Full Text Available 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.

  7. Integrated dual-tomography for refractive index analysis of free-floating single living cell with isotropic superresolution.

    Science.gov (United States)

    B, Vinoth; Lai, Xin-Ji; Lin, Yu-Chih; Tu, Han-Yen; Cheng, Chau-Jern

    2018-04-13

    Digital holographic microtomography is a promising technique for three-dimensional (3D) measurement of the refractive index (RI) profiles of biological specimens. Measurement of the RI distribution of a free-floating single living cell with an isotropic superresolution had not previously been accomplished. To the best of our knowledge, this is the first study focusing on the development of an integrated dual-tomographic (IDT) imaging system for RI measurement of an unlabelled free-floating single living cell with an isotropic superresolution by combining the spatial frequencies of full-angle specimen rotation with those of beam rotation. A novel 'UFO' (unidentified flying object) like shaped coherent transfer function is obtained. The IDT imaging system does not require any complex image-processing algorithm for 3D reconstruction. The working principle was successfully demonstrated and a 3D RI profile of a single living cell, Candida rugosa, was obtained with an isotropic superresolution. This technology is expected to set a benchmark for free-floating single live sample measurements without labeling or any special sample preparations for the experiments.

  8. Startup, testing, and operation of the Santa Clara 2MW direct carbonate fuel cell demonstration plant

    Energy Technology Data Exchange (ETDEWEB)

    Skok, A.J.; Leo, A.J. [Fuel Cell Engineering Corp., Danbury, CT (United States); O`Shea, T.P. [Santa Clara Demonstration Project, CA (United States)

    1996-12-31

    The Santa Clara Demonstration Project (SCDP) is a collaboration between several utility organizations, Fuel Cell Engineering Corporation (FCE), and the U.S. Dept. Of Energy aimed at the demonstration of Energy Research Corporation`s (ERC) direct carbonate fuel cell (DFC) technology. ERC has been pursuing the development of the DFC for commercialization near the end of this decade, and this project is an integral part of the ERC commercialization effort. The objective of the Santa Clara Demonstration Project is to provide the first full, commercial scale demonstration of this technology. The approach ERC has taken in the commercialization of the DFC is described in detail elsewhere. An aggressive core technology development program is in place which is focused by ongoing interaction with customers and vendors to optimize the design of the commercial power plant. ERC has selected a 2.85 MW power plant unit for initial market entry. Two ERC subsidiaries are supporting the commercialization effort: the Fuel Cell Manufacturing Corporation (FCMC) and the Fuel Cell Engineering Corporation (FCE). FCMC manufactures carbonate stacks and multi-stack modules, currently from its production facility in Torrington, CT. FCE is responsible for power plant design, integration of all subsystems, sales/marketing, and client services. FCE is serving as the prime contractor for the design, construction, and testing of the SCDP Plant. FCMC has manufactured the multi-stack submodules used in the DC power section of the plant. Fluor Daniel Inc. (FDI) served as the architect-engineer subcontractor for the design and construction of the plant and provided support to the design of the multi-stack submodules. FDI is also assisting the ERC companies in commercial power plant design.

  9. Hollow fiber: a biophotonic implant for live cells

    Science.gov (United States)

    Silvestre, Oscar F.; Holton, Mark D.; Summers, Huw D.; Smith, Paul J.; Errington, Rachel J.

    2009-02-01

    The technical objective of this study has been to design, build and validate biocompatible hollow fiber implants based on fluorescence with integrated biophotonics components to enable in fiber kinetic cell based assays. A human osteosarcoma in vitro cell model fiber system has been established with validation studies to determine in fiber cell growth, cell cycle analysis and organization in normal and drug treated conditions. The rationale for implant development have focused on developing benchmark concepts in standard monolayer tissue culture followed by the development of in vitro hollow fiber designs; encompassing imaging with and without integrated biophotonics. Furthermore the effect of introducing targetable biosensors into the encapsulated tumor implant such as quantum dots for informing new detection readouts and possible implant designs have been evaluated. A preliminary micro/macro imaging approach has been undertaken, that could provide a mean to track distinct morphological changes in cells growing in a 3D matrix within the fiber which affect the light scattering properties of the implant. Parallel engineering studies have showed the influence of the optical properties of the fiber polymer wall in all imaging modes. Taken all together, we show the basic foundation and the opportunities for multi-modal imaging within an in vitro implant format.

  10. Infrared microspectroscopy of live cells in microfluidic devices (MD-IRMS): toward a powerful label-free cell-based assay.

    Science.gov (United States)

    Vaccari, L; Birarda, G; Businaro, L; Pacor, S; Grenci, G

    2012-06-05

    Until nowadays most infrared microspectroscopy (IRMS) experiments on biological specimens (i.e., tissues or cells) have been routinely carried out on fixed or dried samples in order to circumvent water absorption problems. In this paper, we demonstrate the possibility to widen the range of in-vitro IRMS experiments to vibrational analysis of live cellular samples, thanks to the development of novel biocompatible IR-visible transparent microfluidic devices (MD). In order to highlight the biological relevance of IRMS in MD (MD-IRMS), we performed a systematic exploration of the biochemical alterations induced by different fixation protocols, ethanol 70% and formaldehyde solution 4%, as well as air-drying on U937 leukemic monocytes by comparing their IR vibrational features with the live U937 counterpart. Both fixation and air-drying procedures affected lipid composition and order as well as protein structure at a different extent while they both induced structural alterations in nucleic acids. Therefore, only IRMS of live cells can provide reliable information on both DNA and RNA structure and on their cellular dynamic. In summary, we show that MD-IRMS of live cells is feasible, reliable, and biologically relevant to be recognized as a label-free cell-based assay.

  11. Measuring the acoustophoretic contrast factor of living cells in microchannels

    DEFF Research Database (Denmark)

    Augustsson, P.; Barnkob, Rune; Grenvall, C.

    2010-01-01

    We report a new method, which allows for accurate measurement of the acostophoretic contrast factor Φ of different cell types, an acousto-physical parameter of fundamental importance in microchip acoustophoresis. As a test case the Φ factor is measured for undifferentiated and four-days different......We report a new method, which allows for accurate measurement of the acostophoretic contrast factor Φ of different cell types, an acousto-physical parameter of fundamental importance in microchip acoustophoresis. As a test case the Φ factor is measured for undifferentiated and four...

  12. Detection of constitutive heterodimerization of the integrin Mac-1 subunits by fluorescence resonance energy transfer in living cells

    International Nuclear Information System (INIS)

    Fu Guo; Yang Huayan; Wang Chen; Zhang Feng; You Zhendong; Wang Guiying; He Cheng; Chen Yizhang; Xu Zhihan

    2006-01-01

    Macrophage differentiation antigen associated with complement three receptor function (Mac-1) belongs to β 2 subfamily of integrins that mediate important cell-cell and cell-extracellular matrix interactions. Biochemical studies have indicated that Mac-1 is a constitutive heterodimer in vitro. Here, we detected the heterodimerization of Mac-1 subunits in living cells by means of two fluorescence resonance energy transfer (FRET) techniques (fluorescence microscopy and fluorescence spectroscopy) and our results demonstrated that there is constitutive heterodimerization of the Mac-1 subunits and this constitutive heterodimerization of the Mac-1 subunits is cell-type independent. Through FRET imaging, we found that heterodimers of Mac-1 mainly localized in plasma membrane, perinuclear, and Golgi area in living cells. Furthermore, through analysis of the estimated physical distances between cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) fused to Mac-1 subunits, we suggested that the conformation of Mac-1 subunits is not affected by the fusion of CFP or YFP and inferred that Mac-1 subunits take different conformation when expressed in Chinese hamster ovary (CHO) and human embryonic kidney (HEK) 293T cells, respectively

  13. Zero Emission Bay Area (ZEBA) Fuel Cell Bus Demonstration Results: Third Report

    Energy Technology Data Exchange (ETDEWEB)

    Eudy, L.; Post, M.

    2014-05-01

    This report presents results of a demonstration of 12 fuel cell electric buses (FCEB) operating in Oakland, California. The 12 FCEBs operate as a part of the Zero Emission Bay Area (ZEBA) Demonstration, which also includes two new hydrogen fueling stations. This effort is the largest FCEB demonstration in the United States and involves five participating transit agencies. The ZEBA partners are collaborating with the U.S. Department of Energy (DOE) and DOE's National Renewable Energy Laboratory (NREL) to evaluate the buses in revenue service. NREL has published two previous reports, in August 2011 and July 2012, describing operation of these buses. New results in this report provide an update covering eight months through October 2013.

  14. Zero Emission Bay Area (ZEBA) Fuel Cell Bus Demonstration: Second Results Report

    Energy Technology Data Exchange (ETDEWEB)

    Eudy, L.; Chandler, K.

    2012-07-01

    This report presents results of a demonstration of 12 new fuel cell electric buses (FCEB) operating in Oakland, California. The 12 FCEBs operate as a part of the Zero Emission Bay Area (ZEBA) Demonstration, which also includes two new hydrogen fueling stations. This effort is the largest FCEB demonstration in the United States and involves five participating transit agencies. The ZEBA partners are collaborating with the U.S. Department of Energy (DOE) and DOE's National Renewable Energy Laboratory (NREL) to evaluate the buses in revenue service. The first results report was published in August 2011, describing operation of these new FCEBs from September 2010 through May 2011. New results in this report provide an update through April 2012.

  15. Progenitor cells in auricular cartilage demonstrate cartilage-forming capacity in 3D hydrogel culture

    Directory of Open Access Journals (Sweden)

    IA Otto

    2018-02-01

    Full Text Available Paramount for the generation of auricular structures of clinically-relevant size is the acquisition of a large number of cells maintaining an elastic cartilage phenotype, which is the key in producing a tissue capable of withstanding forces subjected to the auricle. Current regenerative medicine strategies utilize chondrocytes from various locations or mesenchymal stromal cells (MSCs. However, the quality of neo-tissues resulting from these cell types is inadequate due to inefficient chondrogenic differentiation and endochondral ossification, respectively. Recently, a subpopulation of stem/progenitor cells has been identified within the auricular cartilage tissue, with similarities to MSCs in terms of proliferative capacity and cell surface biomarkers, but their potential for tissue engineering has not yet been explored. This study compared the in vitro cartilage-forming ability of equine auricular cartilage progenitor cells (AuCPCs, bone marrow-derived MSCs and auricular chondrocytes in gelatin methacryloyl (gelMA-based hydrogels over a period of 56 d, by assessing their ability to undergo chondrogenic differentiation. Neocartilage formation was assessed through gene expression profiling, compression testing, biochemical composition and histology. Similar to MSCs and chondrocytes, AuCPCs displayed a marked ability to generate cartilaginous matrix, although, under the applied culture conditions, MSCs outperformed both cartilage-derived cell types in terms of matrix production and mechanical properties. AuCPCs demonstrated upregulated mRNA expression of elastin, low expression of collagen type X and similar levels of proteoglycan production and mechanical properties as compared to chondrocytes. These results underscored the AuCPCs’ tissue-specific differentiation potential, making them an interesting cell source for the next generation of elastic cartilage tissue-engineered constructs.

  16. Examining live cell cultures during apoptosis by digital holographic phase imaging and Raman spectroscopy

    Science.gov (United States)

    Khmaladze, Alexander

    2017-11-01

    Cellular apoptosis is a unique, organized series of events, leading to programmed cell death. In this work, we present a combined digital holography/Raman spectroscopy technique to study live cell cultures during apoptosis. Digital holographic microscopy measurements of live cell cultures yield information about cell shape and volume, changes to which are indicative of alterations in cell cycle and initiation of cell death mechanisms. Raman spectroscopic measurements provide complementary information about cells, such as protein, lipid and nucleic acid content, and the spectral signatures associated with structural changes in molecules. Our work indicates that the chemical changes in proteins, which were detected by Raman measurements, preceded morphological changes, which were seen with digital holographic microscopy.

  17. Semi-automated quantification of living cells with internalized nanostructures

    KAUST Repository

    Margineanu, Michael B.; Julfakyan, Khachatur; Sommer, Christoph; Perez, Jose E.; Contreras, Maria F.; Khashab, Niveen M.; Kosel, Jü rgen; Ravasi, Timothy

    2016-01-01

    novel method for the quantification of cells that internalize a specific type of nanostructures. This approach is suitable for high-throughput and real-time data analysis and has the potential to be used to study the interaction of different types

  18. Enhanced 3D fluorescence live cell imaging on nanoplasmonic substrate

    International Nuclear Information System (INIS)

    Gartia, Manas Ranjan; Hsiao, Austin; Logan Liu, G; Sivaguru, Mayandi; Chen Yi

    2011-01-01

    We have created a randomly distributed nanocone substrate on silicon coated with silver for surface-plasmon-enhanced fluorescence detection and 3D cell imaging. Optical characterization of the nanocone substrate showed it can support several plasmonic modes (in the 300-800 nm wavelength range) that can be coupled to a fluorophore on the surface of the substrate, which gives rise to the enhanced fluorescence. Spectral analysis suggests that a nanocone substrate can create more excitons and shorter lifetime in the model fluorophore Rhodamine 6G (R6G) due to plasmon resonance energy transfer from the nanocone substrate to the nearby fluorophore. We observed three-dimensional fluorescence enhancement on our substrate shown from the confocal fluorescence imaging of chinese hamster ovary (CHO) cells grown on the substrate. The fluorescence intensity from the fluorophores bound on the cell membrane was amplified more than 100-fold as compared to that on a glass substrate. We believe that strong scattering within the nanostructured area coupled with random scattering inside the cell resulted in the observed three-dimensional enhancement in fluorescence with higher photostability on the substrate surface.

  19. Self-assembled fluorescent organic nanoparticles for live cell imaging

    NARCIS (Netherlands)

    Fischer, I.; Petkau, K.; Dorland, Y.L.; Schenning, A.P.H.J.; Brunsveld, L.

    2013-01-01

    Fluorescent, cell-permeable, organic nanoparticles based on self-assembled p-conjugated oligomers with high absorption cross-sections and high quantum yields have been developed. The nanoparticles are generated with a tuneable density of amino groups for charge-mediated cellular uptake by a

  20. Green light for quantitative live-cell imaging in plants

    NARCIS (Netherlands)

    Grossmann, Guido; Krebs, Melanie; Maizel, Alexis; Stahl, Yvonne; Vermeer, Joop E.M.; Ott, Thomas

    2018-01-01

    Plants exhibit an intriguing morphological and physiological plasticity that enables them to thrive in a wide range of environments. To understand the cell biological basis of this unparalleled competence, a number ofmethodologies have been adapted or developed over the last decades that allow

  1. Understanding of Protein Synthesis in a Living Cell

    Science.gov (United States)

    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…

  2. Enhanced 3D fluorescence live cell imaging on nanoplasmonic substrate

    Energy Technology Data Exchange (ETDEWEB)

    Gartia, Manas Ranjan [Department of Nuclear, Plasma and Radiological Engineering, University of Illinois, Urbana, IL 61801 (United States); Hsiao, Austin; Logan Liu, G [Department of Bioengineering, University of Illinois, Urbana, IL 61801 (United States); Sivaguru, Mayandi [Institute for Genomic Biology, University of Illinois, Urbana, IL 61801 (United States); Chen Yi, E-mail: loganliu@illinois.edu [Department of Electrical and Computer Engineering, University of Illinois, Urbana, IL 61801 (United States)

    2011-09-07

    We have created a randomly distributed nanocone substrate on silicon coated with silver for surface-plasmon-enhanced fluorescence detection and 3D cell imaging. Optical characterization of the nanocone substrate showed it can support several plasmonic modes (in the 300-800 nm wavelength range) that can be coupled to a fluorophore on the surface of the substrate, which gives rise to the enhanced fluorescence. Spectral analysis suggests that a nanocone substrate can create more excitons and shorter lifetime in the model fluorophore Rhodamine 6G (R6G) due to plasmon resonance energy transfer from the nanocone substrate to the nearby fluorophore. We observed three-dimensional fluorescence enhancement on our substrate shown from the confocal fluorescence imaging of chinese hamster ovary (CHO) cells grown on the substrate. The fluorescence intensity from the fluorophores bound on the cell membrane was amplified more than 100-fold as compared to that on a glass substrate. We believe that strong scattering within the nanostructured area coupled with random scattering inside the cell resulted in the observed three-dimensional enhancement in fluorescence with higher photostability on the substrate surface.

  3. Visualizing how cancer chromosome abnormalities form in living cells

    Science.gov (United States)

    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

  4. Antibodies against alpha-synuclein reduce oligomerization in living cells.

    Directory of Open Access Journals (Sweden)

    Thomas Näsström

    Full Text Available Recent research implicates soluble aggregated forms of α-synuclein as neurotoxic species with a central role in the pathogenesis of Parkinson's disease and related disorders. The pathway by which α-synuclein aggregates is believed to follow a step-wise pattern, in which dimers and smaller oligomers are initially formed. Here, we used H4 neuroglioma cells expressing α-synuclein fused to hemi:GFP constructs to study the effects of α-synuclein monoclonal antibodies on the early stages of aggregation, as quantified by Bimolecular Fluorescence Complementation assay. Widefield and confocal microscopy revealed that cells treated for 48 h with monoclonal antibodies internalized antibodies to various degrees. C-terminal and oligomer-selective α-synuclein antibodies reduced the extent of α-synuclein dimerization/oligomerization, as indicated by decreased GFP fluorescence signal. Furthermore, ELISA measurements on lysates and conditioned media from antibody treated cells displayed lower α-synuclein levels compared to untreated cells, suggesting increased protein turnover. Taken together, our results propose that extracellular administration of monoclonal antibodies can modify or inhibit early steps in the aggregation process of α-synuclein, thus providing further support for passive immunization against diseases with α-synuclein pathology.

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

    Directory of Open Access Journals (Sweden)

    Neil O Carragher

    2011-04-01

    Full Text Available 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.

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

    Science.gov (United States)

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

    2009-06-01

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

  7. Calibration and quantification of fast intracellular motion (FIM) in living cells using correlation analysis

    Czech Academy of Sciences Publication Activity Database

    Veselý, Pavel; Mikš, A.; Novák, J.; Boyde, A.

    2003-01-01

    Roč. 25, - (2003), s. 230-239 ISSN 0161-0457 R&D Projects: GA ČR GA304/99/0368 Institutional research plan: CEZ:AV0Z5052915 Keywords : fast intracellular motion * living cell ů video rate confocal laser scanning microscopy Subject RIV: EA - Cell Biology Impact factor: 0.733, year: 2003

  8. Optical imaging of non-fluorescent nanodiamonds in live cells using transient absorption microscopy.

    Science.gov (United States)

    Chen, Tao; Lu, Feng; Streets, Aaron M; Fei, Peng; Quan, Junmin; Huang, Yanyi

    2013-06-07

    We directly observe non-fluorescent nanodiamonds in living cells using transient absorption microscopy. This label-free technology provides a novel modality to study the dynamic behavior of nanodiamonds inside the cells with intrinsic three-dimensional imaging capability. We apply this method to capture the cellular uptake of nanodiamonds under various conditions, confirming the endocytosis mechanism.

  9. Overview about the fuel cell bus demonstration programs CUTE, ECTOS and STEP

    International Nuclear Information System (INIS)

    Faltenbacher, M.; Fischer, M.; Eyerer, P.; Binder, M.; Schuckert, M.

    2004-01-01

    'Full text:' The paper will give an overview about the CUTE, ECTOS and STEP projects. The aim of the projects is to develop and demonstrate a emission-free and low-noise transport system, including the accompanying energy infrastructure, which has great potential for reducing the global greenhouse effect according to the Kyoto protocol, improving the quality of the atmosphere and life in densely populated areas and conserving fossil resources. For this purpose the application of the innovative hydrogen-based fuel cell technology is established by using fuel cell powered buses in an urban environment together with novel hydrogen production and support systems as part of a European Union wide demonstration scheme. The project demonstrates also to European Society the availability of the FC technology as a safe and reliable transportation technology. The major objectives are as follows: Demonstration of more than 20 fuel cell powered regular service buses over a period of two years in several European inner city areas to illustrate the different operating conditions to be found in Europe; Design, construction and operation of the necessary infrastructure for hydrogen production, including the required refuelling stations; Collection of findings concerning the construction and operating behaviour of hydrogen production for mobile use, and exchange of experiences including bus operation under differing conditions among the numerous participating companies; and, the research work of IKP and PE comprises the ecological analysis of the entire life cycle and comparison with conventional alternatives (diesel driven buses, CNG-buses). It also includes the economical analysis of the hydrogen infrastructure. First experiences from CUTE and ECTOS were presented. (author)

  10. Biophysical Techniques for Detection of cAMP and cGMP in Living Cells

    Directory of Open Access Journals (Sweden)

    Viacheslav O. Nikolaev

    2013-04-01

    Full Text Available 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.

  11. An intramolecular charge transfer process based fluorescent probe for monitoring subtle pH fluctuation in living cells.

    Science.gov (United States)

    Sun, Mingtai; Du, Libo; Yu, Huan; Zhang, Kui; Liu, Yang; Wang, Suhua

    2017-01-01

    It is crucial to monitor intracellular pH values and their fluctuation since the organelles of cells have different pH distribution. Herein we construct a new small molecule fluorescent probe HBT-O for monitoring the subtle pH values within the scope of neutral to acid in living cells. The probe exhibited good water solubility, a marked turquoise to olivine emission color change in response to pH, and tremendous fluorescence hypochromatic shift of ∼50nm (1718cm -1 ) as well as the increased fluorescence intensity when the pH value changed from neutral to acid. Thus, the probe HBT-O can distinguish the subtle changes in the range of normal pH values from neutral to acid with significant fluorescence changes. These properties can be attributed to the intramolecular charge transfer (ICT) process of the probe upon protonation in buffer solutions at varied pH values. Moreover, the probe was reversible and nearly non-toxic for living cells. Then the probe was successfully used to detect pH fluctuation in living cells by exhibiting different fluorescence colors and intensity. These findings demonstrate that the probe will find useful applications in biology and biomedical research. Copyright © 2016 Elsevier B.V. All rights reserved.

  12. Controllable synthesis of green and blue fluorescent carbon nanodots for pH and Cu(2+) sensing in living cells.

    Science.gov (United States)

    Shi, Lihong; Li, Yanyan; Li, Xiaofeng; Zhao, Bo; Wen, Xiangping; Zhang, Guomei; Dong, Chuan; Shuang, Shaomin

    2016-03-15

    We report a controllable strategy for fabrication of green and blue fluorescent carbon nanodots (CDs), and demonstrate their applications for pH and Cu(2+) sensing in living cells. Green and blue fluorescent CDs have been synthesized by hydrothermal method and pyrolysis of leeks, respectively, providing an easy way for the production of CDs without the request of tedious synthetic methodology or the use of toxic/expensive solvents and starting materials. Green fluorescent CDs (G-CDs) exhibit high tolerance to pH values and external cations. Blue fluorescent CDs (B-CDs) can be applied to pH and Cu(2+) sensing. The linear range of Cu(2+) detection is 0.01-10.00 μM and the detection limit is 0.05 μM. For pH detection, there is a good linearity in the pH range of 3.5-10.0. The linear and rapid response of B-CDs to Cu(2+) and pH is valuable for Cu(2+) and pH sensing in living cells. Confocal fluorescent imaging of human cervical carcinoma cells indicates that B-CDs could visualize Cu(2+) and pH fluctuations in living cells with negligible autofluorescence. Copyright © 2015 Elsevier B.V. All rights reserved.

  13. Dynamic nano-imaging of label-free living cells using electron beam excitation-assisted optical microscope

    Science.gov (United States)

    Fukuta, Masahiro; Kanamori, Satoshi; Furukawa, Taichi; Nawa, Yasunori; Inami, Wataru; Lin, Sheng; Kawata, Yoshimasa; Terakawa, Susumu

    2015-01-01

    Optical microscopes are effective tools for cellular function analysis because biological cells can be observed non-destructively and non-invasively in the living state in either water or atmosphere condition. Label-free optical imaging technique such as phase-contrast microscopy has been analysed many cellular functions, and it is essential technology for bioscience field. However, the diffraction limit of light makes it is difficult to image nano-structures in a label-free living cell, for example the endoplasmic reticulum, the Golgi body and the localization of proteins. Here we demonstrate the dynamic imaging of a label-free cell with high spatial resolution by using an electron beam excitation-assisted optical (EXA) microscope. We observed the dynamic movement of the nucleus and nano-scale granules in living cells with better than 100 nm spatial resolution and a signal-to-noise ratio (SNR) around 10. Our results contribute to the development of cellular function analysis and open up new bioscience applications. PMID:26525841

  14. Dynamic nano-imaging of label-free living cells using electron beam excitation-assisted optical microscope.

    Science.gov (United States)

    Fukuta, Masahiro; Kanamori, Satoshi; Furukawa, Taichi; Nawa, Yasunori; Inami, Wataru; Lin, Sheng; Kawata, Yoshimasa; Terakawa, Susumu

    2015-11-03

    Optical microscopes are effective tools for cellular function analysis because biological cells can be observed non-destructively and non-invasively in the living state in either water or atmosphere condition. Label-free optical imaging technique such as phase-contrast microscopy has been analysed many cellular functions, and it is essential technology for bioscience field. However, the diffraction limit of light makes it is difficult to image nano-structures in a label-free living cell, for example the endoplasmic reticulum, the Golgi body and the localization of proteins. Here we demonstrate the dynamic imaging of a label-free cell with high spatial resolution by using an electron beam excitation-assisted optical (EXA) microscope. We observed the dynamic movement of the nucleus and nano-scale granules in living cells with better than 100 nm spatial resolution and a signal-to-noise ratio (SNR) around 10. Our results contribute to the development of cellular function analysis and open up new bioscience applications.

  15. LONG-LIVED BONE MARROW PLASMA CELLS DURING IMMUNE RESPONSE TO ALPHA (1→3 DEXTRAN

    Directory of Open Access Journals (Sweden)

    I. N. Chernyshova

    2015-01-01

    Full Text Available Production kinetics and some functional properties of long-lived marrow plasma cells were studied in mice immunized with T-independent type 2 antigens. Alpha (1→3 dextran was used as an antigen for immunization. The mice were immunized by dextran, and the numbers of IgM antibody producing cells were determined by ELISPOT method. The cell phenotype was determined by cytofluorimetric technique. In the area of normal bone marrow lymphocytes ~4% of T and ~85% of B cells were detected. About 35% of the cells expressed a plasmocyte marker (CD138; 3% were CD138+IgM+, and about 6% of the lymphocytes were double-positive for CD138+IgA+. Among spleen lymphocytes, 50% of T and 47% of B cells were detected. About 1.5% lymphocytes were CD138+, and 0.5% were positive for CD138 and IgM. Time kinetics of antibody-producing cells in bone marrow and spleen was different. In spleen populations, the peak amounts of antibody-secreting cells have been shown on the day 4; the process abated by the day 28. Vice versa, the numbers of the antibody-producing cells in bone marrow started to increase on the day 4. The process reached its maximum on day 14, and after 28th day became stationary. The in vitro experiments have shown that supplementation of bone marrow cells from immune mice with dextran did not influence their functional activity. It was previously shown for cells responding to T-dependent antigens only. A specific marker for the long-lived plasma cells is still unknown. However, these cells possess a common CD138 marker specific for all plasma cells. A method for isolation of bone marrow CD138+ cells was developed. The CD138+ cells were of 87-97% purity, being enriched in long-lived bone marrow cells, and produced monospecific antibodies.

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

    International Nuclear Information System (INIS)

    Hasegawa, Urara; Nomura, Shin-ichiro M.; Kaul, Sunil C.; Hirano, Takashi; Akiyoshi, Kazunari

    2005-01-01

    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 2 ). The CHPNH 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

  17. Development of analytical cell support for vitrification at the West Valley Demonstration Project. Topical report

    Energy Technology Data Exchange (ETDEWEB)

    Barber, F.H.; Borek, T.T.; Christopher, J.Z. [and others

    1997-12-01

    Analytical and Process Chemistry (A&PC) support is essential to the high-level waste vitrification campaign at the West Valley Demonstration Project (WVDP). A&PC characterizes the waste, providing information necessary to formulate the recipe for the target radioactive glass product. High-level waste (HLW) samples are prepared and analyzed in the analytical cells (ACs) and Sample Storage Cell (SSC) on the third floor of the main plant. The high levels of radioactivity in the samples require handling them in the shielded cells with remote manipulators. The analytical hot cells and third floor laboratories were refurbished to ensure optimal uninterrupted operation during the vitrification campaign. New and modified instrumentation, tools, sample preparation and analysis techniques, and equipment and training were required for A&PC to support vitrification. Analytical Cell Mockup Units (ACMUs) were designed to facilitate method development, scientist and technician training, and planning for analytical process flow. The ACMUs were fabricated and installed to simulate the analytical cell environment and dimensions. New techniques, equipment, and tools could be evaluated m in the ACMUs without the consequences of generating or handling radioactive waste. Tools were fabricated, handling and disposal of wastes was addressed, and spatial arrangements for equipment were refined. As a result of the work at the ACMUs the remote preparation and analysis methods and the equipment and tools were ready for installation into the ACs and SSC m in July 1995. Before use m in the hot cells, all remote methods had been validated and four to eight technicians were trained on each. Fine tuning of the procedures has been ongoing at the ACs based on input from A&PC technicians. Working at the ACs presents greater challenges than had development at the ACMUs. The ACMU work and further refinements m in the ACs have resulted m in a reduction m in analysis turnaround time (TAT).

  18. Development of analytical cell support for vitrification at the West Valley Demonstration Project. Topical report

    International Nuclear Information System (INIS)

    Barber, F.H.; Borek, T.T.; Christopher, J.Z.

    1997-12-01

    Analytical and Process Chemistry (A ampersand PC) support is essential to the high-level waste vitrification campaign at the West Valley Demonstration Project (WVDP). A ampersand PC characterizes the waste, providing information necessary to formulate the recipe for the target radioactive glass product. High-level waste (HLW) samples are prepared and analyzed in the analytical cells (ACs) and Sample Storage Cell (SSC) on the third floor of the main plant. The high levels of radioactivity in the samples require handling them in the shielded cells with remote manipulators. The analytical hot cells and third floor laboratories were refurbished to ensure optimal uninterrupted operation during the vitrification campaign. New and modified instrumentation, tools, sample preparation and analysis techniques, and equipment and training were required for A ampersand PC to support vitrification. Analytical Cell Mockup Units (ACMUs) were designed to facilitate method development, scientist and technician training, and planning for analytical process flow. The ACMUs were fabricated and installed to simulate the analytical cell environment and dimensions. New techniques, equipment, and tools could be evaluated m in the ACMUs without the consequences of generating or handling radioactive waste. Tools were fabricated, handling and disposal of wastes was addressed, and spatial arrangements for equipment were refined. As a result of the work at the ACMUs the remote preparation and analysis methods and the equipment and tools were ready for installation into the ACs and SSC m in July 1995. Before use m in the hot cells, all remote methods had been validated and four to eight technicians were trained on each. Fine tuning of the procedures has been ongoing at the ACs based on input from A ampersand PC technicians. Working at the ACs presents greater challenges than had development at the ACMUs. The ACMU work and further refinements m in the ACs have resulted m in a reduction m in

  19. Demonstration of a novel HIV-1 restriction phenotype from a human T cell line.

    Directory of Open Access Journals (Sweden)

    Yanxing Han

    2008-07-01

    Full Text Available Although retroviruses may invade host cells, a productive infection can be established only after the virus counteracts inhibition from different types of host restriction factors. Fv1, APOBEC3G/F, TRIM5alpha, ZAP, and CD317 inhibit the replication of different retroviruses by interfering with viral uncoating, reverse transcription, nuclear import, RNA stability, and release. In humans, although APOBEC3G/3F and CD317 block HIV-1 replication, their antiviral activities are neutralized by viral proteins Vif and Vpu. So far, no human gene has been found to effectively block wild type HIV-1 replication under natural condition. Thus, identification of such a gene product would be of great medical importance for the development of HIV therapies.In this study, we discovered a new type of host restriction against the wild type HIV-1 from a CD4/CXCR4 double-positive human T cell line. We identified a CEM-derived cell line (CEM.NKR that is highly resistant to productive HIV-1 infection. Viral production was reduced by at least 1000-fold when compared to the other permissive human T cell lines such as H9, A3.01, and CEM-T4. Importantly, this resistance was evident at extremely high multiplicity of infection. Further analyses demonstrated that HIV-1 could finish the first round of replication in CEM.NKR cells, but the released virions were poorly infectious. These virions could enter the target cells, but failed to initiate reverse transcription. Notably, this restriction phenotype was also present in CEM.NKR and 293T heterokaryons.These results clearly indicate that CEM.NKR cells express a HIV inhibitory gene(s. Further characterization of this novel gene product(s will reveal a new antiretroviral mechanism that directly inactivates wild type HIV-1.

  20. Quantitative live imaging of endogenous DNA replication in mammalian cells.

    Directory of Open Access Journals (Sweden)

    Andrew Burgess

    Full Text Available 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.

  1. A minimal rupture cascade model for living cell plasticity

    Science.gov (United States)

    Polizzi, Stefano; Laperrousaz, Bastien; Perez-Reche, Francisco J.; Nicolini, Franck E.; Maguer Satta, Véronique; Arneodo, Alain; Argoul, Françoise

    2018-05-01

    Under physiological and pathological conditions, cells experience large forces and deformations that often exceed the linear viscoelastic regime. Here we drive CD34+ cells isolated from healthy and leukemic bone marrows in the highly nonlinear elasto-plastic regime, by poking their perinuclear region with a sharp AFM cantilever tip. We use the wavelet transform mathematical microscope to identify singular events in the force-indentation curves induced by local rupture events in the cytoskeleton (CSK). We distinguish two types of rupture events, brittle failures likely corresponding to irreversible ruptures in a stiff and highly cross-linked CSK and ductile failures resulting from dynamic cross-linker unbindings during plastic deformation without loss of CSK integrity. We propose a stochastic multiplicative cascade model of mechanical ruptures that reproduces quantitatively the experimental distributions of the energy released during these events, and provides some mathematical and mechanistic understanding of the robustness of the log-normal statistics observed in both brittle and ductile situations. We also show that brittle failures are relatively more prominent in leukemia than in healthy cells suggesting their greater fragility.

  2. Phase imaging of mechanical properties of live cells (Conference Presentation)

    Science.gov (United States)

    Wax, Adam

    2017-02-01

    The mechanisms by which cells respond to mechanical stimuli are essential for cell function yet not well understood. Many rheological tools have been developed to characterize cellular viscoelastic properties but these typically require direct mechanical contact, limiting their throughput. We have developed a new approach for characterizing the organization of subcellular structures using a label free, noncontact, single-shot phase imaging method that correlates to measured cellular mechanical stiffness. The new analysis approach measures refractive index variance and relates it to disorder strength. These measurements are compared to cellular stiffness, measured using the same imaging tool to visualize nanoscale responses to flow shear stimulus. The utility of the technique is shown by comparing shear stiffness and phase disorder strength across five cellular populations with varying mechanical properties. An inverse relationship between disorder strength and shear stiffness is shown, suggesting that cell mechanical properties can be assessed in a format amenable to high throughput studies using this novel, non-contact technique. Further studies will be presented which include examination of mechanical stiffness in early carcinogenic events and investigation of the role of specific cellular structural proteins in mechanotransduction.

  3. Influence of the environment and phototoxicity of the live cell imaging system at IMP microbeam facility

    Science.gov (United States)

    Liu, Wenjing; Du, Guanghua; Guo, Jinlong; Wu, Ruqun; Wei, Junzhe; Chen, Hao; Li, Yaning; Zhao, Jing; Li, Xiaoyue

    2017-08-01

    To investigate the spatiotemporal dynamics of DNA damage and repair after the ion irradiation, an online live cell imaging system has been established based on the microbeam facility at Institute of Modern Physics (IMP). The system could provide a sterile and physiological environment by making use of heating plate and live cell imaging solution. The phototoxicity was investigated through the evaluation of DNA repair protein XRCC1 foci formed in HT1080-RFP cells during the imaging exposure. The intensity of the foci induced by phototoxicity was much lower compared with that of the foci induced by heavy ion hits. The results showed that although spontaneous foci were formed due to RFP exposure during live cell imaging, they had little impact on the analysis of the recruitment kinetics of XRCC1 in the foci induced by the ion irradiation.

  4. Live Cell Refractometry Using Hilbert Phase Microscopy and Confocal Reflectance Microscopy†

    Science.gov (United States)

    Lue, Niyom; Choi, Wonshik; Popescu, Gabriel; Yaqoob, Zahid; Badizadegan, Kamran; Dasari, Ramachandra R.; Feld, Michael S.

    2010-01-01

    Quantitative chemical analysis has served as a useful tool for understanding cellular metabolisms in biology. Among many physical properties used in chemical analysis, refractive index in particular has provided molecular concentration that is an important indicator for biological activities. In this report, we present a method of extracting full-field refractive index maps of live cells in their native states. We first record full-field optical thickness maps of living cells by Hilbert phase microscopy and then acquire physical thickness maps of the same cells using a custom-built confocal reflectance microscope. Full-field and axially averaged refractive index maps are acquired from the ratio of optical thickness to physical thickness. The accuracy of the axially averaged index measurement is 0.002. This approach can provide novel biological assays of label-free living cells in situ. PMID:19803506

  5. Live cell refractometry using Hilbert phase microscopy and confocal reflectance microscopy.

    Science.gov (United States)

    Lue, Niyom; Choi, Wonshik; Popescu, Gabriel; Yaqoob, Zahid; Badizadegan, Kamran; Dasari, Ramachandra R; Feld, Michael S

    2009-11-26

    Quantitative chemical analysis has served as a useful tool for understanding cellular metabolisms in biology. Among many physical properties used in chemical analysis, refractive index in particular has provided molecular concentration that is an important indicator for biological activities. In this report, we present a method of extracting full-field refractive index maps of live cells in their native states. We first record full-field optical thickness maps of living cells by Hilbert phase microscopy and then acquire physical thickness maps of the same cells using a custom-built confocal reflectance microscope. Full-field and axially averaged refractive index maps are acquired from the ratio of optical thickness to physical thickness. The accuracy of the axially averaged index measurement is 0.002. This approach can provide novel biological assays of label-free living cells in situ.

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

    International Nuclear Information System (INIS)

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

    2014-01-01

    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. (paper)

  7. Rapid labeling of intracellular His-tagged proteins in living cells.

    Science.gov (United States)

    Lai, Yau-Tsz; Chang, Yuen-Yan; Hu, Ligang; Yang, Ya; Chao, Ailun; Du, Zhi-Yan; Tanner, Julian A; Chye, Mee-Len; Qian, Chengmin; Ng, Kwan-Ming; Li, Hongyan; Sun, Hongzhe

    2015-03-10

    Small molecule-based fluorescent probes have been used for real-time visualization of live cells and tracking of various cellular events with minimal perturbation on the cells being investigated. Given the wide utility of the (histidine)6-Ni(2+)-nitrilotriacetate (Ni-NTA) system in protein purification, there is significant interest in fluorescent Ni(2+)-NTA-based probes. Unfortunately, previous Ni-NTA-based probes suffer from poor membrane permeability and cannot label intracellular proteins. Here, we report the design and synthesis of, to our knowledge, the first membrane-permeable fluorescent probe Ni-NTA-AC via conjugation of NTA with fluorophore and arylazide followed by coordination with Ni(2+) ions. The probe, driven by Ni(2+)-NTA, binds specifically to His-tags genetically fused to proteins and subsequently forms a covalent bond upon photoactivation of the arylazide, leading to a 13-fold fluorescence enhancement. The arylazide is indispensable not only for fluorescence enhancement, but also for strengthening the binding between the probe and proteins. Significantly, the Ni-NTA-AC probe can rapidly enter different types of cells, even plant tissues, to target His-tagged proteins. Using this probe, we visualized the subcellular localization of a DNA repair protein, Xeroderma pigmentosum group A (XPA122), which is known to be mainly enriched in the nucleus. We also demonstrated that the probe can image a genetically engineered His-tagged protein in plant tissues. This study thus offers a new opportunity for in situ visualization of large libraries of His-tagged proteins in various prokaryotic and eukaryotic cells.

  8. Complete study demonstrating the absence of rhabdovirus in a distinct Sf9 cell line.

    Directory of Open Access Journals (Sweden)

    Yoshifumi Hashimoto

    Full Text Available A putative novel rhabdovirus (SfRV was previously identified in a Spodoptera frugiperda cell line (Sf9 cells [ATCC CRL-1711 lot 58078522] by next generation sequencing and extensive bioinformatic analysis. We performed an extensive analysis of our Sf9 cell bank (ATCC CRL-1711 lot 5814 [Sf9L5814] to determine whether this virus was already present in cells obtained from ATCC in 1987. Inverse PCR of DNA isolated from Sf9 L5814 cellular DNA revealed integration of SfRV sequences in the cellular genome. RT-PCR of total RNA showed a deletion of 320 nucleotides in the SfRV RNA that includes the transcriptional motifs for genes X and L. Concentrated cell culture supernatant was analyzed by sucrose density gradient centrifugation and revealed a single band at a density of 1.14 g/ml. This fraction was further analysed by electron microscopy and showed amorphous and particulate debris that did not resemble a rhabdovirus in morphology or size. SDS-PAGE analysis confirmed that the protein composition did not contain the typical five rhabdovirus structural proteins and LC-MS/MS analysis revealed primarily of exosomal marker proteins, the SfRV N protein, and truncated forms of SfRV N, P, and G proteins. The SfRV L gene fragment RNA sequence was recovered from the supernatant after ultracentrifugation of the 1.14 g/ml fraction treated with diethyl ether suggesting that the SfRV L gene fragment sequence is not associated with a diethyl ether resistant nucleocapsid. Interestingly, the 1.14 g/ml fraction was able to transfer baculovirus DNA into Sf9L5814 cells, consistent with the presence of functional exosomes. Our results demonstrate the absence of viral particles in ATCC CRL-1711 lot 5814 Sf9 cells in contrast to a previous study that suggested the presence of infectious rhabdoviral particles in Sf9 cells from a different lot. This study highlights how cell lines with different lineages may present different virosomes and therefore no general conclusions can

  9. Background fluorescence estimation and vesicle segmentation in live cell imaging with conditional random fields.

    Science.gov (United States)

    Pécot, Thierry; Bouthemy, Patrick; Boulanger, Jérôme; Chessel, Anatole; Bardin, Sabine; Salamero, Jean; Kervrann, Charles

    2015-02-01

    Image analysis applied to fluorescence live cell microscopy has become a key tool in molecular biology since it enables to characterize biological processes in space and time at the subcellular level. In fluorescence microscopy imaging, the moving tagged structures of interest, such as vesicles, appear as bright spots over a static or nonstatic background. In this paper, we consider the problem of vesicle segmentation and time-varying background estimation at the cellular scale. The main idea is to formulate the joint segmentation-estimation problem in the general conditional random field framework. Furthermore, segmentation of vesicles and background estimation are alternatively performed by energy minimization using a min cut-max flow algorithm. The proposed approach relies on a detection measure computed from intensity contrasts between neighboring blocks in fluorescence microscopy images. This approach permits analysis of either 2D + time or 3D + time data. We demonstrate the performance of the so-called C-CRAFT through an experimental comparison with the state-of-the-art methods in fluorescence video-microscopy. We also use this method to characterize the spatial and temporal distribution of Rab6 transport carriers at the cell periphery for two different specific adhesion geometries.

  10. Highly stable loading of Mcm proteins onto chromatin in living cells requires replication to unload

    Science.gov (United States)

    Kuipers, Marjorie A.; Stasevich, Timothy J.; Sasaki, Takayo; Wilson, Korey A.; Hazelwood, Kristin L.; McNally, James G.; Davidson, Michael W.

    2011-01-01

    The heterohexameric minichromosome maintenance protein complex (Mcm2-7) functions as the eukaryotic helicase during DNA replication. Mcm2-7 loads onto chromatin during early G1 phase but is not converted into an active helicase until much later during S phase. Hence, inactive Mcm complexes are presumed to remain stably bound from early G1 through the completion of S phase. Here, we investigated Mcm protein dynamics in live mammalian cells. We demonstrate that Mcm proteins are irreversibly loaded onto chromatin cumulatively throughout G1 phase, showing no detectable exchange with a gradually diminishing soluble pool. Eviction of Mcm requires replication; during replication arrest, Mcm proteins remained bound indefinitely. Moreover, the density of immobile Mcms is reduced together with chromatin decondensation within sites of active replication, which provides an explanation for the lack of colocalization of Mcm with replication fork proteins. These results provide in vivo evidence for an exceptionally stable lockdown mechanism to retain all loaded Mcm proteins on chromatin throughout prolonged cell cycles. PMID:21220507

  11. Hot Cell Installation and Demonstration of the Severe Accident Test Station

    Energy Technology Data Exchange (ETDEWEB)

    Linton, Kory D. [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Burns, Zachary M. [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Terrani, Kurt A. [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Yan, Yong [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)

    2017-08-01

    A Severe Accident Test Station (SATS) capable of examining the oxidation kinetics and accident response of irradiated fuel and cladding materials for design basis accident (DBA) and beyond design basis accident (BDBA) scenarios has been successfully installed and demonstrated in the Irradiated Fuels Examination Laboratory (IFEL), a hot cell facility at Oak Ridge National Laboratory. The two test station modules provide various temperature profiles, steam, and the thermal shock conditions necessary for integral loss of coolant accident (LOCA) testing, defueled oxidation quench testing and high temperature BDBA testing. The installation of the SATS system restores the domestic capability to examine postulated and extended LOCA conditions on spent fuel and cladding and provides a platform for evaluation of advanced fuel and accident tolerant fuel (ATF) cladding concepts. This document reports on the successful in-cell demonstration testing of unirradiated Zircaloy-4. It also contains descriptions of the integral test facility capabilities, installation activities, and out-of-cell benchmark testing to calibrate and optimize the system.

  12. Microscopic visualization of metabotropic glutamate receptors on the surface of living cells using bifunctional magnetic resonance imaging probes.

    Science.gov (United States)

    Mishra, Anurag; Mishra, Ritu; Gottschalk, Sven; Pal, Robert; Sim, Neil; Engelmann, Joern; Goldberg, Martin; Parker, David

    2014-02-19

    A series of bimodal metabotropic glutamate-receptor targeted MRI contrast agents has been developed and evaluated, based on established competitive metabotropic Glu receptor subtype 5 (mGluR5) antagonists. In order to directly visualize mGluR5 binding of these agents on the surface of live astrocytes, variations in the core structure were made. A set of gadolinium conjugates containing either a cyanine dye or a fluorescein moiety was accordingly prepared, to allow visualization by optical microscopy in cellulo. In each case, surface receptor binding was compromised and cell internalization observed. Another approach, examining the location of a terbium analogue via sensitized emission, also exhibited nonspecific cell uptake in neuronal cell line models. Finally, biotin derivatives of two lead compounds were prepared, and the specificity of binding to the mGluR5 cell surface receptors was demonstrated with the aid of their fluorescently labeled avidin conjugates, using both total internal reflection fluorescence (TIRF) and confocal microscopy.

  13. Quantum measurement and orientation tracking of fluorescent nanodiamonds inside living cells

    Science.gov (United States)

    McGuinness, L. P.; Yan, Y.; Stacey, A.; Simpson, D. A.; Hall, L. T.; MacLaurin, D.; Prawer, S.; Mulvaney, P.; Wrachtrup, J.; Caruso, F.; Scholten, R. E.; Hollenberg, L. C. L.

    2011-06-01

    Fluorescent particles are routinely used to probe biological processes. The quantum properties of single spins within fluorescent particles have been explored in the field of nanoscale magnetometry, but not yet in biological environments. Here, we demonstrate optically detected magnetic resonance of individual fluorescent nanodiamond nitrogen-vacancy centres inside living human HeLa cells, and measure their location, orientation, spin levels and spin coherence times with nanoscale precision. Quantum coherence was measured through Rabi and spin-echo sequences over long (>10 h) periods, and orientation was tracked with effective 1° angular precision over acquisition times of 89 ms. The quantum spin levels served as fingerprints, allowing individual centres with identical fluorescence to be identified and tracked simultaneously. Furthermore, monitoring decoherence rates in response to changes in the local environment may provide new information about intracellular processes. The experiments reported here demonstrate the viability of controlled single spin probes for nanomagnetometry in biological systems, opening up a host of new possibilities for quantum-based imaging in the life sciences.

  14. Presenilin is the molecular target of acidic γ-secretase modulators in living cells.

    Directory of Open Access Journals (Sweden)

    Thorsten Jumpertz

    Full Text Available The intramembrane-cleaving protease γ-secretase catalyzes the last step in the generation of toxic amyloid-β (Aβ peptides and is a principal therapeutic target in Alzheimer's disease. Both preclinical and clinical studies have demonstrated that inhibition of γ-secretase is associated with prohibitive side effects due to suppression of Notch processing and signaling. Potentially safer are γ-secretase modulators (GSMs, which are small molecules that selectively lower generation of the highly amyloidogenic Aβ42 peptides but spare Notch processing. GSMs with nanomolar potency and favorable pharmacological properties have been described, but the molecular mechanism of GSMs remains uncertain and both the substrate amyloid precursor protein (APP and subunits of the γ-secretase complex have been proposed as the molecular target of GSMs. We have generated a potent photo-probe based on an acidic GSM that lowers Aβ42 generation with an IC(50 of 290 nM in cellular assays. By combining in vivo photo-crosslinking with affinity purification, we demonstrated that this probe binds the N-terminal fragment of presenilin (PSEN, the catalytic subunit of the γ-secretase complex, in living cells. Labeling was not observed for APP or any of the other γ-secretase subunits. Binding was readily competed by structurally divergent acidic and non-acidic GSMs suggesting a shared mode of action. These findings indicate that potent acidic GSMs target presenilin to modulate the enzymatic activity of the γ-secretase complex.

  15. Near-Infrared Light Activation of Proteins Inside Living Cells Enabled by Carbon Nanotube-Mediated Intracellular Delivery.

    Science.gov (United States)

    Li, He; Fan, Xinqi; Chen, Xing

    2016-02-01

    Light-responsive proteins have been delivered into the cells for controlling intracellular events with high spatial and temporal resolution. However, the choice of wavelength is limited to the UV and visible range; activation of proteins inside the cells using near-infrared (NIR) light, which has better tissue penetration and biocompatibility, remains elusive. Here, we report the development of a single-walled carbon nanotube (SWCNT)-based bifunctional system that enables protein intracellular delivery, followed by NIR activation of the delivered proteins inside the cells. Proteins of interest are conjugated onto SWCNTs via a streptavidin-desthiobiotin (SA-DTB) linkage, where the protein activity is blocked. SWCNTs serve as both a nanocarrier for carrying proteins into the cells and subsequently a NIR sensitizer to photothermally cleave the linkage and release the proteins. The released proteins become active and exert their functions inside the cells. We demonstrated this strategy by intracellular delivery and NIR-triggered nuclear translocation of enhanced green fluorescent protein, and by intracellular delivery and NIR-activation of a therapeutic protein, saporin, in living cells. Furthermore, we showed that proteins conjugated onto SWCNTs via the SA-DTB linkage could be delivered to the tumors, and optically released and activated by using NIR light in living mice.

  16. Live cell refractometry based on non-SPR microparticle sensor.

    Science.gov (United States)

    Liu, Chang; Chen, David D Y; Yu, Lirong; Luo, Yong

    2013-06-01

    Unlike the nanoparticles with surface plasmon resonance, the optical response of polystyrene microparticles (PSMPs) is insensitive to the chemical components of the surrounding medium under the wavelength-dependent differential interference contrast microscopy. This fact is exploited for the measurement of the refractive index of cytoplasm in this study. PSMPs of 400 nm in diameter were loaded into the cell to contact cytoplasm seamlessly, and the refractive index information of cytoplasm could be extracted by differential interference contrast microscopy operated at 420 nm illumination wavelength through the contrast analysis of PSMPs images. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Optofluidics for handling and analysis of single living cells

    KAUST Repository

    Perozziello, Gerardo

    2017-12-07

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

  18. Optofluidics for handling and analysis of single living cells

    KAUST Repository

    Perozziello, Gerardo; Candeloro, Patrizio; Coluccio, Maria Laura; Di Fabrizio, Enzo M.

    2017-01-01

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

  19. Relationships between Cargo, Cell Penetrating Peptides and Cell Type for Uptake of Non-Covalent Complexes into Live Cells

    Directory of Open Access Journals (Sweden)

    Andrea-Anneliese Keller

    2013-02-01

    Full Text Available Modulating signaling pathways for research and therapy requires either suppression or expression of selected genes or internalization of proteins such as enzymes, antibodies, nucleotide binding proteins or substrates including nucleoside phosphates and enzyme inhibitors. Peptides, proteins and nucleotides are transported by fusing or conjugating them to cell penetrating peptides or by formation of non-covalent complexes. The latter is often preferred because of easy handling, uptake efficiency and auto-release of cargo into the live cell. In our studies complexes are formed with labeled or readily detectable cargoes for qualitative and quantitative estimation of their internalization. Properties and behavior of adhesion and suspension vertebrate cells as well as the protozoa Leishmania tarentolae are investigated with respect to proteolytic activity, uptake efficiency, intracellular localization and cytotoxicity. Our results show that peptide stability to membrane-bound, secreted or intracellular proteases varies between different CPPs and that the suitability of individual CPPs for a particular cargo in complex formation by non-covalent interactions requires detailed studies. Cells vary in their sensitivity to increasing concentrations of CPPs. Thus, most cells can be efficiently transduced with peptides, proteins and nucleotides with intracellular concentrations in the low micromole range. For each cargo, cell type and CPP the optimal conditions must be determined separately.

  20. Introducing micrometer-sized artificial objects into live cells: a method for cell-giant unilamellar vesicle electrofusion.

    Directory of Open Access Journals (Sweden)

    Akira C Saito

    Full Text Available Here, we report a method for introducing large objects of up to a micrometer in diameter into cultured mammalian cells by electrofusion of giant unilamellar vesicles. We prepared GUVs containing various artificial objects using a water-in-oil (w/o emulsion centrifugation method. GUVs and dispersed HeLa cells were exposed to an alternating current (AC field to induce a linear cell-GUV alignment, and then a direct current (DC pulse was applied to facilitate transient electrofusion. With uniformly sized fluorescent beads as size indexes, we successfully and efficiently introduced beads of 1 µm in diameter into living cells along with a plasmid mammalian expression vector. Our electrofusion did not affect cell viability. After the electrofusion, cells proliferated normally until confluence was reached, and the introduced fluorescent beads were inherited during cell division. Analysis by both confocal microscopy and flow cytometry supported these findings. As an alternative approach, we also introduced a designed nanostructure (DNA origami into live cells. The results we report here represent a milestone for designing artificial symbiosis of functionally active objects (such as micro-machines in living cells. Moreover, our technique can be used for drug delivery, tissue engineering, and cell manipulation.

  1. Acid base activity of live bacteria: Implications for quantifying cell wall charge

    Science.gov (United States)

    Claessens, Jacqueline; van Lith, Yvonne; Laverman, Anniet M.; Van Cappellen, Philippe

    2006-01-01

    To distinguish the buffering capacity associated with functional groups in the cell wall from that resulting from metabolic processes, base or acid consumption by live and dead cells of the Gram-negative bacterium Shewanella putrefaciens was measured in a pH stat system. Live cells exhibited fast consumption of acid (pH 4) or base (pH 7, 8, 9, and 10) during the first few minutes of the experiments. At pH 5.5, no acid or base was required to maintain the initial pH constant. The initial amounts of acid or base consumed by the live cells at pH 4, 8, and 10 were of comparable magnitudes as those neutralized at the same pHs by intact cells killed by exposure to gamma radiation or ethanol. Cells disrupted in a French press required higher amounts of acid or base, due to additional buffering by intracellular constituents. At pH 4, acid neutralization by suspensions of live cells stopped after 50 min, because of loss of viability. In contrast, under neutral and alkaline conditions, base consumption continued for the entire duration of the experiments (5 h). This long-term base neutralization was, at least partly, due to active respiration by the cells, as indicated by the build-up of succinate in solution. Qualitatively, the acid-base activity of live cells of the Gram-positive bacterium Bacillus subtilis resembled that of S. putrefaciens. The pH-dependent charging of ionizable functional groups in the cell walls of the live bacteria was estimated from the initial amounts of acid or base consumed in the pH stat experiments. From pH 4 to 10, the cell wall charge increased from near-zero values to about -4 × 10 -16 mol cell -1 and -6.5 × 10 -16 mol cell -1 for S. putrefaciens and B. subtilis, respectively. The similar cell wall charging of the two bacterial strains is consistent with the inferred low contribution of lipopolysaccharides to the buffering capacity of the Gram-negative cell wall (of the order of 10%).

  2. Planar Optical Nanoantennas Resolve Cholesterol-Dependent Nanoscale Heterogeneities in the Plasma Membrane of Living Cells

    Science.gov (United States)

    Regmi, Raju; Winkler, Pamina M.; Flauraud, Valentin; Borgman, Kyra J. E.; Manzo, Carlo; Brugger, Jürgen; Rigneault, Hervé; Wenger, Jérôme; García-Parajo, María F.

    2017-10-01

    Optical nanoantennas can efficiently confine light into nanoscopic hotspots, enabling single-molecule detection sensitivity at biological relevant conditions. This innovative approach to breach the diffraction limit offers a versatile platform to investigate the dynamics of individual biomolecules in living cell membranes and their partitioning into cholesterol-dependent lipid nanodomains. Here, we present optical nanoantenna arrays with accessible surface hotspots to study the characteristic diffusion dynamics of phosphoethanolamine (PE) and sphingomyelin (SM) in the plasma membrane of living cells at the nanoscale. Fluorescence burst analysis and fluorescence correlation spectroscopy performed on nanoantennas of different gap sizes show that, unlike PE, SM is transiently trapped in cholesterol-enriched nanodomains of 10 nm diameter with short characteristic times around 100 {\\mu}s. The removal of cholesterol led to the free diffusion of SM, consistent with the dispersion of nanodomains. Our results are consistent with the existence of highly transient and fluctuating nanoscale assemblies enriched by cholesterol and sphingolipids in living cell membranes, also known as lipid rafts. Quantitative data on sphingolipids partitioning into lipid rafts is crucial to understand the spatiotemporal heterogeneous organization of transient molecular complexes on the membrane of living cells at the nanoscale. The proposed technique is fully biocompatible and thus provides various opportunities for biophysics and live cell research to reveal details that remain hidden in confocal diffraction-limited measurements.

  3. Dynamics of Corticosteroid Receptors: Lessons from Live Cell Imaging

    International Nuclear Information System (INIS)

    Nishi, Mayumi

    2011-01-01

    Adrenal corticosteroids (cortisol in humans or corticosterone in rodents) exert numerous effects on the central nervous system that regulates the stress response, mood, learning and memory, and various neuroendocrine functions. Corticosterone (CORT) actions in the brain are mediated via two receptor systems: the glucocorticoid receptor (GR) and the mineralocorticoid receptor (MR). It has been shown that GR and MR are highly colocalized in the hippocampus. These receptors are mainly distributed in the cytoplasm without hormones and translocated into the nucleus after treatment with hormones to act as transcriptional factors. Thus the subcellular dynamics of both receptors are one of the most important issues. Given the differential action of MR and GR in the central nervous system, it is of great consequence to clarify how these receptors are trafficked between cytoplasm and nucleus and their interactions are regulated by hormones and/or other molecules to exert their transcriptional activity. In this review, we focus on the nucleocytoplasmic and subnuclear trafficking of GR and MR in neural cells and non-neural cells analyzed by using molecular imaging techniques with green fluorescent protein (GFP) including fluorescence recovery after photobleaching (FRAP) and fluorescence resonance energy transfer (FRET), and discuss various factors affecting the dynamics of these receptors. Furthermore, we discuss the future directions of in vivo molecular imaging of corticosteroid receptors at the whole brain level

  4. A simple optical fiber device for quantitative fluorescence microscopy of single living cells

    OpenAIRE

    van Graft, M.; van Graft, Marja; Oosterhuis, B.; Oosterhuis, Bernard; van der Werf, Kees; de Grooth, B.G.; Greve, Jan

    1993-01-01

    simple and relatively inexpensive system is described for obtaining quantitative fluorescence measurements on single living cells loaded with a fluorescent probe to study cell physiological processes. The light emitted from the fluorescent cells is captured by and transported through an optical fiber. After passage through appropriate filters the light is measured using a photomultiplier tube. The optical fiber is mounted in one of the microscope outlets. Signals derived from the photomultipl...

  5. Effects of high-gradient magnetic fields on living cell machinery

    Czech Academy of Sciences Publication Activity Database

    Zablotskyy, Vitaliy A.; Lunov, Oleg; Kubinová, Šárka; Polyakova, Tetyana; Syková, E.; Dejneka, Alexandr

    2016-01-01

    Roč. 49, č. 49 (2016), s. 1-23, č. článku 493003. ISSN 0022-3727 R&D Projects: GA MŠk LO1409 Grant - others:FUNBIO(XE) CZ.2.16/3.1.00/21568 Institutional support: RVO:68378271 Keywords : living cell * magnetic gradient force * cell mechanics * stem cell * magnetic field Subject RIV: BO - Biophysics Impact factor: 2.588, year: 2016

  6. Cell lineage analysis demonstrates an endodermal origin of the distal urethra and perineum.

    Science.gov (United States)

    Seifert, Ashley W; Harfe, Brian D; Cohn, Martin J

    2008-06-01

    Congenital malformations of anorectal and genitourinary (collectively, anogenital) organs occur at a high frequency in humans, however the lineage of cells that gives rise to anogenital organs remains poorly understood. The penile urethra has been reported to develop from two cell populations, with the proximal urethra developing from endoderm and the distal urethra forming from an apical ectodermal invagination, however this has never been tested by direct analysis of cell lineage. During gut development, endodermal cells express Sonic hedgehog (Shh), which is required for normal patterning of digestive and genitourinary organs. We have taken advantage of the properties of Shh expression to genetically label and follow the fate of posterior gut endoderm during anogenital development. We report that the entire urethra, including the distal (glandar) region, is derived from endoderm. Cloacal endoderm also gives rise to the epithelial linings of the bladder, rectum and anterior region of the anus. Surprisingly, the lineage map also revealed an endodermal origin of the perineum, which is the first demonstration that endoderm differentiates into skin. In addition, we fate mapped genital tubercle ectoderm and show that it makes no detectable contribution to the urethra. In males, formation of the urethral tube involves septation of the urethral plate by continued growth of the urorectal septum. Analysis of cell lineage following disruption of androgen signaling revealed that the urethral plate of flutamide-treated males does not undergo this septation event. Instead, urethral plate cells persist to the ventral margin of the tubercle, mimicking the pattern seen in females. Based on these spatial and temporal fate maps, we present a new model for anogenital development and suggest that disruptions at specific developmental time points can account for the association between anorectal and genitourinary defects.

  7. Residential Fuel Cell Demonstration Handbook: National Rural Electric Cooperative Association Cooperative Research Network

    Energy Technology Data Exchange (ETDEWEB)

    Torrero, E.; McClelland, R.

    2002-07-01

    This report is a guide for rural electric cooperatives engaged in field testing of equipment and in assessing related application and market issues. Dispersed generation and its companion fuel cell technology have attracted increased interest by rural electric cooperatives and their customers. In addition, fuel cells are a particularly interesting source because their power quality, efficiency, and environmental benefits have now been coupled with major manufacturer development efforts. The overall effort is structured to measure the performance, durability, reliability, and maintainability of these systems, to identify promising types of applications and modes of operation, and to assess the related prospect for future use. In addition, technical successes and shortcomings will be identified by demonstration participants and manufacturers using real-world experience garnered under typical operating environments.

  8. Characterization of the Process Mechanical Cell at the West Valley Demonstration Project

    International Nuclear Information System (INIS)

    Drake, John; Schneider, Ken; Choroser, Jeff; Hughes, Karl

    2003-01-01

    The West Valley Demonstration Project has initiated decontamination and dismantlement (D and D) of the most highly radioactive and contaminated cells in a former spent nuclear fuel reprocessing plant. The goals of the D and D project are to remove loose debris in the cells and estimate the residual radioactivity level of legacy plant equipment. To support accomplishment of these goals, a unique characterization approach was developed to gather the information to meet anticipated Waste Isolation Pilot Plant (WIPP) acceptance criteria for remote-handled transuranic waste, and to facilitate segregation and packaging operations. Implementation of the characterization approach included the development and use of innovative, remote technology for measuring gamma radiation within the hot cell. The technology was used to identify and quantify radiation from individual debris items in radiation fields up to 2,000 R/hr (20 sieverts/hr). Sampling and analysis of the debris were also performed via remote handling means. Significant challenges associated with characterizing the highly radioactive and highly contaminated hot cells were encountered. The innovative solutions for meeting these challenges are applicable throughout the Department of Energy Complex and help support the goal of targeting D and D efforts toward reducing risks to public health and the environment

  9. Watch Out for the “Living Dead”: Cell-Free Enzymes and Their Fate

    Directory of Open Access Journals (Sweden)

    Federico Baltar

    2018-01-01

    Full Text Available Microbes are the engines driving biogeochemical cycles. Microbial extracellular enzymatic activities (EEAs are the “gatekeepers” of the carbon cycle. The total EEA is the sum of cell-bound (i.e., cell-attached, and dissolved (i.e., cell-free enzyme activities. Cell-free enzymes make up a substantial proportion (up to 100% of the total marine EEA. Although we are learning more about how microbial diversity and function (including total EEA will be affected by environmental changes, little is known about what factors control the importance of the abundant cell-free enzymes. Since cell-attached EEAs are linked to the cell, their fate will likely be linked to the factors controlling the cell’s fate. In contrast, cell-free enzymes belong to a kind of “living dead” realm because they are not attached to a living cell but still are able to perform their function away from the cell; and as such, the factors controlling their activity and fate might differ from those affecting cell-attached enzymes. This article aims to place cell-free EEA into the wider context of hydrolysis of organic matter, deal with recent studies assessing what controls the production, activity and lifetime of cell-free EEA, and what their fate might be in response to environmental stressors. This perspective article advocates the need to go “beyond the living things,” studying the response of cells/organisms to different stressors, but also to study cell-free enzymes, in order to fully constrain the future and evolution of marine biogeochemical cycles.

  10. Watch Out for the “Living Dead”: Cell-Free Enzymes and Their Fate

    Science.gov (United States)

    Baltar, Federico

    2018-01-01

    Microbes are the engines driving biogeochemical cycles. Microbial extracellular enzymatic activities (EEAs) are the “gatekeepers” of the carbon cycle. The total EEA is the sum of cell-bound (i.e., cell-attached), and dissolved (i.e., cell-free) enzyme activities. Cell-free enzymes make up a substantial proportion (up to 100%) of the total marine EEA. Although we are learning more about how microbial diversity and function (including total EEA) will be affected by environmental changes, little is known about what factors control the importance of the abundant cell-free enzymes. Since cell-attached EEAs are linked to the cell, their fate will likely be linked to the factors controlling the cell’s fate. In contrast, cell-free enzymes belong to a kind of “living dead” realm because they are not attached to a living cell but still are able to perform their function away from the cell; and as such, the factors controlling their activity and fate might differ from those affecting cell-attached enzymes. This article aims to place cell-free EEA into the wider context of hydrolysis of organic matter, deal with recent studies assessing what controls the production, activity and lifetime of cell-free EEA, and what their fate might be in response to environmental stressors. This perspective article advocates the need to go “beyond the living things,” studying the response of cells/organisms to different stressors, but also to study cell-free enzymes, in order to fully constrain the future and evolution of marine biogeochemical cycles. PMID:29354095

  11. Live attenuated measles virus vaccine therapy for locally established malignant glioblastoma tumor cells

    Directory of Open Access Journals (Sweden)

    Al-Shammari AM

    2014-05-01

    Full Text Available Ahmed M Al-Shammari,1 Farah E Ismaeel,2 Shahlaa M Salih,2 Nahi Y Yaseen11Experimental Therapy Department, Iraqi Center for Cancer and Medical Genetic Researches, Mustansiriya University, 2Departments of Biotechnology, College of Science, Al-Nahrain University, Baghdad, IraqAbstract: Glioblastoma multiforme is the most aggressive malignant primary brain tumor in humans, with poor prognosis. A new glioblastoma cell line (ANGM5 was established from a cerebral glioblastoma multiforme in a 72-year-old Iraqi man who underwent surgery for an intracranial tumor. This study was carried out to evaluate the antitumor effect of live attenuated measles virus (MV Schwarz vaccine strain on glioblastoma multiforme tumor cell lines in vitro. Live attenuated MV Schwarz strain was propagated on Vero, human rhabdomyosarcoma, and human glioblastoma-multiform (ANGM5 cell lines. The infected confluent monolayer appeared to be covered with syncytia with granulation and vacuolation, as well as cell rounding, shrinkage, and large empty space with cell debris as a result of cell lysis and death. Cell lines infected with virus have the ability for hemadsorption to human red blood cells after 72 hours of infection, whereas no hemadsorption of uninfected cells is seen. Detection of MV hemagglutinin protein by monoclonal antibodies in infected cells of all cell lines by immunocytochemistry assay gave positive results (brown color in the cytoplasm of infected cells. Cell viability was measured after 72 hours of infection by 3-(4,5-Dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide assay. Results showed a significant cytotoxic effect for MV (P≤0.05 on growth of ANGM5 and rhabdomyosarcoma cell lines after 72 hours of infection. Induction of apoptosis by MV was assessed by measuring mitochondrial membrane potentials in tumor cells after 48, 72, and 120 hours of infection. Apoptotic cells were counted, and the mean percentage of dead cells was significantly higher after 48, 72

  12. Pre-exposure prophylaxis for HIV-negative persons with partners living with HIV: uptake, use, and effectiveness in an open-label demonstration project in East Africa.

    Science.gov (United States)

    Heffron, Renee; Ngure, Kenneth; Odoyo, Josephine; Bulya, Nulu; Tindimwebwa, Edna; Hong, Ting; Kidoguchi, Lara; Donnell, Deborah; Mugo, Nelly R; Bukusi, Elizabeth A; Katabira, Elly; Asiimwe, Stephen; Morton, Jennifer; Morrison, Susan; Haugen, Harald; Mujugira, Andrew; Haberer, Jessica E; Ware, Norma C; Wyatt, Monique A; Marzinke, Mark A; Frenkel, Lisa M; Celum, Connie; Baeten, Jared M

    2017-11-06

    Introduction : Pre-exposure prophylaxis (PrEP) can provide high protection against HIV infection and is a recommended intervention for HIV-negative persons with substantial HIV risk, such as individuals with a partner living with HIV.  Demonstration projects of PrEP have been conducted in diverse settings worldwide to illustrate practical examples of how PrEP can be delivered.  Methods : We evaluated delivery of PrEP for HIV-negative partners within heterosexual HIV serodiscordant couples in an open-label demonstration project in East Africa.  The delivery model integrated PrEP into HIV treatment services, prioritizing PrEP for HIV-negative partners within serodiscordant couples prior to and during the first 6 months after the partner living with HIV initiated antiretroviral therapy (ART).  We measured adherence to PrEP through medication event monitoring system (MEMS) bottle caps and quantification of tenofovir in plasma among a random sample of participants. We estimated HIV infections prevented using a counterfactual cohort simulated from the placebo arm of a previous PrEP clinical trial. Results : We enrolled 1,010 HIV serodiscordant couples that were naïve to ART and PrEP.  Ninety-seven percent (97%) of HIV-negative partners initiated PrEP, and when PrEP was dispensed, objective measures suggest high adherence: 71% of HIV-negative participants took ≥80% of expected doses, as recorded via MEMS, and 81% of plasma samples had tenofovir detected.  A total of 4 incident HIV infections were observed (incidence rate=0.24 per 100 person-years), a 95% reduction (95% CI 86-98%, pproject for African HIV-negative individuals whose partners were known to be living with HIV.  Delivery of PrEP to HIV-negative partners within HIV serodiscordant couples was feasible and should be prioritized for wide-scale implementation.

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

    Science.gov (United States)

    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. Copyright © 2014 Elsevier Inc. All rights reserved.

  14. Monitoring of living cell attachment and spreading using reverse symmetry waveguide sensing

    DEFF Research Database (Denmark)

    Horvath, R.; Pedersen, H.C.; Skivesen, N.

    2005-01-01

    The effect of the attachment and spreading of living cells on the modes of a grating coupled reverse symmetry waveguide sensor is investigated in real time. The reverse symmetry design has an increased probing depth into the sample making it well suited for the monitoring of cell morphology....... As a result, significant changes in the incoupling peak height and peak shape were observed during cell attachment and spreading. It is suggested that the area under the incoupling peaks reflects the initial cell attachment process, while the mean peak position is mostly governed by the spreading of the cells...

  15. Detecting infrared luminescence and non-chemical signaling of living cells: single cell mid-IR spectroscopy in cryogenic environments

    Science.gov (United States)

    Pereverzev, Sergey

    2017-02-01

    Many life-relevant interaction energies are in IR range, and it is reasonable to believe that some biochemical reactions inside cells can results in emission of IR photons. Cells can use this emission for non-chemical and non-electrical signaling. Detecting weak infrared radiation from live cells is complicated because of strong thermal radiation background and absorption of radiation by tissues. A microfluidic device with live cells inside a vacuum cryogenic environment should suppress this background, and thereby permit observation of live cell auto-luminescence or signaling in the IR regime. One can make IR-transparent windows not emitting in this range, so only the cell and a small amount of liquid around it will emit infrared radiation. Currently mid-IR spectroscopy of single cells requires the use of a synchrotron source to measure absorption or reflection spectra. Decreasing of thermal radiation background will allow absorption and reflection spectroscopy of cells without using synchrotron light. Moreover, cell auto-luminescence can be directly measured. The complete absence of thermal background radiation for cryogenically cooled samples allows the use IR photon-sensitive detectors and obtaining single molecule sensitivity in IR photo-luminescence measurements. Due to low photon energies, photo-luminescence measurements will be non-distractive for pressures samples. The technique described here is based upon US patent 9366574.

  16. Mitochondria Targeted Nanoscale Zeolitic Imidazole Framework-90 for ATP Imaging in Live Cells.

    Science.gov (United States)

    Deng, Jingjing; Wang, Kai; Wang, Ming; Yu, Ping; Mao, Lanqun

    2017-04-26

    Zeolitic imidazole frameworks (ZIFs) are an emerging class of functional porous materials with promising biomedical applications such as molecular sensing and intracellular drug delivery. We report herein the first example of using nanoscale ZIFs (i.e., ZIF-90), self-assembled from Zn 2+ and imidazole-2-carboxyaldehyde, to target subcellular mitochondria and image dynamics of mitochondrial ATP in live cells. Encapsulation of fluorescent Rhodamine B (RhB) into ZIF-90 suppresses the emission of RhB, while the competitive coordination between ATP and the metal node of ZIF-90 dissembles ZIFs, resulting in the release of RhB for ATP sensing. With this method, we are able to image mitochondrial ATP in live cells and study the ATP level fluctuation in cellular glycolysis and apoptosis processes. The strategy reported here could be further extended to tune nanoscale ZIFs inside live cells for targeted delivery of therapeutics to subcellular organelles for advanced biomedical applications.

  17. Magnetogenetic control of protein gradients inside living cells with high spatial and temporal resolution.

    Science.gov (United States)

    Etoc, Fred; Vicario, Chiara; Lisse, Domenik; Siaugue, Jean-Michel; Piehler, Jacob; Coppey, Mathieu; Dahan, Maxime

    2015-05-13

    Tools for controlling the spatial organization of proteins are a major prerequisite for deciphering mechanisms governing the dynamic architecture of living cells. Here, we have developed a generic approach for inducing and maintaining protein gradients inside living cells by means of biofunctionalized magnetic nanoparticles (MNPs). For this purpose, we tailored the size and surface properties of MNPs in order to ensure unhindered mobility in the cytosol. These MNPs with a core diameter below 50 nm could be rapidly relocalized in living cells by exploiting biased diffusion at weak magnetic forces in the femto-Newton range. In combination with MNP surface functionalization for specific in situ capturing of target proteins as well as efficient delivery into the cytosplasm, we here present a comprehensive technology for controlling intracellular protein gradients with a temporal resolution of a few tens of seconds.

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

    Science.gov (United States)

    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

  19. A turn-on fluorescent probe for endogenous formaldehyde in the endoplasmic reticulum of living cells

    Science.gov (United States)

    Tang, Yonghe; Ma, Yanyan; Xu, An; Xu, Gaoping; Lin, Weiying

    2017-06-01

    As the simplest aldehyde compounds, formaldehyde (FA) is implicated in nervous system diseases and cancer. Endoplasmic reticulum is an organelle that plays important functions in living cells. Accordingly, the development of efficient methods for FA detection in the endoplasmic reticulum (ER) is of great biomedical importance. In this work, we developed the first ER-targeted fluorescent FA probe Na-FA-ER. The detection is based on the condensation reaction of the hydrazine group and FA to suppress the photo-induced electron transfer (PET) pathway, resulting in a fluorescence increase. The novel Na-FA-ER showed high sensitivity to FA. In addition, the Na-FA-ER enabled the bio-imaging of exogenous and endogenous FA in living HeLa cells. Most significantly, the new Na-FA-ER was employed to visualize the endogenous FA in the ER in living cells for the first time.

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

    Science.gov (United States)

    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.

  1. Demonstration-Scale High-Cell-Density Fermentation of Pichia pastoris.

    Science.gov (United States)

    Liu, Wan-Cang; Zhu, Ping

    2018-01-01

    Pichia pastoris has been one of the most successful heterologous overexpression systems in generating proteins for large-scale production through high-cell-density fermentation. However, optimizing conditions of the large-scale high-cell-density fermentation for biochemistry and industrialization is usually a laborious and time-consuming process. Furthermore, it is often difficult to produce authentic proteins in large quantities, which is a major obstacle for functional and structural features analysis and industrial application. For these reasons, we have developed a protocol for efficient demonstration-scale high-cell-density fermentation of P. pastoris, which employs a new methanol-feeding strategy-biomass-stat strategy and a strategy of increased air pressure instead of pure oxygen supplement. The protocol included three typical stages of glycerol batch fermentation (initial culture phase), glycerol fed-batch fermentation (biomass accumulation phase), and methanol fed-batch fermentation (induction phase), which allows direct online-monitoring of fermentation conditions, including broth pH, temperature, DO, anti-foam generation, and feeding of glycerol and methanol. Using this protocol, production of the recombinant β-xylosidase of Lentinula edodes origin in 1000-L scale fermentation can be up to ~900 mg/L or 9.4 mg/g cells (dry cell weight, intracellular expression), with the specific production rate and average specific production of 0.1 mg/g/h and 0.081 mg/g/h, respectively. The methodology described in this protocol can be easily transferred to other systems, and eligible to scale up for a large number of proteins used in either the scientific studies or commercial purposes.

  2. Label-free and live cell imaging by interferometric scattering microscopy.

    Science.gov (United States)

    Park, Jin-Sung; Lee, Il-Buem; Moon, Hyeon-Min; Joo, Jong-Hyeon; Kim, Kyoung-Hoon; Hong, Seok-Cheol; Cho, Minhaeng

    2018-03-14

    Despite recent remarkable advances in microscopic techniques, it still remains very challenging to directly observe the complex structure of cytoplasmic organelles in live cells without a fluorescent label. Here we report label-free and live-cell imaging of mammalian cell, Escherischia coli , and yeast, using interferometric scattering microscopy, which reveals the underlying structures of a variety of cytoplasmic organelles as well as the underside structure of the cells. The contact areas of the cells attached onto a glass substrate, e.g. , focal adhesions and filopodia, are clearly discernible. We also found a variety of fringe-like features in the cytoplasmic area, which may reflect the folded structures of cytoplasmic organelles. We thus anticipate that the label-free interferometric scattering microscopy can be used as a powerful tool to shed interferometric light on in vivo structures and dynamics of various intracellular phenomena.

  3. The use of fluorescent intrabodies to detect endogenous gankyrin in living cancer cells

    International Nuclear Information System (INIS)

    Rinaldi, Anne-Sophie; Freund, Guillaume; Desplancq, Dominique; Sibler, Annie-Paule; Baltzinger, Mireille; Rochel, Natacha; Mély, Yves; Didier, Pascal; Weiss, Etienne

    2013-01-01

    Expression of antibody fragments in mammalian cells (intrabodies) is used to probe the target protein or interfere with its biological function. We previously described the in vitro characterisation of a single-chain Fv (scFv) antibody fragment (F5) isolated from an intrabody library that binds to the oncoprotein gankyrin (GK) in solution. Here, we have isolated several other scFvs that interact with GK in the presence of F5 and tested whether they allow, when fused to fluorescent proteins, to detect by FRET endogenous GK in living cells. The binding of pairs of scFvs to GK was analysed by gel filtration and the ability of each scFv to mediate nuclear import/export of GK was determined. Binding between scFv-EGFP and RFP-labelled GK in living cells was detected by fluorescence lifetime imaging microscopy (FLIM). After co-transfection of two scFvs fused to EGFP and RFP, respectively, which form a tri-molecular complex with GK in vitro, FRET signal was measured. This system allowed us to observe that GK is monomeric and distributed throughout the cytoplasm and nucleus of several cancer cell lines. Our results show that pairs of fluorescently labelled intrabodies can be monitored by FLIM–FRET microscopy and that this technique allows the detection of lowly expressed endogenous proteins in single living cells. Highlights: ► Endogenous GK in living cells was targeted with pairs of fluorescently-tagged scFvs. ► Tri-molecular complexes containing two scFvs and one molecule GK were formed. ► GK was detected using fluorescence lifetime-based FRET imaging. ► GK is monomeric and homogeneously distributed in several cancer cell lines. ► This technique may have many applications in live-cell imaging of endogenous proteins

  4. The use of fluorescent intrabodies to detect endogenous gankyrin in living cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Rinaldi, Anne-Sophie; Freund, Guillaume; Desplancq, Dominique; Sibler, Annie-Paule; Baltzinger, Mireille [Ecole Supérieure de Biotechnologie de Strasbourg, UMR 7242, CNRS/Université de Strasbourg, boulevard Sébastien Brant, 67412 Illkirch (France); Rochel, Natacha [Institut de Génétique et de Biologie Moléculaire et Cellulaire, UMR 7104, CNRS/INSERM/Université de Strasbourg, rue Laurent Fries, 67404 Illkirch (France); Mély, Yves; Didier, Pascal [Faculté de Pharmacie, UMR 7213, CNRS/Université de Strasbourg, route du Rhin, 67401 Illkirch (France); Weiss, Etienne, E-mail: eweiss@unistra.fr [Ecole Supérieure de Biotechnologie de Strasbourg, UMR 7242, CNRS/Université de Strasbourg, boulevard Sébastien Brant, 67412 Illkirch (France)

    2013-04-01

    Expression of antibody fragments in mammalian cells (intrabodies) is used to probe the target protein or interfere with its biological function. We previously described the in vitro characterisation of a single-chain Fv (scFv) antibody fragment (F5) isolated from an intrabody library that binds to the oncoprotein gankyrin (GK) in solution. Here, we have isolated several other scFvs that interact with GK in the presence of F5 and tested whether they allow, when fused to fluorescent proteins, to detect by FRET endogenous GK in living cells. The binding of pairs of scFvs to GK was analysed by gel filtration and the ability of each scFv to mediate nuclear import/export of GK was determined. Binding between scFv-EGFP and RFP-labelled GK in living cells was detected by fluorescence lifetime imaging microscopy (FLIM). After co-transfection of two scFvs fused to EGFP and RFP, respectively, which form a tri-molecular complex with GK in vitro, FRET signal was measured. This system allowed us to observe that GK is monomeric and distributed throughout the cytoplasm and nucleus of several cancer cell lines. Our results show that pairs of fluorescently labelled intrabodies can be monitored by FLIM–FRET microscopy and that this technique allows the detection of lowly expressed endogenous proteins in single living cells. Highlights: ► Endogenous GK in living cells was targeted with pairs of fluorescently-tagged scFvs. ► Tri-molecular complexes containing two scFvs and one molecule GK were formed. ► GK was detected using fluorescence lifetime-based FRET imaging. ► GK is monomeric and homogeneously distributed in several cancer cell lines. ► This technique may have many applications in live-cell imaging of endogenous proteins.

  5. Simple and fast spectral domain algorithm for quantitative phase imaging of living cells with digital holographic microscopy

    Science.gov (United States)

    Min, Junwei; Yao, Baoli; Ketelhut, Steffi; Kemper, Björn

    2017-02-01

    The modular combination of optical microscopes with digital holographic microscopy (DHM) has been proven to be a powerful tool for quantitative live cell imaging. The introduction of condenser and different microscope objectives (MO) simplifies the usage of the technique and makes it easier to measure different kinds of specimens with different magnifications. However, the high flexibility of illumination and imaging also causes variable phase aberrations that need to be eliminated for high resolution quantitative phase imaging. The existent phase aberrations compensation methods either require add additional elements into the reference arm or need specimen free reference areas or separate reference holograms to build up suitable digital phase masks. These inherent requirements make them unpractical for usage with highly variable illumination and imaging systems and prevent on-line monitoring of living cells. In this paper, we present a simple numerical method for phase aberration compensation based on the analysis of holograms in spatial frequency domain with capabilities for on-line quantitative phase imaging. From a single shot off-axis hologram, the whole phase aberration can be eliminated automatically without numerical fitting or pre-knowledge of the setup. The capabilities and robustness for quantitative phase imaging of living cancer cells are demonstrated.

  6. Accurate live and dead bacterial cell enumeration using flow cytometry (Conference Presentation)

    Science.gov (United States)

    Ou, Fang; McGoverin, Cushla; Swift, Simon; Vanholsbeeck, Frédérique

    2017-03-01

    Flow cytometry (FCM) is based on the detection of scattered light and fluorescence to identify cells with particular characteristics of interest. However most FCM cannot precisely control the flow through its interrogation point and hence the volume and concentration of the sample cannot be immediately obtained. The easiest, most reliable and inexpensive way of obtaining absolute counts with FCM is by using reference beads. We investigated a method of using FCM with reference beads to measure live and dead bacterial concentration over the range of 106 to 108 cells/mL and ratio varying from 0 to 100%. We believe we are the first to use this method for such a large cell concentration range while also establishing the effect of varying the live/dead bacteria ratios. Escherichia coli solutions with differing ratios of live:dead cells were stained with fluorescent dyes SYTO 9 and propidium iodide (PI), which label live and dead cells, respectively. Samples were measured using a LSR II Flow Cytometer (BD Biosciences); using 488 nm excitation with 20 mW power. Both SYTO 9 and PI fluorescence were collected and threshold was set to side scatter. Traditional culture-based plate count was done in parallel to the FCM analysis. The concentration of live bacteria from FCM was compared to that obtained by plate counts. Preliminary results show that the concentration of live bacteria obtained by FCM and plate counts correlate well with each other and indicates this may be extended to a wider concentration range or for studying other cell characteristics.

  7. Live cell imaging techniques to study T cell trafficking across the blood-brain barrier in vitro and in vivo

    Directory of Open Access Journals (Sweden)

    Coisne Caroline

    2013-01-01

    Full Text Available Abstract Background The central nervous system (CNS is an immunologically privileged site to which access for circulating immune cells is tightly controlled by the endothelial blood–brain barrier (BBB located in CNS microvessels. Under physiological conditions immune cell migration across the BBB is low. However, in neuroinflammatory diseases such as multiple sclerosis, many immune cells can cross the BBB and cause neurological symptoms. Extravasation of circulating immune cells is a multi-step process that is regulated by the sequential interaction of different adhesion and signaling molecules on the immune cells and on the endothelium. The specialized barrier characteristics of the BBB, therefore, imply the existence of unique mechanisms for immune cell migration across the BBB. Methods and design An in vitro mouse BBB model maintaining physiological barrier characteristics in a flow chamber and combined with high magnification live cell imaging, has been established. This model enables the molecular mechanisms involved in the multi-step extravasation of T cells across the in vitro BBB, to be defined with high-throughput analyses. Subsequently these mechanisms have been verified in vivo using a limited number of experimental animals and a spinal cord window surgical technique. The window enables live observation of the dynamic interaction between T cells and spinal cord microvessels under physiological and pathological conditions using real time epifluorescence intravital imaging. These in vitro and in vivo live cell imaging methods have shown that the BBB endothelium possesses unique and specialized mechanisms involved in the multi-step T cell migration across this endothelial barrier under physiological flow. The initial T cell interaction with the endothelium is either mediated by T cell capture or by T cell rolling. Arrest follows, and then T cells polarize and especially CD4+ T cells crawl over long distances against the direction of

  8. Atomic force microscopy as a tool for the investigation of living cells.

    Science.gov (United States)

    Morkvėnaitė-Vilkončienė, Inga; Ramanavičienė, Almira; Ramanavičius, Arūnas

    2013-01-01

    Atomic force microscopy is a valuable and useful tool for the imaging and investigation of living cells in their natural environment at high resolution. Procedures applied to living cell preparation before measurements should be adapted individually for different kinds of cells and for the desired measurement technique. Different ways of cell immobilization, such as chemical fixation on the surface, entrapment in the pores of a membrane, or growing them directly on glass cover slips or on plastic substrates, result in the distortion or appearance of artifacts in atomic force microscopy images. Cell fixation allows the multiple use of samples and storage for a prolonged period; it also increases the resolution of imaging. Different atomic force microscopy modes are used for the imaging and analysis of living cells. The contact mode is the best for cell imaging because of high resolution, but it is usually based on the following: (i) image formation at low interaction force, (ii) low scanning speed, and (iii) usage of "soft," low resolution cantilevers. The tapping mode allows a cell to behave like a very solid material, and destructive shear forces are minimized, but imaging in liquid is difficult. The force spectroscopy mode is used for measuring the mechanical properties of cells; however, obtained results strongly depend on the cell fixation method. In this paper, the application of 3 atomic force microscopy modes including (i) contact, (ii) tapping, and (iii) force spectroscopy for the investigation of cells is described. The possibilities of cell preparation for the measurements, imaging, and determination of mechanical properties of cells are provided. The applicability of atomic force microscopy to diagnostics and other biomedical purposes is discussed.

  9. Fluorescent peptide biosensor for probing the relative abundance of cyclin-dependent kinases in living cells.

    Directory of Open Access Journals (Sweden)

    Laetitia Kurzawa

    Full Text Available Cyclin-dependant kinases play a central role in coordinating cell growth and division, and in sustaining proliferation of cancer cells, thereby constituting attractive pharmacological targets. However, there are no direct means of assessing their relative abundance in living cells, current approaches being limited to antigenic and proteomic analysis of fixed cells. In order to probe the relative abundance of these kinases directly in living cells, we have developed a fluorescent peptide biosensor with biligand affinity for CDKs and cyclins in vitro, that retains endogenous CDK/cyclin complexes from cell extracts, and that bears an environmentally-sensitive probe, whose fluorescence increases in a sensitive fashion upon recognition of its targets. CDKSENS was introduced into living cells, through complexation with the cell-penetrating carrier CADY2 and applied to assess the relative abundance of CDK/Cyclins through fluorescence imaging and ratiometric quantification. This peptide biosensor technology affords direct and sensitive readout of CDK/cyclin complex levels, and reports on differences in complex formation when tampering with a single CDK or cyclin. CDKSENS further allows for detection of differences between different healthy and cancer cell lines, thereby enabling to distinguish cells that express high levels of these heterodimeric kinases, from cells that present decreased or defective assemblies. This fluorescent biosensor technology provides information on the overall status of CDK/Cyclin complexes which cannot be obtained through antigenic detection of individual subunits, in a non-invasive fashion which does not require cell fixation or extraction procedures. As such it provides promising perspectives for monitoring the response to therapeutics that affect CDK/Cyclin abundance, for cell-based drug discovery strategies and fluorescence-based cancer diagnostics.

  10. Analysis of surface properties of fixed and live cells using derivatized agarose beads.

    Science.gov (United States)

    Navarro, Vanessa M; Walker, Sherri L; Badali, Oliver; Abundis, Maria I; Ngo, Lylla L; Weerasinghe, Gayani; Barajas, Marcela; Zem, Gregory; Oppenheimer, Steven B

    2002-01-01

    A novel assay has been developed for the histochemical characterization of surface properties of cells based on their adhesion to agarose beads derivatized with more than 100 types of molecules, including sugars, lectins and other proteins, and amino acids. The assay simply involves mixing small quantities of washed cells and beads in droplets on glass microscope slides and determining to which beads various cell types adhere. Distilled water was found to be the best medium for this assay because added ions or molecules in other media inhibit adhesion in some cases. Many cells, however, cannot tolerate distilled water. Here we show that cells fixed with either of two fixatives (1% formaldehyde or Prefer fixative) displayed similar bead-binding properties as did live cells. Specificity of cell-bead binding was tested by including specific free molecules in the test suspensions in hapten-type inhibition experiments. If a hapten compound inhibited live-cell adhesion to a specific bead, it also inhibited fixed-cell adhesion to a specific bead. The results of these experiments suggest that fixed cells display authentic surface properties, opening the door for the use of this assay with many cell types that cannot tolerate distilled water.

  11. [Non-invasive analysis of proteins in living cells using NMR spectroscopy].

    Science.gov (United States)

    Tochio, Hidehito; Murayama, Shuhei; Inomata, Kohsuke; Morimoto, Daichi; Ohno, Ayako; Shirakawa, Masahiro

    2015-01-01

    NMR spectroscopy enables structural analyses of proteins and has been widely used in the structural biology field in recent decades. NMR spectroscopy can be applied to proteins inside living cells, allowing characterization of their structures and dynamics in intracellular environments. The simplest "in-cell NMR" approach employs bacterial cells; in this approach, live Escherichia coli cells overexpressing a specific protein are subjected to NMR. The cells are grown in an NMR active isotope-enriched medium to ensure that the overexpressed proteins are labeled with the stable isotopes. Thus the obtained NMR spectra, which are derived from labeled proteins, contain atomic-level information about the structure and dynamics of the proteins. Recent progress enables us to work with higher eukaryotic cells such as HeLa and HEK293 cells, for which a number of techniques have been developed to achieve isotope labeling of the specific target protein. In this review, we describe successful use of electroporation for in-cell NMR. In addition, (19)F-NMR to characterize protein-ligand interactions in cells is presented. Because (19)F nuclei rarely exist in natural cells, when (19)F-labeled proteins are delivered into cells and (19)F-NMR signals are observed, one can safely ascertain that these signals originate from the delivered proteins and not other molecules.

  12. Time series modeling of live-cell shape dynamics for image-based phenotypic profiling.

    Science.gov (United States)

    Gordonov, Simon; Hwang, Mun Kyung; Wells, Alan; Gertler, Frank B; Lauffenburger, Douglas A; Bathe, Mark

    2016-01-01

    Live-cell imaging can be used to capture spatio-temporal aspects of cellular responses that are not accessible to fixed-cell imaging. As the use of live-cell imaging continues to increase, new computational procedures are needed to characterize and classify the temporal dynamics of individual cells. For this purpose, here we present the general experimental-computational framework SAPHIRE (Stochastic Annotation of Phenotypic Individual-cell Responses) to characterize phenotypic cellular responses from time series imaging datasets. Hidden Markov modeling is used to infer and annotate morphological state and state-switching properties from image-derived cell shape measurements. Time series modeling is performed on each cell individually, making the approach broadly useful for analyzing asynchronous cell populations. Two-color fluorescent cells simultaneously expressing actin and nuclear reporters enabled us to profile temporal changes in cell shape following pharmacological inhibition of cytoskeleton-regulatory signaling pathways. Results are compared with existing approaches conventionally applied to fixed-cell imaging datasets, and indicate that time series modeling captures heterogeneous dynamic cellular responses that can improve drug classification and offer additional important insight into mechanisms of drug action. The software is available at http://saphire-hcs.org.

  13. Traceless affinity labeling of endogenous proteins for functional analysis in living cells.

    Science.gov (United States)

    Hayashi, Takahiro; Hamachi, Itaru

    2012-09-18

    Protein labeling and imaging techniques have provided tremendous opportunities to study the structure, function, dynamics, and localization of individual proteins in the complex environment of living cells. Molecular biology-based approaches, such as GFP-fusion tags and monoclonal antibodies, have served as important tools for the visualization of individual proteins in cells. Although these techniques continue to be valuable for live cell imaging, they have a number of limitations that have only been addressed by recent progress in chemistry-based approaches. These chemical approaches benefit greatly from the smaller probe sizes that should result in fewer perturbations to proteins and to biological systems as a whole. Despite the research in this area, so far none of these labeling techniques permit labeling and imaging of selected endogenous proteins in living cells. Researchers have widely used affinity labeling, in which the protein of interest is labeled by a reactive group attached to a ligand, to identify and characterize proteins. Since the first report of affinity labeling in the early 1960s, efforts to fine-tune the chemical structures of both the reactive group and ligand have led to protein labeling with excellent target selectivity in the whole proteome of living cells. Although the chemical probes used for affinity labeling generally inactivate target proteins, this strategy holds promise as a valuable tool for the labeling and imaging of endogenous proteins in living cells and by extension in living animals. In this Account, we summarize traceless affinity labeling, a technique explored mainly in our laboratory. In our overview of the different labeling techniques, we emphasize the challenge of designing chemical probes that allow for dissociation of the affinity module (often a ligand) after the labeling reaction so that the labeled protein retains its native function. This feature distinguishes the traceless labeling approach from the traditional

  14. Nanograting-based plasmon enhancement for total internal reflection fluorescence microscopy of live cells

    International Nuclear Information System (INIS)

    Kim, Kyujung; Cho, Eun-Jin; Suh, Jin-Suck; Huh, Yong-Min; Kim, Donghyun; Kim, Dong Jun

    2009-01-01

    We investigated evanescent field enhancement based on subwavelength nanogratings for improved sensitivity in total internal reflection microscopy of live cells. The field enhancement is associated with subwavelength-grating-coupled plasmon excitation. An optimum sample employed a silver grating on a silver film and an SF10 glass substrate. Field intensity was enhanced by approximately 90% when measured by fluorescent excitation of microbeads relative to that on a bare prism as a control, which is in good agreement with numerical results. The subwavelength-grating-mediated field enhancement was also applied to live cell imaging of quantum dots, which confirmed the sensitivity enhancement qualitatively.

  15. Demonstration of NK cell-mediated lysis of varicella-zoster virus (VZV)-infected cells: characterization of the effector cells

    Energy Technology Data Exchange (ETDEWEB)

    Tilden, A.B.; Cauda, R.; Grossi, C.E.; Balch, C.M.; Lakeman, A.D.; Whitley, R.J.

    1986-06-01

    Infection with varicella-zoster virus (VZV) rendered RAJI cells more susceptible to lysis by non-adherent blood lymphocytes. At an effector to target ratio of 80:1 the mean percentage of /sup 51/Cr release of VZV-infected RAJI cells was 41 +/- 12%, whereas that of uninfected RAJI cells was 15 +/- 6%. The increased susceptibility to lysis was associated with increased effector to target conjugate formation in immunofluorescence binding assays. The effector cells cytotoxic for VZV-infected RAJI cells were predominantly Leu-11a/sup +/ Leu-4/sup -/ granular lymphocytes as demonstrated by fluorescence-activated cell sorting. The effector cell active against VZV-infected RAJI cells appeared similar to those active against herpes simplex virus (HSV)-infected cells, because in cold target competition experiments the lysis of /sup 51/Cr-labeled VZV-infected RAJI cells was efficiently inhibited by either unlabeled VZV-infected RAJI cells (mean 71% inhibition, 2:1 ratio unlabeled to labeled target) or HSV-infected RAJI cells (mean 69% inhibition) but not by uninfected RAJI cells (mean 10% inhibition). In contrast, competition experiments revealed donor heterogeneity in the overlap between effector cells for VZV- or HSV-infected RAJI vs K-562 cells.

  16. Demonstration of the SeptiStrand benthic microbial fuel cell powering a magnetometer for ship detection

    Science.gov (United States)

    Arias-Thode, Y. Meriah; Hsu, Lewis; Anderson, Greg; Babauta, Jerome; Fransham, Roy; Obraztsova, Anna; Tukeman, Gabriel; Chadwick, D. Bart

    2017-07-01

    The Navy has a need for monitoring conditions and gathering information in marine environments. Sensors can monitor and report environmental parameters and potential activities such as animal movements, ships, or personnel. However, there has to be a means to power these sensors. One promising enabling technology that has been shown to provide long-term power production in underwater environments is the benthic microbial fuel cells (BMFC). BMFCs are devices that generate energy by coupling bioanodes and biocathodes through an external energy harvester. Recent studies have demonstrated success for usage of BMFCs in powering small instruments and other devices on the seafloor over limited periods of time. In this effort, a seven-stranded BMFC linear array of 30 m was designed to power a seafloor magnetometer to detect passing ship movements through Pearl Harbor, Hawaii. The BMFC system was connected to a flyback energy harvesting circuit that charged the battery powering the magnetometer. The deployment was demonstrated the BMFC supplied power to the battery for approximately 38 days. This is the first large-scale demonstration system for usage of the SeptiStrand BMFC technology to power a relevant sensor.

  17. In-vitro analysis of APA microcapsules for oral delivery of live bacterial cells.

    Science.gov (United States)

    Chen, H; Ouyang, W; Jones, M; Haque, T; Lawuyi, B; Prakash, S

    2005-08-01

    Oral administration of microcapsules containing live bacterial cells has potential as an alternative therapy for several diseases. This article evaluates the suitability of the alginate-poly-L-lysine-alginate (APA) microcapsules for oral delivery of live bacterial cells, in-vitro, using a dynamic simulated human gastro-intestinal (GI) model. Results showed that the APA microcapsules were morphologically stable in the simulated stomach conditions, but did not retain their structural integrity after a 3-day exposure in simulated human GI media. The microbial populations of the tested bacterial cells and the activities of the tested enzymes in the simulated human GI suspension were not substantially altered by the presence of the APA microcapsules, suggesting that there were no significant adverse effects of oral administration of the APA microcapsules on the flora of the human gastrointestinal tract. When the APA microcapsules containing Lactobacillus plantarum 80 (LP80) were challenged in the simulated gastric medium (pH = 2.0), 80.0% of the encapsulated cells remained viable after a 5-min incubation; however, the viability decreased considerably (8.3%) after 15 min and dropped to 2.6% after 30 min and lower than 0.2% after 60 min, indicating the limitations of the currently obtainable APA membrane for oral delivery of live bacteria. Further in-vivo studies are required before conclusions can be made concerning the inadequacy of APA microcapsules for oral delivery of live bacterial cells.

  18. Live Imaging of Influenza Infection of the Trachea Reveals Dynamic Regulation of CD8+ T Cell Motility by Antigen.

    Science.gov (United States)

    Lambert Emo, Kris; Hyun, Young-Min; Reilly, Emma; Barilla, Christopher; Gerber, Scott; Fowell, Deborah; Kim, Minsoo; Topham, David J

    2016-09-01

    During a primary influenza infection, cytotoxic CD8+ T cells need to infiltrate the infected airways and engage virus-infected epithelial cells. The factors that regulate T cell motility in the infected airway tissue are not well known. To more precisely study T cell infiltration of the airways, we developed an experimental model system using the trachea as a site where live imaging can be performed. CD8+ T cell motility was dynamic with marked changes in motility on different days of the infection. In particular, significant changes in average cell velocity and confinement were evident on days 8-10 during which the T cells abruptly but transiently increase velocity on day 9. Experiments to distinguish whether infection itself or antigen affect motility revealed that it is antigen, not active infection per se that likely affects these changes as blockade of peptide/MHC resulted in increased velocity. These observations demonstrate that influenza tracheitis provides a robust experimental foundation to study molecular regulation of T cell motility during acute virus infection.

  19. Live Imaging of Influenza Infection of the Trachea Reveals Dynamic Regulation of CD8+ T Cell Motility by Antigen.

    Directory of Open Access Journals (Sweden)

    Kris Lambert Emo

    2016-09-01

    Full Text Available During a primary influenza infection, cytotoxic CD8+ T cells need to infiltrate the infected airways and engage virus-infected epithelial cells. The factors that regulate T cell motility in the infected airway tissue are not well known. To more precisely study T cell infiltration of the airways, we developed an experimental model system using the trachea as a site where live imaging can be performed. CD8+ T cell motility was dynamic with marked changes in motility on different days of the infection. In particular, significant changes in average cell velocity and confinement were evident on days 8-10 during which the T cells abruptly but transiently increase velocity on day 9. Experiments to distinguish whether infection itself or antigen affect motility revealed that it is antigen, not active infection per se that likely affects these changes as blockade of peptide/MHC resulted in increased velocity. These observations demonstrate that influenza tracheitis provides a robust experimental foundation to study molecular regulation of T cell motility during acute virus infection.

  20. Intracellular imaging of docosanol in living cells by coherent anti-Stokes Raman scattering microscopy

    Science.gov (United States)

    You, Sixian; Liu, Yuan; Arp, Zane; Zhao, Youbo; Chaney, Eric J.; Marjanovic, Marina; Boppart, Stephen A.

    2017-07-01

    Docosanol is an over-the-counter topical agent that has proved to be one of the most effective therapies for treating herpes simplex labialis. However, the mechanism by which docosanol suppresses lesion formation remains poorly understood. To elucidate its mechanism of action, we investigated the uptake of docosanol in living cells using coherent anti-Stokes Raman scattering microscopy. Based on direct visualization of the deuterated docosanol, we observed highly concentrated docosanol inside living cells 24 h after drug treatment. In addition, different spatial patterns of drug accumulation were observed in different cell lines. In keratinocytes, which are the targeted cells of docosanol, the drug molecules appeared to be docking at the periphery of the cell membrane. In contrast, the drug molecules in fibroblasts appeared to accumulate in densely packed punctate regions throughout the cytoplasm. These results suggest that this molecular imaging approach is suitable for the longitudinal tracking of drug molecules in living cells to identify cell-specific trafficking and may also have implications for elucidating the mechanism by which docosanol suppresses lesion formation.

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

    Science.gov (United States)

    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.

  2. Self-Assembled Fluorescent Bovine Serum Albumin Nanoprobes for Ratiometric pH Measurement inside Living Cells.

    Science.gov (United States)

    Yang, Qiaoyu; Ye, Zhongju; Zhong, Meile; Chen, Bo; Chen, Jian; Zeng, Rongjin; Wei, Lin; Li, Hung-wing; Xiao, Lehui

    2016-04-20

    In this work, we demonstrated a new ratiometric method for the quantitative analysis of pH inside living cells. The structure of the nanosensor comprises a biofriendly fluorescent bovine serum albumin (BSA) matrix, acting as a pH probe, and pH-insensitive reference dye Alexa 594 enabling ratiometric quantitative pH measurement. The fluorescent BSA matrix was synthesized by cross-linking of the denatured BSA proteins in ethanol with glutaraldehyde. The size of the as-synthesized BSA nanoparticles can be readily manipulated from 30 to 90 nm, which exhibit decent fluorescence at the peak wavelength of 535 nm with a pH response range of 6-8. The potential of this pH sensor for intracellular pH monitoring was demonstrated inside living HeLa cells, whereby a significant change in fluorescence ratio was observed when the pH of the cell was switched from normal to acidic with anticancer drug treatment. The fast response of the nanosensor makes it a very powerful tool in monitoring the processes occurring within the cytosol.

  3. A Novel Technique to Follow Consequences of Exogenous Factors, Including Therapeutic Drugs, on Living Human Breast Epithelial Cells

    Science.gov (United States)

    1999-07-01

    and lipid vectors, are being tested. Concurrent with the development of procedures for live - cell imaging , we are examining the distribution of proteins...dimensional matrix. These studies have not yet begun. There are a number of procedures that must be developed and perfected in the live - cell imaging , as...components of the Wnt signaling pathway are too preliminary and require additional research prior to publication. (9) CONCLUSIONS Live cell imaging of

  4. Quantitative live-cell imaging of human immunodeficiency virus (HIV-1) assembly.

    Science.gov (United States)

    Baumgärtel, Viola; Müller, Barbara; Lamb, Don C

    2012-05-01

    Advances in fluorescence methodologies make it possible to investigate biological systems in unprecedented detail. Over the last few years, quantitative live-cell imaging has increasingly been used to study the dynamic interactions of viruses with cells and is expected to become even more indispensable in the future. Here, we describe different fluorescence labeling strategies that have been used to label HIV-1 for live cell imaging and the fluorescence based methods used to visualize individual aspects of virus-cell interactions. This review presents an overview of experimental methods and recent experiments that have employed quantitative microscopy in order to elucidate the dynamics of late stages in the HIV-1 replication cycle. This includes cytosolic interactions of the main structural protein, Gag, with itself and the viral RNA genome, the recruitment of Gag and RNA to the plasma membrane, virion assembly at the membrane and the recruitment of cellular proteins involved in HIV-1 release to the nascent budding site.

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

    Directory of Open Access Journals (Sweden)

    Aseefhali Bankapur

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

  6. Efficient FRET-based fuorescent ratiometric chemosensors for Fe3+ and its application in living cells

    International Nuclear Information System (INIS)

    Wang, Cuicui; Liu, Yaqi; Cheng, Junye; Song, Jianhua; Zhao, Yufen; Ye, Yong

    2015-01-01

    A series of novel FRET-based fluorescent ratiometric chemosensors (L 1 –L 6 ) were designed and synthesized. Sensor L 2 showed reversible and the best selective recognition toward Fe 3+ over other metal ions with a detection limit of 0.418 ppm, which can meet the selective requirements for practical application. Experiment results showed that the response behavior of L 2 toward Fe 3+ is pH independent in weak acid condition (pH 4.0–6.0). In addition, sensor L 2 was successfully applied for ratiometric visualization of Fe 3+ in living cells. - Highlights: • The detection limit of a new FRET probe for Fe 3+ was 0.418 ppm. • The probe exhibited high selectivity and sensitivity detection to Fe 3+ with a pH span of 4.0–6.0. • The significant changes in color could be used for naked-eye detection • The fluorescence imaging experiment demonstrated its value of practical application

  7. Colorimetric and Fluorescent Bimodal Ratiometric Probes for pH Sensing of Living Cells.

    Science.gov (United States)

    Liu, Yuan-Yuan; Wu, Ming; Zhu, Li-Na; Feng, Xi-Zeng; Kong, De-Ming

    2015-06-01

    pH measurement is widely used in many fields. Ratiometric pH sensing is an important way to improve the detection accuracy. Herein, five water-soluble cationic porphyrin derivatives were synthesized and their optical property changes with pH value were investigated. Their pH-dependent assembly/disassembly behaviors caused significant changes in both absorption and fluorescence spectra, thus making them promising bimodal ratiometric probes for both colorimetric and fluorescent pH sensing. Different substituent identity and position confer these probes with different sensitive pH-sensing ranges, and the substituent position gives a larger effect. By selecting different porphyrins, different signal intensity ratios and different fluorescence excitation wavelengths, sensitive pH sensing can be achieved in the range of 2.1-8.0. Having demonstrated the excellent reversibility, good accuracy and low cytotoxicity of the probes, they were successfully applied in pH sensing inside living cells. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Quantifying Force and Viscoelasticity Inside Living Cells Using an Active–Passive Calibrated Optical Trap

    DEFF Research Database (Denmark)

    Ritter, Christine M.; Maes, Josep; Oddershede, Lene

    2017-01-01

    As described in the previous chapters, optical tweezers have become a tool of precision for in vitro single-molecule investigations, where the single molecule of interest most often is studied in purified form in an experimental assay with a well-controlled fluidic environment. A well-controlled ...... is that the size and refractive properties of the trapped object and the viscoelastic properties of its environment need not be known. We explain the protocol and demonstrate its use with experiments of trapped granules inside live S.pombe cells.......As described in the previous chapters, optical tweezers have become a tool of precision for in vitro single-molecule investigations, where the single molecule of interest most often is studied in purified form in an experimental assay with a well-controlled fluidic environment. A well......-controlled fluidic environment implies that the physical properties of the liquid, most notably the viscosity, are known and the fluidic environment can, for calibrational purposes, be treated as a simple liquid. In vivo, however, optical tweezers have primarily been used as a tool of manipulation and not so often...

  9. Live-cell imaging of post-golgi transport vesicles in cultured hippocampal neurons

    DEFF Research Database (Denmark)

    Jensen, Camilla Stampe; Misonou, Hiroaki

    2015-01-01

    compartments of neurons. In the past two decades, the establishment and advancement of fluorescent protein technology have provided us with opportunities to study how proteins are trafficked in living cells. However, live imaging of trafficking processes in neurons necessitate imaging tools to distinguish...... the several different routes that neurons use for protein trafficking. Here we provide a novel protocol to selectively visualize post-Golgi transport vesicles carrying fluorescent-labeled ion channel proteins in living neurons. Further, we provide a number of analytical tools we developed to quantify...... mechanisms by which post-Golgi vesicles are trafficked in neurons. Our protocol uniquely combines the classic temperature-block with close monitoring of the transient expression of transfected protein tagged with fluorescent proteins, and provides a quick and easy way to study protein trafficking in living...

  10. Targeted Gene Deletion Demonstrates that Cell Adhesion MoleculeICAM-4 is Critical for Erythroblastic Island Formation

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Gloria; Lo, Annie; Short, Sarah A.; Mankelow, Tosti J.; Spring, Frances; Parsons, Stephen F.; Mohandas, Narla; Anstee, David J.; Chasis, Joel Anne

    2006-02-15

    Erythroid progenitors differentiate in erythroblastic islands, bone marrow niches composed of erythroblasts surrounding a central macrophage. Evidence suggests that within islands adhesive interactions regulate erythropoiesis and apoptosis. We are exploring whether erythroid intercellular adhesion molecule-4 (ICAM-4), animmunoglobulin superfamily member, participates in island formation. Earlier, we identified alpha V integrins as ICAM-4 counter receptors. Since macrophages express alpha V, ICAM-4 potentially mediates island attachments. To test this, we generated ICAM-4 knockout mice and developed quantitative, live cell techniques for harvesting intact islands and for reforming islands in vitro. We observed a 47 percent decrease in islands reconstituted from ICAM-4 null marrow compared to wild type. We also found a striking decrease in islands formed in vivo in knockout mice. Further, peptides that block ICAM-4 alpha V adhesion produced a 53-57 percent decrease in reconstituted islands, strongly suggesting that ICAM-4 binding to macrophage alpha V functions in island integrity. Importantly, we documented that alpha V integrin is expressed in macrophages isolated from erythro blastic islands. Collectively, these data provide convincing evidence that ICAM-4 is critical in erythroblastic island formation via ICAM-4/alpha V adhesion and also demonstrate that the novel experimental strategies we developed will be valuable in exploring molecular mechanisms of erythroblastic island formation and their functional role in regulating erythropoiesis.

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

    OpenAIRE

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

    2007-01-01

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

  12. A new image correction method for live cell atomic force microscopy

    International Nuclear Information System (INIS)

    Shen, Y; Sun, J L; Zhang, A; Hu, J; Xu, L X

    2007-01-01

    During live cell imaging via atomic force microscopy (AFM), the interactions between the AFM probe and the membrane yield distorted cell images. In this work, an image correction method was developed based on the force-distance curve and the modified Hertzian model. The normal loading and lateral forces exerted on the cell membrane by the AFM tip were both accounted for during the scanning. Two assumptions were made in modelling based on the experimental measurements: (1) the lateral force on the endothelial cells was linear to the height; (2) the cell membrane Young's modulus could be derived from the displacement measurement of a normal force curve. Results have shown that the model could be used to recover up to 30% of the actual cell height depending on the loading force. The accuracy of the model was also investigated with respect to the loading force and mechanical property of the cell membrane

  13. A new image correction method for live cell atomic force microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Shen, Y; Sun, J L; Zhang, A; Hu, J; Xu, L X [College of Life Science and Biotechnology, Shanghai Jiao Tong University, Shanghai 200030 (China)

    2007-04-21

    During live cell imaging via atomic force microscopy (AFM), the interactions between the AFM probe and the membrane yield distorted cell images. In this work, an image correction method was developed based on the force-distance curve and the modified Hertzian model. The normal loading and lateral forces exerted on the cell membrane by the AFM tip were both accounted for during the scanning. Two assumptions were made in modelling based on the experimental measurements: (1) the lateral force on the endothelial cells was linear to the height; (2) the cell membrane Young's modulus could be derived from the displacement measurement of a normal force curve. Results have shown that the model could be used to recover up to 30% of the actual cell height depending on the loading force. The accuracy of the model was also investigated with respect to the loading force and mechanical property of the cell membrane.

  14. Clonal expansion under the microscope: studying lymphocyte activation and differentiation using live-cell imaging.

    Science.gov (United States)

    Polonsky, Michal; Chain, Benjamin; Friedman, Nir

    2016-03-01

    Clonal expansion of lymphocytes is a hallmark of vertebrate adaptive immunity. A small number of precursor cells that recognize a specific antigen proliferate into expanded clones, differentiate and acquire various effector and memory phenotypes, which promote effective immune responses. Recent studies establish a large degree of heterogeneity in the level of expansion and in cell state between and within expanding clones. Studying these processes in vivo, while providing insightful information on the level of heterogeneity, is challenging due to the complex microenvironment and the inability to continuously track individual cells over extended periods of time. Live cell imaging of ex vivo cultures within micro fabricated arrays provides an attractive methodology for studying clonal expansion. These experiments facilitate continuous acquisition of a large number of parameters on cell number, proliferation, death and differentiation state, with single-cell resolution on thousands of expanding clones that grow within controlled environments. Such data can reveal stochastic and instructive mechanisms that contribute to observed heterogeneity and elucidate the sequential order of differentiation events. Intercellular interactions can also be studied within these arrays by following responses of a controlled number of interacting cells, all trapped within the same microwell. Here we describe implementations of live-cell imaging within microwell arrays for studies of lymphocyte clonal expansion, portray insights already gained from these experiments and outline directions for future research. These tools, together with in vivo experiments tracking single-cell responses, will expand our understanding of adaptive immunity and the ways by which it can be manipulated.

  15. Effect of infrared light on live blood cells: Role of β-carotene.

    Science.gov (United States)

    Barkur, Surekha; Bankapur, Aseefhali; Chidangil, Santhosh; Mathur, Deepak

    2017-06-01

    We have utilized Raman tweezers to measure and assign micro-Raman spectra of optically trapped, live red blood cells (RBCs), white blood cells (WBCs) and platelets. Various types of WBCs- both granulocytes, lymphocytes, and their different types have been studied. The Raman bands are assigned to different biomolecules of blood cells. The Raman spectra thus obtained has been enabled detection of β-carotene in these blood cells, the spectral features of which act as a signature that facilitates experimental probing of the effect of 785nm laser light on different blood cells as a function of incident laser power in the mW range. The spectral changes that we obtain upon laser irradiation indicate that, both haemoglobin as well as the cell membrane sustains damage. In case of lymphocytes and platelets the peaks corresponding to β-carotene showed drastic changes. Thorough analysis of the spectral changes indicates possibility of free radical induced damage of β-carotene in lymphocytes and platelets. Among different blood cells, RBCs have a power threshold of only 10mW. The power threshold for other types of blood cells is somewhat higher, but always below about 30mW. These values are likely to serve as useful guides for Raman tweezers based experiments on live cells. Copyright © 2017. Published by Elsevier B.V.

  16. Live-cell super-resolution imaging of intrinsically fast moving flagellates

    International Nuclear Information System (INIS)

    Glogger, M; Subota, I; Spindler, M-C; Engstler, M; Fenz, S F; Stichler, S; Bertlein, S; Teßmar, J; Groll, J

    2017-01-01

    Recent developments in super-resolution microscopy make it possible to resolve structures in biological cells at a spatial resolution of a few nm and observe dynamical processes with a temporal resolution of ms to μ s. However, the optimal structural resolution requires repeated illumination cycles and is thus limited to chemically fixed cells. For live cell applications substantial improvement over classical Abbe-limited imaging can already be obtained in adherent or slow moving cells. Nonetheless, a large group of cells are fast moving and thus could not yet be addressed with live cell super-resolution microscopy. These include flagellate pathogens like African trypanosomes, the causative agents of sleeping sickness in humans and nagana in livestock. Here, we present an embedding method based on a in situ forming cytocompatible UV-crosslinked hydrogel. The fast cross-linking hydrogel immobilizes trypanosomes efficiently to allow microscopy on the nanoscale. We characterized both the trypanosomes and the hydrogel with respect to their autofluorescence properties and found them suitable for single-molecule fluorescence microscopy (SMFM). As a proof of principle, SMFM was applied to super-resolve a structure inside the living trypanosome. We present an image of a flagellar axoneme component recorded by using the intrinsic blinking behavior of eYFP. (paper)

  17. Hoechst tagging: a modular strategy to design synthetic fluorescent probes for live-cell nucleus imaging.

    Science.gov (United States)

    Nakamura, Akinobu; Takigawa, Kazumasa; Kurishita, Yasutaka; Kuwata, Keiko; Ishida, Manabu; Shimoda, Yasushi; Hamachi, Itaru; Tsukiji, Shinya

    2014-06-11

    We report a general strategy to create small-molecule fluorescent probes for the nucleus in living cells. Our strategy is based on the attachment of the DNA-binding Hoechst compound to a fluorophore of interest. Using this approach, simple fluorescein, BODIPY, and rhodamine dyes were readily converted to novel turn-on fluorescent nucleus-imaging probes.

  18. The spatio-temporal organization of DNA-repair: a live cell study

    NARCIS (Netherlands)

    D. Hoogstraten (Deborah)

    2003-01-01

    textabstractThe aim of the work outlined in this thesis is to gain more insight into the organization, dynamic properties and differential reaction kinetics of NER factors within living mammalian cell nuclei. To accomplish this, we made use of the green fluorescent protein technology to study TFIIH

  19. Using in Vitro live-cell imaging to explore chemotherapeutics delivered by lipid-based nanoparticles

    NARCIS (Netherlands)

    A.L.B. Seynhaeve (Ann); T.L.M. ten Hagen (Timo)

    2017-01-01

    textabstractConventional imaging techniques can provide detailed information about cellular processes. However, this information is based on static images in an otherwise dynamic system, and successive phases are easily overlooked or misinterpreted. Live-cell imaging and time-lapse microscopy, in

  20. Fungicidal mechanisms of cathelicidins LL-37 and CATH-2 revealed by live-cell imaging

    NARCIS (Netherlands)

    Ordonez Alvarez, Soledad; Amarullah, Ilham H; Wubbolts, Richard W; Veldhuizen, Edwin J A; Haagsman, Henk P

    2014-01-01

    Antifungal mechanisms of action of two cathelicidins, chicken CATH-2 and human LL-37, were studied and compared with the mode of action of the salivary peptide histatin 5 (Hst5). Candida albicans was used as a model organism for fungal pathogens. Analysis by live-cell imaging showed that the

  1. Live cell CRISPR-imaging in plants reveals dynamic telomere movements

    KAUST Repository

    Dreissig, Steven; Schiml, Simon; Schindele, Patrick; Weiss, Oda; Rutten, Twan; Schubert, Veit; Gladilin, Evgeny; Mette, Michael F.; Puchta, Holger; Houben, Andreas

    2017-01-01

    of the bacterial CRISPR-Cas9 system. By fusing eGFP/mRuby2 to the catalytically inactive version of Streptococcus pyogenes and Staphylococcus aureus Cas9, we show robust visualization of telomere repeats in live leaf cells of Nicotiana benthamiana. By tracking

  2. Synthesis, biological evaluation, and live cell imaging of novel fluorescent duocarmycin analogs.

    Science.gov (United States)

    Tietze, Lutz F; Behrendt, Frank; Pestel, Galina F; Schuberth, Ingrid; Mitkovski, Mišo

    2012-11-01

    For a better understanding of the mode of action of duocarmycin and its analogs, the novel fluorescent duocarmycin derivatives 13-15 and 17b-19b were synthesized, and their bioactivity as well as their cellular uptake investigated using confocal laser scanning microscopy (CLSM) in live-cell imaging experiments. Copyright © 2012 Verlag Helvetica Chimica Acta AG, Zürich.

  3. Caveolae-mediated endocytosis of biocompatible gold nanoparticles in living Hela cells

    DEFF Research Database (Denmark)

    Hao, Xian; Wu, Jiazhen; Shan, Yuping

    2012-01-01

    the internalization mechanism of small-size AuNPs by living Hela cells. Herein, we found that the caveolae-mediated endocytosis was the dominant pathway for the intracellular delivery of small-size AuNPs. The intracellular delivery was suppressed when we depleted the cholesterol with methyl-β-cyclodextrin (M beta CD...

  4. Live-cell calcium imaging of adherent and non-adherent GL261 cells reveals phenotype-dependent differences in drug responses.

    Science.gov (United States)

    Strong, Averey D; Daniels, Richard L

    2017-08-02

    The tumor-derived GL261 cell line is used as a model for studying glioblastoma and other high-grade gliomas, and can be cultured adherently or as free-floating aggregates known as neurospheres. These different culture conditions give rise to distinct phenotypes, with increased tumorigenicity displayed by neurosphere-cultured cells. An important technique for understanding GL261 pathobiology is live cell fluorescent imaging of intracellular calcium. However, live cell imaging of GL261 neurospheres presents a technical challenge, as experimental manipulations where drugs are added to the extracellular media cause the cells to move during analysis. Here we present a method to immobilize GL261 neurospheres with low melting point agarose for calcium imaging using the fluorescent calcium sensor fura-2. GL261 cells were obtained from the NCI-Frederick Cancer Research Tumor Repository and cultured as adherent cells or induced to form neurospheres by placing freshly trypsinized cells into serum-free media containing fibroblast growth factor 2, epidermal growth factor, and B-27 supplement. Prior to experiments, adherent cells were loaded with fura-2 and cultured on 8-well chamber slides. Non-adherent neurospheres were first loaded with fura-2, placed in droplets onto an 8-well chamber slide, and finally covered with a thin layer of low melting point agarose to immobilize the cells. Ratiometric pseudocolored images were obtained during treatment with ATP, capsaicin, or vehicle control. Cells were marked as responsive if fluorescence levels increased more than 30% above baseline. Differences between treatment groups were tested using Student's t-tests and one-way ANOVA. We found that cellular responses to pharmacological treatments differ based on cellular phenotype. Adherent cells and neurospheres both responded to ATP with a rise in intracellular calcium. Notably, capsaicin treatment led to robust responses in GL261 neurospheres but not adherent cells. We demonstrate the use

  5. Automatic analysis of dividing cells in live cell movies to detect mitotic delays and correlate phenotypes in time.

    Science.gov (United States)

    Harder, Nathalie; Mora-Bermúdez, Felipe; Godinez, William J; Wünsche, Annelie; Eils, Roland; Ellenberg, Jan; Rohr, Karl

    2009-11-01

    Live-cell imaging allows detailed dynamic cellular phenotyping for cell biology and, in combination with small molecule or drug libraries, for high-content screening. Fully automated analysis of live cell movies has been hampered by the lack of computational approaches that allow tracking and recognition of individual cell fates over time in a precise manner. Here, we present a fully automated approach to analyze time-lapse movies of dividing cells. Our method dynamically categorizes cells into seven phases of the cell cycle and five aberrant morphological phenotypes over time. It reliably tracks cells and their progeny and can thus measure the length of mitotic phases and detect cause and effect if mitosis goes awry. We applied our computational scheme to annotate mitotic phenotypes induced by RNAi gene knockdown of CKAP5 (also known as ch-TOG) or by treatment with the drug nocodazole. Our approach can be readily applied to comparable assays aiming at uncovering the dynamic cause of cell division phenotypes.

  6. Mapping photothermally induced gene expression in living cells and tissues by nanorod-locked nucleic acid complexes.

    Science.gov (United States)

    Riahi, Reza; Wang, Shue; Long, Min; Li, Na; Chiou, Pei-Yu; Zhang, Donna D; Wong, Pak Kin

    2014-04-22

    The photothermal effect of plasmonic nanostructures has numerous applications, such as cancer therapy, photonic gene circuit, large cargo delivery, and nanostructure-enhanced laser tweezers. The photothermal operation can also induce unwanted physical and biochemical effects, which potentially alter the cell behaviors. However, there is a lack of techniques for characterizing the dynamic cell responses near the site of photothermal operation with high spatiotemporal resolution. In this work, we show that the incorporation of locked nucleic acid probes with gold nanorods allows photothermal manipulation and real-time monitoring of gene expression near the area of irradiation in living cells and animal tissues. The multimodal gold nanorod serves as an endocytic delivery reagent to transport the probes into the cells, a fluorescence quencher and a binding competitor to detect intracellular mRNA, and a plasmonic photothermal transducer to induce cell ablation. We demonstrate the ability of the gold nanorod-locked nucleic acid complex for detecting the spatiotemporal gene expression in viable cells and tissues and inducing photothermal ablation of single cells. Using the gold nanorod-locked nucleic acid complex, we systematically characterize the dynamic cellular heat shock responses near the site of photothermal operation. The gold nanorod-locked nucleic acid complex enables mapping of intracellular gene expressions and analyzes the photothermal effects of nanostructures toward various biomedical applications.

  7. Intracellular water exchange for measuring the dry mass, water mass and changes in chemical composition of living cells.

    Directory of Open Access Journals (Sweden)

    Francisco Feijó Delgado

    Full Text Available We present a method for direct non-optical quantification of dry mass, dry density and water mass of single living cells in suspension. Dry mass and dry density are obtained simultaneously by measuring a cell's buoyant mass sequentially in an H2O-based fluid and a D2O-based fluid. Rapid exchange of intracellular H2O for D2O renders the cell's water content neutrally buoyant in both measurements, and thus the paired measurements yield the mass and density of the cell's dry material alone. Utilizing this same property of rapid water exchange, we also demonstrate the quantification of intracellular water mass. In a population of E. coli, we paired these measurements to estimate the percent dry weight by mass and volume. We then focused on cellular dry density - the average density of all cellular biomolecules, weighted by their relative abundances. Given that densities vary across biomolecule types (RNA, DNA, protein, we investigated whether we could detect changes in biomolecular composition in bacteria, fungi, and mammalian cells. In E. coli, and S. cerevisiae, dry density increases from stationary to exponential phase, consistent with previously known increases in the RNA/protein ratio from up-regulated ribosome production. For mammalian cells, changes in growth conditions cause substantial shifts in dry density, suggesting concurrent changes in the protein, nucleic acid and lipid content of the cell.

  8. Correlation between live attenuated measles viral load and growth inhibition percentage in non-small cell lung cancer cell line

    Directory of Open Access Journals (Sweden)

    Rasha Fadhel Obaid

    2018-03-01

    Conclusion Live attenuated measles virus strain induced cytotoxic effect against human lung cancer cell line (A549 by induction of apoptosis as an important mechanism of anti-tumor activity, in addition, it indicates a correlation between the quantity of MV genomesand percentage of growth inhibition. This relation  has proved that measles virus had anticancer effect.

  9. In vivo MRI discrimination between live and lysed iron-labelled cells using balanced steady state free precession

    International Nuclear Information System (INIS)

    Ribot, E.J.; Foster, P.J.

    2012-01-01

    The goal of this study was to evaluate the ability of balanced steady state free precession (b-SSFP) magnetic resonance imaging sequence to distinguish between live and lysed iron-labelled cells. Human breast cancer cells were labelled with iron oxide nanoparticles. Cells were lysed using sonication. Imaging was performed at 3 T. The timing parameters for b-SSFP and the number of iron-labelled cells in samples were varied to optimise the b-SSFP signal difference between live and lysed iron-labelled cell samples. For in vivo experiments, cells were mixed with Matrigel and implanted into nude mice. Three mice implanted with live labelled cancer cells were irradiated to validate this method. Lysed iron-labelled cells have a significantly higher signal compared with live, intact iron-labelled cells in bSSFP images. The contrast between live and dead cells can be maximised by careful optimisation of timing parameters. A change in the b-SSFP signal was measured 6 days after irradiation, reflecting cell death in vivo. Histology confirmed the presence of dead cells in the implant. Our results show that the b-SSFP sequence can be optimised to allow for the discrimination of live iron-labelled cells and lysed iron-labelled cells in vitro and in vivo. (orig.)

  10. In vivo MRI discrimination between live and lysed iron-labelled cells using balanced steady state free precession

    Energy Technology Data Exchange (ETDEWEB)

    Ribot, E.J. [University of Western Ontario, Imaging Research Laboratories, Robarts Research Institute, London, ON (Canada); Foster, P.J. [University of Western Ontario, Imaging Research Laboratories, Robarts Research Institute, London, ON (Canada); University of Western Ontario, Department of Medical Biophysics, London, ON (Canada)

    2012-09-15

    The goal of this study was to evaluate the ability of balanced steady state free precession (b-SSFP) magnetic resonance imaging sequence to distinguish between live and lysed iron-labelled cells. Human breast cancer cells were labelled with iron oxide nanoparticles. Cells were lysed using sonication. Imaging was performed at 3 T. The timing parameters for b-SSFP and the number of iron-labelled cells in samples were varied to optimise the b-SSFP signal difference between live and lysed iron-labelled cell samples. For in vivo experiments, cells were mixed with Matrigel and implanted into nude mice. Three mice implanted with live labelled cancer cells were irradiated to validate this method. Lysed iron-labelled cells have a significantly higher signal compared with live, intact iron-labelled cells in bSSFP images. The contrast between live and dead cells can be maximised by careful optimisation of timing parameters. A change in the b-SSFP signal was measured 6 days after irradiation, reflecting cell death in vivo. Histology confirmed the presence of dead cells in the implant. Our results show that the b-SSFP sequence can be optimised to allow for the discrimination of live iron-labelled cells and lysed iron-labelled cells in vitro and in vivo. (orig.)

  11. Lateral motion and bending of microtubules studied with a new single-filament tracking routine in living cells.

    Science.gov (United States)

    Pallavicini, Carla; Levi, Valeria; Wetzler, Diana E; Angiolini, Juan F; Benseñor, Lorena; Despósito, Marcelo A; Bruno, Luciana

    2014-06-17

    The cytoskeleton is involved in numerous cellular processes such as migration, division, and contraction and provides the tracks for transport driven by molecular motors. Therefore, it is very important to quantify the mechanical behavior of the cytoskeletal filaments to get a better insight into cell mechanics and organization. It has been demonstrated that relevant mechanical properties of microtubules can be extracted from the analysis of their motion and shape fluctuations. However, tracking individual filaments in living cells is extremely complex due, for example, to the high and heterogeneous background. We introduce a believed new tracking algorithm that allows recovering the coordinates of fluorescent microtubules with ∼9 nm precision in in vitro conditions. To illustrate potential applications of this algorithm, we studied the curvature distributions of fluorescent microtubules in living cells. By performing a Fourier analysis of the microtubule shapes, we found that the curvatures followed a thermal-like distribution as previously reported with an effective persistence length of ∼20 μm, a value significantly smaller than that measured in vitro. We also verified that the microtubule-associated protein XTP or the depolymerization of the actin network do not affect this value; however, the disruption of intermediate filaments decreased the persistence length. Also, we recovered trajectories of microtubule segments in actin or intermediate filament-depleted cells, and observed a significant increase of their motion with respect to untreated cells showing that these filaments contribute to the overall organization of the microtubule network. Moreover, the analysis of trajectories of microtubule segments in untreated cells showed that these filaments presented a slower but more directional motion in the cortex with respect to the perinuclear region, and suggests that the tracking routine would allow mapping the microtubule dynamical organization in cells

  12. Live cell imaging combined with high-energy single-ion microbeam

    Energy Technology Data Exchange (ETDEWEB)

    Guo, Na; Du, Guanghua, E-mail: gh-du@impcas.ac.cn; Liu, Wenjing; Wu, Ruqun; Wei, Junzhe [Institute of Modern Physics, Chinese Academy of Sciences, Lanzhou (China); Guo, Jinlong [Institute of Modern Physics, Chinese Academy of Sciences, Lanzhou (China); Northwest Normal University, Lanzhou (China); Chen, Hao [Institute of Modern Physics, Chinese Academy of Sciences, Lanzhou (China); Institute of Nuclear Science and Technology, University of Lanzhou, Lanzhou (China)

    2016-03-15

    DNA strand breaks can lead to cell carcinogenesis or cell death if not repaired rapidly and efficiently. An online live cell imaging system was established at the high energy microbeam facility at the Institute of Modern Physics to study early and fast cellular response to DNA damage after high linear energy transfer ion radiation. The HT1080 cells expressing XRCC1-RFP were irradiated with single high energy nickel ions, and time-lapse images of the irradiated cells were obtained online. The live cell imaging analysis shows that strand-break repair protein XRCC1 was recruited to the ion hit position within 20 s in the cells and formed bright foci in the cell nucleus. The fast recruitment of XRCC1 at the ion hits reached a maximum at about 200 s post-irradiation and then was followed by a slower release into the nucleoplasm. The measured dual-exponential kinetics of XRCC1 protein are consistent with the proposed consecutive reaction model, and the measurements obtained that the reaction rate constant of the XRCC1 recruitment to DNA strand break is 1.2 × 10{sup −3} s{sup −1} and the reaction rate constant of the XRCC1 release from the break-XRCC1 complex is 1.2 × 10{sup −2} s{sup −1}.

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

    Directory of Open Access Journals (Sweden)

    Ken-Ichi Wada

    2017-12-01

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

  14. Live cell imaging combined with high-energy single-ion microbeam

    International Nuclear Information System (INIS)

    Guo, Na; Du, Guanghua; Liu, Wenjing; Wu, Ruqun; Wei, Junzhe; Guo, Jinlong; Chen, Hao

    2016-01-01

    DNA strand breaks can lead to cell carcinogenesis or cell death if not repaired rapidly and efficiently. An online live cell imaging system was established at the high energy microbeam facility at the Institute of Modern Physics to study early and fast cellular response to DNA damage after high linear energy transfer ion radiation. The HT1080 cells expressing XRCC1-RFP were irradiated with single high energy nickel ions, and time-lapse images of the irradiated cells were obtained online. The live cell imaging analysis shows that strand-break repair protein XRCC1 was recruited to the ion hit position within 20 s in the cells and formed bright foci in the cell nucleus. The fast recruitment of XRCC1 at the ion hits reached a maximum at about 200 s post-irradiation and then was followed by a slower release into the nucleoplasm. The measured dual-exponential kinetics of XRCC1 protein are consistent with the proposed consecutive reaction model, and the measurements obtained that the reaction rate constant of the XRCC1 recruitment to DNA strand break is 1.2 × 10"−"3 s"−"1 and the reaction rate constant of the XRCC1 release from the break-XRCC1 complex is 1.2 × 10"−"2 s"−"1.

  15. Live cell imaging compatible immobilization of Chlamydomonas reinhardtii in microfluidic platform for biodiesel research.

    Science.gov (United States)

    Park, Jae Woo; Na, Sang Cheol; Nguyen, Thanh Qua; Paik, Sang-Min; Kang, Myeongwoo; Hong, Daewha; Choi, Insung S; Lee, Jae-Hyeok; Jeon, Noo Li

    2015-03-01

    This paper describes a novel surface immobilization method for live-cell imaging of Chlamydomonas reinhardtii for continuous monitoring of lipid droplet accumulation. Microfluidics allows high-throughput manipulation and analysis of single cells in precisely controlled microenvironment. Fluorescence imaging based quantitative measurement of lipid droplet accumulation in microalgae had been difficult due to their intrinsic motile behavior. We present a simple surface immobilization method using gelatin coating as the "biological glue." We take advantage of hydroxyproline (Hyp)-based non-covalent interaction between gelatin and the outer cell wall of microalgae to anchor the cells inside the microfluidic device. We have continuously monitored single microalgal cells for up to 6 days. The immobilized microalgae remain viable (viability was comparable to bulk suspension cultured controls). When exposed to wall shear stress, most of the cells remain attached up to 0.1 dyne/cm(2) . Surface immobilization allowed high-resolution, live-cell imaging of mitotic process in real time-which followed previously reported stages in mitosis of suspension cultured cells. Use of gelatin coated microfluidics devices can result in better methods for microalgae strain screening and culture condition optimization that will help microalgal biodiesel become more economically viable. © 2014 Wiley Periodicals, Inc.

  16. Live cell imaging combined with high-energy single-ion microbeam

    Science.gov (United States)

    Guo, Na; Du, Guanghua; Liu, Wenjing; Guo, Jinlong; Wu, Ruqun; Chen, Hao; Wei, Junzhe

    2016-03-01

    DNA strand breaks can lead to cell carcinogenesis or cell death if not repaired rapidly and efficiently. An online live cell imaging system was established at the high energy microbeam facility at the Institute of Modern Physics to study early and fast cellular response to DNA damage after high linear energy transfer ion radiation. The HT1080 cells expressing XRCC1-RFP were irradiated with single high energy nickel ions, and time-lapse images of the irradiated cells were obtained online. The live cell imaging analysis shows that strand-break repair protein XRCC1 was recruited to the ion hit position within 20 s in the cells and formed bright foci in the cell nucleus. The fast recruitment of XRCC1 at the ion hits reached a maximum at about 200 s post-irradiation and then was followed by a slower release into the nucleoplasm. The measured dual-exponential kinetics of XRCC1 protein are consistent with the proposed consecutive reaction model, and the measurements obtained that the reaction rate constant of the XRCC1 recruitment to DNA strand break is 1.2 × 10-3 s-1 and the reaction rate constant of the XRCC1 release from the break-XRCC1 complex is 1.2 × 10-2 s-1.

  17. Long-term live cell imaging and automated 4D analysis of drosophila neuroblast lineages.

    Directory of Open Access Journals (Sweden)

    Catarina C F Homem

    Full Text Available The developing Drosophila brain is a well-studied model system for neurogenesis and stem cell biology. In the Drosophila central brain, around 200 neural stem cells called neuroblasts undergo repeated rounds of asymmetric cell division. These divisions typically generate a larger self-renewing neuroblast and a smaller ganglion mother cell that undergoes one terminal division to create two differentiating neurons. Although single mitotic divisions of neuroblasts can easily be imaged in real time, the lack of long term imaging procedures has limited the use of neuroblast live imaging for lineage analysis. Here we describe a method that allows live imaging of cultured Drosophila neuroblasts over multiple cell cycles for up to 24 hours. We describe a 4D image analysis protocol that can be used to extract cell cycle times and growth rates from the resulting movies in an automated manner. We use it to perform lineage analysis in type II neuroblasts where clonal analysis has indicated the presence of a transit-amplifying population that potentiates the number of neurons. Indeed, our experiments verify type II lineages and provide quantitative parameters for all cell types in those lineages. As defects in type II neuroblast lineages can result in brain tumor formation, our lineage analysis method will allow more detailed and quantitative analysis of tumorigenesis and asymmetric cell division in the Drosophila brain.

  18. A device for real-time live-cell microscopy during dynamic dual-modal mechanostimulation

    Science.gov (United States)

    Lorusso, D.; Nikolov, H. N.; Chmiel, T.; Beach, R. J.; Sims, S. M.; Dixon, S. J.; Holdsworth, D. W.

    2017-03-01

    Mechanotransduction - the process by which cells sense and respond to mechanical stimuli - is essential for several physiological processes including skeletal homeostasis. Mammalian cells are thought to be sensitive to different modes of mechanical stimuli, including vibration and fluid shear. To better understand the mechanisms underlying the early stages of mechanotransduction, we describe the development of devices for mechanostimulation (by vibration and fluid shear) of live cells that can be integrated with real-time optical microscopy. The integrated system can deliver up to 3 Pa of fluid shear simultaneous with high-frequency sinusoidal vibrations up to 1 g. Stimuli can be applied simultaneously or independently to cells during real-time microscopic imaging. A custom microfluidic chamber was prepared from polydimethylsiloxane on a glass-bottom cell culture dish. Fluid flow was applied with a syringe pump to induce shear stress. This device is compatible with a custom-designed motion control vibration system. A voice coil actuates the system that is suspended on linear air bushings. Accelerations produced by the system were monitored with an on-board accelerometer. Displacement was validated optically using particle tracking digital high-speed imaging (1200 frames per second). During operation at nominally 45 Hz and 0.3 g, displacements were observed to be within 3.56% of the expected value. MC3T3-E1 osteoblast like cells were seeded into the microfluidic device and loaded with the calcium sensitive fluorescent probe fura-2, then mounted onto the dual-modal mechanostimulation platform. Cells were then imaged and monitored for fluorescence emission. In summary, we have developed a system to deliver physiologically relevant vibrations and fluid shear to live cells during real-time imaging and photometry. Monitoring the behavior of live cells loaded with appropriate fluorescent probes will enable characterization of the signals activated during the initial

  19. Endogenous Fluorescence Signatures in Living Pluripotent Stem Cells Change with Loss of Potency

    Science.gov (United States)

    Squirrell, Jayne M.; Fong, Jimmy J.; Ariza, Carlos A.; Mael, Amber; Meyer, Kassondra; Shevde, Nirupama K.; Roopra, Avtar; Lyons, Gary E.; Kamp, Timothy J.; Eliceiri, Kevin W.; Ogle, Brenda M.

    2012-01-01

    The therapeutic potential of stem cells is limited by the non-uniformity of their phenotypic state. Thus it would be advantageous to noninvasively monitor stem cell status. Driven by this challenge, we employed multidimensional multiphoton microscopy to quantify changes in endogenous fluorescence occurring with pluripotent stem cell differentiation. We found that global and cellular-scale fluorescence lifetime of human embryonic stem cells (hESC) and murine embryonic stem cells (mESC) consistently decreased with differentiation. Less consistent were trends in endogenous fluorescence intensity with differentiation, suggesting intensity is more readily impacted by nuances of species and scale of analysis. What emerges is a practical and accessible approach to evaluate, and ultimately enrich, living stem cell populations based on changes in metabolism that could be exploited for both research and clinical applications. PMID:22952742

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

    Directory of Open Access Journals (Sweden)

    Qi Tang

    2016-04-01

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

  1. Single Molecule Detection in Living Biological Cells using Carbon Nanotube Optical Probes

    Science.gov (United States)

    Strano, Michael

    2009-03-01

    Nanoscale sensing elements offer promise for single molecule analyte detection in physically or biologically constrained environments. Molecular adsorption can be amplified via modulation of sharp singularities in the electronic density of states that arise from 1D quantum confinement [1]. Single-walled carbon nanotubes (SWNT), as single molecule optical sensors [2-3], offer unique advantages such as photostable near-infrared (n-IR) emission for prolonged detection through biological media, single-molecule sensitivity and, nearly orthogonal optical modes for signal transduction that can be used to identify distinct classes of analytes. Selective binding to the SWNT surface is difficult to engineer [4]. In this lecture, we will briefly review the immerging field of fluorescent diagnostics using band gap emission from SWNT. In recent work, we demonstrate that even a single pair of SWNT provides at least four optical modes that can be modulated to uniquely fingerprint chemical agents by the degree to which they alter either the emission band intensity or wavelength. We validate this identification method in vitro by demonstrating detection and identification of six genotoxic analytes, including chemotherapeutic drugs and reactive oxygen species (ROS), which are spectroscopically differentiated into four distinct classes. We also demonstrate single-molecule sensitivity in detecting hydrogen peroxide, one of the most common genotoxins and an important cellular signal. Finally, we employ our sensing and fingerprinting method of these analytes in real time within live 3T3 cells, demonstrating the first multiplexed optical detection from a nanoscale biosensor and the first label-free tool to optically discriminate between genotoxins. We will also discuss our recent efforts to fabricate biomedical sensors for real time detection of glucose and other important physiologically relevant analytes in-vivo. The response of embedded SWNT in a swellable hydrogel construct to

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

    Science.gov (United States)

    Peng, Cheng

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

  3. Multifocus confocal Raman microspectroscopy for fast multimode vibrational imaging of living cells.

    Science.gov (United States)

    Okuno, Masanari; Hamaguchi, Hiro-o

    2010-12-15

    We have developed a multifocus confocal Raman microspectroscopic system for the fast multimode vibrational imaging of living cells. It consists of an inverted microscope equipped with a microlens array, a pinhole array, a fiber bundle, and a multichannel Raman spectrometer. Forty-eight Raman spectra from 48 foci under the microscope are simultaneously obtained by using multifocus excitation and image-compression techniques. The multifocus confocal configuration suppresses the background generated from the cover glass and the cell culturing medium so that high-contrast images are obtainable with a short accumulation time. The system enables us to obtain multimode (10 different vibrational modes) vibrational images of living cells in tens of seconds with only 1 mW laser power at one focal point. This image acquisition time is more than 10 times faster than that in conventional single-focus Raman microspectroscopy.

  4. Live-cell imaging of conidial anastomosis tube fusion during colony initiation in Fusarium oxysporum.

    Directory of Open Access Journals (Sweden)

    Smija M Kurian

    Full Text Available Fusarium oxysporum exhibits conidial anastomosis tube (CAT fusion during colony initiation to form networks of conidial germlings. Here we determined the optimal culture conditions for this fungus to undergo CAT fusion between microconidia in liquid medium. Extensive high resolution, confocal live-cell imaging was performed to characterise the different stages of CAT fusion, using genetically encoded fluorescent labelling and vital fluorescent organelle stains. CAT homing and fusion were found to be dependent on adhesion to the surface, in contrast to germ tube development which occurs in the absence of adhesion. Staining with fluorescently labelled concanavalin A indicated that the cell wall composition of CATs differs from that of microconidia and germ tubes. The movement of nuclei, mitochondria, vacuoles and lipid droplets through fused germlings was observed by live-cell imaging.

  5. Living biointerfaces based on non-pathogenic bacteria to direct cell differentiation

    Science.gov (United States)

    Rodrigo-Navarro, Aleixandre; Rico, Patricia; Saadeddin, Anas; Garcia, Andres J.; Salmeron-Sanchez, Manuel

    2014-07-01

    Genetically modified Lactococcus lactis, non-pathogenic bacteria expressing the FNIII7-10 fibronectin fragment as a protein membrane have been used to create a living biointerface between synthetic materials and mammalian cells. This FNIII7-10 fragment comprises the RGD and PHSRN sequences of fibronectin to bind α5β1 integrins and triggers signalling for cell adhesion, spreading and differentiation. We used L. lactis strain to colonize material surfaces and produce stable biofilms presenting the FNIII7-10 fragment readily available to cells. Biofilm density is easily tunable and remains stable for several days. Murine C2C12 myoblasts seeded over mature biofilms undergo bipolar alignment and form differentiated myotubes, a process triggered by the FNIII7-10 fragment. This biointerface based on living bacteria can be further modified to express any desired biochemical signal, establishing a new paradigm in biomaterial surface functionalisation for biomedical applications.

  6. Combination of Small Molecule Microarray and Confocal Microscopy Techniques for Live Cell Staining Fluorescent Dye Discovery

    Directory of Open Access Journals (Sweden)

    Attila Bokros

    2013-08-01

    Full Text Available Discovering new fluorochromes is significantly advanced by high-throughput screening (HTS methods. In the present study a combination of small molecule microarray (SMM prescreening and confocal laser scanning microscopy (CLSM was developed in order to discover novel cell staining fluorescent dyes. Compounds with high native fluorescence were selected from a 14,585-member library and further tested on living cells under the microscope. Eleven compartment-specific, cell-permeable (or plasma membrane-targeted fluorochromes were identified. Their cytotoxicity was tested and found that between 1–10 micromolar range, they were non-toxic even during long-term incubations.

  7. Concentration-dependent fluorescence live-cell imaging and tracking of intracellular nanoparticles

    Energy Technology Data Exchange (ETDEWEB)

    Seo, Ji Hye; Joo, Sang-Woo [Department of Chemistry, Soongsil University, Seoul 156-743 (Korea, Republic of); Cho, Keunchang [Logos Biosystems, Incorporated, Anyang 431-070 (Korea, Republic of); Lee, So Yeong, E-mail: leeso@snu.ac.kr, E-mail: sjoo@ssu.ac.kr [Laboratory of Pharmacology, College of Veterinary Medicine and Research Institute for Veterinary Science, Seoul National University, Seoul 151-742 (Korea, Republic of)

    2011-06-10

    Using live-cell imaging techniques we investigated concentration-dependent intracellular movements of fluorescence nanoparticles (NPs) in real-time after their entry into HeLa cells via incubation. Intracellular particle traces appeared to be a mixture of both random and fairly unidirectional movements of the particles. At rather low concentrations of NPs, a majority of the non-random intracellular particle trajectories are assumed to mostly go along microtubule networks after endocytosis, as evidenced from the inhibition test with nocodazole. On the other hand, as the concentrations of NPs increased, random motions were more frequently observed inside the cells.

  8. Concentration-dependent fluorescence live-cell imaging and tracking of intracellular nanoparticles

    International Nuclear Information System (INIS)

    Seo, Ji Hye; Joo, Sang-Woo; Cho, Keunchang; Lee, So Yeong

    2011-01-01

    Using live-cell imaging techniques we investigated concentration-dependent intracellular movements of fluorescence nanoparticles (NPs) in real-time after their entry into HeLa cells via incubation. Intracellular particle traces appeared to be a mixture of both random and fairly unidirectional movements of the particles. At rather low concentrations of NPs, a majority of the non-random intracellular particle trajectories are assumed to mostly go along microtubule networks after endocytosis, as evidenced from the inhibition test with nocodazole. On the other hand, as the concentrations of NPs increased, random motions were more frequently observed inside the cells.

  9. Horizontal gene transfers with or without cell fusions in all categories of the living matter.

    Science.gov (United States)

    Sinkovics, Joseph G

    2011-01-01

    This article reviews the history of widespread exchanges of genetic segments initiated over 3 billion years ago, to be part of their life style, by sphero-protoplastic cells, the ancestors of archaea, prokaryota, and eukaryota. These primordial cells shared a hostile anaerobic and overheated environment and competed for survival. "Coexist with, or subdue and conquer, expropriate its most useful possessions, or symbiose with it, your competitor" remain cellular life's basic rules. This author emphasizes the role of viruses, both in mediating cell fusions, such as the formation of the first eukaryotic cell(s) from a united crenarchaeon and prokaryota, and the transfer of host cell genes integrated into viral (phages) genomes. After rising above the Darwinian threshold, rigid rules of speciation and vertical inheritance in the three domains of life were established, but horizontal gene transfers with or without cell fusions were never abolished. The author proves with extensive, yet highly selective documentation, that not only unicellular microorganisms, but the most complex multicellular entities of the highest ranks resort to, and practice, cell fusions, and donate and accept horizontally (laterally) transferred genes. Cell fusions and horizontally exchanged genetic materials remain the fundamental attributes and inherent characteristics of the living matter, whether occurring accidentally or sought after intentionally. These events occur to cells stagnating for some 3 milliard years at a lower yet amazingly sophisticated level of evolution, and to cells achieving the highest degree of differentiation, and thus functioning in dependence on the support of a most advanced multicellular host, like those of the human brain. No living cell is completely exempt from gene drains or gene insertions.

  10. Modeling minor actinide multiple recycling in a lead-cooled fast reactor to demonstrate a fuel cycle without long-lived nuclear waste

    Directory of Open Access Journals (Sweden)

    Stanisz Przemysław

    2015-09-01

    Full Text Available The concept of closed nuclear fuel cycle seems to be the most promising options for the efficient usage of the nuclear energy resources. However, it can be implemented only in fast breeder reactors of the IVth generation, which are characterized by the fast neutron spectrum. The lead-cooled fast reactor (LFR was defined and studied on the level of technical design in order to demonstrate its performance and reliability within the European collaboration on ELSY (European Lead-cooled System and LEADER (Lead-cooled European Advanced Demonstration Reactor projects. It has been demonstrated that LFR meets the requirements of the closed nuclear fuel cycle, where plutonium and minor actinides (MA are recycled for reuse, thereby producing no MA waste. In this study, the most promising option was realized when entire Pu + MA material is fully recycled to produce a new batch of fuel without partitioning. This is the concept of a fuel cycle which asymptotically tends to the adiabatic equilibrium, where the concentrations of plutonium and MA at the beginning of the cycle are restored in the subsequent cycle in the combined process of fuel transmutation and cooling, removal of fission products (FPs, and admixture of depleted uranium. In this way, generation of nuclear waste containing radioactive plutonium and MA can be eliminated. The paper shows methodology applied to the LFR equilibrium fuel cycle assessment, which was developed for the Monte Carlo continuous energy burnup (MCB code, equipped with enhanced modules for material processing and fuel handling. The numerical analysis of the reactor core concerns multiple recycling and recovery of long-lived nuclides and their influence on safety parameters. The paper also presents a general concept of the novel IVth generation breeder reactor with equilibrium fuel and its future role in the management of MA.

  11. Secretory vesicles in live cells are not free-floating but tethered to filamentous structures: A study using photonic force microscopy

    International Nuclear Information System (INIS)

    Abu-Hamdah, Rania; Cho, Won Jin; Hoerber, J.K.H.; Jena, Bhanu P.

    2006-01-01

    It is well established that actin and microtubule cytoskeletal systems are involved in organelle transport and membrane trafficking in cells. This is also true for the transport of secretory vesicles in neuroendocrine cells and neurons. It was however unclear whether secretory vesicles remain free-floating, only to associate with such cytoskeletal systems when needing transport. This hypothesis was tested using live pancreatic acinar cells in physiological buffer solutions, using the photonic force microscope (PFM). When membrane-bound secretory vesicles (0.2-1.2 μm in diameter) in live pancreatic acinar cells were trapped at the laser focus of the PFM and pulled, they were all found tethered to filamentous structures. Mild exposure of cells to nocodazole and cytochalasin B, disrupts the tether. Immunoblot analysis of isolated secretory vesicles, further demonstrated the association of actin, myosin V, and kinesin. These studies demonstrate for the first time that secretory vesicles in live pancreatic acinar cells are tethered and not free-floating, suggesting that following vesicle biogenesis, they are placed on their own railroad track, ready to be transported to their final destination within the cell when required. This makes sense, since precision and regulation are the hallmarks of all cellular process, and therefore would hold true for the transport and localization of subcellular organelles such as secretory vesicles

  12. Secretory vesicles in live cells are not free-floating but tethered to filamentous structures: A study using photonic force microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Abu-Hamdah, Rania [Department of Physiology, Wayne State University School of Medicine, 5245 Scott Hall, 540 E. Canfield, Detroit, MI 48201 (United States); Cho, Won Jin [Department of Physiology, Wayne State University School of Medicine, 5245 Scott Hall, 540 E. Canfield, Detroit, MI 48201 (United States); Hoerber, J.K.H. [Department of Physics, University of Bristol, Bristol BS8 1TD (United Kingdom); Jena, Bhanu P. [Department of Physiology, Wayne State University School of Medicine, 5245 Scott Hall, 540 E. Canfield, Detroit, MI 48201 (United States)]. E-mail: bjena@med.wayne.edu

    2006-06-15

    It is well established that actin and microtubule cytoskeletal systems are involved in organelle transport and membrane trafficking in cells. This is also true for the transport of secretory vesicles in neuroendocrine cells and neurons. It was however unclear whether secretory vesicles remain free-floating, only to associate with such cytoskeletal systems when needing transport. This hypothesis was tested using live pancreatic acinar cells in physiological buffer solutions, using the photonic force microscope (PFM). When membrane-bound secretory vesicles (0.2-1.2 {mu}m in diameter) in live pancreatic acinar cells were trapped at the laser focus of the PFM and pulled, they were all found tethered to filamentous structures. Mild exposure of cells to nocodazole and cytochalasin B, disrupts the tether. Immunoblot analysis of isolated secretory vesicles, further demonstrated the association of actin, myosin V, and kinesin. These studies demonstrate for the first time that secretory vesicles in live pancreatic acinar cells are tethered and not free-floating, suggesting that following vesicle biogenesis, they are placed on their own railroad track, ready to be transported to their final destination within the cell when required. This makes sense, since precision and regulation are the hallmarks of all cellular process, and therefore would hold true for the transport and localization of subcellular organelles such as secretory vesicles.

  13. Melanosomal dynamics assessed with a live-cell fluorescent melanosomal marker.

    Directory of Open Access Journals (Sweden)

    Jan M Bruder

    Full Text Available Melanocytes present in skin and other organs synthesize and store melanin pigment within membrane-delimited organelles called melanosomes. Exposure of human skin to ultraviolet radiation (UV stimulates melanin production in melanosomes, followed by transfer of melanosomes from melanocytes to neighboring keratinocytes. Melanosomal function is critical for protecting skin against UV radiation, but the mechanisms underlying melanosomal movement and transfer are not well understood. Here we report a novel fluorescent melanosomal marker, which we used to measure real-time melanosomal dynamics in live human epidermal melanocytes (HEMs and transfer in melanocyte-keratinocyte co-cultures. A fluorescent fusion protein of Ocular Albinism 1 (OA1 localized to melanosomes in both B16-F1 cells and HEMs, and its expression did not significantly alter melanosomal distribution. Live-cell tracking of OA1-GFP-tagged melanosomes revealed a bimodal kinetic profile, with melanosomes exhibiting combinations of slow and fast movement. We also found that exposure to UV radiation increased the fraction of melanosomes exhibiting fast versus slow movement. In addition, using OA1-GFP in live co-cultures, we monitored melanosomal transfer using time-lapse microscopy. These results highlight OA1-GFP as a specific and effective melanosomal marker for live-cell studies, reveal new aspects of melanosomal dynamics and transfer, and are relevant to understanding the skin's physiological response to UV radiation.

  14. Short-lived, transitory cell-cell interactions foster migration-dependent aggregation.

    Directory of Open Access Journals (Sweden)

    Melissa D Pope

    Full Text Available During embryonic development, motile cells aggregate into cohesive groups, which give rise to tissues and organs. The role of cell migration in regulating aggregation is unclear. The current paradigm for aggregation is based on an equilibrium model of differential cell adhesivity to neighboring cells versus the underlying substratum. In many biological contexts, however, dynamics is critical. Here, we provide evidence that multicellular aggregation dynamics involves both local adhesive interactions and transport by cell migration. Using time-lapse video microscopy, we quantified the duration of cell-cell contacts among migrating cells that collided and adhered to another cell. This lifetime of cell-cell interactions exhibited a monotonic decreasing dependence on substratum adhesivity. Parallel quantitative measurements of cell migration speed revealed that across the tested range of adhesive substrata, the mean time needed for cells to migrate and encounter another cell was greater than the mean adhesion lifetime, suggesting that aggregation dynamics may depend on cell motility instead of the local differential adhesivity of cells. Consistent with this hypothesis, aggregate size exhibited a biphasic dependence on substratum adhesivity, matching the trend we observed for cell migration speed. Our findings suggest a new role for cell motility, alongside differential adhesion, in regulating developmental aggregation events and motivate new design principles for tuning aggregation dynamics in tissue engineering applications.

  15. Synchrotron infrared spectromicroscopy as a novel bioanalytical microprobe for individual living cells: Cytotoxicity considerations

    Energy Technology Data Exchange (ETDEWEB)

    Holman, Hoi-Ying N.; Bjornstad, Kathleen A.; McNamara, Morgan P.; Martin, Michael C.; McKinney, Wayne R.; Blakely, Eleanor A.

    2001-12-12

    Synchrotron radiation-based Fourier transform infrared (SR-FTIR) spectromicroscopy is a newly emerging analytical tool capable of monitoring the biochemistry within an individual living mammalian cell in real time. This unique technique provides infrared (IR)spectra, hence chemical information, with high signal-to-noise at spatial resolutions as fine as 3 to 10 microns. Mid-IR photons are too low in energy (0.05-0.5 eV) to either break bonds or to cause ionization, and the synchrotron IR beam has been shown to produce minimal sample heating. However, an important question remains, ''Does the intense synchrotron beam induce any cytotoxic effects in living cells?'' In this work, we present the results from a series of standard biological assays to evaluate any short-and/or long-term effects on cells exposed to the synchrotron radiation-based infrared (SR-IR) beam. Cell viability was tested using alcian blue dye-exclusion and colony formation assays. Cell-cycle progression was tested with bromodeoxyuridine (BrdU) uptake during DNA synthesis. Cell metabolism was tested using an 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay. All control, 5-, 10-, and 20-minute SR-IR exposure tests (267 total and over 1000 controls) show no evidence of cytotoxic effects. Concurrent infrared spectra obtained with each experiment confirm no detectable chemistry changes between control and exposed cells.

  16. Antigen modulation of the immune response. III. Evaluation of the hypothetical short-lived memory cell

    International Nuclear Information System (INIS)

    Feldbush, T.L.; Lande, I.; Bryan, B.; O'Neill, E.

    1974-01-01

    The putative short-lived memory cells, whose existence has been suggested by the results of secondary adoptive transfer experiments, were investigated. On the basis of the following evidences we have concluded that the short-lived memory cell is probably an artifact of the adoptive transfer technique: when immune thoracic duct lymphocytes, known to consist predominantly of long-lived memory cells, were transferred to irradiated recipients and challenged at various times after transfer, approximately 80 to 90 percent of the initial response was absent by Day 14 challenge; preirradiating adoptive recipients with increasing dose of x-irradiation tended to lengthen the observed half life of memory cells; single or multiple treatments of immune donors with 0.3 mg Vinblastin before transfer resulted in neither a depression of the initial secondary response nor an alteration in the rate of decline of the memory potential; reconstitution of irradiated hosts with normal spleen cells one day before transfer of memory cells and challenge resulted in inhibition of the adoptive secondary response; and the transfer of memory cells to antigen free intermediate hosts, in which they were allowed to reside for one day or fourteen days before transfer to irradiated recipients, resulted in only a slight decline in their capacity to respond. We propose that the rapid decline of memory potential in adoptive recipients challenged at various times after transfer is due to modulating effects by the hosts as it recovers from irradiation. These effects may be the result of cell crowding or the loss of irradiation-produced stimulatory factors. The relevance of these findings to adoptive transfer systems in general and the secondary response of intact animals is discussed

  17. The magnetic introduction of magnetite nanoparticles into live cells for radiosensibility enhancement

    Energy Technology Data Exchange (ETDEWEB)

    Yurenya, Anton Y., E-mail: antonyurenya@gmail.com [National Research Center “Kurchatov Institute”, Moscow (Russian Federation); Faculty of Physics, Lomonosov Moscow State University, Moscow (Russian Federation); Polikarpov, Mikhail A. [National Research Center “Kurchatov Institute”, Moscow (Russian Federation); Chukalova, Aynur A. [National Research Center “Kurchatov Institute”, Moscow (Russian Federation); Moscow Institute of Physics and Technology, Moscow (Russian Federation); Moskaleva, Elizaveta Y.; Taldenkov, Alexander N. [National Research Center “Kurchatov Institute”, Moscow (Russian Federation); Panchenko, Vladislav Y. [National Research Center “Kurchatov Institute”, Moscow (Russian Federation); Faculty of Physics, Lomonosov Moscow State University, Moscow (Russian Federation)

    2017-04-01

    Earlier we proposed a new radiotherapy enhancement method that entails the administration of {sup 57}Fe iron-oxide nanoparticles into the cells . Within this work we were prompt to investigate the capability of iron oxide nanoparticles with monolayer coating to penetrate into live cells. Magnetite particle samples were synthesized and stabilized with HCl or citric acid. The cells were incubated in the presence of nanoparticles for 1 h, washed and dried. To distinguish inside-cell particles from outside ones a set of experiments with low temperature incubation was carried out. Several cell samples were prepared in the presence of an external magnetic field in order to study the possibility of the nanoparticle uptake enhancement. To evaluate the amount of particles in each cell sample we used a SQUID-magnetometer. The nanoparticle suspension with HCl stabilization turned to be inadequate for intracellular introduction. Approximately 2·10{sup 5} particles with citric acid covering conjugated with each cell after incubation at normal conditions. An application of an external magnetic field increased this amount up to 10{sup 7} particles/cell. Most probably much of these particles penetrated into cells. - Highlights: • Uncoated magnetite nanoparticle suspension is unusable for intracellular introduction. • Magnetite particles stabilized with citric acid penetrate into cells via endocytosis. • An application of a magnetic field enhances cellular uptake of magnetite particles. • The amount of particles in cell samples can be evaluated with a SQUID-magnetometer.

  18. Selenium Incorporated Cationic Organochalcogen: Live Cell Compatible and Highly Photostable Molecular Stain for Imaging and Localization of Intracellular DNA.

    Science.gov (United States)

    Gaur, Pankaj; Kumar, Ajay; Dey, Gourab; Kumar, Rajendra; Bhattacharyya, Shalmoli; Ghosh, Subrata

    2016-05-04

    Successful integration of selenium unit into a newly designed cationic chemical architecture led to the development of a highly photostable molecular maker PA5 to be used in fluorescence microscopy as cellular nucleus staining agent for longer duration imaging under continuous laser illumination. Adaptation of a targeted single-atom modification strategy led to the development of a series of proficient DNA light-up probes (PA1-PA5). Further, their comparative photophysical studies in the presence of DNA revealed the potential of electron rich heteroatoms of chalcogen family in improving binding efficiency and specificity of molecular probes toward DNA. The findings of cell studies confirmed the outstanding cell compatibility of probe PA5 in terms of cell permeability, biostability, and extremely low cytotoxicity. Moreover, the photostability experiment employing continuous laser illumination in solution phase as well as in cell assay (both fixed and live cells) revealed the admirable photobleaching resistance of PA5. Finally, while investigating the phototoxicity of PA5, the probe was found not to exhibit light-induced toxicity even when irradiated for longer duration. All these experimental results demonstrated the promising standing of PA5 as a futuristic cell compatible potential stain for bioimaging and temporal profiling of DNA.

  19. Uptake of silica covered Quantum Dots into living cells: Long term vitality and morphology study on hyaluronic acid biomaterials

    International Nuclear Information System (INIS)

    D'Amico, Michele; Fiorica, Calogero; Palumbo, Fabio Salvatore; Militello, Valeria; Leone, Maurizio; Dubertret, Benoit; Pitarresi, Giovanna; Giammona, Gaetano

    2016-01-01

    Quantum Dots (QDs) are promising very bright and stable fluorescent probes for optical studies in the biological field but water solubility and possible metal bio-contamination need to be addressed. In this work, a simple silica-QD hybrid system is prepared and the uptake in bovine chondrocytes living cells without any functionalization of the external protective silica shield is demonstrated. Moreover, long term treated cells vitality (up to 14 days) and the transfer of silica-QDs to the next cell generations are here reported. Confocal fluorescence microscopy was also used to determine the morphology of the so labelled cells and the relative silica-QDs distribution. Finally, we employ silica-QD stained chondrocytes to characterize, as proof of concept, hydrogels obtained from an amphiphilic derivative of hyaluronic acid (HA-EDA-C _1_8) functionalized with different amounts of the RGD peptide. - Highlights: • Non functionalized silica-quantum dots fluorescent nanoparticles uptake is observed. • Morphology studies of such cells could be done by confocal fluorescence microscopy. • Labelled chondrocytes are viable until at least 14 days. • RGD functionalized Hyaluronic Acid hydrogels are studied as cell scaffolds. • Chondrocyte are promptly attached on RGD-functionalized hydrogels.